Metabolic Reprogramming by Hexosamine Biosynthetic and. Golgi N-Glycan Branching Pathways. Michael Christopher Ryczko

Transcription

1 Metabolic Reprogramming by Hexosamine Biosynthetic and Golgi N-Glycan Branching Pathways By Michael Christopher Ryczko A thesis submitted in conformity with the requirements for the Degree of Doctor of Philosophy Graduate Department of Molecular Genetics University of Toronto Copyright by Michael Ryczko 2015

2 Metabolic Reprogramming by Hexosamine Biosynthetic and Golgi N-Glycan Branching Pathways Michael Christopher Ryczko Doctor of Philosophy Department of Molecular Genetics University of Toronto 2015 Abstract Most mammalian growth factor receptors and solute transporters are co-translationally N- glycosylated. N-glycans are branched and elongated in the Golgi, and their interaction with lectins regulates cell surface residency and activity of transmembrane glycoproteins. The Golgi branching N-acetylglucosaminyltransferases, Mgat1, 2, 4, 5 and 6, require a common donor substrate uridine-diphosphate-n-acetylglucosamine (UDP-GlcNAc) generated de novo by the hexosamine biosynthetic pathway (HBP) from glucose, glutamine and acetyl-coa, as well as from GlcNAc salvage pathway. In this thesis I describe evidence for cell-autonomous regulation of cellular metabolism by the Golgi N-glycan branching pathway. Induced expression of Mgat1, Mgat5 or Mgat6, and GlcNAc supplementation to the HBP, increased central metabolites in an additive manner. I show that UDP-GlcNAc levels are also sensitive to dietary GlcNAc supplementation in vivo, increasing nutrient uptake and promoting anabolic metabolism via the Golgi N-glycan branching pathway. Chronic oral GlcNAc supplementation in C57BL/6 mice ii

3 increased hepatic UDP-GlcNAc and N-glycan branching on liver glycoproteins. Furthermore, GlcNAc supplementation altered levels of numerous hepatic metabolites, insulin and glucagon balance, and promoted excess lipid storage, as well as body-weight increase, without affecting food intake or energy expenditure. In cultured cells, GlcNAc enhanced uptake of glucose, glutamine and fatty acids, and elevated fatty acid synthesis and storage in an N-glycan-dependent manner. Mgat5 -/- mice exhibit a lean phenotype, and oral GlcNAc rescued fat accumulation, consistent with functional redundancy of N-glycan branches. However, fat accumulation in this context was rescued at the expense of lean mass, suggesting that Mgat5 plays a role in lean to fat body composition. These results suggest that GlcNAc reprograms cellular metabolism by enhancing nutrient uptake and lipid storage through the HBP and UDP-GlcNAc supply to the Golgi N-glycan branching pathway. iii

4 There are more things in heaven and earth, Horatio, Than are dreamt of in our philosophy. Hamlet Act 1, Scene 5 William Shakespeare iv

5 Acknowledgments First I would like to thank my supervisor Jim Dennis for his support and motivation during completion of this thesis. Jim s dedication and enthusiasm for science is remarkable. I would like to thank all members of the Dennis and Swallow labs, both past and present, who have helped me during my graduate odyssey. In particular, I would like to acknowledge Judy Pawling for her moral support and excellent technical contributions. Special thanks also goes to Ryan Williams, Anita Johswich, Wendy Johnston, Carol Swallow, and Roland Xu for their friendship, moral support and advice during my graduate tenure. Since science is a collaborative endeavor, I also want to thank my collaborators: Judy Pawling, Anas M. Abdel Rahman, Rui Chan (from Daniel Figeys lab), Miyako Nakano (from Naoyuki Taniguchi s lab), Kevin Yau, Anita Johswich, Tania Rodrigues, Aldis Krizus and Cunjie Zhang, who contributed to work presented in chapters 2 and 3. I also want to thank Sean Egan and Anne-Claude Gingras for serving as my supervisory committee members. I am grateful for your input and guidance throughout my Ph.D. Last but not least, I would like to thank my beloved wife, Rubeena Khan, my dearest mom, Wandziunia Ryczko, our little potlu Goldie, and the rest of my family for unconditional love and understanding, unwavering support, and personal sacrifice. Thank you for always being there for me. I am grateful for having you in my life and I would not have been able to reach this stage without you. v

6 Table of Contents Abstract... ii Acknowledgments...v Table of Contents... vi List of Figures... xi List of Tables... xii List of Abbreviations... xiii Chapter 1. Introduction Glycobiology Glycans Glycans Synthesis and Function N-Glycosylation N-Glycan Branching and Glycan-Galectin Lattice Golgi N-Glycan Branching Pathway UDP-GlcNAc Mgat Branching Enzymes Mgat1 in N-Glycan Branching Mgat2 in N-Glycan Branching Mgat4 in N-Glycan Branching Mgat5 and β1,6-linked GlcNAc Branching Mgat6 in N-Glycan Branching Functional Redundancy of N-Glycan Branches...20 vi

7 HBP and UDP-GlcNAc Formation de novo UDP-GlcNAc Biosynthesis Fate of UDP-GlcNAc Precursor Metabolites for UDP-GlcNAc Biosynthesis HBP and UDP-GlcNAc in Insulin Resistance and Diabetes UDP-GlcNAc from Salvage Pathway GlcNAc Supplementation in vitro and in vivo GlcNAc Increases UDP-GlcNAc and β1,6-branched N-Glycans GlcNAc Supplementation for N-Glycan Compensation GlcNAc Safety and Efficacy Dietary Monosaccharide Supplementation as Therapy Metabolism Glycolysis and the Tricarboxylic Acid (TCA) Cycle Insulin, Glucagon and Liver Function in Glucose Homeostasis Liver Glycogen Feeding and Fasting Glycoprotein Receptors for Glucagon and Insulin, and Glucose Transporters Glucagon Receptor (Gcgr) Insulin Receptor (Insr) Glucose Transporters (Gluts) Nutrient Sensing and Signaling Pathways mtor AMPK Fatty Acid Synthesis...44 vii

8 Lipid Storage and Breakdown Body Weight and Obesity Body Weight Regulation Leptin Determinants of Body Weight and Obesity Energy Balance The Carbohydrate Hypothesis of Obesity Rationale, Objectives and Summary...53 Chapter 2. Golgi N-Glycan Branching N-Acetylglucosaminyltransferases I, V and VI Promote Nutrient Uptake and Metabolism Summary Introduction Materials and Methods Materials and Chemicals Cell Culture Western Blotting Enzyme Assays Quantitative Lectin Fluorescence Imaging Cell Viability Assay Cell Membrane Preparation for N-Glycan Profiling Enzymatic Release and Purification of N-Glycans LC-ESI MS for Analysis of N-Glycan Alditols Metabolite Analysis by LC-MS/MS LC-MS/MS Measurement of 15 N 15 N-Glutamine Uptake and Metabolism Results...70 viii

9 Tet-Inducible Expression of Mgat1, Mgat5 and Mgat6 in Human Cells Tet-Inducible Mgat1, Mgat5 and Mgat6 N-Glycan Branching Regulation of Cellular Metabolite Levels by Mgat1, Mgat5, Mgat6 and HBP Tet-Inducible Mgat5 Enhances Amino Acid Uptake and Growth in Nutrient-Poor Conditions Discussion Mgat6 Enhances Functionality of N-Glycan Branching in Mammalian cells Mgat5 Enhances Metabolism Under Glutamine-Deprived Conditions...79 Chapter 3. Metabolic Reprogramming by the Hexosamine Biosynthetic Pathway and Golgi N-Glycan Branching Summary Introduction Materials and Methods Chemicals and Materials Mice Phenotyping in vivo Targeted Metabolomics Biochemical Studies and Histology Site-Specific Characterization of Hepatic N-Glycosylation Cell Culture Statistical Analysis Results Oral GlcNAc Supplementation Alters Liver Metabolism Oral GlcNAc Increases Body-Weight Without Increasing Food Consumption Oral GlcNAc Increases Lipid Accumulation and Catabolism ix

10 Oral GlcNAc Does Not Alter Physical Activity or Energy Expenditure Oral GlcNAc Reprograms Fasting Metabolism Oral GlcNAc Increases Complex N-Glycan Branching in Liver Glycoproteins GlcNAc Increases Nutrient Uptake and Lipid Accumulation in Cultured Cells Oral GlcNAc Partially Restores Anabolic Metabolism in Mgat5 -/- Mice Discussion Chapter 4. Discussion and Future Directions GlcNAc and N-Glycan Branching in Mgat5 Null Mice HBP and N-Glycan Branching Reprogram Metabolism to Promote Fat Accumulation Consequences of Long-Term Daily Oral GlcNAc Intake GlcNAc Salvage into HBP GlcNAc Supply and Increased N-Glycan Branching Promote Fat Accumulation Increased HBP Activity Promotes Fat Accumulation HBP and the Thrifty Genotype/Phenotype Hypothesis N-Glycan Branching and Nutrient Uptake HBP Interacts with Gene Polymorphisms in N-Glycan Branching Pathway GlcNAc Supplementation and O-GlcNAcylation GlcNAc Supplementation and Gut Microbiota References x

11 List of Figures Figure 1.1 Mgats and Their N-Glycan Branches...8 Figure 1.2 Mgat5 in N-Glycan Branching...8 Figure 1.3 Glycan-Galectin Lattice Dynamics...10 Figure 1.4 N-Glycan Branching Pathway...11 Figure 1.5 Hexosamine Pathway for Biosynthesis of UDP-GlcNAc...23 Figure 1.6 Glucose Production in Liver During Fasting...38 Figure 2.1 Branching pathway and inducible expression of branching enzymes...81 Figure 2.2 N-glycan profiles of transgenic Flp-In-TREx HeLa cells...82 Figure 2.3 Figure 2.4 Figure 2.5 Figure 2.6 N-glycan profiles of transgenic Flp-In-TREx Hek293 cells by LC-ESI MS...84 Metabolite levels are sensitive to tet-induced branching and HBP stimulation...86 Growth of Mgat5 Flp-In-TREx Hek293 cells in defined Glc and Gln conditions...88 Amino acid levels increase with Mgat5 expression under Gln-deprived conditions...89 Figure 2.7 Tet-induced Mgat5 increases TCA cycle intermediates...91 Figure 2.8 Figure 2.9 Tet-induced Mgat5 increases HBP and glycolysis metabolites under Gln/Glc limiting conditions...92 Summary of metabolite changes with tet-induced Mgat5 in Hek293 cells under low Gln/Glc conditions...93 Figure 2.10 Tet-induced Mgat5 branching stimulates Gln uptake...94 Figure 3.1 Oral GlcNAc is rapidly absorbed by gut to enter bloodstream and be taken up by tissue from circulation xi

12 Figure 3.2 Oral GlcNAc increases UDP-GlcNAc level and promotes weight-gain in mice Figure 3.3 Oral GlcNAc promotes weight-gain and lipid accumulation Figure 3.4 Oral GlcNAc promotes fatty acid oxidation without affecting activity or energy expenditure Figure 3.5 Oral GlcNAc alters fasting liver metabolism Figure 3.6 Figure 3.7 Oral GlcNAc increases tri-antennary N-glycan structures on glycosite Asn 89 of CEACAM1 hepatic transmembrane glycoprotein GlcNAc increases UDP-GlcNAc, β-1,6-glcnac branched N-glycans and lipid accumulation Figure 3.8 HBP and N-glycan dependent regulation of cellular metabolism Figure 3.9 Oral GlcNAc promotes lipid storage in male and female Mgat5 wild-type and null mice List of Tables Table 3.1 Phenotypic differences in serum biochemistry between GlcNAc treated and untreated mice on 9% fat diet, under fasted and fed conditions Table 3.2 Global analysis of relative GlcNAc content in liver N-glycans xii

13 List of Abbreviations 3&2-PG 3- and 2-Phosphoglycerate ACC Acetyl-CoA Carboxylase A-CoA Acetyl-CoA Akt Protein Kinase B ALT Alanine Aminotransferase AMPK AMP-Activated Protein Kinase ANOVA Analysis of Variance Asn Asparagine ATP Adenosine Triphosphate AUC Area Under the Curve BMI Body-Mass Index CDG Congenital Disorders of Glycosylation CEACAM1 Carcinoembryonic Antigen-Related Cell Adhesion Molecule 1 ConA Concanavalin A DEXA Dual-Energy X-Ray Absorptiometry DHAP Dihydroxyacetone Phosphate DNA Deoxyribonucleic Acid EE Energy Expenditure EGFR Epidermal Growth Factor Receptor ER Endoplasmic Reticulum FA Fatty Acid FASN Fatty Acid Synthase FFA Free Fatty Acid Fru-1,6BP Fructose-1,6-Bisphosphate Fru-6P Fructose-6-Phosphate Fuc Fucose GAG Glycosaminoglycans Gal Galactose GALE UDP-Galactose-4-Epimerase xiii

14 GalNAc N-acetylgalactosamine Gcgr Glucagon Receptor GFAT Glutamine-Fructose 6-Phosphate Amidotransferase Glc Glucose Glc-6P Glucose-6-Phosphate GlcNAc N-acetylglucosamine GlcNAc-P N-acetylglucosamine-Phosphate Gln Glutamine Glu Glutamate Glut Glucose Transporter GNE UDP-GlcNAc-2-Epimerase/N-Acetylmannosamine Kinase GNPNAT1 Glucosamine-6-Phosphate Acetyltransferase GS Glycogen Synthase GSH Glutathione (reduced) GSSG Glutathione (oxidized) HBP Hexosamine Biosynthetic Pathway I/G Insulin to Glucagon Ratio Insr Insulin Receptor IL-3 Interleukin 3 Iso/Leu Isoleucine/Leucine LC-MS/MS Liquid Chromatography Tandem Mass Spectrometry L-PHA Lectin Phaseolus Vulgaris Leukoagglutinatin m/z Mass to Charge Ratio Man Mannose ManNAc N-Acetylmannosamine MFI Mean Fluorescent Intensity Mgat Mannosyl α-1,6-glycoprotein N-acetylglucosaminyltransferase MMTV Murine Mammary Tumor Virus MRI Magnetic Resonance Imaging MS Mass Spectrometry mtorc1 Mammalian target of rapamycin complex 1 xiv

15 mtorc2 Mammalian target of rapamycin complex 2 NAD+ Nicotinamide Adenine Dinucleotide (oxidized) NADH Nicotinamide Adenine Dinucleotide (reduced) NADP+ Nicotinamide Adenine Dinucleotide Phosphate (oxidized) NAGK N-Acetylglucosamine Kinase NeuAc N-Acetylneuraminic Acid N-X-S/T Asparagine-X-Serine/Threonine sequon OAA Oxaloacetate p Phosphate PAGE Polyacrylamide Gel Electrophoresis PC Principle Component PCA Principal Component Analysis PEP Phosphophenolpyruvic Acid PGM3 Phosphoacetyl Glucosamine Mutase Phe Phenylalanine PI3K Phosphoinositide-3 Kinase Pro Proline Pten Phosphatase and Tensin Homolog PyMT Polyoma Virus Middle T Oncoprotein Ras Rat sarcoma oncogene RER Respiratory Exchange Ratio rer Rough Endoplasmic Reticulum S6 Ribosomal Protein S6 SEM Standard Error of the Mean Ser Serine Sia Sialic Acid Slc Solute Carrier Protein SNP Single Nucleotide Polymorphism SREBP Sterol regulatory element-binding protein SW Swainsonine TCA cycle Tricarboxylic Acid Cycle xv

16 Tet TG TGF-β Thr Trp Tyr UAP1 UDP UDP-GalNAc UDP-Glc UDP-GlcNAc UMP UTP VCO2 VO2 wt Tetracycline Triglycerides Transforming Growth Factor Beta Threonine Tryptophan Tyrosine UDP-N-Acetylglucosamine Pyrophosphorylase Uridine-diphosphate Uridine-Diphosphate N-Acetylgalactosamine Uridine-Diphosphate Glucose Uridine-Diphosphate N-Acetylglucosamine Uridine Monophosphate Uridine Triphosphate Volume of Carbon Dioxide Volume of Oxygen Wild-Type xvi

17 Metabolic Reprogramming by Hexosamine Biosynthetic and Golgi N-Glycan Branching Pathways By Michael Christopher Ryczko A thesis submitted in conformity with the requirements for the Degree of Doctor of Philosophy Graduate Department of Molecular Genetics University of Toronto Copyright by Michael Ryczko 2015

18 Metabolic Reprogramming by Hexosamine Biosynthetic and Golgi N-Glycan Branching Pathways Michael Christopher Ryczko Doctor of Philosophy Department of Molecular Genetics University of Toronto 2015 Abstract Most mammalian growth factor receptors and solute transporters are co-translationally N- glycosylated. N-glycans are branched and elongated in the Golgi, and their interaction with lectins regulates cell surface residency and activity of transmembrane glycoproteins. The Golgi branching N-acetylglucosaminyltransferases, Mgat1, 2, 4, 5 and 6, require a common donor substrate uridine-diphosphate-n-acetylglucosamine (UDP-GlcNAc) generated de novo by the hexosamine biosynthetic pathway (HBP) from glucose, glutamine and acetyl-coa, as well as from GlcNAc salvage pathway. In this thesis I describe evidence for cell-autonomous regulation of cellular metabolism by the Golgi N-glycan branching pathway. Induced expression of Mgat1, Mgat5 or Mgat6, and GlcNAc supplementation to the HBP, increased central metabolites in an additive manner. I show that UDP-GlcNAc levels are also sensitive to dietary GlcNAc supplementation in vivo, increasing nutrient uptake and promoting anabolic metabolism via the Golgi N-glycan branching pathway. Chronic oral GlcNAc supplementation in C57BL/6 mice ii

19 increased hepatic UDP-GlcNAc and N-glycan branching on liver glycoproteins. Furthermore, GlcNAc supplementation altered levels of numerous hepatic metabolites, insulin and glucagon balance, and promoted excess lipid storage, as well as body-weight increase, without affecting food intake or energy expenditure. In cultured cells, GlcNAc enhanced uptake of glucose, glutamine and fatty acids, and elevated fatty acid synthesis and storage in an N-glycan-dependent manner. Mgat5 -/- mice exhibit a lean phenotype, and oral GlcNAc rescued fat accumulation, consistent with functional redundancy of N-glycan branches. However, fat accumulation in this context was rescued at the expense of lean mass, suggesting that Mgat5 plays a role in lean to fat body composition. These results suggest that GlcNAc reprograms cellular metabolism by enhancing nutrient uptake and lipid storage through the HBP and UDP-GlcNAc supply to the Golgi N-glycan branching pathway. iii

20 Acknowledgments First I would like to thank my supervisor Jim Dennis for his support and motivation during completion of this thesis. Jim s dedication and enthusiasm for science is remarkable. I would like to thank all members of the Dennis and Swallow labs, both past and present, who have helped me during my graduate odyssey. I would like to particularly acknowledge Judy Pawling for her scientific contributions and moral support. Special thanks also goes to Ryan Williams, Anita Johswich, Wendy Johnston, Carol Swallow, and Roland Xu for their friendship, moral support and advice during my graduate tenure. Since science is a collaborative endeavor, I also want to thank my scientific collaborators: Judy Pawling, Anas Abdel Rahman, Rui Chan (from Daniel Figeys lab), Miyako Nakano (from Naoyuki Taniguchi s lab), Kevin Yau, Anita Johswich, Tania Rodrigues, Aldis Krizus and Cunjie Zhang, who contributed to work presented in chapters 2 and 3. I also want to thank Sean Egan and Anne-Claude Gingras for serving as my supervisory committee members. I am grateful for your comments, input and guidance throughout my Ph.D. Last but not least, I would like to thank my beloved wife, Rubeena Khan, my dearest mom, Wandziunia Ryczko, Goldie little potlu, and the rest of my family for unconditional love and understanding, unwavering support, and personal sacrifice. Thank you for always being there for me. I am grateful for having you in my life and I would not have been able to reach this stage without you. iv

21 Table of Contents Abstract... ii Acknowledgments... iv Table of Contents...v List of Figures...x List of Tables... xi List of Abbreviations... xii Chapter 1. Introduction Glycobiology Glycans Glycans Synthesis and Function N-Glycosylation N-Glycan Branching and Glycan-Galectin Lattice Golgi N-Glycan Branching Pathway UDP-GlcNAc Mgat Branching Enzymes Mgat1 in N-Glycan Branching Mgat2 in N-Glycan Branching Mgat4 in N-Glycan Branching Mgat5 and β1,6-linked GlcNAc Branching Mgat6 in N-Glycan Branching Functional Redundancy of N-Glycan Branches...20 v

22 HBP and UDP-GlcNAc Formation de novo UDP-GlcNAc Biosynthesis Fate of UDP-GlcNAc Precursor Metabolites for UDP-GlcNAc Biosynthesis HBP and UDP-GlcNAc in Insulin Resistance and Diabetes UDP-GlcNAc from Salvage Pathway GlcNAc Supplementation in vitro and in vivo GlcNAc Increases UDP-GlcNAc and β1,6-branched N-Glycans GlcNAc Supplementation for N-Glycan Compensation GlcNAc Safety and Efficacy Dietary Monosaccharide Supplementation as Therapy Metabolism Glycolysis and the Tricarboxylic Acid (TCA) Cycle Insulin, Glucagon and Liver Function in Glucose Homeostasis Liver Glycogen Feeding and Fasting Glycoprotein Receptors for Glucagon and Insulin, and Glucose Transporters Glucagon Receptor (Gcgr) Insulin Receptor (Insr) Glucose Transporters (Gluts) Nutrient Sensing and Signaling Pathways mtor AMPK Fatty Acid Synthesis...44 vi

23 Lipid Storage and Breakdown Body Weight and Obesity Body Weight Regulation Leptin Determinants of Body Weight and Obesity Energy Balance The Carbohydrate Hypothesis of Obesity Rationale, Objectives and Summary...53 Chapter 2. Golgi N-Glycan Branching N-Acetylglucosaminyltransferases I, V and VI Promote Nutrient Uptake and Metabolism Summary Introduction Materials and Methods Materials and Chemicals Cell Culture Western Blotting Enzyme Assays Quantitative Lectin Fluorescence Imaging Cell Viability Assay Cell Membrane Preparation for N-Glycan Profiling Enzymatic Release and Purification of N-Glycans LC-ESI MS for Analysis of N-Glycan Alditols Metabolite Analysis by LC-MS/MS LC-MS/MS Measurement of 15 N 15 N-Glutamine Uptake and Metabolism Results...70 vii

24 Tet-Inducible Expression of Mgat1, Mgat5 and Mgat6 in Human Cells Tet-Inducible Mgat1, Mgat5 and Mgat6 N-Glycan Branching Regulation of Cellular Metabolite Levels by Mgat1, Mgat5, Mgat6 and HBP Tet-Inducible Mgat5 Enhances Amino Acid Uptake and Growth in Nutrient-Poor Conditions Discussion Mgat6 Enhances Functionality of N-Glycan Branching in Mammalian cells Mgat5 Enhances Metabolism Under Glutamine-Deprived Conditions...79 Chapter 3. Metabolic Reprogramming by the Hexosamine Biosynthetic Pathway and Golgi N-Glycan Branching Summary Introduction Materials and Methods Chemicals and Materials Mice Phenotyping in vivo Targeted Metabolomics Biochemical Studies and Histology Site-Specific Characterization of Hepatic N-Glycosylation Cell Culture Statistical Analysis Results Oral GlcNAc Supplementation Alters Liver Metabolism Oral GlcNAc Increases Body-Weight Without Increasing Food Consumption Oral GlcNAc Increases Lipid Accumulation and Catabolism viii

25 Oral GlcNAc Does Not Alter Physical Activity or Energy Expenditure Oral GlcNAc Reprograms Fasting Metabolism Oral GlcNAc Increases Complex N-Glycan Branching in Liver Glycoproteins GlcNAc Increases Nutrient Uptake and Lipid Accumulation in Cultured Cells Oral GlcNAc Partially Restores Anabolic Metabolism in Mgat5 -/- Mice Discussion Chapter 4. Discussion and Future Directions GlcNAc and N-Glycan Branching in Mgat5 Null Mice HBP and N-Glycan Branching Reprogram Metabolism to Promote Fat Accumulation Consequences of Long-Term Daily Oral GlcNAc Intake GlcNAc Salvage into HBP GlcNAc Supply and Increased N-Glycan Branching Promote Fat Accumulation Increased HBP Activity Promotes Fat Accumulation HBP and the Thrifty Genotype/Phenotype Hypothesis N-Glycan Branching and Nutrient Uptake HBP Interacts with Gene Polymorphisms in N-Glycan Branching Pathway GlcNAc Supplementation and O-GlcNAcylation GlcNAc Supplementation and Gut Microbiota References ix

26 List of Figures Figure 1.1 Mgats and Their N-Glycan Branches...8 Figure 1.2 Mgat5 in N-Glycan Branching...8 Figure 1.3 Glycan-Galectin Lattice Dynamics...10 Figure 1.4 N-Glycan Branching Pathway...11 Figure 1.5 Hexosamine Pathway for Biosynthesis of UDP-GlcNAc...23 Figure 1.6 Glucose Production in Liver During Fasting...38 Figure 2.1 Branching pathway and inducible expression of branching enzymes...81 Figure 2.2 N-glycan profiles of transgenic Flp-In-TREx HeLa cells...82 Figure 2.3 Figure 2.4 Figure 2.5 Figure 2.6 N-glycan profiles of transgenic Flp-In-TREx Hek293 cells by LC-ESI MS...84 Metabolite levels are sensitive to tet-induced branching and HBP stimulation...86 Growth of Mgat5 Flp-In-TREx Hek293 cells in defined Glc and Gln conditions...88 Amino acid levels increase with Mgat5 expression under Gln-deprived conditions...89 Figure 2.7 Tet-induced Mgat5 increases TCA cycle intermediates...91 Figure 2.8 Figure 2.9 Tet-induced Mgat5 increases HBP and glycolysis metabolites under Gln/Glc limiting conditions...92 Summary of metabolite changes with tet-induced Mgat5 in Hek293 cells under low Gln/Glc conditions...93 Figure 2.10 Tet-induced Mgat5 branching stimulates Gln uptake...94 Figure 3.1 Oral GlcNAc is rapidly absorbed by gut to enter bloodstream and be taken up by tissue from circulation x

27 Figure 3.2 Oral GlcNAc increases UDP-GlcNAc level and promotes weight-gain in mice Figure 3.3 Oral GlcNAc promotes weight-gain and lipid accumulation Figure 3.4 Oral GlcNAc promotes fatty acid oxidation without affecting activity or energy expenditure Figure 3.5 Oral GlcNAc alters fasting liver metabolism Figure 3.6 Figure 3.7 Oral GlcNAc increases tri-antennary N-glycan structures on glycosite Asn 89 of CEACAM1 hepatic transmembrane glycoprotein GlcNAc increases UDP-GlcNAc, β-1,6-glcnac branched N-glycans and lipid accumulation Figure 3.8 HBP and N-glycan dependent regulation of cellular metabolism Figure 3.9 Oral GlcNAc promotes lipid storage in male and female Mgat5 wild-type and null mice List of Tables Table 3.1 Phenotypic differences in serum biochemistry between GlcNAc treated and untreated mice on 9% fat diet, under fasted and fed conditions Table 3.2 Global analysis of relative GlcNAc content in liver N-glycans xi

28 List of Abbreviations 3&2-PG 3- and 2-Phosphoglycerate ACC Acetyl-CoA Carboxylase A-CoA Acetyl-CoA Akt Protein Kinase B ALT Alanine Aminotransferase AMPK AMP-Activated Protein Kinase ANOVA Analysis of Variance Asn Asparagine ATP Adenosine Triphosphate AUC Area Under the Curve BMI Body-Mass Index CDG Congenital Disorders of Glycosylation CEACAM1 Carcinoembryonic Antigen-Related Cell Adhesion Molecule 1 ConA Concanavalin A DEXA Dual-Energy X-Ray Absorptiometry DHAP Dihydroxyacetone Phosphate DNA Deoxyribonucleic Acid EE Energy Expenditure EGFR Epidermal Growth Factor Receptor ER Endoplasmic Reticulum FA Fatty Acid FASN Fatty Acid Synthase FFA Free Fatty Acid Fru-1,6BP Fructose-1,6-Bisphosphate Fru-6P Fructose-6-Phosphate Fuc Fucose GAG Glycosaminoglycans Gal Galactose GALE UDP-Galactose-4-Epimerase xii

29 GalNAc N-acetylgalactosamine Gcgr Glucagon Receptor GFAT Glutamine-Fructose 6-Phosphate Amidotransferase Glc Glucose Glc-6P Glucose-6-Phosphate GlcNAc N-acetylglucosamine GlcNAc-P N-acetylglucosamine-Phosphate Gln Glutamine Glu Glutamate Glut Glucose Transporter GNE UDP-GlcNAc-2-Epimerase/N-Acetylmannosamine Kinase GNPNAT1 Glucosamine-6-Phosphate Acetyltransferase GS Glycogen Synthase GSH Glutathione (reduced) GSSG Glutathione (oxidized) HBP Hexosamine Biosynthetic Pathway I/G Insulin to Glucagon Ratio Insr Insulin Receptor IL-3 Interleukin 3 Iso/Leu Isoleucine/Leucine LC-MS/MS Liquid Chromatography Tandem Mass Spectrometry L-PHA Lectin Phaseolus Vulgaris Leukoagglutinatin m/z Mass to Charge Ratio Man Mannose ManNAc N-Acetylmannosamine MFI Mean Fluorescent Intensity Mgat Mannosyl α-1,6-glycoprotein N-acetylglucosaminyltransferase MMTV Murine Mammary Tumor Virus MRI Magnetic Resonance Imaging MS Mass Spectrometry mtorc1 Mammalian target of rapamycin complex 1 xiii

30 mtorc2 Mammalian target of rapamycin complex 2 NAD+ Nicotinamide Adenine Dinucleotide (oxidized) NADH Nicotinamide Adenine Dinucleotide (reduced) NADP+ Nicotinamide Adenine Dinucleotide Phosphate (oxidized) NAGK N-Acetylglucosamine Kinase NeuAc N-Acetylneuraminic Acid N-X-S/T Asparagine-X-Serine/Threonine sequon OAA Oxaloacetate p Phosphate PAGE Polyacrylamide Gel Electrophoresis PC Principle Component PCA Principal Component Analysis PEP Phosphophenolpyruvic Acid PGM3 Phosphoacetyl Glucosamine Mutase Phe Phenylalanine PI3K Phosphoinositide-3 Kinase Pro Proline Pten Phosphatase and Tensin Homolog PyMT Polyoma Virus Middle T Oncoprotein Ras Rat sarcoma oncogene RER Respiratory Exchange Ratio rer Rough Endoplasmic Reticulum S6 Ribosomal Protein S6 SEM Standard Error of the Mean Ser Serine Sia Sialic Acid Slc Solute Carrier Protein SNP Single Nucleotide Polymorphism SREBP Sterol regulatory element-binding protein SW Swainsonine TCA cycle Tricarboxylic Acid Cycle xiv

31 Tet TG TGF-β Thr Trp Tyr UAP1 UDP UDP-GalNAc UDP-Glc UDP-GlcNAc UMP UTP VCO2 VO2 wt Tetracycline Triglycerides Transforming Growth Factor Beta Threonine Tryptophan Tyrosine UDP-N-Acetylglucosamine Pyrophosphorylase Uridine-diphosphate Uridine-Diphosphate N-Acetylgalactosamine Uridine-Diphosphate Glucose Uridine-Diphosphate N-Acetylglucosamine Uridine Monophosphate Uridine Triphosphate Volume of Carbon Dioxide Volume of Oxygen Wild-Type xv

32 Chapter 1. Introduction 1

33 1.1 Glycobiology Glycans Along with nucleic acids, proteins and lipids, glycans are one of the four major macromolecules in all living organism (Marth, 2008). Biological systems and their complexity emerge from the layered functional interaction of the genome, proteome, lipidome, glycome, and their biosynthesis from small-molecule metabolites. Glycans are oligosaccharides or polysaccharides consisting of a large number of different monosaccharides ligated via a glycosidic bond, and comprising the carbohydrate portion of glycoconjugates such as glycoproteins, glycolipids and proteoglycans (Varki, 1993). The potential structural diversity of glycans in mammalian cells is very large, due to the possible combination of monosaccharide constituents, anomeric bond configurations, different linkages formed, and extent of branching (Ohtsubo & Marth, 2006). Approximately 5% of the genome encodes proteins involved in glycan biosynthesis, degradation and recognition (2009). Glycosylation is the enzyme-catalyzed covalent attachment of glycans to a polypeptide or other organic compound, typically catalyzed by glycosyltransferases utilizing specific activated sugar-nucleotide donor substrates. The vast majority of cell surface proteins are glycosylated. Indeed, the surface of eukaryotic cells is covered with a dense coating of a myriad sugar chains or glycans covalently attached to either membrane proteins or lipids, giving rise to the glycocalyx, a complex network of glycoconjugates (Varki, 1993). Glycan synthesis depends on monosaccharides converted from other types of sugars inside the cells, salvaged from degraded glycoconjugates, or transported into the cell from extracellular environment (Du et al, 2009). The most abundant monosaccharides found in glycans are glucose (Glc), galactose (Gal), mannose (Man), N-acetylglucosamine (GlcNAc), N-acetylgalactosamine (GalNAc), sialic acid (Sia), and fucose (Fuc) (Ohtsubo & 2

34 Marth, 2006). The extent of glycan biosynthesis is controlled by enzyme concentrations, as well as the availability of precursor metabolites and activated donor substrates (Du et al, 2009). Consequently, glycan biosynthesis is very sensitive to the nutrient status of a cell Glycan Synthesis and Function Unlike the biosynthesis of nucleic acids or proteins, glycan formation is not a deterministic template-dependent process, but rather a stochastic event resulting from the cooperative and competitive interaction of enzymes, transporters and substrates available (Lauc et al, 2010). Since glycans are generated through combinatorial action of different glycosidases and glycosyltransferases in the endoplasmic reticulum (ER) and the Golgi apparatus, their final structure depends on the level of enzyme expression and activity, acceptor accessibility, and substrate availability (Schachter, 1986). Thus, glycan biosynthesis is dynamically and reciprocally linked to both an organism s genome and its environment, which affect directly the enzymatic processes, or indirectly the induction of epigenetic changes that modify gene expression. This makes glycan biosynthesis sensitive to environmental conditions. Moreover, unlike the linearity of DNA and protein, glycan branching, length and diversity of secondary modifications of monosaccharides confers on them an extra degree of variation and unsurpassed structural complexity (Ohtsubo & Marth, 2006). The broad repertoire of biological functions of glycans can be categorized as having intramolecular and intermolecular functions. Intermolecular functions include regulation of processes dependent on carbohydrate binding proteins, regulation of cell-tocell contact and cell-matrix adhesion, and even interaction with pathogenic molecules (Ohtsubo & Marth, 2006). Intramolecular functions can influence the properties of glycoproteins such as their activity, biological half-life, thermal stability, and protease sensitivity (Ohtsubo & Marth, 2006). 3

35 Additional intramolecular functions of glycans include regulation and monitoring of folding in the ER, and maintenance of three-dimensional conformation (Ohtsubo & Marth, 2006). Glycan complexity increases with vertebrate phylogeny, suggesting involvement in multicellular development and morphogenesis, i.e. the flow and exchange of information required to transform a collection of cells into a coherent society of different interacting components (Ohtsubo & Marth, 2006). A large-scale N-glycoproteomic study revealed that more than 10% of the mouse proteome is N-glycosylated (Zielinska et al, 2010). Intracellularly, glycans within the secretory pathway regulate protein maturation, quality control, turnover, and trafficking of molecules to organelles (Ohtsubo & Marth, 2006). The abundance, diversity and ubiquity of N- glycan structures at the cell surface suggest a significant role for encoded information that is distinct from the genome. Cell surface N-glycans on glycoproteins serve as ligands for a number of evolutionarily conserved carbohydrate-binding protein families, such as C-type lectins, galectins, and siglecs, whose function is to decipher biological information conveyed by the vast array of N-glycans (Dennis et al, 2009). Functional interactions of galectins with cell surface glycoconjugates can modulate cellular functions such as cell signaling and cell adhesion (Dennis et al, 2009). Located at the interface of the inside and outside of the cell, N-glycans are perfectly positioned to interact with the extracellular environment of the cell, and to mediate a variety of recognition events and biological processes in health and disease through protein-glycan interactions (Dennis et al, 2009). Changes in patters of cell surface N-glycans often accompany development of cancer and metastasis. In fact, altered glycosylation, either increase or decrease of specific N-glycans, or the appearance of glycans normally restricted to embryonic expression, is frequently observed in tumours (Dennis et al, 2009). These structural alterations are often the result of changes in expression and activity of glycosyltransferases, and their substrate availability. 4

36 1.1.3 N-Glycosylation N-linked glycosylation occurs through co- and post-translational modification of membrane and secreted glycoproteins in eukaryotic cells. N-glycans are attached to the nitrogen atom of asparagine (Asn) residues in the peptide consensus sequon Asn-X-Ser/Thr, where X corresponds to any amino acid except Pro (Dennis et al, 2009). The presence of a sequon is necessary but not sufficient for N-glycosylation to occur, as some sequons are not glycosylated (Zielinska et al, 2010). Thus, when Asn-X-Ser/Thr motifs are present in the amino acid sequence of a protein they are not identified categorically as N-glycan sites, but rather are referred to as potential N-glycan sites. The enzyme oligosaccharyltransferase initiates protein N-glycosylation by transferring a pre-assembled sugar oligosaccharide from dolichol to a nascent protein. Factors influencing oligosaccharyltransferase catalysis include availability of precursors, enzyme activity, the number of sequons in a glycopeptide, and their conformational accessibility (Schachter, 1986). N-glycosylation is catalyzed by a series of enzymes functioning in a sequential and competitive manner in the rough ER (rer) and Golgi apparatus, where it assumes an assemblyline style of manufacturing (Schachter, 2010). Different glycosyltransferases reside in different compartments of the Golgi, where they act in a specific order during glycoprotein transit through the secretory pathway (Dennis et al, 2009). Moreover, many glycosyltransferases are expressed in a tissue and time specific manner (Ohtsubo & Marth, 2006). Glycosyltransferases are single-pass type II integral membrane proteins with a short cytoplasmic amino terminal domain, a transmembrane anchor domain, a Proline-rich neck or stem region, and carboxyl terminal catalytic domain (Schachter, 2010). Glycosyltransferases use activated high-energy sugar-nucleotides as donor substrates, attaching them to polypeptides or to the growing glycan chain (Ohtsubo & Marth, 5

37 2006). N-linked glycosylation is required for proper function of numerous glycoproteins. For instance, cell surface delivery and retention of receptors and nutrient transporters depends on their N-glycan branching, which is both under genetic and metabolic control (Dennis et al, 2009). The extent of N-glycan branching is dependent on the expression and kinetics of medial Golgi enzymes - the mannosidases and acetylglucosaminyltransferases, the metabolic flux through the hexosamine biosynthetic pathway (HBP) to generate the high-energy sugar-nucleotide donor uridine-diphosphate-n-acetylglucosamine (UDP-GlcNAc), as well as protein synthesis rate and availability of glycoprotein acceptors (Dennis et al, 2009). N-glycosylation of proteins starts with an en block transfer of pre-assembled lipid-carrierlinked Glc3Man9GlcNAc2 donor to Asn residue in receptive Asn-X-Ser/Thr sequons of nascent proteins by oligosaccharyltransferase in the lumen of rer (Schachter, 2010). The transferred preassembled Asn-linked glycan is then extensively remodeled by removal and addition of various monosaccharides during transition through the rer and Golgi apparatus en route to the cell surface (Dennis et al, 2009). Remodeling, or lack thereof, results in formation of three main types of N- linked glycans on mature glycoproteins: high-mannose, hybrid, and complex (Schachter, 2010). All N-glycans contain a single conserved core structure consisting of two GlcNAc residues (chitobiose core), and three Man residues (tri-mannosyl core), forming a common penta-saccharide (Man3GlcNAc2) core structure (Schachter, 2010). This core is further linked to other sugars to form a variety of different branched N-glycans. UDP-GlcNAc:dolichyl-phosphate GlcNAc-1- phosphate transferase (GNPTA/DPAGT) is the enzyme in the first committed step of the dolichollinked oligosaccharide pathway for N-glycan biosynthesis to utilize UDP-GlcNAc as a substrate (Schachter, 2010). This initial step can be blocked by the drug tunicamycin, an analog of UDP- GlcNAc that inhibits GNPTA (Schachter, 2010). 6

38 In metazoans, trimming is performed by glucosidases and mannosidases, while extension is carried out by Golgi N-acetylglucosaminyltransferases (Mgats) (Dennis et al, 2009). The highmannose structures are formed from the oligosaccharide precursor by α-mannosidase-mediated cleavage of Man residues, without addition of any other monosaccharides (Schachter, 2010). Complex-type N-glycans are formed by successive removal and addition of monosaccharides, and are characterized according to the number of GlcNAc branches attached to terminal Man residues on the core penta-saccharide structure (Taniguchi & Korekane, 2011). Depending on the number of branches attached, complex-type N-glycans are subdivided into bi-, tri-, tetra-, and pentaantennary structures. The hybrid-type N-glycans share structural features found in both highmannose and complex-type N-glycans (Schachter, 2010) N-Glycan Branching and Glycan-Galectin Lattice Biosynthesis and branching of complex N-glycans proceeds via linkage of GlcNAc by mannosyl glycoprotein N-acetylglucosaminyltransferase (Mgat) enzymes, also known as GlcNActransferases, to the conserved core Man residues (Dennis et al, 2009). Each Mgat transfers GlcNAc in a specific linkage (Taniguchi & Korekane, 2011) (Figure 1.1). GlcNAc branches in turn are further extended through sequential addition of Gal and other monosaccharides to complete elongation of glycan antennas that act as ligands for galectins (Dennis et al, 2009) (Figure 1.2). Galectins are a family of secreted proteins. Most of them are bivalent or multivalent with regard to their carbohydrate-binding activities (Dennis et al, 2009). Membrane glycoproteins carrying N- glycan branches offer binding sites for galectins to facilitate transmembrane glycoprotein crosslinking, thus forming a glycan-galectin lattice that regulates cell surface residency and activity of numerous receptors, cell adhesion proteins, and solute transporters (Dennis et al, 2009) 7

39 Figure 1.1 Mgats and Their N-Glycan Branches An idealized N-glycan showing different possible branches, with specific linkages and the glycosyltransferase enzymes (Mgat1, Mgat2, Mgat3, Mgat4, Mgat5 and Mgat6) responsible for their formation. Each GlcNAc branch may be elongated with galactose, poly-nacetyllactosamine, sialic acid and fucose (Taniguchi & Korekane, 2011). Figure 1.2 Mgat5 in N-Glycan Branching The branched N-glycans attached at N-X-S/T sequons in mammalian glycoproteins. Mono, bi, tri, and tetra refer to the number of branches in an N-glycan. The glycosyltransferase Mgat5 catalyses the addition of a β1,6-linked GlcNAc branch (green arrows) to form tri- or tetraantennary N-glycans, the most complex types of branched N-glycans in mammals. In general, the more GlcNAc branches per N-glycan, the more Gal residues are added and elongated to form poly-n-lactosamine [Galβ1,4GlcNAc]n. Galectins bind Gal and form specific cross-linked lattices with glycoproteins, which increases their cell surface expression (Stanley, 2007). 8

40 (Figure 1.3). Galectins are N-acetyllactosamine (Gal and GlcNAc) binding proteins whose major ligands are Golgi-remodeled N-glycans common to cell surface glycoproteins (Dennis et al, 2009). Poly-N-acetyllactosamine glycan structures serve as high affinity ligands for galectins, which bind to N-glycans of glycoproteins with affinities proportional to GlcNAc content (Dennis et al, 2009). Glycoproteins with N-acetyllactosamine glycans interact with galectins in a cross-linking manner, which leads to their retention at the cell surface by slowing later mobility, and delaying loss by constitutive endocytosis (Dennis et al, 2009). Increased cell surface residency in turn leads to greater sensitivity to extracellular cues and promotes receptor mediated signaling (Johswich et al, 2014; Lau et al, 2007; Mendelsohn et al, 2007). Sustained surface exposure and clustering of signaling receptors creates a platform to multiply ligand-induced signal intensity Golgi N-Glycan Branching Pathway UDP-GlcNAc UDP-GlcNAc is an essential common donor substrate required by all Mgat enzymes. UDP- GlcNAc is synthesized in the cytosol by the HBP and transported through sugar-nucleotide transporters into the medial Golgi apparatus (Dennis et al, 2009) (Figure 1.4). The Golgi N-glycan branching pathway is characterized by multistep ultrasensitivity to UDP-GlcNAc for branching enzymes Mgat1, Mgat2, Mgat4, and Mgat5 (Dennis et al, 2009). The relative affinity of branching enzymes for UDP-GlcNAc declines sequentially by ~300 fold (0.04 to 10 mm) moving down the N-glycan branching pathway from Mgat1 to Mgat5, while this trend is reversed for their respective glycoprotein N-glycan acceptors (Dennis et al, 2009). Thus, activities of enzymes Mgat1 and Mgat2 are limited by low affinity for acceptor glycoproteins, while activities of enzymes Mgat4 9

41 Figure 1.3 Glycan-Galectin Lattice Dynamics The glycocalyx is the thick carbohydrate layer surrounding the cell. Glycan structures generated in the Golgi differ in affinities for galectins. Galectins cross-link glycoprotein receptors and oppose (1) loss of EGFR to Caveolin 1-positive microdomains, (2) coated-pit endocytosis, (3) precocious clustering of receptors, and (4) F-actin-mediated entry of T-cell receptor into and exit of CD45 from ganglioside GM1-positive microdomains (blue). (5) Nutrient supply and growth signaling increase membrane remodeling, regulate metabolite flux through the hexosamine biosynthetic pathway to UDP-GlcNAc and Golgi N-glycan branching on receptors and transporters to promote surface retention by the glycan-galectin lattice (Dennis et al, 2009). 10

42 Figure 1.4 N-Glycan Branching Pathway Oligosaccharyltransterase (OST) utilizes the preassembled donor Glc3Man9GlcNAc2-ppdolichol to transfer the glycan to N-X-S/T sequons on glycoproteins in the ER. In the secretory pathway, glycoproteins transit from the ER to cis, medial, and trans Golgi apparatus, en route to the cell surface. The N-acetylglucosaminyltransferases enzymes, designated by their gene names (Mgat1, Mgat2, Mgat4, and Mgat5) generate branched N-glycans that display a range of affinities for galectins. The Km values for Mgat1, Mgat2, Mgat4, and Mgat5 are indicated as measured in vitro for UDP-GlcNAc and acceptor glycoproteins. The Golgi UDP-GlcNAc antiporter exchanges uridine monophosphate (UMP) for UDP-GlcNAc and establishes the steady state amounts of UDP-GlcNAc inside the Golgi (Dennis et al, 2009). 11

43 and Mgat5 are limited by UDP-GlcNAc concentrations generated by the HBP. This implies that initial branching by Mgat1 and 2 depends on the rate of protein synthesis, while N-glycan branching by Mgat4 and 5 is determined mostly by UDP-GlcNAc availability. Indeed, supplementation of extracellular GlcNAc has been shown to increase intracellular UDP-GlcNAc levels, Mgat5-mediated N-glycan branching, and glycoprotein retention at the cells surface, with increased sensitivity of cells to growth factors and cytokines (Johswich et al, 2014; Lau et al, 2007; Mendelsohn et al, 2007) Mgat Branching Enzymes The Man5GlcNAc2 glycan is substrate for the first N-acetylglucosaminyltransferase, or GlcNAc-transferase enzyme Mgat1 (GlcNAc-TI) (Schachter, 2010). Through transfer of GlcNAc from UDP-GlcNAc in the medial Golgi, Mgat1 modifies the high-mannose structure to a hybrid N-glycan. This is an essential step required for synthesis of either hybrid or complex-type glycans, and cells deficient in Mgat1 can only generate high-mannose type structures (Schachter, 2010). The product of Mgat1 can then be further stripped of remaining Man residues by α-mannosidase II, rendering it a substrate for Mgat2 (GlcNAc-TII) (Schachter, 2010). The conversion of a monoantennary to a complex bi-antennary structure requires addition of GlcNAc by Mgat2. Specific Mgat activity requires prior action of a distinct Mgat for catalysis to occur. One exception to this is Mgat3, which catalyzes the transfer of GlcNAc residue to the core to form a bisected N-glycan. However, due to the steric hindrance resulting from the presence of a bisecting GlcNAc, this structure cannot be used as an acceptor by other Mgats, thus preventing further branching reactions and formation of tri- and tetra-antennary N-glycans (Taniguchi & Korekane, 2011). Since Mgat3 12

44 inhibits further N-glycan branch formation it has been suggested to play a regulatory role in biosynthesis of complex and hybrid-type N-glycans (Taniguchi & Korekane, 2011). In instances where Mgat3 is not involved, additional N-glycan structures can be generated by Mgat5 (GlcNAc-V) and Mgat4a/b/c (GlcNAc-IVa/b/c) isoenzymes (Taniguchi & Korekane, 2011). Therefore, the number of antennas formed on an N-glycan depends on expression and dynamic action of different GlcNAc-transferases, the concentration of common donor UDP- GlcNAc, and acceptor glycoprotein with its immature N-glycan moving through the medial-golgi. The abundance of these factors and execution of these processes dictates the ultimate N-glycan structure produced. Indeed, this varies among species, tissues, cells, glycoproteins, and even varies with respect to different glycosylation sites on the same glycoprotein. For instance, when protein synthesis slows down UDP-GlcNAc is spared, providing greater opportunity for the late branching enzymes Mgat4 and Mgat5 to act and increase branching. Indeed, low glucose media conditions increase surface β1,6-glcnac-branched N-glycans in mouse embryonic fibroblasts (Cheung et al. 2007). N-glycan products of Mgat1, 2, 4 and 5 can be extended further through sequential addition of Gal, and terminal capping with Fuc and Sia (Dennis et al, 2009). N-linked glycans represent the most complex and functionally diverse covalent modification characterized (Ohtsubo & Marth, 2006). N-glycosylation is an inherently noisy and variable process, with potential N-glycan sites not always being occupied with glycans, and even those that are occupied often varying in their structure. The structural variability of N-glycans has been described in terms of microheterogeneity and macroheterogeneity. Microheterogeneity refers to the site-specific composition, chain-length, and branching pattern variability that occurs at a specific glycosite amongst different molecules of the same glycoprotein (Schachter, 1986). On the other hand, 13

45 macroheterogeneity results from variable Asn-X-Ser/Thr sequon usage, suggesting that local three-dimensional conformation of the polypeptide, and the immediate microenvironment in the vicinity of the potential glycosite, influences its accessibility to oligosaccharyltransferase (Schachter, 1986) Mgat1 in N-Glycan Branching Although N-glycosylation is dispensable for survival of isolated cells in vitro, it is crucial for proper functioning in vivo (Schachter, 2010). To elaborate on the N-glycan branching pathway and impairments caused when its synthesis is perturbed at different points, I will review phenotypes associated with mutations in genes coding for Mgat enzymes. Mgat1 is the first branching enzyme to act in the Golgi N-glycan branching pathway, which then allows additional branches to be added through action of other Mgats. The gene Mgat1 encodes for the enzyme which starts the branching process by adding GlcNAc to the trimmed core producing the hybrid N-glycan required for recognition by α-mannosidase II in the medial Golgi (Dennis et al, 2009). Mgat1 activity is essential for the synthesis of complex-type N-glycans. Genetic ablation studies in mice have proved informative concerning the structure-function relationship of Mgats. Mgat1-dependent N-glycans are required for normal mammalian development, as evidenced by systemic Mgat1 null mouse embryos dying in utero at around embryonic day 9.5, and presenting underdevelopment including fewer somites, a tube-like heart, defective vascularisation, and an open neural tube (Schachter, 2010). The phenotype would have been more severe, and Mgat1 null mice would most likely die much earlier if it was not for maternally derived Mgat1 gene transcripts, which rescue early embryos (Shi et al, 2004). This suggests that hybrid 14

46 and/or complex N-glycans might be required for blastocyst formation or implantation (Shi et al, 2004). Lack of Mgat1 affects N-glycan structures at the cell surface, with all hybrid and complex N-glycans being replaced by Man5GlcNAc2 (Schachter, 2010). Since Mgat1-dependent N- glycosylated glycoproteins on the cell surface are required for normal cell to cell interaction, as well as cell surface residency and activity of growth factor receptors and nutrient transporters, combined disturbance in these fundamental biological processes are most likely responsible for the lethality of Mgat1 null mice. Mgat1 loss of function mutation in rice Oryza sativa causes severe developmental defect with early lethality due to reduced sensitivity to the plant growth hormones cytokinins (Fanata et al, 2013). Deletion of Mgat1 in Drosophila melanogaster results in viable flies exhibiting defects in locomotion, brain abnormalities, and severely shortened lifespan (Sarkar et al, 2010). This phenotype is rescued by neuronal Mgat1 expression, which also increases lifespan in wild-type flies (Sarkar et al, 2010). These results imply that neuronal glycoproteins dependent on Mgat1 modification play a role in control of fly lifespan by affecting global metabolic changes (Sarkar et al, 2010). Knockdown of Mgat1 in prostate cancer cells reduces tumor progression both in terms of tumor size and metastasis (Beheshti Zavareh et al, 2012). This is interesting, as a link between cancer and metabolism has been rediscovered in recent years (Vander Heiden et al, 2009) Mgat2 in N-Glycan Branching Mgat2 is required for synthesis of complex N-glycans, and is widely expressed in mammalian cells and tissues (Wang et al, 2001). Mgat2 encodes the enzyme that transfers GlcNAc onto the tri-mannosyl core. Homozygous deletion of Mgat2, which abolishes complex-type N- 15

47 glycans but retains hybrid branched structures, results in prenatal and perinatal death with few null mice surviving to adulthood (Wang et al, 2001). These mice displayed postnatal phenotype similar to that observed in human patients with congenital disorders of glycosylation IIa (CDG-IIa), including failure to thrive, dysmorphic facial features, and poor psychomotor development (Wang et al, 2001). Furthermore, Mgat2 null mice were runted in comparison to wild-type littermates, had decreased blood glucose, showed gastrointestinal abnormalities, and exhibited reduced bodyweight at all developmental stages (Wang et al, 2001) Mgat4 in N-Glycan Branching Bi-antennary N-glycans can be further modified through addition of GlcNAc onto the trimannosyl core by Mgat4 isoenzymes (Taniguchi & Korekane, 2011). Interestingly, Mgat4b is upregulated in the liver of fast-growing chickens, and is thought to promote fat deposition in this context (Claire D'Andre et al, 2013). Mgat4a is expressed in most mouse and human tissues, but its levels are much higher in pancreas and small intestine (Ohtsubo et al, 2011; Ohtsubo et al, 2005). In pancreatic β-cells, Mgat4a-dependent N-glycosylation is necessary for generating multiantennary N-glycans on the glucose transporter 2 (Glut2), which enables galectin-glycan binding to maintain cell surface residency of Glut2 for proper glucose transport and sensing (Ohtsubo et al, 2005). Indeed, Mgat4a null mice are hyperglycemic, displaying elevated free fatty acids and triglycerides, with reduced insulin levels and impaired glucose-stimulated insulin secretion (Ohtsubo et al, 2005). With aging, glucose intolerance in these mice progressed to metabolic dysfunction, insulin resistance, and liver steatosis (Ohtsubo et al, 2005). 16

48 Furthermore, a high-fat diet strongly attenuated Mgat4a expression in pancreatic β-cells of wild-type mice. This resulted in reduced branching of Glut2 N-glycans required for proper cell surface retention, which in turn lead to impairment of insulin secretion, and eventually type 2 diabetes and hepatic steatosis (Ohtsubo et al, 2005). In pancreatic β-cells, Mgat4a expression is transcriptionally regulated by FoxA2 and Hnf1-α, whose intracellular distribution is regulated by cellular redox balance that might be affected by high-fat diet induced oxidative stress (Ohtsubo et al, 2011). Protection from high-fat diet-induced metabolic disease was conferred by ectopic Mgat4a constitutive expression in β-cell of transgenic mice, in which Glut2 glycosylation and its cell surface residence was maintained (Ohtsubo et al, 2011). Mgat4a was also reduced in enterocytes from human obese subjects, who exhibited endosomal Glut2 accumulation, most likely resulting from altered N-glycan branching on Glut2 (Ait-Omar et al, 2011) Mgat5 and β1,6-linked GlcNAc Branching Mgat5 encodes for a medial-golgi N-glycan branching enzyme responsible for catalyzing addition of β1,6-linked GlcNAc to Man residue, thereby forming complex-type tri- or tetraantennary N-glycan structures on glycoproteins (Dennis et al, 2009). The red kidney bean (Phaseolus vulgaris) lectin L-phytohemagglutinin (L-PHA) exhibits specific and selective reactivity toward β1-6glcnac-branched N-glycans (Grigorian et al, 2009). Since Mgat5 has a high Km value for UDP-GlcNAc, in comparison to enzymes Mgat1 or Mgat2, the intracellular concentration of this substrate determines the amount of complex tri- and tetra-antennary N- glycans found on glycoproteins (Dennis et al, 2009; Lau et al, 2007). Mgat5 products are the preferred substrate for elongation with N-acetyllactosamine and poly-n-acetyllactosamine, which is comprised of repeating units of GlcNAc and Gal of variable length, allowing for further 17

49 modification of the N-glycan chain by fucosylation and sialylation (Dennis et al, 2009; Grigorian et al, 2009). Mice deficient in Mgat5 lack tri- and tetra-antennary N-glycans. This results in altered distribution or clustering of glycoproteins at the cell surface, due to reduced affinity for galectins, and diminished signal transduction intensities from certain growth factor receptors and adhesion proteins (Dennis et al, 2009; Grigorian et al, 2009). Mgat5 null mice are viable and appear similar to wild-type littermates at birth. However, later on they display metabolic phenotypes such as leaner body composition and smaller size, resistance to weight-gain on high-fat diet, hypoglycemia, sensitivity to fasting in terms of exaggerated glycogen depletion and lipid mobilization, as well as increased oxidative respiration by reliance on fatty acid oxidation (Cheung et al, 2007). Glucose uptake is impaired in mouse embryonic fibroblasts derived from Mgat5 null mice, especially under conditions of low-glucose or serum-free medium, suggesting that Mgat5- modified N-glycans promote glucose uptake and anabolic metabolism (Cheung et al, 2007). Mgat5 null mice also display adult phenotypes that may be linked in part through metabolism, including delayed oncogene-induced tumorigenesis, sensitivity to autoimmune disease, loss of adult stem cells, and premature aging with accelerated loss of bone and muscle mass (Cheung et al, 2007; Dennis et al, 2009; Grigorian et al, 2009). The levels of β1,6-branch on N-linked glycans regulates the cell surface residency of glycoproteins to affect their function (Dennis et al, 2009). Biological processes regulated by Mgat5-dependent N-glycan modification include metabolic homeostasis, cell adhesion, motility and migration, cytokine and growth factor signaling, and cellular proliferation and differentiation (Dennis et al, 2009). Also, alterations in Mgat5 activity or function, leading to changes in N-glycan branching pattern on glycoproteins, have been linked to cancer progression and invasive 18

50 metastastic dissemination. In different types of cancer, Mgat5 transcript levels and enzymatic activity are upregulated, which in turn leads to increased β1,6-branched N-linked glycan structures and poly-n-acetyllactosamine content on membrane glycoproteins (Dennis et al, 2009). Mgat5 transcript levels are regulated by a Ras-Raf-Mek-Erk-Ets oncogenic signaling pathway, and typically show a three to five fold increase when this pathway is activated through transformation (Taniguchi & Korekane, 2011). The phenotypic effects of overexpression of Mgat5 are decreased cell cell and cell matrix adhesion, and promotion of motility and invasiveness (Taniguchi & Korekane, 2011). Transgenic mice expressing the polyoma virus middle T (PyMT) oncoprotein under control of the murine mammary tumor virus (MMTV) long terminal repeat develop multifocal mammary epithelium carcinomas that metastasize to lungs. Mgat5 null mice intercrossed with transgenic mice expressing PyMT show a considerable delay in mammary tumor development, and a reduction in tumor growth and lung metastasis, compared to those observed in MMTV-PyMT;Mgat5 +/- or MMTV-PyMT;Mgat5 +/+ mice (Dennis et al, 2009). Mgat5 -/- adult mice also showed suppression of tumor progression in HER2/neu-induced mammary oncogenesis model, and Pten +/- lymphoma and carcinoma tumor model (Cheung & Dennis, 2007; Granovsky et al, 2000; Guo et al, 2010). In these models, the effects of Mgat5 deletion were traced to alterations in PI3K and ERK signaling pathways Mgat6 in N-Glycan Branching Mgat6 (GlcNAc-VI) catalyzes the formation of the most highly branched penta-antennary complex-type N-glycan, the β1,4-linked branch (Sakamoto et al, 2000; Watanabe et al, 2006). Prior action of Mgat5 is a prerequisite for Mgat6 activity (Brockhausen et al, 1989). Mgat6 enzymatic activity was found in avian tissues, such as in the hen oviduct, chicken liver, duck colon 19

51 and turkey intestine (Brockhausen et al, 1989). This activity has also been seen in fish ovaries (Brockhausen et al, 1989). The gene coding for Mgat6 is expressed in various chicken (Gallus gallus) tissues (Sakamoto et al, 2000). However, Mgat6 gene and its enzymatic activity have not been detected in mammalian tissues (Brockhausen et al, 1989; Sakamoto et al, 2000) Functional Redundancy of N-Glycan Branches Genetic studies with Mgat knockout mice revealed that loss of an Mgat enzyme is more severe the earlier that enzyme acts in the N-glycan branching pathway. In later stages of the branching pathway a degree of redundancy between N-glycan structures generated by Mgat4 and Mgat5 may offer compensation for functional defects (Dennis & Brewer, 2013). This is contingent on substrate availability and spatiotemporal expression of genes coding for each enzyme (Dennis & Brewer, 2013). Experiments with Mgat4a and Mgat4b single and double knockout mice show induced glycomic biosynthetic compensation (Dennis & Brewer, 2013). This occurs through compensatory induction of other Mgats to generate similar N-glycan epitopes, as means of maintaining overall expression of N-glycan ligands on glycoproteins for cross-linking galectins at the cell surface (Dennis & Brewer, 2013). Furthermore, functional redundancy or compensation between N-acetyllactosamine branches in structurally related N-glycans can be promoted by UDP- GlcNAc, the rate-limiting substrate in their biosynthesis in the Golgi N-glycan branching pathway (Dennis & Brewer, 2013; Lau et al, 2007). The N-acetyllactosamine units, although repositioned within N-glycans with GlcNAc treatment, have been shown to substitute and compensate functionally by rescuing affinity for galectins' association with N-glycan branches at the cell surface (Dennis & Brewer, 2013; Lau et al, 2007). In fact, GlcNAc supplementation in Mgat5 -/- cells doubled the tri-antennary N-glycan levels, and rescued levels of EGF and TGF-β cell surface 20

52 receptors and their signaling, suggesting that less-branched N-glycans in larger quantities are sufficient to restore galectin binding (Dennis & Brewer, 2013; Lau et al, 2007) HBP and UDP-GlcNAc Formation The general route for incorporating monosaccharides into a glycan starts with their conversion into activated sugar-nucleotides, which serve as donors for glycosylation reactions (Ohtsubo & Marth, 2006). Activated sugar-nucleotides are synthesized by covalently linking a monosaccharide to a nucleotide, typically nucleoside diphosphate, and in many cases one sugarnucleotide can be converted into another (Du et al, 2009). The de novo biosynthetic steps to generate UDP-GlcNAc follow a variant of the Leloir pathway, elucidated biochemically in the 1950s (Marshall, 2006). However HBP, also called the hexosamine signaling pathway, was only well delineated and formally described based on genetic and biochemical evidence in the early 1990s (Marshall, 2006). UDP-GlcNAc is the major product of the HBP, and is the primary substrate in virtually all glycoprotein processing pathways, including Golgi N-glycan branching pathway de novo UDP-GlcNAc Biosynthesis HBP is one of several pathways that divert minor amounts of glucose from glycolysis or glycogen storage (Buse, 2006; Hardiville & Hart, 2014). Fructose 6-phosphate (Fru-6P), the obligatory intermediate in glycolysis, enters the HBP through conversion to glutamine-6- phosphate (GlcN-6P) (Figure 1.5). The rate-limiting enzyme in HBP responsible for catalysis of this reaction is glutamine-fructose 6-phosphate amidotransferase (GFAT or GFPT), which 21

53 transfers ammonia from glutamine to Fru-6P, and isomerizes the resulting fructosimine-6p to GlcN-6P (Buse, 2006; Hardiville & Hart, 2014). UDP-GlcNAc inhibits GFAT allosterically, thereby regulating its own synthesis through competitive feedback inhibition, and controlling the amount of glucose entering HBP (Du et al, 2009; Love & Hanover, 2005). The next reaction in UDP-GlcNAc synthesis involves transfer of an acetyl group from acetyl coenzyme A (acetyl-coa) by GlcN-6P acetyltransferase (GNPNAT1 or GNA1), to obtain GlcNAc-6P (Hardiville & Hart, 2014). Isomerization of GlcNAc-6P to GlcNAc-1P is catalyzed by GlcNAc phosphomutase (PGM3 or AGM1), which is autophosphorylated and dephosphorylated during the reaction cycle. Finally, activation of monosaccharides as sugar-nucleotides is an essential step in the biosynthetic pathway. Hence, uridylation of GlcNAc-1P by UDP-GlcNAc pyrophosphorylase (UAP1 or AGX1) results in formation of the end-product UDP-GlcNAc, which is then transported into the rer and Golgi apparatus and used as a donor for N-glycosylation by a number of GlcNActransferases (Dennis et al, 2009) Fate of UDP-GlcNAc In the secretory pathway, UDP-GlcNAc serves as a basic building block for synthesis of glycans on secreted and membrane-bound glycoproteins. UDP-GlcNAc is also used to make glycolipids, cell surface O-glycans, glycosaminoglycan (GAG) chains used in proteoglycans of the extracellular matrix, and hyaluronic acid (2009). In addition, some UDP-GlcNAc is epimerized to generate UDP-N-acetylgalactosamine (UDP-GalNAc), by the enzyme UDP-galactose-4- epimerase (GALE), or cleaved into UDP and ManNAc by UDP-GlcNAc-2-epimerase/Nacetylmannosamine kinase (GNE), the bi-functional and rate-limiting enzyme of sialic acid biosynthesis (Galeano et al, 2007). The same pool of UDP-GlcNAc generated by HBP also 22

54 Figure 1.5 Hexosamine Pathway for Biosynthesis of UDP-GlcNAc An offshoot of the glycolytic pathway, the HBP integrates the metabolism of carbohydrates (glucose), amino acids (glutamine), fats (acetyl-coa), and nucleotides (uridine-diphosphate) in the synthesis of UDP-GlcNAc. The enzymes and metabolites involved in the synthesis of UDP- GlcNAc are presented, with different precursors color-coded to denote their contribution to UDP-GlcNAc synthesis. 23

55 functions as the obligatory substrate for intracellular O-GlcNAcylation (Love & Hanover, 2005). O-GlcNAcylation is a dynamic and reversible posttranslational modification where a single GlcNAc moiety is attached to the hydroxyl group of serine or threonine residue in cytoplasmic and nuclear proteins (Hardiville & Hart, 2014). Enzyme O-GlcNAc transferase (OGT) catalyzes the addition, while O-GlcNAcase the removal of these single GlcNAc moieties (Hardiville & Hart, 2014) Precursor Metabolites for UDP-GlcNAc Biosynthesis The precursors for UDP-GlcNAc synthesis are nutrient derived. UDP-GlcNAc has been suggested as an ideal sensor for the metabolic state of a cell, since its levels are sensitive to the availability and fluctuations in concentrations of various nutrients and metabolites from different metabolic pathways: glucose (glycolysis, gluconeogenesis, and glycogenolysis), acetyl-coa (pyruvate or fatty acid oxidation), glutamine (nitrogen metabolism), and uridine-triphosphate (energy charge and nucleotide metabolism) (Hardiville & Hart, 2014; Love & Hanover, 2005; Marshall, 2006). Therefore, through its dependence on HBP, N-glycosylation is also dependent on intermediary metabolism. Since metabolic pathways are deeply interconnected, their interdependency explains why a perturbation or imbalance in one pathway often affects the function in another, such as the HBP. For instance, in a calorie-matched experiment rats fed a highglucose diet for a week had higher UDP-GlcNAc liver content than rats fed a high-lard diet (Tepperman et al, 1981). Sustained or chronic hyperglycemia promotes increased HBP flux and ultimately results in elevated formation of UDP-GlcNAc (Buse, 2006). In cell culture, intracellular UDP-GlcNAc levels and cell surface β1,6glcnac-branched N-glycans were found to be sensitive 24

56 to the external supply of metabolites Glc, Gln, ammonia, acetoacetate and uridine, as well as to GlcNAc supplementation (Abdel Rahman et al, 2013; Grigorian et al, 2007; Wellen et al, 2010) HBP and UDP-GlcNAc in Insulin Resistance and Diabetes Several laboratories have proposed that flux through the HBP plays a role in development of insulin resistance and other pathologies and complications associated with diabetes, primarily through O-GlcNAcylation (Love & Hanover, 2005; McClain, 2002). Hyperglycemia, commonly seen in diabetes mellitus, increases glucose flux through the HBP resulting in increased biosynthesis of UDP-GlcNAc (Buse, 2006). Mice injected with a low-dose of streptozotocin and fed a high-fat diet develop hyperglycemia, hyperinsulinemia and obesity, and are used as a model for type 2 diabetes mellitus (Fricovsky et al, 2012). As the disease progresses, these mice show increased GFAT levels and elevated concentration of UDP-GlcNAc in the diabetic heart tissue (Fricovsky et al, 2012). Transgenic mouse models suggest that increased flux through the HBP plays a role in development of insulin resistance and type 2 diabetes (Love & Hanover, 2005). Overexpression of GFAT in muscle and fat leads to whole body insulin resistance, as does overexpression of GFAT solely in pancreatic β-islet cells or in the liver (Love & Hanover, 2005). The liver-targeted GFAT overexpression in transgenic mice results in obese animals displaying glucose intolerance, hyperinsulinemia and hyperglycemia as the mice age (McClain, 2002). GFAT activity and UDP-GlcNAc concentrations were also increased in insulin-resistant genetically obese mice, while UDP-GlcNAc concentrations were reduced in rats with chronic calorie restriction and enhanced insulin sensitivity (Buse, 2006). 25

57 UDP-GlcNAc from Salvage Pathway In addition to de novo HBP synthesis of UDP-GlcNAc from nutritional sources and precursors, the salvage amino sugar metabolism pathway is also capable of generating UDP- GlcNAc from intracellularly recycled constituents (Dennis et al, 2009). Free cellular GlcNAc becomes available either as a result of endogenous lysosomal degradation and catabolism of glycoconjugates, by direct uptake, or through degradation of dietary glycoconjugates by glycosidases (Dennis et al, 2009). Thus, UDP-GlcNAc can be obtained or derived from catabolic and anabolic pathways. The cytosolic enzyme N-acetylglucosamine kinase (NAGK) catalyzes phosphorylation of free cytoplasmic GlcNAc to GlcNAc-6P, which is then salvaged into HBP to generate UDP-GlcNAc (Dennis et al, 2009). In cultured mammalian cells, increased intracellular UDP-GlcNAc, via extracellular GlcNAc supplementation, enhances N-glycan biosynthesis and branching, and improves association of glycoprotein receptors and transporters with galectins, thereby increasing their cell surface retention, and sensitivity to extracellular cues (Dennis et al, 2009) GlcNAc Supplementation in vitro and in vivo One of the oldest and most abundant organic compounds on the planet, GlcNAc is a monosaccharide primarily found in chitin of arthropods, insects and fungi, as well as bacterial peptidoglycan (Chen et al, 2010; Koropatkin et al, 2012). GlcNAc is also a constituent of heterogeneous polysaccharides such as hyaluronic acid and GAG, as well as being a major and essential building block for various forms of glycosylation. GlcNAc is a commercially available dietary or nutritional supplement used by people and pets all over the world, with numerous uses 26

58 and abuses attributed to it in modern pharmacopoeia (Chen et al, 2010). Unregulated and readily available in health and wellness stores, GlcNAc is recommended by doctors, veterinarians, as well as complementary and alternative medical practitioners. These recommendations, for numerous conditions and claiming various health benefits, span the whole range from theoretically sound and encouraging but clinically unproven, to those based on preliminary animal studies, to wellintentioned but unfounded and speculative. GlcNAc supplements have also been promulgated by unscrupulous charlatans in guise of glyconutrients (Schnaar & Freeze, 2008). Because of the latter, research into GlcNAc and related monosaccharides is at times tainted and viewed with skepticism, especially if the studies are sponsored by a company profiting from positive results associated with the compound examined (Schnaar & Freeze, 2008). Nevertheless, numerous independent basic science and clinical research studies utilizing GlcNAc have been conducted, yielding legitimate, verifiable and interesting results GlcNAc Increases UDP-GlcNAc and β1,6-branched N-Glycans Defects arising from mutations in the early de novo HBP enzyme GNPNAT1 (GNA1) can be partially reversed by restoring UDP-GlcNAc accumulation through GlcNAc or UDP-GlcNAc supplementation in biological systems as diverse as mouse embryonic fibroblasts, Arabidopsis thaliana and Caenorhabditis elegans (Boehmelt et al, 2000; Johnston et al, 2006; Nozaki et al, 2012). Numerous lines of evidence demonstrate that metabolic flux through HBP and availability of UDP-GlcNAc regulate Golgi N-glycan branching enzyme activities to control cell surface level and retention of transmembrane glycoproteins (Dennis et al, 2009; Grigorian et al, 2009; Johswich et al, 2014; Lau et al, 2007; Mendelsohn et al, 2007; Sasai et al, 2002). In different mammalian immortalized cells, as well as in primary T-cells and hepatocytes, increased flux through the HBP 27

59 via extracellular GlcNAc supplementation has been shown to increase intracellular UDP-GlcNAc levels, glycoprotein β1,6-glcnac-branched N-glycans, and transmembrane glycoprotein cell surface residency, by enhancing association with galectins to increase sensitivity to extracellular factors (Dennis et al, 2009; Grigorian et al, 2009; Johswich et al, 2014; Lau et al, 2007; Mendelsohn et al, 2007; Sasai et al, 2002). In addition, by increasing the degree of N-glycan branching, GlcNAc supplementation also increased cell surface expression level of voltage-gated potassium channels involved in modulating membrane excitability and action potential (Zhu et al, 2012). Furthermore, GlcNAc was reported to modulate the neuronal ionotropic glutamate receptor function and ensuing electrophysiological activity by enhancing the receptor's N-glycan-galectin interaction to alter neuronal excitability (Copits et al, 2014). GlcNAc has been reported to enter the mammalian cell through passive diffusion or pinocytosis, without deacetylation (Tesoriere et al, 1972). In cultured mammalian cells salvaged GlcNAc does not appear to be converted to glucose and used as a fuel source for energy production in glycolysis, but instead contributes almost exclusively to the UDP-GlcNAc pool for glycoconjugate formation (Abdel Rahman et al, 2013; Chertov et al, 2011; Wellen et al, 2010). Treatment of glucose-starved hematopoietic cells with GlcNAc, to maintain HBP flux, promoted IL-3 surface expression and signaling in a manner dependent on N-glycan branching, which in turn allowed glutamine uptake for energy production, lipid biosynthesis, cell growth, and uptake of the amino acid leucine (Wellen et al, 2010). Like GlcNAc, UDP-GlcNAc was also shown to promote nutrient uptake in cell culture. Treatment of mouse embryonic fibroblasts with UDP- GlcNAc resulted in increased uptake of glucose, amino acids and uridine, concomitant with GlcNAc incorporation into N-glycans of cell surface glycoproteins (Natraj & Datta, 1978). 28

60 GlcNAc Supplementation for N-Glycan Compensation Mgat5 -/- mice display increased levels of circulating hormone glucagon but are glucagon receptor deficient (Cheung et al, 2007; Johswich et al, 2014). This is linked to a lack of Mgat5- mediated N-glycan branching on the glucagon receptor, which affects its cell surface residency and downstream signaling (Johswich et al, 2014). Mgat5-branched N-glycans on hepatic glucagon receptor increase affinity for galectin binding to counteract cell surface lateral mobility, and increase responsiveness to glucagon (Johswich et al, 2014). GlcNAc supplementation rescued glucagon receptor sensitivity, signaling and dynamics in Mgat5 -/- primary hepatocytes, and improved glucagon tolerance response in Mgat5 -/- mice (Johswich et al, 2014). This is consistent with the notion of N-glycan redundancy, where increasing UDP-GlcNAc levels drives compensating increases in N-glycan branching by other Mgat enzymes (Dennis & Brewer, 2013; Lau et al, 2007). Adult Mgat5 -/- mice are also hypersensitive to autoimmune disease, and a number of T-cell glycoproteins are known to suppress autoimmunity in this context (Grigorian et al, 2009). Multiple sclerosis has been linked to genetic and environmental dysregulation of the Golgi N-glycan branching pathway, and GlcNAc has been shown to suppress its progression in a mouse model of this disease (Grigorian et al, 2011). Oral GlcNAc in drinking water increased Mgat5-modified β1,6-glcnac-branched N-glycans on cell surface glycoproteins of T-cells, suppressing inflammatory T-cell response and clinical course of experimental autoimmune encephalomyelitis in mice (Grigorian et al, 2011; Grigorian et al, 2007; Grigorian et al, 2009). This suppression is seen even when oral GlcNAc administration is initiated after disease onset (Grigorian et al, 2011). These results suggests that oral GlcNAc supplementation in mice increases UDP-GlcNAc supply to the Golgi N-glycan branching pathway. Furthermore, oral GlcNAc in drinking water suppressed 29

61 spontaneous autoimmune diabetes in non-obese diabetic (NOD) mice when initiated prior to disease onset (Grigorian et al, 2007). Collectively, these data suggest that UDP-GlcNAcdependent biosynthesis regulates T-cell-mediated autoimmunity by altering GlcNAc branching in N-glycans (Grigorian et al, 2009) GlcNAc Safety and Efficacy Importantly, orally administered GlcNAc showed no significant adverse effects or obvious indications of toxicity or carcinogenesis in experiments with rats at concentration of ~2500 mg/kg/day (Lee et al, 2004; Takahashi et al, 2009). Also, infusion of GlcNAc into the third cerebroventricle of rats did not affect feeding, drinking or ambulation, suggesting that the primary site of action of GlcNAc may be in peripheral cells and organs. (Sakata & Kurokawa, 1992). Studies with orally administered radioactive GlcNAc in rats found that GlcNAc is not deacetylated within the intestinal lumen, and that absorbed GlcNAc was incorporated into tissues such as the liver, stomach and intestine, as well as in serum proteins (Capps et al, 1966). Furthermore, GlcNAc appeared to be specifically incorporated into glycan components of glycoproteins and GAGs (Capps et al, 1966). In a mouse model of rheumatoid arthritis, oral GlcNAc decreased the severity of arthritis symptoms, such as joint swelling, and histopathological joint indicators associated with disease (Azuma et al, 2012). In normal human subjects acute intravenous administration of high amount of GlcNAc did not appear to affect blood sugar homeostasis or insulin secretion (Gaulden & Keating, 1964). Furthermore, unlike for glucose, insulin had no obvious effect on disappearance of circulating GlcNAc administered intravenously in human subjects (Levin et al, 1961). In humans, single oral dose of 1 g of GlcNAc per day, the equivalent to ~15 mg/kg/day, resulted in its absorption from the intestinal track and elevation of serum GlcNAc (Talent & Gracy, 1996). 30

62 Finally, a pilot study involving treatment of children with severe treatment-resistant inflammatory bowel disease with oral GlcNAc as adjunct therapy at 3-6 g/day, or ~ mg/kg/day, resulted in improvement of clinical symptoms in some cases (Salvatore et al, 2000) Dietary Monosaccharide Supplementation as Therapy There are many examples of monosaccharide supplementation as therapy in humans. For some genetic metabolic diseases, where de novo synthesis of specific monosaccharides is defective, oral monosaccharide supplementation to salvage pathways can rescue sugar-nucleotide levels and impaired glycoprotein production, with therapeutic benefit at biochemical and clinical levels. For example, the congenital disorder of glycosylation Ib (CDG-Ib), a deficiency in phosphomannose isomerase decreases the supply of guanosine diphosphate mannose (GDP-Man) for dolichol-pp-oligosaccharide synthesis (Niehues et al, 1998). Oral mannose (Man) supplementation was found to alleviate chronic postnatal CDG-Ib phenotypes of failure to thrive, coagulopathies, protein-losing enteropathy, and liver fibrosis (Niehues et al, 1998). In this case, oral Man administration elevated serum Man concentration increasing the import of Man into cell, where Man salvage pathway bypassed the defective step - conversion of Fru-6P to mannose-6- phosphate (Man-6P), to increase Man-6P and GDP-Man (Niehues et al, 1998). Another CDG, the leukocyte adhesion deficiency type II (CDG-IId) is caused by a hypomorphic mutation in the Golgi GDP-fucose transporter, with patients suffering recurrent infections due to a deficiency in selectin ligands (Jaeken & Matthijs, 2007). The fucose (Fuc) salvage pathway can generate higher than normal concentrations of GDP-Fuc, sufficient to rescue CDG-IId patients' deficit in GDP-Fuc transport (Marquardt et al, 1999). Similarly, Fuc fed to mice with a deficiency in GDP-keto-4-31

63 keto-6-deoxymannose 3,5-epimerase-4-reductase (FX) restored Fuc content in glycoproteins and rescued associated deficits in postnatal physiology (Smith et al, 2002). Mutations in the UDP-GlcNAc 2-epimerase/ManNAc kinase (GNE) gene, which codes for a key enzyme in sialic acid biosynthesis, results in a rare autosomal recessive neuromuscular disorder of adult-onset in humans, and early postnatal death in mice (Hinderlich et al, 1997). Supplementation of pregnant mice with N-acetylmannosamine (ManNAc) or mannosamine extended survival of Gne mutant pups by enhancing sialic acid production, restoring renal glycoprotein sialylation, and improving renal physiology (Galeano et al, 2007). Peracetylated ManNAc and sialic acid are effective prophylactic therapeutic treatments that prevent development of biochemical defects and myopathic phenotype in Gne mutant mice expressing the GNE mutation associated with human disease (Malicdan et al, 2010; Malicdan et al, 2012). In humans this mutation leads to hereditary inclusion body myopathy. Furthermore, ManNAc and sialic acid administration are effective in rats and mice, respectively, in significantly decreasing albuminuria associated with nephrotic syndrome by increasing sialylation of glomerular glycoproteins (Clement et al, 2011; Ito et al, 2012). Sialic acid enriched diet also proved effective at influencing neuronal function in old rats by rejuvenating salivation and restoring brain ganglioside bound sialic acid levels, both of which decline with age (Sprenger et al, 2009). Finally, further supporting the effectiveness of monosaccharide treatment as metabolic intervention, galactose supplementation of fibroblasts derived from patients with markedly diminished activity of phosphoglucomutase 1, as well as dietary supplementation in patients themselves, resulted in restoration of protein glycosylation and clinical improvement (Tegtmeyer et al, 2014). 1.2 Metabolism 32

64 1.2.1 Glycolysis and Tricarboxylic Acid (TCA) Cycle Glycolysis is a catabolic pathway that through a series of intricate consecutive metabolic steps converts hexose sugars, such as glucose, to pyruvate. In animal cells, pyruvate can then be converted to lactate, or in the presence of oxygen be oxidized in the TCA cycle, also known as the citric acid cycle or Krebs cycle. Glycolysis takes place in almost all living cells, where it is the key metabolic pathway for adenosine 5'-triphosphate (ATP) production, generation of reducing equivalents, and conversion of carbohydrates into compounds which can undergo terminal oxidation, or be used for biosynthesis (Vander Heiden et al, 2009). Glycolysis occurs in the cytoplasm and starts with glucokinase or hexokinase-mediated phosphorylation of glucose to glucose-6-phoshate (Glc-6P). Glycolysis can function under aerobic or anaerobic conditions. Under anaerobic conditions, or in a situation without sufficient reducing equivalents due either to the lack of oxygen or high cellular metabolism, pyruvate is converted to lactate (Vander Heiden et al, 2009). Lactate produced under anaerobic conditions can diffuse from cells into the bloodstream (Postic et al, 2004). Under aerobic conditions, pyruvate formed during glycolysis is transported into the mitochondrial matrix, where it is oxidatively decarboxylated by the pyruvate dehydrogenase complex to generate acetyl-coa (Vander Heiden et al, 2009). The acetyl residues formed are subsequently oxidized to carbon dioxide and water in the TCA cycle (Vander Heiden et al, 2009). Another source of acetyl-coa supplied to the TCA is derived from β-oxidation of fatty acids (Postic et al, 2004). Complete oxidation in the TCA cycle is accompanied by release of relatively large amounts of energy, much of which is salvaged by reducing equivalents through oxidative phosphorylation to aerobically synthesize ATP (Vander Heiden et al, 2009). The TCA cycle is a multi-enzyme cyclic metabolic pathway in the mitochondrial matrix involving eight discrete steps. Each step is catalyzed by a different enzyme, and while some 33

65 reactions are reversible, others are unidirectional. TCA has numerous functions, both anabolic and catabolic, and consequently is often described as the hub of intermediary metabolism (Vander Heiden et al, 2009). The catabolic aspect of TCA initiates terminal oxidation of energy substrates, since many catabolic pathways generate intermediates of the TCA cycle or supply metabolites such as pyruvate and acetyl-coa that can enter the cycle. The TCA cycle also supplies important precursors for anabolic pathways such as oxaloacetate and malate for gluconeogenesis, oxaloacetate and 2-oxogluterate for synthesis of amino acids such as glutamate, and citrate for fatty acids biosynthesis (Vander Heiden et al, 2009). Thus, the TCA not only takes up acetyl-coa from fatty acid degradation, but also supplies material for its synthesis by condensing the acetyl residue with oxaloacetate to form citrate and exporting it into the cytoplasm. In the cytosol, citrate is cleaved by ATP-dependent citrate lyase into acetyl-coa and oxaloacetate (Vander Heiden et al, 2009). The rate-determining enzyme acetyl-coa carboxylase (ACC) carboxylates acetyl-coa to malonyl-coa, which can then be used as a substrate in lipogenesis, through the action of fatty acid synthase (FASN) complex (Postic et al, 2004) Insulin, Glucagon and Liver Function in Glucose Homeostasis Liver is a vital organ responsible for maintaining whole-body nutrient homeostasis, including maintenance of a relatively stable blood glucose concentration. The pancreatic hormones insulin and glucagon are involved in coordinating this process of organismal metabolism (Schwartz et al, 2013). Plasma glucose must remain within a narrow concentration range during periods of feeding and fasting to avoid hyperglycemia and hypoglycemia, respectively. This tight control is exerted through coordinated regulation of glucose absorption from the intestine, uptake and metabolism by peripheral tissues, as well as storage, release and production by the liver. Insulin 34

66 serves as the primary regulator of blood glucose concentration by promoting glucose uptake in muscle and adipose tissue, as well as glycogen formation and gluconeogenesis inhibition in the liver (Schwartz et al, 2013). Following a meal, increasing concentrations of glucose in the blood stimulate pancreatic β-cells to secrete insulin. The fundamental role of insulin is to coordinate use of metabolic fuels in the body, by partitioning them for oxidation or storage. Insulin accelerates glycolysis, stimulates glucose conversion to glycogen, and lipid biosynthesis in the liver, muscle, and adipose tissue (Schwartz et al, 2013). To this end, insulin regulates gluconeogenic and lipogenic enzyme accumulation and function through regulation of gene expression, translation and through posttranslational modifications of select cytosolic proteins (Lin & Accili, 2011). Glucagon is a counterregulatory hormone that decreases glycolysis and raises plasma glucose levels in response to insulin-induced hypoglycemia (Lin & Accili, 2011). High glucose uptake favours glucose storage as glycogen, as well as glycolysis to generate pyruvate, which in liver and adipose tissue is then converted to fatty acid and triglyceride (Postic et al, 2004). Glucose is also metabolized by the pentose phosphate pathway to generate ribose 5-phosphate, a precursor in nucleotide biosynthesis, and NADPH, which is required for biosynthesis of fatty acids (Postic et al, 2004) Liver Glycogen Once absorbed, carbohydrates are carried to the liver where they are converted to glucose. Glycogen, a branched polymer of glucose consisting of up to 50,000 residues, is the primary form of stored carbohydrate in animal tissue. Liver is the central organ involved in glycogen formation (glycogenesis), storage and breakdown (glycogenolysis) (Lin & Accili, 2011). These processes are 35

67 coordinated to maintain normal glucose levels in the blood during glycemic fluctuations associated with states of feeding and starving. Due to its hydrated state, glycogen requires ample storage space, and as such liver can only store a limited amount of glucose in this form. Liver glycogen represents a short-term reserve of glucose for peripheral organs and tissues. Indeed, glycogenmetabolizing enzymes have the property that enables the liver to act as a sensor of blood glucose and to store or mobilize glycogen according to peripheral needs. Glycogen synthase (GS) is the rate-limiting enzyme for glycogen synthesis and deposition, responsible for transfer of glucose from UDP-glucose, its nucleotide-sugar donor form (Postic et al, 2004). GS phosphorylation on Ser641 leads to inactivation, while glycogenesis is stimulated by activating GS through dephosphorylation by protein phosphatase 1, which is controlled by insulin (Postic et al, 2004) Feeding and Fasting All cells and organs in the body have a constant requirement for nutrients and metabolites, which serve as energy substrates. Since nutrient supply in nature is irregular and variable in amount and type, organs such as the liver and adipose tissue act as temporary reservoirs storing energy bearing metabolites and nutrients that can be readily deployed. In metabolism, a distinction is often made between the fed state, also known as the absorptive state, immediately following a meal, and the fasting or starving state, which develops later if food is not consumed. The two phases operate on different metabolic programs, which are dictated by plasma levels of various metabolites and the hormonal signaling cascades they trigger (Schwartz et al, 2013). During the absorptive state, the insulin to glucagon ratio increases and the availability of substrates trigger an anabolic phase, whereby liver forms increased amounts of glycogen and fats, muscle synthesizes proteins from amino acids and stores glycogen, and adipose tissue synthesizes triglycerides and stores them in 36

68 lipid droplets (Lin & Accili, 2011). Without food intake the post-absorptive state gradually evolves into the fasting state (Figure 1.6). During fasting, blood glucose and insulin levels decrease, and glycogen synthesis is inhibited, as is fatty acid synthesis (Schwartz et al, 2013). In response to lowered blood glucose concentration, pancreatic α-cells release increased amounts of glucagon, which act on the liver to stimulate glucose release by glycogenolysis to maintain blood glucose homeostasis and provide other tissues with an energy source (Lin & Accili, 2011). Glucagon also plays an important role in initiating and maintaining hyperglycemic conditions in diabetes (Lin & Accili, 2011). In fact, insulin resistance is manifested by hyperinsulinemia, increased hepatic glucose production, and decreased glucose disposal. The reduced insulin to glucagon ratio results in a switch to different fuel metabolism, as the body engages in using its energy reserves by breaking down glycogen, fats and proteins, and distributing these energy supplying metabolites between organs (Lin & Accili, 2011). Increased level of glucagon relative to insulin also stimulates the mobilization of fatty acids from adipose tissue. Glucagon release also activates hormone-sensitive lipases, which catalyze triglyceride hydrolysis in adipocytes to release glycerol and free fatty acids into the circulation (Lin & Accili, 2011). Liver is a major site for fatty acid oxidation to acetyl-coa and fat-derived ketone body formation (Lin & Accili, 2011). Free fatty acids and ketone bodies released into the blood serve as important energy sources during hunger. After prolonged glucose starvation, muscles and other tissues begin to degrade protein to amino acids, which are then oxidized or secreted and used in gluconeogenesis by the liver. Gluconeogenesis is the formation of glucose from non-carbohydrate sources, primarily glycerol produced through degradation of fats, and lactate, alanine, glutamine and other glucogenic amino acids derived from muscles (Lin & Accili, 2011). In animals 37

69 Figure 1.6 Glucose Production in Liver During Fasting During fasting, when plasma glucose levels drop due to lack of new supplies or rapid use, the pancreatic islet cells increase glucagon secretion, and decrease insulin release. To maintain plasma glucose homeostasis, glucagon stimulates liver glycogenolysis and increases the enzymatic activity required for gluconeogenesis, utilizing glycerol, lactate and amino acids released by adipocytes and muscle cells into the bloodstream. 38

70 gluconeogenesis takes place primarily in the liver, and its main function is to generate sufficient glucose supply for organs such as brain and muscles during fasting or starvation (Lin & Accili, 2011). Typically this system is activated once hepatic glycogen has been depleted. Most of the reaction steps involved in gluconeogenesis represent a reversal of glycolysis reactions, catalyzed by the same enzymes that are used in glycolysis (Postic et al, 2004). Other enzymes are specific to gluconeogenesis and are only synthesized under the influence of glucagon when needed (Lin & Accili, 2011) Glycoprotein Receptors for Glucagon and Insulin, and Glucose Transporters Transmembrane receptors for the hormones glucagon and insulin are N-glycosylated, as are glucose transporters. All of these are specialized glycoproteins residing at the cell surface, where they respond to the extracellular environment and trigger changes in function of the cell Glucagon Receptor (Gcgr) The glucagon receptor (Gcgr) is a G-protein coupled receptor with four and five N-glycan sequons in man and mice, respectively. Glucagon binding to Gcgr results in a conformational change that activates adenylate cyclase, forming the secondary messenger cyclic AMP (camp), which in turn activates protein kinase A (PKA) (Johswich et al, 2014). PKA then phosphorylates and inactivates GS, thereby terminating the synthesis of glycogen (Lin & Accili, 2011). Recently, the Dennis laboratory has shown in primary hepatocytes that Mgat5-branched N-glycans on Gcgr increase receptor binding to galectin-9, which slows its membrane mobility and enhances 39

71 sensitivity to glucagon (Johswich et al, 2014). This study suggests that N-glycan branching on Gcgr acts as a positive regulator of glucagon responsiveness Insulin Receptor (Insr) The insulin receptor (Insr) belongs to a family of related tyrosine kinase receptors. Interestingly, human and murine Insr both have eighteen N-glycan sequon motifs. A natural variant of human Insr has a substitution of Lysine for Asparagine at a position immediately preceding the first N-glycan sequon (Kadowaki et al, 1990). This was found to decrease the affinity of insulin binding to the receptor (Kadowaki et al, 1990). Furthermore, site-directed mutagenesis of the first and second Insr N-glycan sequons significantly reduced insulin binding to the receptor on the cell surface (Caro et al, 1994). Insulin binding to Insr leads to tyrosine phosphorylation of several insulin receptor substrates (IRSs), which then act as docking proteins for other signaling proteins (Lin & Accili, 2011). Phosphorylation of IRSs proteins activates the phosphatidylinositol-3-kinase (PI3K) and Akt signaling pathway, which is responsible for most of the metabolic actions of insulin (Lin & Accili, 2011). One of these is to increase translocation of glucose transporters (Gluts) from cytoplasmic vesicles to the cell surface, where they can mediate glucose import from the blood via facilitated diffusion (Lin & Accili, 2011) Glucose Transporters (Gluts) Most Glut proteins, encoded by Slc2a genes, typically have a single N-glycan sequon. Dependence of Gluts on N-glycan branching for increased residency and activity at the cell surface has been shown for Glut1, Glut2 and Glut4 (Haga et al, 2011; Kitagawa et al, 1995; Lau et al, 40

72 2007; Ohtsubo et al, 2005; Zhu et al, 2012). In both mouse and human pancreatic β-cells, cell surface expression of Glut2 depends on binding of its Mgat4-mediated N-glycan structure to galectin-9, which slows mobility at the cell surface and loss to endocytosis, thereby enhancing glucose transport (Ohtsubo et al, 2011; Ohtsubo et al, 2005). There is also pharmacological evidence for N-glycan requirement on nutrient transporters at the cell surface. Indeed, membrane transport of three major classes of nutrients, namely glucose, amino acids and uridine, was inhibited by tunicamycin, an inhibitor of protein N-glycosylation in the ER (Olden et al, 1979). A similar result was obtained using a different tool to perturb N-glycan function, suggesting that interfering with N-glycans on glycoproteins alters plasma membrane transport systems to impair nutrient uptake. In this case, wheat germ agglutinin, a lectin that binds GlcNAc within N-glycans on cell surface glycoproteins, has been shown to have an inhibitory effect on transport of amino acids through the plasma membrane (Li & Kronfeld, 1977) Nutrient Sensing and Signaling Pathways Cells have evolved several sensory systems and homeostatic regulatory mechanisms to sense nutrient levels, cellular energy and metabolic status, as well as to respond to fluctuations by adjusting flux through metabolic pathways. Pathways that detect differences in extracellular and intracellular levels of nutrients and metabolites are often integrated and coordinated. The mammalian target of rapamycin (mtor) and the AMP-activated protein kinase (AMPK) pathways are two well characterized nutrient sensing and signaling pathways mtor 41

73 mtor controls cell growth and metabolism in response to nutrients, growth factors and cellular energy, by positively and negatively regulating several anabolic and catabolic processes, respectively, to collectively determine mass accumulation (Cornu et al, 2013; Zoncu et al, 2011). mtor is a Ser/Thr protein kinase that interacts with several distinct proteins to form two complexes named mtor complex 1 (mtorc1) and mtor complex 2 (mtorc2) (Cornu et al, 2013). mtorc1 activates p70 S6 kinase, which in turn phosphorylates ribosomal protein S6, thus activating translation and transcriptional up-regulation of anabolic pathways (Cornu et al, 2013). The abundance of phosphorylated ribosomal protein S6 is directly related to mtorc1 activity, and inversely related to level of cellular autophagy (Zoncu et al, 2011). Autophagy is involved in regulating intracellular lipid metabolism in the liver through macrolipophagy (Singh et al, 2009). Macrolipophagy promotes breakdown of intracellular lipids stored in lipid droplets to supply fatty acids for β-oxidation or other uses (Singh et al, 2009). Hepatic mtorc1 activity negatively regulates autophagy and production of ketone bodies for peripheral tissues to use as energy source in response to fasting (Zoncu et al, 2011). Hence, fasting response typically suppresses mtorc1 activity, however its chronic activation by overabundance of nutrients and/or hyperinsulinemia can drive ectopic accumulation of lipids in the liver (Zoncu et al, 2011). Indeed, mtorc1 activity is significantly elevated in liver, muscle, and adipose tissue of both genetically induced and high-fat diet induced obese mice, suggesting its involvement in the pathogenesis of obesity and obesity-associated metabolic disorders (Rui, 2007). In addition to its key role in lipid metabolism within the liver, hepatic mtorc1 signaling also affects systemic glucose and insulin homeostasis, most likely due to its effects on Akt and hepatic glucose uptake (Albert & Hall, 2014). Hyper-activation of mtorc1 signaling upon fasting results in metabolic stress due to systemic and hepatic glutamine depletion, and thereby an inability of glutaminolysis to sustain the TCA 42

74 cycle (Albert & Hall, 2014). This is consistent with the finding that mtorc1 inhibition by rapamycin increases intracellular glutamine levels (Albert & Hall, 2014). In certain settings, such as low-nutrient conditions, AMPK acts as a negative regulator of mtorc1 signaling (Hardie, 2014) AMPK AMPK is a master regulator of energy homeostasis, as well as cellular and organismal metabolism (Hardie, 2014). It senses low ATP levels and restores cellular energy by inhibiting energy consuming anabolic while stimulating catabolic pathways such as autophagy and fatty acid oxidation (Hardie, 2014). To maintain energy homeostasis, a delicate balance between fat storage and breakdown is crucial. The adipose tissue hormone adiponectin, whose actions are mediated by AMPK, inhibits fatty acid synthesis, stimulates fatty acid uptake and oxidation, and sensitizes liver and muscle tissues to insulin (Unger et al, 2010). AMPK activity can be measured through its phosphorylation at Thr172, as well as by phosphorylation of its downstream target acetyl-coa carboxylase (ACC) at Ser79 (Hardie, 2014). ACC catalyzes the pivotal step in fatty acid synthesis, the formation of malonyl-coa, by attaching a bicarbonate ion to acetyl-coa (Jump, 2011). Malonyl-CoA inhibits transport of acyl-coa into the mitochondrial matrix and its subsequent breakdown (Jump, 2011). Thus, de novo fatty acid synthesis is in part negatively regulated by AMPK. Dephosphorylated ACC is enzymatically active, while phosphorylation inactivates its enzymatic activity and promotes fatty acid oxidation instead (Hardie, 2012). The inactivating phosphorylation of ACC is catalyzed by AMPK, which in turn is regulated by an activating phosphorylation by camp dependent PKA, while the reactivating dephosphorylation of ACC is catalyzed by protein phosphatase 2A (Hardie, 2014). 43

75 1.2.6 Fatty Acid Synthesis The rate of fatty acid synthesis can be influenced by diet. In well-fed state with an ample supply of glucose, insulin is secreted, which causes a low degree of ACC phosphorylation, and increased formation of malonyl-coa (Jump, 2011). The biosynthesis of fatty acids, or de novo lipogenesis, occurs in many tissues, however post-absorptively this occurs primarily in the liver and fatty tissues. Fatty acid synthase (FASN) is a key biosynthetic multi-enzyme protein complex responsible for de novo lipogenesis (Jump, 2011). By catalyzing the synthesis of long-chain fatty acids from acetyl-coa and malonyl-coa, in the presence of NADPH as a reducing agent, FASN produces long-chain saturated fatty acids for storage in the liver or export to other tissues (Jump, 2011). FASN uses acetyl-coa as a starter molecule, and in a cyclic reaction, acetyl residue is elongated by two carbon units at a time for seven cycles, until formation of sixteen carbon palmitic acid as the major product (Jump, 2011). The liver is the most important site for synthesis of fatty acids and triglycerides, most of which are subsequently released into the blood. The cytosolic buildup of acetyl-coa encourages de novo hepatic lipogenesis, which over time can lead to triglyceride accumulation and even development of fatty liver disease, or hepatic steatosis (Jump, 2011). FASN is transcriptionally regulated by sterol regulatory element binding protein-1c (SREBP-1c) in response to feeding and insulin (Jump, 2011). Long-term adaptive control of lipogenesis occurs though changes in the rate of synthesis at the transcriptional level, and degradation of the participating enzymes (Jump, 2011) Lipid Storage and Breakdown 44

76 Due to the limited storage capacity of glycogen, all carbohydrates consumed beyond energy needs are converted to and stored as fat. Fats are the most important energy reserves in an animal. This reserve pool is practically almost unlimited. Triglycerides are neutral lipids, each composed of three fatty acids in ester linkage with a single glycerol (Walther & Farese, 2012). In animals, triglycerides are highly concentrated stores of metabolic fuel, and yield more energy per weight upon oxidation than either proteins or carbohydrates (Speakman & O'Rahilly, 2012). Although most tissues are capable of producing triglycerides, their primary site of accumulation is the adipose tissue. In vertebrates, lipid droplets are the major cytoplasmic storage organelles for triglycerides (Walther & Farese, 2012). They are characterized as ubiquitous components of different cell types, even those that, unlike adipocytes, only have a limited capacity for triglyceride storage (Walther & Farese, 2012). Lipid droplets play a crucial role in regulating cellular lipid levels through hydrolysis and trafficking, and provide energy and substrates for synthesis and repair of cell membranes (Walther & Farese, 2012). During fasting or starvation the body s strategy is to minimise the use of carbohydrate and protein, and to obtain as much energy as possible from fat stores. Hence, lipogenesis is inhibited, and fatty acids activated in a process requiring ATP to form acyl-coa, which can then be transported into the mitochondrial matrix for β-oxidation and acetyl-coa generation (Walther & Farese, 2012). Resulting acetyl residues can be oxidized to carbon-dioxide in the TCA cycle, producing reduced coenzyme and ATP derived through oxidative phosphorylation (Vander Heiden et al, 2009). Fatty acid breakdown in the liver is increased when levels of free fatty acids are elevated, such as after consumption of high-fat foods, and increased lipolysis during fasting or starvation (Jump, 2011). Short chain fatty acids are dissolved in plasma, while longer chain fatty acids are bound to albumin. Fatty acids are transported across the cell membrane by membrane- 45

77 associated fatty acid-binding proteins, or fatty acid transporters, which not only facilitate but also regulate cellular fatty acid uptake (Schwenk et al, 2010). A number of fatty acid transporters have been identified, including CD36, which is extensively glycosylated. Indeed, mouse CD36 has eight N-glycan sequons, while human CD36 has ten. These are situated in a region coding for the large extracellular loop. Insulin treatment stimulates simultaneous recruitment of CD36 and glucose transporter Glut4 from the recycling endosomal storage compartment to the cell membrane in order to increase fatty acid and glucose uptake, respectively (Schwenk et al, 2010). 1.3 Body Weight and Obesity Body weight is a simple and effective way to measure tissue mass and estimate body composition of an animal. In humans, the body mass index (BMI) provides a more accurate measurement, since it is independent of height and correlates fairly well with the total body fat (Speakman & O'Rahilly, 2012). BMI is calculated from body weight in kilograms divided by the square of height in meters. To specifically measure body composition in terms of fat and lean muscle mass, various imaging modalities are available. Dual-energy X-ray absorptiometry (DEXA) passes two photons of varied energy intensity through body tissues, and body fat percentage is estimated based on the attenuation patterns of these photons. Alternatively, to obtain even more accurate measures of fat mass, computed tomography (CT) or magnetic resonance imaging (MRI) can be used. In humans, obesity is clinically defined as BMI of greater than 30 kg/m 2, while a number of 25 up to 30 indicates that a person is overweight. Around the globe, in both industrialized and developing countries, there has been an increase in adiposity among people, often progressing to obesity. It is estimated that currently about ~2 billion people worldwide are overweight and ~700 million are obese (Speakman & O'Rahilly, 2012). These are staggering 46

78 numbers compared to 50 years ago (Speakman & O'Rahilly, 2012). Obesity is a risk factor for chronic diseases such insulin resistance, diabetes, metabolic syndrome, cardio-vascular disease, fatty-liver disease, and even some forms of cancer (Speakman & O'Rahilly, 2012; Unger et al, 2010). The organs affected in these conditions, including the liver, pancreatic islets, skeletal muscle, heart and kidney, display ectopic lipid accumulation (steatosis), which can ultimately lead to lipotoxicity, apoptosis, and gradual organ failure (Unger et al, 2010). The increased prevalence of obesity and these chronic diseases related to nutrition and metabolism have prompted considerable efforts to understand their origin and to identify effective prevention strategies or treatments Body Weight Regulation The biology of body weight regulation is complex and operates through effects on energy intake, metabolic activity, energy expenditure, and caloric partitioning into tissues such as fat and muscle. Since there is a constant flux in the nutritional supply and energy needs of an organism, strategies that maintain a steady state under varying nutritional conditions are crucial for healthy functioning of an organism. Although body weights across a population vary considerably, individuals show a remarkable weight stability, suggesting that body weight is physiologically regulated (Flier & Maratos-Flier, 2007). Regulation of fat depots involves a coordinated interplay between central regulators of feeding behavior in the hypothalamus, neuroendocrine signals, and metabolic regulators of energy expenditure and fat storage (Flier & Maratos-Flier, 2007). Dramatic alterations in body weight have been shown to result from mechanical or pharmacological lesions of the hypothalamus (Flier & Maratos-Flier, 2007). Peripheral tissues such as the gut, white and brown adipose, and skeletal muscles also regulate body weight and its composition (Flier & 47

79 Maratos-Flier, 2007). One of the most dramatic illustration of this comes from animals with a mutation in myostatin, or animals treated with compounds that block or antagonize the activity of myostatin such as follistatin, which results in significantly larger skeletal muscle mass and increased muscle strength (Gangopadhyay, 2013) Leptin The adipose-derived hormone leptin regulates systemic energy homeostasis by linking an individual s fat stores with caloric intake and expenditure (Flier & Maratos-Flier, 2007). The receptors for leptin are located in the hypothalamus, and have eighteen N-glycan sequons in humans, and seventeen and sixteen in rat and mouse, respectively. Leptin functions as a satiety signal released from adipose tissue in proportion to fat stores, and is part of a signaling pathway that acts to maintain the size of body fat depot (Flier & Maratos-Flier, 2007). Leptin activity impinges on the hypothalamic regulatory neuro-circuitry to increase energy expenditure and inhibit feeding (Flier & Maratos-Flier, 2007). Both leptin and insulin have been characterized as adiposity signals, since their plasma levels positively correlate with body weight and are in direct proportion to the amount of energy stores in adipose tissue (Schwartz et al, 2013). Leptin also stimulates fatty acid oxidation in non-adipose tissues, so as to minimize ectopic lipid accumulation and protect against lipotoxicity (Unger et al, 2010). Humans with a rare congenital loss-of-function mutations in the leptin gene are clinically obese, due to abnormalities in energy expenditure and increased food intake (Speakman & O'Rahilly, 2012). These symptoms are reversed by administration of recombinant human leptin (Speakman & O'Rahilly, 2012). Mice deficient in leptin or in the leptin receptor exhibit overfeeding and develop obesity and diabetes (Flier & Maratos-Flier, 2007). 48

80 1.3.2 Determinants of Body Weight and Obesity Factors affecting weight regulation and obesity have been identified at a number of different levels of analysis. These span the whole spectrum, from social to economic, psychological, behavioral, developmental, physiological, biochemical, metabolic, genetic and molecular, with interaction and feedback across and between factors. Adiposity is a highly heritable trait, and genetics appears to explain around 65% of weight variation between individuals (Speakman & O'Rahilly, 2012). Although many polymorphisms associate robustly with obesity, much of the genetic variation that underpins differences in adiposity across the normal population remains unexplained (Speakman & O'Rahilly, 2012). It appears that body weight and composition is a phenotype at the interface of genes and environment, where genes most likely determine susceptibility to environmental factors. Environmental factors such as gut microbiota, stress, endocrine disruptors have also been linked to risk of developing obesity (Speakman & O'Rahilly, 2012). The most widely accepted explanation for the rise in adiposity around the globe involves increased availability and access to palatable calorie-dense foods, which leads to overeating and excess calorie intake; combined with decreased energy expenditure through sedentary lifestyle resulting from modernization and technological advances, which have greatly reduced labour intensive activities (Speakman & O'Rahilly, 2012; Unger et al, 2010). The idea of a toxicenvironment and exposure to chemical obesogens, such as bisphenol-a and pesticides, has also been proposed to account for the rise in obesity rates in the last few decades (Grun, 2010). This theory argues that our environment has become contaminated due to pharmaceutical drugs, chemical pollutants, as well as hormones and antibiotics used in farming, which disrupt the energy 49

81 balance, endocrine function, lipid metabolism and fat storage by a variety of mechanisms (Grun, 2010). However, the prevailing explanation for the observed increase in body weight and obesity is that of energy imbalance, i.e. eating too many calories and not getting enough physical activity (Speakman & O'Rahilly, 2012) Energy Balance The guiding principle of body regulation bioenergetics is that of energy balance or energy homeostasis, which proclaims that to maintain a constant body weight, energy or calorie intake from food consumed must equal energy or calories burned by the metabolism of the organism (Flier & Maratos-Flier, 2007). Hence, body weight is maintained in a steady state by a balance between caloric intake and energy expenditure. Any imbalance between energy intake and expenditure, which includes total physical activity, metabolic rate and thermogenesis, is reflected in a change in the amount of stored energy as fat (Flier & Maratos-Flier, 2007). Thus, when nutritional intake chronically exceeds the energy needs of an organism, the excess energy is stored in body fat, which in turn leads to weight gain. This dominant theory is firmly rooted in the first law of thermodynamics, i.e. the law of conservation of energy, which states that energy can neither be crated nor destroyed, but only converted from one form to another. However, the dogma of energy imbalance between calories consumed and calories expended being the fundamental cause of obesity is not universally accepted (Lustig et al, 2012; Taubes, 2013) The Carbohydrate Hypothesis of Obesity 50

82 The theory of energy balance has been challenged with arguments that physiology is not physics, and that not all calories are created equal, but rather that nutrient composition affects fat accumulation (Taubes, 2012). In this theory, sugar has been singled out as the toxic culprit responsible for increase in obesity, diabetes and the metabolic syndrome around the globe (Lustig et al, 2012; Taubes, 2013). This alternative hypothesis suggests that adiposity is not caused by excess calories alone, but rather by the quantity and quality of carbohydrates and starches consumed. The logic behind this theory is that since insulin regulates carbohydrate metabolism and stimulates the synthesis and storage of fats in the liver and fat depots, and blood levels of insulin are effectively determined by carbohydrate intake, then consumption of sweet carbohydrates with a high-glycemic index will result in more insulin release and more fat accumulation (Taubes, 2013). With elevated insulin levels (hyperinsulinemia) in the bloodstream for prolonged periods, adipocytes respond by accumulating more fat, thus promoting more weight gain (Taubes, 2013). A variant of this theory suggests that it is specifically excess consumption of fructose, found in sucrose and high-fructose corn-syrup, that is responsible for the current epidemic of obesity and chronic metabolic disease (Lustig, 2013). Indeed, fructose consumption has increased worldwide, as it is often used as a sweetener during food processing, paralleling the increase in obesity and chronic metabolic diseases over the last few decades (Lustig et al, 2012). Unlike glucose, fructose is very sweet, and does not generate an insulin response (Lustig, 2013). Its hepatic metabolism in the post-absorptive state impairs β-oxidation, and promotes de novo lipogenesis, triglyceride formation and hepatic steatosis (Lustig, 2013). This is often followed by hepatic insulin resistance, hyperglycemia, as well as formation of reactive oxygen species, resulting in cellular toxicity and dysfunction (Lustig, 2013). Furthermore, fructose also has 51

83 dependence-producing properties, which promote changes in the brain s reward system, leading to excessive consumption (Lustig, 2013). Consequently, fructose has been deemed toxic and a danger to individuals and society requiring prompt regulation, since it exerts its detrimental health effects beyond its calories, and in ways that are similar to alcohol (Lustig et al, 2012). However, it should also be noted that the idea that carbohydrates are mostly to blame for making animals fat has many detractors, who instead point to fat as the real culprit. Most nutrients consumed in excess of energy needs are converted to fat, however since carbohydrates must first be metabolized for this purpose, their conversion is actually more ineffective than fats, which can be directly integrated into triglycerides and lipid droplets. Moreover, there is plenty of evidence from mouse experimental research to support the claim that calorie enriched fatty diet leads to body weight increase (Cheung et al, 2007). The incompleteness in our understanding of body weight regulation and what causes obesity is illustrated by the lack of a general consensus on the issue, or a universally accepted explanation. Indeed, competing diet programs promising success and results are often polar opposites of one another. Furthermore, the fact that dieting, weight-loss and fad diets are a billion dollar industry, with most being ineffective, and those that do lose weight initially not being able to maintain the weigh-loss they have obtained either through hard-work or deprivation, is a testament to the fact that we do not fully understand the underlying biology of body weight regulation, and hence lack actionable knowledge for impactful intervention in public health advice. 52

84 1.4 Rationale, Objectives and Summary N-glycosylation and Golgi N-glycan branching are essential modifications of proteins translated in the secretory pathway. Glycoproteins traffic through the Golgi apparatus, where exposure to N-glycan branching enzymes is dependent on enzyme expression and UDP-GlcNAc supply. Glucose transporters Glut1, Glut2 and Glut4 are dependent on N-glycan branching pathway and galectin binding for optimal cell surface retention and glucose transport (Dennis et al, 2009). Mutations in genes encoding branching enzymes Mgat4a and Mgat5 in the N-glycan branching pathway disrupt glucose homeostasis in mice (Cheung et al, 2007; Ohtsubo et al, 2005). When I began my research project, Mgat4a expression in the pancreas was reported to be regulated at the level of gene expression, and both Mgat5 and Mgat4a mutant mice displayed aberrant regulation of body-weight on high-fat diet (Cheung et al, 2007; Ohtsubo et al, 2005). Relatively little consideration had been given to the contribution of metabolic feedback through the HBP or GlcNAc salvage to UDP-GlcNAc, the common donor substrate used by Mgat enzymes, until a publication from the Dennis laboratory reported that elevated UDP-GlcNAc supply to the N- glycan branching pathway enhanced cell surface residency and activity of growth factor receptors and Glut4 (Lau et al, 2007). The main objective of my thesis was to determine whether metabolic input through the HBP to UDP-GlcNAc contributed to control of N-glycan branching pathway in vivo, and whether this could be seen to occur cell-autonomous in vitro. Another objective was to examine the effect of increased expression of N-glycan branching enzymes and/or UDP-GlcNAc supply on cellular metabolism using targeted mass-spectrometry based metabolomics, and to explore the potential mechanisms by which HBP-mediated increase in N-glycan branching exerts its effect on metabolism. 53

85 Effects of overexpressing Mgat1, Mgat5 or Mgat6 on central metabolites were investigated in cells growing under normal cell culture media conditions supplemented with GlcNAc. Induced overexpression of Mgat enzymes increased respective N-glycan branches on cell surface glycoproteins, and increased central metabolites in different metabolic pathways. Further increases were observed by using GlcNAc to supplement the HBP and increase UDP-GlcNAc pool for N- glycan branching enzymes. Importantly, GlcNAc supplementation and Mgat5 overexpression displayed synergistic increase in Mgat5-mediated N-glycan branching. Moreover, my findings suggest that N-glycan branching cooperates with the HBP to regulate nutrient import and metabolism in a cell-autonomous manner. I also examined whether UDP-GlcNAc levels are sensitive to dietary GlcNAc supplementation in vivo, and might increase nutrient uptake and promote anabolic metabolism via Golgi N-glycan branching. Chronic oral GlcNAc supplementation in C57BL/6 mice increased hepatic UDP-GlcNAc and N-glycan branching on liver glycoproteins. GlcNAc supplementation altered hepatic metabolism resulting in excess lipid storage and body-weight increase. In cultured cells, GlcNAc enhanced nutrient uptake and promoted fatty-acid synthesis and storage in an N- glycan-dependent manner. These results suggest that GlcNAc salvage promotes nutrient uptake and lipid storage by enhancing UDP-GlcNAc supply to the Golgi N-glycan branching pathway to modify N-glycan branches on transmembrane glycoproteins. GlcNAc supplementation also restored fat accumulation in Mgat5 null mice, which exhibit a lean phenotype with reduced fat depots. In both genders of Mgat5 null mice oral GlcNAc increased fat accumulation, a result consistent with functional redundancy of N-glycan branches. The rescue of fat accumulation in Mgat5 null mice occurred at the expense of lean mass, indicating a requirement for Mgat5 in the normal lean to fat balance. 54

86 Chapter 2 Golgi N-Glycan Branching N-Acetylglucosaminyltransferases I, V and VI Promote Nutrient Uptake and Metabolism A version of this chapter was accepted for publication in Glycobiology 2015 Feb;25(2): Anas Abdel Rahman *, Michael C. Ryczko *, Miyako Nakano *, Judy Pawling, Tania Rodrigues, Anita Johswich, Naoyuki Taniguchi, and James W. Dennis * co-first authors Attributions: The LC ESI-MS chromatogram results for N-glycan analysis displayed in Figures 2.2C-E and 2.3D-F were performed by Miyako Nakano. The targeted metabolomics mass spectrometry experiments in Figures 2.4A-C, 2.5C, 2.6A and C, 2.7, 2.8, and 2.10 were performed together with Anas Abdel Rahman. All other experiments were designed and performed by Michael Ryczko. 55

87 2.1 Summary Nutrient transporters are critical gate-keepers of extracellular metabolite entry into the cell. As integral membrane proteins, most transporters are N-glycosylated, and the N-glycans are remodeled in the Golgi apparatus. The Golgi branching enzymes N-acetylglucosaminyltransferase I, II, IV, V, and avian VI (encoded by Mgat1, Mgat2, Mgat4a/b/c, Mgat5 and Mgat6), each catalyze the addition of GlcNAc in N-glycans. Here I asked whether N-glycan branching promotes nutrient transport and metabolism in immortal human HeLa carcinoma and non-malignant Hek293 embryonic kidney cells. Mgat6 is absent in mammals, but ectopic expression can be expected to add an additional β1,4 linked branch to N-glycans, and may provide evidence for functional redundancy of the N-glycan branches. Tetracycline-induced over-expression of Mgat1, Mgat5, and Mgat6 resulted in increased enzyme activity and increased N-glycan branching concordant with the known specificities of these enzymes. Tet-induced Mgat1, Mgat5 and Mgat6 combined with stimulation of the HBP to UDP-GlcNAc, increased cellular metabolite levels, lactate and oxidative metabolism in an additive manner. I then tested the hypothesis that N-glycan branching alone might promote nutrient uptake when glucose and glutamine are limiting. In low glutamine and glucose medium, tet-induced Mgat5 alone increased amino acids uptake, intracellular levels of glycolytic and TCA intermediates, as well as Hek293 cell growth. More specifically, tet-induced Mgat5 and HBP elevated the import of Gln, although transport of other metabolites may be regulated in parallel. Our results suggest that N-glycan branching cooperates with HBP to regulate metabolite import in a cell autonomous manner, and can enhance cell growth in low-nutrient environments. 56

88 2.2 Introduction The medial Golgi N-acetylglucosaminyltransferases I, II, IV, V (Mgat1, Mgat2, Mgat4a/b/c, Mgat5) form a linear pathway that initiate the GlcNAc branches on newly synthesized glycoproteins (Figure 2.1A) (Schachter, 1986). In the trans Golgi, the branches are extended with galactose, fucose and sialic acid to generate sequences recognized by galectins, C-type lectins and siglecs. The N-glycan branching pathway is required for tumor progression, and molecular mechanisms have emerged that intersect with basic metabolism (Lau et al, 2007; Shirato et al, 2011). Tumor growth rates are reduced in Mgat5 -/- mice with a PyMT transgene or in Pten heterozygous mice (Cheung & Dennis, 2007; Granovsky et al, 2000). Suppression of branching by either Mgat1 shrna (Beheshti Zavareh et al, 2012) or over-expression of N- acetylglucosaminyltransferase III (Mgat3) (Song et al, 2010; Yoshimura et al, 1995) also inhibit tumor progression in mice. Furthermore, over-expression of Mgat5 in epithelial cells promotes transformation (Demetriou et al, 1995), and in human mammary and colon cancer, increased Mgat5-branched N-glycans are associated with disease progression (Fernandes et al, 1991; Seelentag et al, 1998). Previously it was shown that galectins bind branched N-glycans and cross-link glycoprotein to form a highly dynamic lattice that slows loss of membrane glycoproteins to endocytosis (Lau & Dennis, 2008). In Mgat5 -/- tumor cells, the retention of surface receptors is reduced along with sensitivity to growth factors and cytokines. The mutant phenotype could be rescued by either (i) Mgat5 re-expression, (ii) inhibition of constitutive endocytosis, or (iii) increased UDP-GlcNAc, the common sugar-nucleotide donor for the Mgat enzymes (Lau et al, 2007; Partridge et al, 2004). In the absence of Mgat5, compensating amounts of N- acetyllactosamine branches are produced by Mgat1, Mgat2 and Mgat4 when cells are 57

89 supplemented with GlcNAc to increase UDP-GlcNAc (Lau et al, 2007). This suggests redundancy of the branches and cooperative action of N-glycans in support of growth factor receptors and other glycoproteins at the cell surface (Dennis & Brewer, 2013). Interestingly, the chicken (Gallus gallus) GnT-VI (Mgat6) enzyme catalyzes the GlcNAcβ1,4 linkage to GlcNAcβ1,6Manα1,6Manβ in the trimannosyl core of N-glycans (Figure 2.1A). Mgat6 is expressed widely in birds (Watanabe et al, 2006), with homologues in frog (Xenopus laevis), duckbilled platypus (Ornithorhynchus anatinus) and a few species of fish. Mgat6 gene and activity are absent in mammals. If N-glycan branching is indeed cooperative and regulates metabolism, it is reasonable to assume that ectopic expression of Mgat6 in mammalian cells may reveal evidence for both. Growth factor receptors activate Akt, Ras and mtor/s6k signaling, which leads to increased Glc and amino acid transporter activity (Frauwirth et al, 2002; Wellen et al, 2010; Wise et al, 2008; Yun et al, 2009). Many of the ~600 mammalian solute transporters have extracellular domains with sites of N-glycosylation, and like receptor kinases, may be regulated by N-glycan branching. Gluts are dependent on N-glycan branching for surface residency of Glut2 in β-cells (Ohtsubo et al, 2005), Glut1 in tumor cells (Kitagawa et al, 1995), and insulin-stimulated or HBPstimulated increases in surface Glut4 (Haga et al, 2011; Lau et al, 2007). The GlcNAc β1,4 branching enzyme GnT-IVa (Mgat4a) is selectively expressed in insulin producing β cells of the pancreas, and Mgat4a -/- mice develop type 2 diabetes (Ohtsubo et al, 2005; Takamatsu et al, 2010). The branched N-glycans on Glut2 bind galectin-9 which promotes surface residency of Glut2, and transport of Glc. N-glycan branching depends on de novo synthesis of UDP-GlcNAc by the HBP and/or salvage of GlcNAc. Moreover, growth factor signaling increases UDP-GlcNAc levels, and shrna 58

90 inhibition of GFAT in the de novo HBP reduced Ras-driven tumor growth (Ying et al, 2012). Ras/Mapk and PI3K signaling also stimulates Mgat5 gene expression (Buckhaults et al, 1997; Dennis et al, 1989; Kang et al, 1996), as well as Mgat4 in human carcinomas (D'Arrigo et al, 2005; Takamatsu et al, 1999). The affinity of Mgat1, Mgat2, Mgat4a/b/c and Mgat5 enzymes for UDP- GlcNAc decreases ~300 fold moving from Mgat1 to Mgat5. As such, Mgat1 functions near saturation and Mgat5 well below its Km value for UDP-GlcNAc (Lau et al, 2007; Sasai et al, 2002). The action of Mgat1 is required to generate substrates for further remodeling by α-mannosidase II, Mgat 2, Mgat4 and Mgat5. Gln supplied to cells above typically used cell culture levels (>4 mm) increases UDP-GlcNAc levels, similar to GlcNAc supplementation in normal culture conditions, while Glc below physiological concentration (<5 mm) reduced UDP-GlcNAc levels (Abdel Rahman et al, 2013). Thus both Glc and Gln flux through HBP regulate UDP-GlcNAc levels, but at the low and high end of their concentration ranges, respectively. These studies offer the intriguing possibility that cellular regulation of N-glycan branching on solute transporters and receptors has evolved as a mechanism of adaptation to changing nutrient and environmental cues. Mgat5-deficient mice are hypoglycemic, hyposensitive to glucagon (Johswich et al, 2014), and resistant to weight-gain on a high-fat diet, although their food intake is equivalent to wild-type littermates (Cheung et al, 2007). This suggests that N-glycan branching may promote a thrifty phenotype, i.e. efficiency in uptake and/or utilization of nutrients (Cheung et al, 2007). As an experimental model, I generated HeLa tumor cells and Hek293 immortalized cells with inducible transgenes for Mgat1, Mgat5, or Mgat6 over-expression. Here I have characterized the effects of short-term (~24 h) transgene over-expression on nutrient levels and central metabolism in cells in culture. I report that Mgat1, Mgat5 or Mgat6 over-expression alone, and with HBP stimulation to UDP-GlcNAc, increases metabolite levels, suggesting cooperative action of the N-glycan 59

91 branching enzymes. Mgat5 transgene over-expression alone restored metabolite levels and Hek293 cell growth to normal in low Gln and Glc culture conditions. The uptake rate of Gln and the intracellular levels of other amino acids were enhanced by Mgat5 over-expression. This is the first evidence of cell autonomous regulation of nutrient uptake and metabolism by N-glycan branching. 2.3 Materials and Methods Materials and Chemicals PhosSTOP Phosphatase Inhibitor Cocktail and Complete Protease Cocktail were purchased from Roche. Alexa Fluor-488 conjugated lectins Concanavalin A (ConA) and L-PHA were purchased from (Invitrogen). Metabolite standards and reagents were obtained from Sigma Chemicals (St. Louis, MO) with minimal purity of 98%. Stable-isotope 15 N 15 N-L-Glutamine was purchased from Cambridge Isotope Laboratories Inc. (Andover, MA). All organic solvents and water used in sample and LC/MS mobile phase preparation were HPLC grade and obtained from Fisher Scientific (Fair Lawn, NJ). N-Glycosidase F (PNGase F, EC , recombinant cloned from Flavobacterium meningosepticum and expressed in E. coli) was purchased from Roche Diagnostics (Mannheim, Germany). PVDF membrane (Immuno-Blot, 0.2 µm, 7.0 x 8.5 cm) was purchased from Bio-Rad (Hercules, CA). Cation-exchange columns were made of (30 mg) Dowex 50W-X mesh resins (Wako Chemical, Osaka, Japan) packed on top of a 10 μl ZipTip u- C18 (Millipore, Billerica, MA). The resins were protonated with 1M HCl (50 µl, 3 times), were rinsed with methanol (50 µl, 3 times), and then equilibrated with water (50 µl, 3 times) before use. Microtiter plates (96-well flat bottom, MaxiSorp) were purchased from Nunc (Roskilde, 60

92 Denmark). All other chemicals were purchased from Sigma-Aldrich, Nacalai Tesque (Kyoto, Japan) or Wako Chemical. Other reagents and solvents were of HPLC or LC/MS grade Cell Culture HeLa Flp-In-TREx cells were a kind gift from Dr. Stephen Taylor (University of Manchester), while Hek293 Flp-In-TREx cells (Ward et al, 2011) were purchased from Invitrogen. Both adherent cell lines were grown in an incubator at 37 C and 5% CO2 in a humidified atmosphere. They were maintained in high-glc DMEM (Sigma) supplemented with 10% fetal bovine serum (FBS), 2 mm Gln, penicillin/streptomycin, and 3ug/ml blastocidin and 100 ug/ml zeocin, unless indicated otherwise. Human Mgat1 cdna, human Mgat5 cdna, and chicken Mgat6 cdna were FLAG-tagged at the N-terminus, and each construct was cloned individually into the pcdna5/frt/to expression vector. Each plasmid was integrated into the genome of the HeLa or Hek293 Flp-In-TREx cells described above, at a pre-integrated FRT recombination site, by co-transfection with Flp recombinase encoding POG44 plasmid, using Lipofectamine (Invitrogen) and OptiMEM media lacking FBS or antibiotics. Following selection in 200 ug/ml of hygromycin, resistant clones were individually picked, expanded and treated with 1 ug/ml tetracycline for 24h. To verify gene expression, cell lysates were analyzed by Western-blotting using monoclonal Anti-FLAG M2 antibody (Sigma) Western Blotting Cells were rinsed with ice-cold PBS and lysed in ice-cold lysis buffer (50 mm HEPES, 120 mm NaCl, 2 mm EDTA, 1.0% NP-40, one tablet of phosphatase inhibitor cocktail per 10 ml, and one tablet of EDTA-free protease inhibitors per 10 ml). The soluble fractions of cell lysates were 61

93 isolated by centrifugation at at 20,000G for 10 min by centrifugation in a microfuge at 4 C. Proteins were denatured by addition of 5X sample buffer, boiled for 5 min, resolved by 8% or 10% SDS-PAGE, and analyzed by immunoblotting for the FLAG-tagged proteins Enzyme Assays Enzyme sources were cell lysates obtained from large plates of tetracycline induced and not induced cells lysed on ice in 0.9% NaCl, 1% Triton X-100, and including protease inhibitors (Beheshti Zavareh et al, 2012; Korczak et al, 2000). Mgat1 enzyme activity was measured using synthetic acceptors. The assays contained 10 µl of cell lysate, 2 mm Man(1,3) Man(1,6) Glc- O(CH2)7CH3 acceptor (Toronto Research Chemicals), 0.5 µci [6-3 H]-UDP-GlcNAc (44,400 dpm/nmol) in 50 mm MES ph 6.5, 2 mm Mn2Cl2, 5mM AMP in total volume of ~20 µl. After 1.5 h of incubation at 37 C, 1 ml of ice-cold water was added to stop further reaction and assays were either frozen or processed immediately. Mgat5 enzyme activity was determined as transfer of UDP-[6-3 H] GlcNAc to the synthetic acceptors. The reaction contained 10 µl of cell lysate, 1 µci of [6-3 H]-UDP-GlcNAc (44,000 dpm/nmol), 50 mm MES ph 6.5, 0.5 M GlcNAc, 1mM UDP- GlcNAc, 25mM AMP, in a final volume of 20 µl, with synthetic acceptors 1 mm βglcnac(1,2)αman(1,6)βglc-o(ch2)7ch3 for Mgat5. Reactions were incubated at 37 C for 180 min for the Mgat5 assay. Endogenous activity for the enzymes was measured in the absence of acceptor, and subtracted from values determined in the presence of added acceptor. Reactions were stopped with 1ml of H2O, and enzyme products were separated from radioactive substrates by binding them to 50 mg C18 cartridges (Alltech), preconditioned with methanol rinsing and water washes. Reactions were loaded and the columns washed 3 times with water. Radio labeled products were eluted with methanol directly into scintillation vials with two separately applied

94 µl aliquots of methanol, and the radioactivity determined by liquid scintillation counting. Mgat6 enzyme activity was assayed according to the method of Taguchi et al (Taguchi et al, 2000) using as acceptor substrate, PA-agalacto [(2,6),(2,4)] tetra-antennary and 100 mm UDP-GlcNAc in buffer 150 mm HEPES-NaOH, 60 mm MnCl2, 200 mm GlcNAc, 0.5% Triton X-100 and 2 mg/ml BSA for 4h at 37 o C. The product was separated by HPLC on TSKgel ODS-80TM (0.46 x 15 cm, Tosoh, Tokyo, Japan), eluted with 20 mm ammonium acetate (ph 4.0)/0.02% 1-butanol at flow rate of 1 ml/min, column temp at 55 and detection by fluorescence (excitation/emission =320/400 nm) Quantitative Lectin Fluorescence Imaging Cells were plated in a 96-well plate at 2000 cells/well in high-glc DMEM supplemented with 10% FBS, 2 mm Gln, and incubated at 37 C and 5% CO2 with and without tetracycline for 24h to induce gene expression. Cells were then fixed for 15min with 4% paraformaldehyde, washed with PBS, and incubated for 1h at room temperature in 50 µl PBS, 1/5000 of Hoechst 33342, and either 1/1000 of 2mg/ml Alexa Fluor-488 conjugated lectin ConA or L-PHA (Invitrogen, Carlsbad, CA). After washing with PBS, cells were imaged and staining per cell quantified using IN Cell Analyzer 1000 automated fluorescence imaging system Cell Viability Assay Following plating in a 96-well cell culture plate, and treatment with various concentrations of Glc, Gln and FBS in DMEM for at least 24h, AlamarBlue cell viability reagent (Invitrogen) was added at 1/10th the volume directly to cells in cell culture media. The plate was then returned to the incubator at 37 C and 5% CO2 in a humidified atmosphere for an additional incubation of 6h. 63

95 Cell viability was measured fluorometrically, as relative fluorescence units (RFU), in a microplate reader (Gemini Fluorescence from Molecular Devices) following the reduction of the nonfluorescent oxidized dye AlamarBlue by viable and metabolically active cells to its fluorescent product, using fluorescent excitation and emission wavelengths of 570 and 585 nm respectively. The number of metabolically active, proliferating, viable cells correlates with the magnitude of dye reduction and fluorescence emission intensity from each well Cell Membrane Preparation for N-Glycan Profiling For N-glycan analysis by LC-MS, HeLa and Hek293 Flp-In-TREx cells were cultured in standard culture medium, DMEM plus 10% FBS at 37 C and 5% CO2 in a humidified atmosphere for 24h, with and without 1ug/ml tetracycline to induce gene expression. ~2x10 7 cells per treatment were rinsed with phosphate buffer saline (PBS), trypsinized, collected in pellets, frozen on dry ice, and stored at -80 C. The frozen pellets were suspended in 2 ml of lysis buffer containing 50 mm Tris-HCl (ph 7.4), 0.1 M NaCl, 1 mm EDTA and protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany) and kept on ice for 20 min and then homogenized using a polytron homogenizer (Omni TH tissue homogenizer, Omni International, Inc., VA; 15 sec, 7 times on ice bath). The following approach was taken, according to the procedure reported by Nakano and colleagues (Nakano et al, 2011). The homogenized cells were centrifuged at 2000g for 20 min at 4 C. The supernatant was diluted with 2 ml of Tris-buffer (50 mm Tris-HCl (ph 7.4), 0.1 M NaCl) and then were sedimented by ultracentrifugation at g for 80 min at 4 C (swing rotors, Himac, Hitachi Koki). The supernatant was discarded, and the membrane pellet was suspended in 100 µl Tris-buffer. After adding 400 µl Tris-buffer containing 1% (v/v) Triton X-114, the suspended mixture was homogenized by pipetting strongly. The homogenate was chilled on ice 64

96 for 10 min and incubated at 37 C for 20 min and then phase partitioned by centrifugation at 1940g for 2 min. The upper aqueous phase was removed. The lower detergent phase was further mixed with 1 ml of ice-cold acetone and kept at -25 C overnight to precipitate proteins and remove any detergent. After centrifugation at 1940g for 2 min, the precipitated cell membrane proteins were stored at -25 C if not used immediately Enzymatic Release and Purification of N-Glycans The precipitated membrane proteins were dissolved with 10 µl 8 M Urea. The solubilized proteins were dotted (2.5 µl x 4 times) onto PVDF membrane pre-wetted with ethanol. After drying the PVDF membrane with cold blast of a dryer for 10 min, the PVDF membrane was rinsed with ethanol for 1 min and then rinsed three times for 1 min with water. The protein on the membrane was stained for 5 min with (800 µl solution A: 0.1% (w/v) Direct Blue 71 (Sigma-Aldrich) in 10 ml solution B: acetic acid : ethanol : water = 1:4:5). After destaining with solution B for 1 min, the PVDF membrane was dried with cold blast of a dryer for 10 min. Protein stained blue were cut from the PVDF membrane and placed in separate wells of a 96-well microtiter plate. The spots were then covered with 100 μl of 1% (w/v) polyvinylpyrrolidone in 50% (v/v) methanol, agitated for 20 min, and rinsed with water (100 µl x 5 times). PNGase F (3U in 10 μl of 30 mm phosphate buffer (ph 7.3)) was added to each well and incubated at 37 C for 15 min. An additional 10 µl of water was added to each well and incubated at 37 C overnight to release N-glycans. During the incubation, the sample wells were sealed with amplification tape to prevent evaporation. To collect the released N-glycans, the samples were sonicated (in the 96-well plate) for 10 min, and the released N-glycans (20 µl) were transferred to 1.5-ml polypropylene tubes. The sample well was rinsed with water (50 µl twice), and the washings combined. To completely 65

97 generate the reducing terminus, ammonium acetate buffer (100 mm, ph 5.0, 20 µl) was added to the released N-glycans for 1 hour at room temperature. After evaporating to dryness, the glycans were resolved with 10 µl of 50 mm KOH and then reduced by adding 10 µl of 2 M NaBH4 in 50 mm KOH at 50 C for 3 hours. 1 µl of acetic acid was added to stop the reaction, and the N-glycan alditols were desalted using a cation-exchange column. The glycan alditols were eluted with water (50 µl twice), dried, and the remaining borate was removed by the addition of (100 μl x 3) methanol and drying under vacuum. To remove sialic acids from the glycans 100 µl of 2 M acetic acid was added to the dried glycan samples and the samples were incubated at 80 o C for 2 hours and then were evaporated to dryness. The desialylated N-glycan alditols were re-suspended in 10 mm NH4HCO3 (20 µl) immediately before glycan analysis by liquid chromatographyelectrospray ionization mass spectrometry (LC-ESI MS) LC-ESI MS for Analysis of N-Glycan Alditols N-Glycan alditols were separated using a HyperCarb porous graphitized carbon column (5 μm HyperCarb, 0.32 mm I.D. 100 mm, Thermo Scientific) under the following gradient conditions. The separation was performed using a sequence of isocratic and two segmented linear gradients: 0 8 min, 10 mm NH4HCO3; 8 53 min, % (v/v) CH3CN in 10 mm NH4HCO3; min, % (v/v) CH3CN in 10 mm NH4HCO3; and increasing to 81% (v/v) CH3CN in 10 mm NH4HCO3 for 6 min and re-equilibrated with 10 mm NH4HCO3 for 15 min at a flow rate of 5μl/min (LC/MSD Trap XCT Plus Series 1100, Agilent Technologies, USA). The injection volume of samples was 5 µl. In the MS (Esquire HCT, Bruker Daltonics GmbsH, Bremen, Germany), the voltage of the capillary outlet was set at 3.5 kv, and the temperature of the transfer capillary was maintained at 300 C. The flow rate of nitrogen gas for drying was 5 66

98 l/min. The MS spectra were obtained in the negative ion mode over the mass range m/z 150 to m/z The scan rates were 8100 amu/s for the MS mode and the MS/MS mode. Monoisotopic masses were assigned with possible monosaccharide compositions using the GlycoMod tool available on the ExPASy server ( mass tolerance for precursor ions is +/- 0.1 Da) and the proposed oligosaccharide structures were provided at UnicarbKB database ( and further verified through annotation using a fragmentation mass matching approach based on the MS/MS data. Validation of the technical reproducibility of the analytical conditions such as retention time and mass number was carried out using known glycans derived from bovine fetuin before analyzing any experimental samples. The relative abundance of each glycan structure on the cell membrane glycoproteins was calculated based on the peak area of the ion chromatogram of the corresponding glycan structure extracted using the mass of the [M-2H] 2- ion, after processing of the peaks (smoothing algorithm; Gauss, smoothing widths; 1pnts, S/N thresholds; 1, no exclusion mass, using Bruker Daltonics DataAnalysis software ver. 3.4) (Nakano et al, 2011) Metabolite Analysis by LC-MS/MS To determine the relative levels of metabolites, HeLa and Hek293 Flp-In-TREx cells were cultured in 6-well plates at 37 C and 5% CO2 in a humidified atmosphere for 24h in various nutrient conditions as described before (Abdel Rahman et al, 2013), with and without tetracycline to induce gene expression. Media was aspirated and cells rinsed on the plates with warm PBS. The plates were snap frozen in liquid N2 and moved to -80 C until extraction. The metabolites were rapidly extracted by addition of 1 ml ice-cold solution of (40% acetonitrile, 40% methanol, and 20% water). After quenching, the cells were scraped and transferred to 1.5 ml tube and shaken for 67

99 1 h at 4 C and 1000 rpm in a Thermomixer (Eppendorf, Germany). The samples were spun down at 14,000 rpm, for 10 min at 4 C (Eppendorf, Germany), and then the supernatant transferred to fresh tubes to be evaporated to dryness in a CentreVap concentrator at 40 C (Labconco, MO). The dry extract samples were stored at -80 C for LC/MS analysis. Cell number for each culture condition was determined for normalization purposes by trypsinization of parallel replicate wells. The dry metabolite extracts were reconstituted in 200 µl of water containing internal standards (500 µg/ml and 300 µg/ml of D7-glucose and 13 C9 15 N-Tyrosine, respectively), for the purpose of calibration, normalization and quantification. The mixture of metabolites was injected twice through the HPLC (Dionex Corporation, CA) in gradient reversed phase column Inertsil ODS-3, 4.6 mm internal diameter, 150 mm length, and 3-µM particle size for positive and negative mode analysis. In positive mode analysis, the mobile phase gradient ramps from 5% to 90% of acetonitrile in 16 min, then after 1 min at 90%, the composition returns to 5% acetonitrile in 0.1% acetic acid in two min. In negative mode, the acetonitrile composition ramped from 5 to 90% in 10 min, then after 1 min at 90%, the gradient ramped back to 5% acetonitrile in buffer-a (0.1% tributylamine, 0.03% acetic acid, 10% methanol). The total runtime in both modes was 20 min, the samples were stored at 4 C, and the injection volume was 10 µl. An automated washing procedure was developed before and after each sample to avoid any sample carryover. The eluted metabolites were analyzed at the optimum polarity in MRM mode on electrospray ionization (ESI) triple-quadrupole mass spectrometer (ABSciex4000Qtrap, Toronto, ON, Canada). The mass spectrometric data acquisition time for each run is 20 min, and the dwell time for each MRM channel is 10 ms. Common mass spectrometric parameters are the same as tuning conditions described below, except: GS1 and GS2 were 50 psi; CUR was 20 psi, and CAD was 3 and 7 for positive and negative modes, respectively, and source temperature (TEM) was 68

100 400 C. Signal was normalized to internal standard and cell number. The LC-MS/MS system does not resolve hexose, hexosamine and sugar-nucleotide stereoisomers, including Glc/Galactose, GlcNAc/GalNAc, GlcNAc-6P/GlcNAc-1P, and UDP-GlcNAc/UDP-GalNAc. To monitor trends in metabolic pathways, I referred to these stereoisomers in their Glc forms. Stock solutions were prepared for each metabolite standard at a concentration of 100 µm in 40/60 methanol/water (v/v), 0.1%NaOH. A final concentration (10µM) of each metabolite was obtained for mass spectrometric tuning. A standard mixture of all metabolites was prepared at 200, 500, and 3000 nm as a sensitivity and specificity quality control. Ten additional serially diluted samples were prepared ranging from 1 nm to 2 µm for linearity assessments. A mixture of all the standard metabolites was used daily to check the LC-MS/MS system for optimal ionization polarity, declustering potential (DP), precursor ion (Q1), product ion (Q3), and collision energy (CE). Ion source potential (ISP) was 4500 V for positive and negative modes. Nebulizer gas (GS1) and bath gas (GS2) were 10 psi, curtain gas (CUR) was 15 psi, and collision gas (CAD) was 4 psi. Source temperature (TEM) was set to zero and interface heater was ON. The mass spectrometer was maintained and calibrated using a special kit designed by the manufacturer (ABSciex, Canada). The raw LC-MS/MS data was imported to MultiQuant version (ABSciex, Toronto, ON, Canada) and the extracted ion chromatogram (XIC) peaks were integrated. The result table contains the area, area ratio (area of analyte/area of internal standard), retention time, and concentration, and is then exported from MultiQuant as text files to MarkerView version (ABSciex, Toronto, ON, Canada) for normalization with cell number and protein content. Cell number normalized data was analyzed using the MetaboAnalyst online software for metabolomic 69

101 analysis ( and the KEGG databases ( (Xia et al, 2012) LC-MS/MS Measurement of 15 N 15 N-Glutamine Uptake and Metabolism Flp-In-TREx Hek293 Mgat5 cells were treated for ~24h with 1 mg/ml of tetracycline, to induce Mgat5 gene expression, in DMEM with 2.5 mm glucose and no glutamine plus 10% FBS, with and without 15 mm GlcNAc. The media was then changed to DMEM with 2.5 mm glucose and 1 mm 15 N 15 N-glutamine without FBS, for 1, 5 and 10 min. After incubation, cells were collected and metabolites extracted using the procedure detailed above. The chromatographic peaks for metabolite interest, and correct transitions for the labelled form of the metabolite were integrated using MultiQuant (ABSciex, Toronto, ON, Canada) (Soliman et al, 2014). 2.4 Results Tet-Inducible Expression of Mgat1, Mgat5 and Mgat6 in Human Cells To generate transgenic cell lines, HeLa and Hek293 cell lines with a single Flp-In-TREx insertion site were transfected with vectors for insertion of tetracycline (tet)-inducible FLAGtagged Mgat1, Mgat5 or Mgat6. The Flp-In-TREx site is designed for controlled expression from a single insertion, and indeed, tet-inducible expression of FLAG-tagged protein was detected in multiple independent HeLa cell clones (Figure 2.1B-E). Independent clones displayed similar levels of tet-inducible expression, with increases in Mgat1 and Mgat5 enzyme activity by 4 and 30 fold, respectively, while Mgat6 introduced a novel activity not present in mammalian cells (Figure 2.1F-H). Changes in N-glycan composition at the cell surface were determined by ConA and L- 70

102 PHA lectin staining. ConA binds high-mannose or mono-antennary N-glycans, while L-PHA binds Mgat5-modified tri- and tetra-antennary N-glycans. In HeLa cells, tet-induced Mgat1 enhanced ConA but not L-PHA binding. L-PHA binding was enhanced in tet-induced Mgat5, while ConA was unchanged (Figure 2.2A and B). Tet-induced Mgat6 reduced L-PHA binding suggesting the addition of the Mgat6 branch to Mgat5-branched N-glycans may interfere with L- PHA binding (Figure 2.2B). To further characterize the tet-induced Mgat1, Mgat5 and Mgat6 changes in N-glycan distributions, structures from the Flp-In-TREx HeLa cell glycoproteins were released by PNGase, and N-glycan alditols analyzed by LC-ESI MS. We interpret this data in light of the known specificity of the enzymes (Schachter, 1986; Watanabe et al, 2006) and prior knowledge of the Golgi pathway in mammalian cells (Schachter, 1986; Watanabe et al, 2006). We limited our interpretation to structures with the N-glycan core plus 1-4 Hex-HexNAc units, and the cartoon structures are drawn without branch linkages. The MS features corresponding to the masses of 16 predicted N-glycan structures were quantified. Twenty-three additional structures are indicated by their m/z values and probable compositions (Figure 2.2C-E). The profile for tet-induced Mgat1 HeLa cells revealed a 4-10 fold increase in the immediate product of Mgat1 (structures 14, 15 and 16) but not the down-stream tri- and tetra- antennary N-glycans (Figure 2.2C). This was consistent with the lectin binding profile of the cells (Figure 2.2A and B), and suggests a backup in N-glycan processing due to insufficient α mannosidase II activity. Tet-induced Mgat5 revealed a small increase in peaks with m/z of the expected products; 2,2,6 tri- and 4,2,2,6 tetra-antennary N- glycans with and without fucose (Figure 2.2D, structures 3, 5, 8 and 11). In tet-induced Mgat6 HeLa cells, novel peaks were observed with m/z predicted to be the 4,2,2,4 tetra- and 4,2,2,4,6 penta-antennary products of Mgat6 with and without fucose (Figure 71

103 2.2E). Structures 10, 12 and 13 are predicted to be tetra- and penta- antennary products of avian Mgat6, while the native 4,2,2,6 tetra-antennary N-glycans (structures 8 and 11) were reduced. This is consistent with our earlier studies showing that Mgat5-modified intermediates are the preferred acceptor for Mgat6 (Watanabe et al, 2006), thus conversion of tri- and tetra- to tetra- and pentabranched N-glycan (Figure 2.2E). Structures 8 and 11 are preferred ligands for L-PHA binding, and their conversion to 10, 12 and 13 is associated with reduced L-PHA binding (Figure 2.2B). A low amount of Mgat6 activity was detected in the absence of tet (Figure 2.1H), as well as a small amounts of structures 10, 12 and 13, suggesting some background (leaky expression) of the Mgat6 transgene (Figure 2.2E). HeLa cells had insufficient α-mannosidase II activity to process the tet-induced Mgat1 product (i.e. HexNAc-Hex5-HexNAc2-Asn), which appeared to be the limiting factor for tet-induced increases in Mgat5 and Mgat6 branched N-glycans Tet-Inducible Mgat1, Mgat5 and Mgat6 N-Glycan Branching Hek293 is an immortalized human embryonic cell line with epithelial morphology previously reported to be growth-sensitive to changes in N-glycan branching and HBP (Lau et al, 2008b). Hek293 cell lines with tet-inducible Mgat1, Mgat5 or Mgat6 were made by the Flp-In- TREx system, and displayed tet-inducible expression of the enzymes, comparable to that seen in the HeLa cell lines (Figure 2.3A). Flp-In-TREx Hek293 clones also displayed very similar induction of enzyme activities and glycan branching, as illustrated for tet-inducible Mgat5 (Figure 2.3B). Tet-induced Mgat5 expression alone increased L-PHA binding by ~13%, 30 mm GlcNAc supplementation to HBP by ~30%, and addition of both by ~83%, clearly displaying a synergistic interaction (Figure 2.3C). 72

104 To characterize the structural effects of tet-induction, N-glycans were released by PNGase and the N-glycan alditols were analyzed by LC-ESI MS. Overall, Hek293 cells displayed a greater fold change in tri- and tetra-antennary N-glycans than HeLa cells (Figure 2.3D-F). The tetraantennary structures 8 and 11 were reduced in tet-induced Mgat1 Hek293 cells (Figure 2.3D). This may seem counter-intuitive, but is consistent with multistep ultrasensitivity to UDP-GlcNAc that defines the Golgi N-glycan branching pathway in non-transformed epithelial cells (Lau et al, 2007). Briefly, multistep ultrasensitivity is due to declining affinities of Mgat1, Mgat2, Mgat4 and Mgat5 enzymes for UDP-GlcNAc (Km values increases from 0.04 mm to 10 mm across the pathway). Mgat1 over-expression has been shown to reduce substrate available to Mgat4 and Mgat5, but rescue is possible by extracellular GlcNAc supplementation to increase UDP-GlcNAc pool (Lau et al, 2007). Over-expression of Mgat4 and Mgat5 in malignant cells reduces pathway dependence on UDP-GlcNAc, and thereby loss of regulatory control by HBP (Lau & Dennis, 2008). Tet-induced Mgat5 Hek293 cells displayed a ~2 fold increase in tri-antennary (structure 3 and 5), and a ~2 fold increase in tetra-antennary (structure 8 and 11) N-glycans (Figure 2.3E). Tet-induced Mgat6 Hek293 cells produced the same novel oligosaccharide features observed in HeLa cells, predicted to be the 2,2,4,6 tetra- (structures 7 and 10), 4,2,2,4 tetra- (structures 9 and 12) and 4,2,2,4,6 penta- (structure 13), while native 4,2,2,6 tetra- (structures 8 and 11) were reduced (Figure 2.3F) Regulation of Cellular Metabolite Levels by Mgat1, Mgat5, Mgat6 and HBP Next, metabolites in HeLa and Hek293 Flp-In-TREx cells were measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS) using multiple reaction monitoring 73

105 (MRM), targeting glycolysis, amino acids, nucleotides, TCA cycle, and HBP metabolites. Supplementing cells with GlcNAc increases UDP-GlcNAc available to the N-glycan branching enzymes, and should interact with tet-induced Mgat1, Mgat5 or Mgat6 to reveal more intense phenotypes (Lau et al, 2007; Sasai et al, 2002). Cells were cultured in standard nutrient-rich medium, with and without tet, in the presence of 0, 15 or 30 mm GlcNAc for 24h, and cellular metabolites were extracted and analyzed by LC-MS/MS. In both Flp-In-TREx HeLa and Hek293 cells, GlcNAc supplementation increased intracellular UDP-GlcNAc up to ~5 fold over control. Unsupervised clustering of metabolite data revealed that GlcNAc and tet-induced Mgat1, Mgat5 or Mgat6 increased the levels of many metabolites in HeLa cells, and Hek293 cells (Figure 2.4A). In the absence of GlcNAc, tet-induced Mgat1, Mgat5 or Mgat6 was associated with modest increases in metabolite levels, which was further enhanced by GlcNAc supplementation. For example, tet-induced Mgat5 and Mgat6 were additive with GlcNAc supplementation for increases in lactate in Hek293, suggesting increased glycolytic flux (Figure 2.4B). Increased lactate production is commonly observed in proliferating cells, along with high rates of oxidative respiration that require mechanisms of stress tolerance (Kaplon et al, 2013; Levine & Puzio-Kuter, 2010). Reactive oxygen species (ROS) are toxic byproducts of oxidative respiration that require buffering by glutathione (GSH) in a reaction that produces glutathione disulfide (GSSG) (Suzuki et al, 2010). The ratio of glutathione (GSH) to glutathione disulfide (GSSG) declined markedly with GlcNAc supplementation in Hek293 cells, with and without tetinduction for all three enzymes (Figure 2.4C). Mgat6 Hek293 cells displayed lower GSH/GSSG ratio without induction, which declined further with tet-induction and GlcNAc treatment, possibly due to low-level expression of Mgat6 in the absence of tet. Taken together, the results suggest that N-glycan branching and HBP increase the levels of many metabolites, including glycolysis and 74

106 TCA cycle intermediates, as well as amino acids, with increased disposition of carbon into oxidative respiration (Figure 2.4D). However, tet-induction and/or GlcNAc supplementation did not increase cell proliferation in the rich-medium conditions used in these experiments (Abdel Rahman et al, 2013) Tet-Inducible Mgat5 Enhances Amino Acid Uptake and Growth in Nutrient-Poor Conditions Nutrient-rich culture medium does not reflect typical in vivo conditions, where glucose and amino acid availability are frequently limiting. Therefore, Glc and Gln were titrated in the medium, and cell growth compared for non-induced and tet-induced N-glycan branching. HeLa tumor cells displayed growth autonomy that did not benefit from tet-induced Mgat5 when Glc and/or Gln supply was limiting. In contrast, tet-induced expression of Mgat5 alone in Hek293 cells increased cell growth under low Glc/Gln conditions (Figure 2.5A). This is also consistent with the greater inducibility of Mgat5-branched N-glycans in the Hek293 cell line (Figure 2.2D and 2.3D). Therefore, Hek293 cells were cultured for 24h in DMEM medium containing four Gln/Glc concentrations (0/2.5, 0/10, 2/2.5, 2/10 mm), selected to reveal growth-sensitive effect of tetinduced Mgat5 (Figure 2.5A and B). Cellular metabolites were analyzed by LC-MS/MS, and principal component analysis (PCA) of data revealed clear separation of experimental groups based on Glc and Gln supply (Figure 2.5C). Using PCA technique, which reduces the dimensionality of data but maintains the variation, we observe Mgat5 overexpression having the greatest effect in low Gln. In 2 mm Gln, separation was observed for high versus low Glc, but no further separation was detected with tet-induced Mgat5, suggesting Glc uptake is not likely the primary effect of tet-induced Mgat5. However, in 0 mm Gln, the tet-treated and untreated cells 75

107 were separated, and with greater distance when Glc supply was also low (Figure 2.5C). Note that in 0 mm Gln without tet, Glc had no effect on separation in the PCA plot. We conclude that in Gln-depleted conditions, tet-induced Mgat5 enhances intracellular metabolite levels and growth by increasing amino acid uptake, while Glc supply acts as a modifying factor. In Gln-depleted conditions, most metabolites that were measured increased with tetinduced Mgat5, while the effect of tet-induced Mgat5 in richer medium (i.e. 2 mm Gln plus 2.5 or 10 mm Glc) was minimal (Figure 2.6 and 2.8). In Gln-depleted medium, intracellular Gln was reduced by ~10 fold, and glutamate (Glu) and α-ketoglutarate by ~2-3 fold (Figure 2.6A and 2.7). Tet-induced Mgat5 increased intracellular amino acid content, while depleting them from the growth media (Figure 2.6B). Increased consumption from the media by tet-induced Mgat5 cells was observed for Arg, His, Met, Phe, Ser, Trp, Tyr, Val, Ileu, Leu, and Cys, which includes the essential amino acids (EAA). The catabolism of amino acids provides NH4 + for Glu and Gln synthesis, as well as anaplerotic support to the TCA cycle. Indeed, tet-induced Mgat5 increased Glu and TCA cycle intermediates including succinyl-coa, succinate, fumarate and malate (Figure 2.7). Glycolysis and HBP intermediates were also increased in low Gln conditions, suggesting a reduced demand for Glc due to the increased uptake and catabolism of amino acids (Figure 2.8). Tet-induced Mgat5 increased GlcN-P, GlcNAc-P and UDP-GlcNAc, which contribute positive feedback to the N-glycan branching enzymes, and downstream effectors. Mgat5 has a low affinity for UDP-GlcNAc, and is therefore sensitive to Glc and Gln flux through HBP (Abdel Rahman et al, 2013). Our results suggest that up-regulation of Mgat5 may generate positive feedback to nutrient uptake by increasing UDP-GlcNAc supply to the Golgi (Figure 2.4D). The effect of tetinduced Mgat5 on central metabolite levels are summarized in Figure

108 Increased Gln uptake and catabolism is a critical feature of metabolism in proliferating cells (Levine & Puzio-Kuter, 2010; Wellen & Thompson, 2012). Branched-chain EAA uptake is mediated by a bidirectional transporter complex (Slc7a5 and Slc3a2), that exports Gln in exchange for the import of branched-chain EAA (Nicklin et al, 2009). To measure Gln uptake directly, cells were pulse-labeled with dual labeled 15 N 15 N-Gln for 1, 5 and 10 min. Tet-induced Mgat5 increased L-PHA staining and modestly increased uptake of 15 N 15 N-Gln, which was further increased in an additive manner by 15 mm GlcNAc (Figure 2.10A). 15 N 15 N-Gln conversion to 15 N-Glu and reamination to single-labeled 15 N-Gln showed similar kinetics (Figure 2.10, right). Tet-induced Mgat1 suppressed L-PHA staining by ~20%, consistent with observed suppression of tetraantennary structures 8 and 11 by mass spectrometry (Figure 2.3D). Tet-induced Mgat1 alone had little effect on 15 N 15 N-Gln uptake (Figure 2.10B). These results demonstrate that Mgat5-modified N-glycan branching and HBP cooperate to up-regulate Gln import. 2.5 Discussion In this report, transgenic Hek293 and HeLa cell lines with single site insertion for tetinducible over-expression of Mgat1, Mgat5, and avian Mgat6 enzymes were generated, and their effects on N-glycan profiles and central metabolism characterized by mass spectrometry. N-glycan profiling revealed unexpected differences between HeLa and Hek293 cell lines following tetinducible transgene over-expression. In HeLa cells, tet-induced Mgat1 markedly increased the expected hybrid N-glycans, but had a much smaller effect on downstream branching, suggesting the cells have insufficient α-mannosidase II activity to process the additional Mgat1 product. In Hek293 cells, tet-induced Mgat1 did not increase hybrid N-glycans, but did partially suppress downstream branching, consistent with previous characterization of the pathway as multistep 77

109 ultrasensitive to UDP-GlcNAc (Lau & Dennis, 2008; Lau et al, 2007). Tet-induced Mgat5 increased tri- and tetra-antennary N-glycans in Hek293 more robustly than in HeLa cells. HBP supplementation was additive with tet-induced Mgat1, Mgat5 and Mgat6 for increasing levels of cellular metabolites in nutrient-rich medium. The nutrients were not a stimulus for cell proliferation, but rather catabolism as indicated by increased lactate and oxidative respiration. GlcNAc supplementation at >15 mm overcame the UDP-GlcNAc-limiting effects of Mgat1 overexpression in Hek293T cells (Lau et al, 2007). Similarly, GlcNAc supplementation acts downstream of α-mannosidase II and Mgat1, and markedly up-regulated metabolite levels in HeLa cells. GlcNAc supplementation might mimic nutrient abundance in glucose, nitrogen and/or fatty-acid metabolism, and such conditions might arise in hyperglycemia, and excess intake or catabolism of amino acids and/or fat Mgat6 Enhances Functionality of N-Glycan Branching in Mammalian Cells The Mgat5 product is the preferred acceptor for Mgat6, which adds a GlcNAcβ1,4 branch that is not present in mammals (Watanabe et al, 2006). The mature N-glycan branches function in an additive manner to regulate galectin binding to growth factor receptors and nutrient transporters at the cell surface (Lau et al, 2007). The addition of an ectopic branch to N-glycans in mammalian cells might display an additive and stronger phenotypic effect compared to over-expression of Mgat5. Tet-induced Mgat6 alone, and with GlcNAc supplementation, in both HeLa and Hek293 cell lines, displayed the strongest enhancement of metabolite levels of the three enzymes tested (Figure 2.4A). It is possible that over-expression of Mgat6 qualitatively changes the interaction between mammalian branching enzymes and HBP, or that Mgat6 has substrate preferences that alter the relationship between nutrient transporters activities. Overall, our results provide 78

110 compelling evidence that functional redundancy of branches and their conditional regulation by HBP is a critical feature of metabolic regulation (Dennis & Brewer, 2013). The emergence of Mgat6 in tetrapod dinosaurs and retention of the gene in modern birds may play a critical role in their metabolic rates and fitness Mgat5 Enhances Metabolism Under Glutamine-Deprived Conditions Instead of using the typical cell culture conditions, which have excess amounts of Glc (25mM) and Gln (4mM), more limiting condition were used to discern the effect of Mgat5 overexpression on metabolism and cell growth. In low Gln/Glc culture conditions, tet-induced Mgat5 alone increased proliferation in cultures of Hek293 cells, but not in HeLa cells, consistent with the relative change in N-glycan branching upon tet-mgat5 induction in the two cell lines. Tetinduced Mgat5 in Hek293 cells stimulated the uptake of essential amino acids, and increased the levels of glycolytic, HBP and TCA pathway intermediates (Figure 2.6 and 2.7). In a more direct assay of uptake, tet-induced Mgat5 and 15 mm GlcNAc stimulated 15 N 15 N-Gln uptake in a cooperative manner (Figure 2.8). GlcNAc supplementation to HBP has been shown to stimulate Gln uptake and catabolism in Bax -/- Bak -/- lymphoma cells in the absence of Glc (Wellen et al, 2010). In these experiments, surface IL-3 receptor was reduced in Glc-starved condition, but could be rescued by GlcNAc supplementation to UDP-GlcNAc and N-glycan branching (Wellen et al, 2010). Furthermore, IL-3 receptor-dependent signaling stimulated transcription of SLC1A5, a high-affinity Gln transporter, and SLC6A19, a transporter of Leu and Gln. However, the present study is the first to show a direct effect of Mgat5-mediated branching on amino acid uptake and metabolic flux, in low Gln/Glc (i.e. 0 / 2.5 mm) +10% FBS culture conditions. 79

111 Gln is not an essential amino acid, but high growth rates in embryonic and cancer cells depend on the import of Gln, and anaplerotic conversion to α-ketoglutarate which supports the TCA cycle (DeBerardinis et al, 2008). SLC7A5/SLC3A2 is a bidirectional transporter that imports branched-chain essential amino acids (BCAA) (Leu, Ile,Val) in exchange for Gln efflux (Nicklin et al, 2009). The Gln transporter, SLC1A5 is widely expressed and required to support BCAA uptake, although in some cells, Gln biosynthesis from α-ketoglutarate and glutamate can support internal needs and SLC7A5/SLC3A2 activity (Hassanein et al, 2013). Tet-induced Mgat5 alone in Hek293 cells was sufficient to increase uptake of Gln and EAA in low Gln/Glc culture conditions. Gln is also a positive regulator of HBP (Abdel Rahman et al, 2013) and may drive reciprocal positive feedback between N-glycan branching and metabolism (Figure 2.4D and 2.9). Mgat5- deficient mice are hypoglycemic and display a reduced sensitivity to glucagon (Johswich et al, 2014), and here we present the first evidence of a cell autonomous regulation of metabolism by N- glycan branching. Up-regulation of Mgat5 in Hek293 cells stimulated amino acid uptake and increased metabolite levels under low Gln/Glc culture conditions. Further work is required to identify the various nutrient transporters regulated by N-glycan branching and their contribution to development, disease and environmental stress. 80

112 Figure 2.1 Branching pathway and inducible expression of branching enzymes. (A) N-glycan branching pathway modified from Essentials of Glycobiology 2 nd edition, Chapter 8 Figure 5 (Varki et al, 2009). GlcNAcT-I (Mgat1), GlcNAcT-V (Mgat5), avian GlcNAcT-VI (Mgat6) are circled. (B-D) Expression of transgene proteins detected by Western blots probed with antibodies to Flag and tubulin. Flag-tagged GlcNAcT-I, GlcNAcT-V (upper band), and GlcNAcT- VI have expected molecular weights of 53kD, 86kD and 54kD, respectively. Flp-In-TREx HeLa clones were cultured in DMEM with 25 mm Glc, 4 mm Gln, 10% FBS (normal culture conditions), with or without 1 ug/ml of tetracycline (tet) for 24h. (E) Tet dose response in clone 8 Mgat5 Flp- In-TREx HeLa cells. (F-H) N-acetylglucosaminyltransferases activities measured in cell lysates from tet-inducible expression of Flag-tagged Mgat1, Mgat5 and Mgat6 in different Flp-In-TREx HeLa cell clones. 81

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114 Figure 2.2 N-glycan profiles of transgenic Flp-In-TREx HeLa cells. (A) ConA and (B) L-PHA lectin binding to Mgat1, Mgat5 and Mgat6 in Flp-In-TREx HeLa clones, with and without 1 ug/ml tet for 24h, measured by fluorescence microscopy, *p<0.05 and **p<0.01 by student t-test. (C-E) LC-ESI MS chromatogram for N-glycans in Flp-In-TREx HeLa cells expressing (C) Mgat1 clone 3, (D) Mgat5 clone 8, and (E) Mgat6 clone 6. The red chromatogram is tet-induced and black is non-induced. Orange circles indicate increases, and blue circles indicate decreases. Cells were grown in DMEM, 25 mm Glc and 4 mm Gln +10% FBS (standard conditions) with and without tet for 24h. 83

115 84

116 Figure 2.3 N-glycan profiles of transgenic Flp-In-TREx Hek293 cells by LC-ESI MS. (A) Expression of transgene proteins detected by western blot probed with anti-flag and tubulin antibodies in three different Flp-In-TREx Flag-tagged Mgat5 Hek293 clones, and Mgat5 enzyme activity measured in cell lysates from tet-inducible expression of Flag-tagged Mgat5 in clone 4. (B) Tet-induced Mgat5 increases complex-type N-glycan branching in Hek293 Mgat5 clones 4 and 8, quantified by Alexa-488 conjugated L-PHA fluorescence imaging. For quantification, mean ±SD, one-way ANOVA with Dunnett's multiple comparison test. (C) L-PHA binding of tri- and tetra-antennary N-glycans displays a synergistic effect for GlcNAc supplementation with tet-induced Mgat5 in clone 4. *p<0.05 and **p<0.01 by student t-test compared to -tet control. (D-F) LC-ESI MS chromatogram of N-glycans in Flp-In-TREx Hek293 cells expressing (D) Mgat1, (E) clone 4 Mgat5, and (F) Mgat6. Experimental conditions as described in Figure 2. 85

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118 Figure 2.4 Metabolite levels are sensitive to tet-induced branching and HBP stimulation. (A) Mgat1, Mgat5 and Mgat6 Flp-In-TREx HeLa and Hek293 cells were grown in standard culture conditions with and without tet (T) and 0, 15 or 30 mm GlcNAc supplementation for 24h. Each row represents a biological replicate (n=4-5). Metabolites in cell lysates were measured by targeted LC-MS/MS and normalized to cell number. Data for 129 metabolites was analyzed by unsupervised clustering and presented as heat maps. (B) Lactate and (C) GSH / GSSG ratios in Flp-In-TREx Hek293 cells with and without tet-induced Mgat1, Mgat5 and Mgat6 induction for 24h. Additive effects of tet and GlcNAc, *p<0.05 by student t-test. (D) Scheme for increased nutrient uptake and flow to catabolism with tet-induced increased N-glycan branching and GlcNAc supplementation. Green and yellow arrows indicate putative positive feedback from central metabolism through de novo HBP to N-glycan branching, which in turns promotes cell surface residency of transporters and more nutrient uptake. 87

119 Figure 2.5 Growth of Mgat5 Flp-In-TREx Hek293 cells in defined Glc and Gln conditions. Mgat5 Flp-In-TREx Hek293 clone 4 cells were cultured with and without tet in medium modified for Glc/Gln content as indicated + 10% FCS for 48h. (A) Cell growth as a function of Glc and Gln concentrations, with and without tet-induced Mgat5 (cell count ± SD, n=3). (B) Growth conditions (grey bars) corresponding to (C) metabolite profiling by LC-MS/MS, and analyzed by principal component analysis (PCA). 88

120 89

121 Figure 2.6 Amino acid levels increase with Mgat5 expression under Gln-deprived conditions. (A) Mgat5 Flp-In-TREx Hek293 clone 4 cells were cultured in the four Gln/Glc conditions indicated, and data was normalized to 2.5/10 Gln/Glc no-tet (second bar from the right, red line) and plotted as fold change (mean ±SD, n=5, *p<0.05). The axis labels are shown for Ala at the top. The pathway scheme is from Chapter 20 (Amino Acid Degradation and Synthesis) Lippincott's Illustrated Reviews: Biochemistry. (B) Heat map of amino acid levels in cells and medium showing data for each of 4-5 replicates. In limiting conditions, tet-induced Mgat5 increased cellular amino acids content, and the depleted growth medium of the same, indicating increased amino acids uptake. Red is high, green is low. White boxed area highlights contrasting effect of +tet on cells and medium. 90

122 Figure 2.7 Tet-induced Mgat5 increases TCA cycle intermediates. Mgat5 Flp-In-TREx Hek293 clone 4 cells were cultured in the four Gln/Glc conditions indicated. The data was normalized to 2.5/10 mm Gln/Glc no-tet conditions (second bar from the right, red line). *p<0.05 by student t-test for tet-induced change at 0/2.5 mm Gln/Glc condition. 91

123 Figure 2.8 Tet-induced Mgat5 increases HBP and glycolysis metabolites under Gln/Glc limiting conditions. Mgat5 Flp-In-TREx Hek293 clone 4 cells were cultured in the four Gln/Glc conditions, and normalized data graphed as fold change. The data was normalized to 2.5/10 mm Gln/Glc notet conditions (second bar from the right, red line). *p<0.05 by student t-test for tet-induced change at 0/2.5 mm Gln/Glc condition. 92

124 Figure 2.9 Summary of metabolite changes with tet-induced Mgat5 in Hek293 cells under low Gln/Glc conditions. Metabolites in bold were measured by LC-MS/MS, and the EAA are marked in blue. * indicates 2 fold or more decrease as a result of growth in low Gln/Glc 0/2.5 mm conditions. Green arrows mark metabolites increased by tet-induced Mgat5. 93

125 Figure 2.10 Tet-induced Mgat5 branching stimulates Gln uptake. Flp-In-TREx Hek293 (A) Mgat5 clone 4 and (B) Mgat1 clone 7 cells, were grown with and without tet and 15 mm GlcNAc in standard culture conditions. Gln was removed for 16 h and cells were pulsed with 1 mm 15 N 15 N Gln for the indicated times. 15 N 15 N Gln and 15 N Gln levels in cell lysates were measured by LC-MS/MS. *p<0.05 and **p<0.01 by student t-test comparing nil to tet or GlcNAc to GlcNAc+tet, with n=5-6 samples. The bar graphs on the left compare branching by L-PHA staining of Mgat1 and Mgat5, with and without tet-induction. 94

126 Chapter 3 Metabolic Reprogramming by the Hexosamine Biosynthetic Pathway and Golgi N-Glycan Branching A version of this chapter is in revision at the Journal of Biological Chemistry Michael C. Ryczko, Judy Pawling, Rui Chen, Anas M. Abdel Rahman, Kevin Yau, Daniel Figeys, and James W. Dennis Attributions: Mouse-work and targeted metabolomics mass-spectrometry experiments in Figures 3.1A-F, 3.2A- G, 3.3A,D-F,I, 3.5E-G, 3.7A,G,I, 3.8A-C,E, and 3.9A-D,G-I were performed together with Judy Pawling. Site-specific characterization of N-glycan structures on liver glycoproteins summarized in Table 3.2 and displayed in Figure 3.6 was performed by Rui Chen using LC-MS/MS. All other experiments were designed, performed and analyzed by Michael Ryczko. 95

127 3.1 Summary UDP-GlcNAc is an essential substrate for protein N-glycosylation and Golgi remodeling of N-glycans, a crucial modification to cell surface receptors and nutrient transporters. de novo synthesis requires glucose, glutamine, acetyl-coa and uridine-triphosphate, and cellular levels of UDP-GlcNAc are sensitive to the supply of these central metabolites. GlcNAc is salvaged from dietary and glycoconjugate turnover into the HBP to generate UDP-GlcNAc. Herein I examined the effects of GlcNAc supplementation and salvage on metabolism in C57BL/6 mice. Fat and body-weight increased without affecting calorie-intake, activity, or energy-expenditure. Chronic oral GlcNAc increased hepatic UDP-GlcNAc, and GlcNAc content in N-glycans on hepatic glycoproteins, indicating increased Golgi N-glycan branching. Furthermore, glucose homeostasis, hepatic glycogen and lipid metabolism were altered with GlcNAc treatment, especially during fasting. In cultured cells, GlcNAc enhanced the uptake of glucose, glutamine and fatty acids, and increased synthesis and lipid accumulation, while inhibition of N-glycan branching blocked GlcNAc-dependent lipid accumulation. The N-acetylglucosaminyltransferase (Mgat1,2,4,5) enzymes of the N-glycan branching pathway display multistep ultrasensitivity to UDP-GlcNAc, due to declining affinity for the common substrate, with Mgat5 operating below its Km, thus making it sensitive to increased UDP-GlcNAc. Mgat5 -/- mice exhibit a lean phenotype, and oral GlcNAc rescued fat accumulation, consistent with functional redundancy of N-glycan branches. However, fat accumulation was rescued at the expense of lean mass, suggesting Mgat5 plays a role in lean to fat body composition. Our results suggest that GlcNAc reprograms cellular metabolism by enhancing nutrient uptake and lipid storage through UDP-GlcNAc supply to N-glycan branching pathway. 96

128 3.2 Introduction GlcNAc is found widely in glycoconjugates, many with ancient origins, notably the GlcNAc polymer chitin found in arthropods, molluscs, insects and fungi, as well as the N-glycans that modify glycoproteins produced in the secretory pathway (Banerjee et al, 2007; Ruiz-Herrera et al, 2002). Biosynthesis of glycoconjugates requires high-energy sugar-nucleotide donors. de novo UDP-GlcNAc biosynthesis by the HBP requires Glc, Gln, Ac-CoA, and UTP, metabolites that are central to carbon, nitrogen, fatty acid, nucleotide, and energy metabolism. In cell culture, starvation levels of Glc suppress UDP-GlcNAc levels, whereas elevated Gln increases UDP- GlcNAc (Abdel Rahman et al, 2013). The rate limiting step in HBP is the conversion of Fru-6P and Gln to GlcN-6P and glutamate by GFAT (Broschat et al, 2002). Overexpression of GFAT increases UDP-GlcNAc, and transgenic mice overexpressing GFAT in the liver displayed obesity, enhanced glycogen storage, impaired glucose tolerance and insulin resistance at 8 months of age (Veerababu et al, 2000). GFAT overexpression in skeletal muscle, adipose tissue, or pancreatic β- cells also resulted in insulin resistance (Cooksey & McClain, 2002). Glucosamine-6P N-acetyltransferase (GNPNAT1/GNA1) converts GlcN-6P and Ac-CoA to GlcNAc-6P (Oikawa et al, 1986), which flows primarily into UDP-GlcNAc in mammalian cells (Wellen et al, 2010). GNA1 is essential for de novo UDP-GlcNAc synthesis, but GNA1-deficient mouse embryo fibroblasts expressed glycoproteins containing GlcNAc, indicating that salvage of GlcNAc from autophagy and exogenous glycoconjugates is a significant source of UDP-GlcNAc (Boehmelt et al, 2000). No mammalian plasma membrane transporter for GlcNAc has been identified, and uptake of extracellular GlcNAc is thought to occur by bulk- or fluid-phase endocytosis (Dennis et al, 2009). Intracellular GlcNAc is converted to GlcNAc-6P by N- acetylglucosamine kinase (NAGK), and enters HBP as substrate for phosphoglucomutase (PGM3) 97

129 and UDP-N-acetylglucosamine pyrophosphorylase (UAP1). GlcNAc-6P N-deacetylase has been reported (AMDHD2) (Bergfeld et al, 2012), but its activity is very low in mammalian cells, and therefore salvaged GlcNAc contributes almost exclusively to the UDP-GlcNAc pool and protein glycosylation (Wellen et al, 2010). The glycosylation pathways utilizing UDP-GlcNAc include N-glycosylation of membrane glycoproteins and O-GlcNAcylation of cytosolic proteins. Cytoplasmic, nuclear and mitochondrial O-GlcNAc modified proteins have been identified as regulators of cell signaling and gene transcription (Hardiville & Hart, 2014). O-GlcNAcylation of proteins in insulin-akt signaling pathway, and transcription factors and cofactors that regulate glycogen synthesis, gluconeogenesis and lipogenesis have been shown to play a role in metabolic homeostasis (Dentin et al, 2008; Ruan et al, 2013; Yang et al, 2008). OGT catalyzes the addition of GlcNAc, while O-GlcNAcase removes it. Interestingly, increasing O-GlcNAcylation globally using a selective inhibitor of O- GlcNAcase did not result in insulin resistance or perturb glucose homeostasis in rodents or 3T3- L1 adipocytes (Macauley et al, 2010a; Macauley et al, 2010b). On the other hand, mutations in genes encoding Golgi N-glycan branching enzymes have been shown to sense glucose and regulate insulin release by pancreatic β-cells, as well as control hepatic glucagon receptor sensitivity (Johswich et al, 2014; Ohtsubo et al, 2005). Enzymes Mgat1, Mgat2, Mgat4a/b/c and Mgat5 each catalyze the addition of GlcNAc in a specific β-linkage to the trimannosyl core of N-glycans (Schachter, 1986), and the branching pathway is characterized by multistep ultrasensitivity to UDP-GlcNAc. Multistep ultrasensitivity arises from a ~300 fold decline in affinity for the common donor substrate UDP-GlcNAc moving down the pathway from Mgat1 to Mgat5 (Lau et al, 2007). Therefore, Mgat5 and the synthesis of tri- and tetra-antennary N-glycans are most sensitive to UDP-GlcNAc levels, and metabolite flux through HBP. 98

130 The GlcNAc branches are further extended with galactose, fucose and sialic acid, generating glycan structures with affinity for galectins, C-type lectins and siglecs at the cell surface (Dennis et al, 2009). Galectins bind N-acetyllactosamine (Galβ1-4GlcNAcβ) branches on N- glycans, and their affinities for membrane glycoproteins are proportional to both the extent of branching and the N-glycan number (NXS/T consensus site) encoded in the protein sequence (Hirabayashi et al, 2002; Lau et al, 2007). For example, Glc transporter Glut4 has a single N- glycan chain and is retained at the cell surface in a UDP-GlcNAc dependent manner (Lau et al, 2007). Galectin-9 binds to the single multi-antennary N-glycan chain on pancreatic Glut2 and prevents its loss to endocytosis, thus allowing glucose import and glucose-stimulated insulin secretion (Ohtsubo et al, 2013; Ohtsubo et al, 2005). Mgat4a -/- mice fail to retain Glut2 on the surface of pancreatic β-cell cells, resulting in reduced secretion of insulin and hyperglycemia (Ohtsubo et al, 2005). N-glycan branches are partially redundant and act cooperatively to support glycoproteins at the cell surface (Dennis & Brewer, 2013). Inducible expression of Mgat5 in Hek293 human embryonic kidney cells growing in low Glc and Gln medium increased N-glycan branching, intracellular metabolites, uptake of amino acids, and cell growth (Abdel Rahman et al, 2014). With a Km value of ~10 mm for UDP-GlcNAc, Mgat5 is very sensitive to HBP activity and output, which may vary with feeding conditions in animals. Here I asked whether GlcNAc supplemented to drinking water of mice can be salvaged in the HBP and interact through N-glycan branching to increase anabolic metabolism and weightgain. In wild-type C57BL/6 mice GlcNAc supplementation increased liver UDP-GlcNAc levels, and enhanced fat accumulation and weight-gain. Chronic GlcNAc intake altered hepatic metabolite levels, decreased respiratory exchange ratio (RER), and increased lipid accumulation and body-weight, without affecting food intake, physical activity or energy expenditure. 99

131 Furthermore, GlcNAc supplementation globally increased the relative GlcNAc content of N- glycans found on hepatic glycoproteins, by increasing Golgi N-glycan branching. Mgat5-deficient mice display adult phenotypes that may be linked in part through metabolism, including delayed oncogene-induced tumor progression (Granovsky et al, 2000), autoimmune sensitivity (Demetriou et al, 2001), depression-like behavior (Soleimani et al, 2008), resistance to weight-gain on a high fat diet, and loss of adult stem cells and early aging (Cheung et al, 2007). GlcNAc supplementation partially restored anabolic metabolism in Mgat5 -/- mice and primary hepatocytes, as indicated by increased lipid accumulation. In cultured cells, GlcNAc enhanced uptake of Glc, Gln and fatty acid, and promoted lipid accumulation in an N-glycan-dependent manner. My results indicate that GlcNAc supplementation increases nutrient uptake and lipid storage by enhancing UDP-GlcNAc supply to the Golgi N-glycan branching pathway. 3.3 Materials and Methods Chemicals and Materials GlcNAc was obtained as Ultimate Glucosamine (Wellesley Therapeutics, Toronto, Ontario, Canada), GlcN was obtained from Sigma Chemicals (St. Louis, MO), and both were dissolved in water. Metabolite standards and reagents were obtained from Sigma Chemicals (St. Louis, MO) with minimal purity of 98%. Stable-isotope 15 N2-L-Glutamine was purchased from Cambridge Isotope Laboratories Inc. (Andover, MA). All organic solvents and water used in sample and LC/MS mobile phase preparation were LC/MS grade and obtained from Fisher Scientific (Fair Lawn, NJ). Antibodies to phospho-thr172 AMPK-α, phospho-ser79 ACC, phospho-ser235/236 S6, phospho-ser473 Akt, phospho-ser641 GS were obtained from Cell Signaling Technology. Antibodies to Tubulin were purchased from Sigma-Aldrich, fatty acid 100

132 synthase from BD Scientific, and mab CTD from Covance. PhosSTOP Phosphatase Inhibitor Cocktail and Complete Protease Cocktail were purchased from Roche. PVDF membrane (Immuno-Blot, 0.2 µm, 7.0 x 8.5 cm) was purchased from Bio-Rad (Hercules, CA). Alexa Fluor- 488 conjugated lectins ConA and L-PHA, BODIPY 493/503 (4,4-Difluoro-1,3,5,7,8-Pentamethyl- 4-Bora-3a,4a-Diaza-s-Indacene), and 2-NBD-Glucose (2-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4- yl)amino)-2-deoxyglucose) were purchased from Invitrogen (Carlsbad, CA). QBT Fatty Acid Uptake Assay Kit containing BODIPY-dodecanoic acid fluorescent fatty acid analog was purchased from Molecular Devices Mice Weight and age-matched young C57BL/6 male mice were used in GlcNAc supplementation experiments. For experiments involving Mgat5 wild-type and null mice, age and sex-matched littermates on the C57BL/6 background were used as described previously (Cheung et al, 2007; Johswich et al, 2014). All mice were maintained in cages of up to 5 mice per cage, in a normal 12 h light/12 h dark cycle on either 4%, 9% or 22% fat food diet (Tekland rodent diet), with or without GlcNAc (0.5, 5.0 or 15 mg/ml) or GlcN (0.5 mg/ml) in the drinking water, as indicated, for the specified duration of time. Bottles with drinking water containing GlcNAc or GlcN were changed twice weekly. Mice were euthanized using CO2 inhalation, and dissections carried out rapidly to remove, weigh, and freeze liver tissue samples on dry-ice, and store in tubes at -80 C until further biochemical and molecular analysis. All experiments using mice were conducted according to protocols and guidelines approved by the Toronto Centre for Phenogenomics animal care committee. 101

133 3.3.3 Phenotyping in vivo Body-weight of mice was measured on a weekly basis. Daily food consumed was determined every 24 h over 10 days, and expressed as calorie intake per body-weight, at 21 weeks following start of GlcNAc supplementation. Body composition, in terms of lean and fat tissue mass, was determined by dual energy X-ray absorptiometry (DEXA) (PIXImus) or magnetic resonance imaging (MRI) (Echo Medical Systems). Whole-body O2 consumption and CO2 production rates were recorded for 20 h with the use of an open-circuit indirect calorimeter (Oxymax Lab Animal Monitoring System, Columbus Instruments). Respiratory exchange ratios (RER) was calculated as the molar ratio of VCO2 to VO2 for 5 mice per group, averaging the measurements for the light and dark cycle. The activity of mice in three spatial dimensions plus time was continuously measured during the same 20 h using infrared photocells attached to the metabolic cage during dark and light cycles. Total activity included ambulatory movement (locomotion), and body movements (grooming and rearing on hind legs). Energy Expenditure (EE) per mouse was calculated as EE = ( VCO2/VO2) VO2. Water and food were available ad libitum in the metabolic chamber and determined for individual mouse. To minimize the potential influence of circadian rhythms on experimental outcomes, standardized periods of fasting or experimental analyses were utilized. For intraperitoneal glucose tolerance test, mice were fasted for 18 h before intraperitoneal injection of 0.01 ml/g of body-weight of a glucose solution containing 150 mg/ml. For the glucagon tolerance test, mice were fasted for 5 h and injected intraperitoneally with a glucagon solution of 1.6 g/ml (0.01 ml/g of body-weight). For both tolerance tests blood samples were drawn from the tail vein at different time intervals over 102

134 the course of 2 h, and blood glucose levels measured using a Glucometer Elite blood glucose meter (Bayer, Toronto, Canada) Targeted Metabolomics Frozen liver tissue (~100 mg per sample) was pulverized using the CellCrusher TM cryogenic tissue pulverizer under liquid nitrogen, which reduces most tissues to a fine, easily recoverable powder. The soluble polar metabolites were extracted by addition of 1 ml of ice-cold extraction solvent consisting of 40% acetonitrile, 40% methanol and 20% water, followed by vortexing for 30 sec, and shaking at 1,000 rpm for 1 h at 4 C in a ThermoMixer (Eppendorf, Germany). For cells grown in cell culture plates, following a PBS wash and flash freezing in liquid nitrogen, metabolites were extracted by adding 1 ml of same ice-cold extraction solvent to the wells of the plate, scraping the cells, collecting in 1.5-ml tubes, and vortexed and shaken as described above. Following extraction, samples were spun down at 20,000G for 10 min at 4 C, and the supernatant transferred to fresh tubes to be evaporated to dryness in a CentriVap concentrator at 40 C (Labconco, MO). The dry extract samples were stored at -80 C for later LC MS/MS analysis. Samples were separated twice on a reversed phase HPLC column Inertsil ODS- 3 of 4.6-mm internal diameter, 150-mm length, and 3 µm particle size (Dionex Corporation, Sunnyvale, CA) for MS analysis in positive and negative modes. The eluted metabolites were analyzed at the optimum polarity in MRM mode on electrospray ionization triple-quadrupole mass spectrometer (4000 QTRAP; ABSciex, Toronto, Canada) as previously described (Abdel Rahman et al, 2013). The LC-MS/MS system does not resolve isomers of hexose or n-acetyl-hexosamine, including glucose/galactose/mannose, GlcNAc/GalNAc/ManNAc, or their phosphorylated or 103

135 sugar-nucleotide forms. In the metabolomic data and throughout the manuscript I refer to the Glc form of these isomers. UDP-GlcNAc/UDP-GalNAc isomerase is widely present in mammalian cells and may maintain homeostatic balance of these sugar-nucleotides Biochemical Studies and Histology Western blot analysis was performed on frozen liver tissue and cells. Protease inhibitor and phosphatase inhibitors were added to all buffers before experiments. Protein were resolved by 8% or 10% SDS-PAGE, analyzed by immunoblotting with primary antibodies incubated overnight at 4 C, followed by incubation with secondary infrared fluorescence antibodies, and visualization of specific proteins using the Odyssey infrared imaging system (LI-COR). To measure relative protein abundance, band intensities were quantified using ImageJ software. Intensity values were normalized to Tubulin. Blood samples at sacrifice were collected via cardiac puncture. For plasma preparation, blood samples were supplemented with trasylol, EDTA, and diprotin and centrifuged at 4,000G at 4 C for 5 min. Basic plasma clinical chemistry analyses were performed. Plasma leptin, insulin and glucagon hormones were measured using blood samples obtained at sacrifice with a mouse endocrine LINCOplex kit (Linco Research) following the manufacturer s protocol. Adiponectin and leptin were measured using ELISA kits (EMD Millipore) according to manufacturer s instructions. To measure glycogen content in the liver, mg of tissue was acid-hydrolyzed in 2 M HCl at 95 C for 2 h and neutralized using 2 M NaOH. The liberated glucose was assayed spectrophotometrically using the glucose assay reagent (hexokinase method) (Amresco) following the manufacturer s protocol. 104

136 Liver lipids were extracted using the Folch-Lees method. The extracts were filtered, and lipids recovered in the chloroform phase. Individual lipid classes were separated by thin layer chromatography using Silica Gel 60 A plates developed in petroleum ether, ethyl ether, acetic acid (80:20:1) and visualized by rhodamine 6G. Phospholipids, diglycerides, triglycerides and cholesteryl esters were scraped from the plates and methylated using BF3/methanol. The methylated fatty acids were extracted and analyzed by gas chromatography. Gas chromatographic analyses were carried out on an Agilent 7890A gas chromatograph equipped with flame ionization detectors, a capillary column (SP2380, 0.25 mm x 30 m, 0.25 µm film, Supelco). Helium was used as a carrier gas. The oven temperature was programmed from 160 C to 230 C at 4 C/min. Fatty acid methyl esters were identified by comparing the retention times to those of known standards. Inclusion of lipid standards with odd chain fatty acids permitted quantitation of the amount of lipid in the sample. For histology, frozen liver sections were stained with oil red O, while formalinfixed liver sections were stained with periodic acid-schiff, and mounted on glass slides for microscopy imaging Site-Specific Characterization of Hepatic N-Glycosylation Frozen mouse liver tissue was cut into pieces (diameter less than 2 mm) and rinsed with cold PBS to remove any residual blood. Liver tissue was homogenized in 2% SDS and 100 mm Tris-HCI lysing buffer (ph=7.4), with protease inhibitor, by blade homogenizer. The homogenate was sonicated to shear the DNA content, and the lysate was then centrifuged at 20,000G for 15 min to remove the insoluble content. A 10 fold volume of cold acetone was added to the supernatant, and the protein was precipitated at -20 o C overnight. Protein precipitant was collected 105

137 by centrifugation at 20,000G for 15 min, and rinsed with cold ethanol/acetone (50%:50%, v/v) twice to remove trace SDS. Dried protein precipitant was dissolved with 8M urea in PBS, and protein concentration was measured by DC Protein Assay (Bio-Rad). Liver lysates from mice in the same group were pooled, and 500 µg of protein from pooled lysate was used as the starting material for the proteomics study. Protein was denatured, reduced by adding 10 mm DTT, and incubated at 56 o C for 45 min. Reduced protein was alkylated by adding 20 mm IAA and incubated at room temperature in the dark for 30 min. The sample solution was diluted 5 times with 100 mm TEAB, and trypsin was added at 1:50 ratio. The protein was digested by incubation at 37 C overnight. After digestion, peptides from control and GlcNAc treated liver samples were labelled with light and heavy reagents respectively, as described elsewhere (Boersema et al, 2009). Labelled peptides were mixed and desalted with a C18 cartridge. 10% of the elution was dried by Speed Vac for total proteome analysis, and the rest was used for glycopeptide enrichment by hydrophilic interaction chromatography (HILIC SPE). The elutions from HILIC SPE were divided into two aliquots: one aliquot was dried by Speed Vac, and the other one was incubated with peptide-n-glycosidase F (PNGase F) for deglycosylation. Both deglycosylated peptides and intact glycopeptides were analyzed by LC-MS/MS with an Orbitrap-Elite mass spectrometer. Quantification of total proteome and N-glycosylated sites were achieved with Maxquant, and results were processed with Perseus (Boersema et al, 2013). Intact glycopeptides were identified and quantified by matching the Y1 ion from the MS/MS spectrum to the deglycosylated peptides identified with Mascot, and extracting the peak of the peptides precursor (Chen et al, 2014). Glycan compositions of intact glycopeptides were validated manually after software analysis Cell Culture 106

138 AML12 immortal hepatocytes were purchased from ATCC and grown in 1:1 mixture of Dulbecco's Modified Eagle's Medium (DMEM) and Ham's F12 medium with mg/ml insulin, mg/ml transferrin, 5 ng/ml selenium, and 40 ng/ml dexamethasone, and 10% fetal bovine serum (FBS). Mouse primary hepatocytes were isolated as previously described (Johswich et al, 2014), seeded in Williams E media supplemented with serum, 2 mm Glutamax, 100 U/ml penicillin, and 100 mg/ml streptomycin on Primaria plates (BD Biosciences, Mississauga, ON), and treated overnight with different concentrations of GlcNAc. Cellular lipid accumulation in lipid droplets was detected using the lipophilic fluorescent probe BODIPY 493/503, while lectin binding to N-glycans was determined using ConA and L-PHA. For quantitative microscopic fluorescence imaging, cells were seeded in a 96-well plate (Costar 3595) at 2000 cells/well in regular media at 37 C and 5% CO2, and treated with GlcNAc for indicated times. Cells were then fixed for 15 min with 4% paraformaldehyde, washed with PBS, and incubated for 1 h at room temperature in 50 µl PBS containing Hoechst 33342, and either BODIPY 493/503, or Alexa Fluor- 488-conjugated ConA or L-PHA. After three washes with PBS, cells were imaged and staining per cell quantified using IN Cell Analyzer 1000 automated fluorescence imaging system and the accompanying software package. For the endpoint measure of glucose uptake, AML12 cells were grown under normal conditions with and without GlcNAc for 20 h. Media was changed to glucose-free media and 100 µm 2-NBD-Glc was added for 1 h at 37 C. The amount of 2-NBD-glucose inside the cells was measured as mean fluorescence intensity (MFI), with cells gated for similar size, using the Beckman Coulter Gallios flow cytometer, and analyzed with Kaluza analysis software. For the LC MS/MS measurement of 15 N2-Gln uptake and metabolism in AML12 and HeLa cells, cells were grown in their respective standard media for 24 h, followed by a switch to respective media 107

139 without Gln or serum but containing 5.5 mm Glc, with or without GlcNAc for an additional 24 h. Cells were then treated with 1 mm 15 N2-Gln containing media for indicated times. After incubation, cells were washed in PBS, quenched in liquid nitrogen, and metabolites extracted using the procedure detailed above to measure the relative intracellular levels of 15 N2-Gln, 15 N-Glu, and 15 N-Glu metabolites by LC MS/MS. 3T3-L1 fibroblast cells were kindly provided by Dr. Amira Klip (Sick Kids Hospital), and maintained in 25 mm Glc containing DMEM with 10% FBS. To differentiate 3T3-L1 cells into adipocytes, cells were grown to confluency, and after 2 days post-confluent cells were stimulated with the differentiation cocktail containing 5 µg/ml insulin, 1 µm dexamethasone, 0.5 mm isobutylmethylxanthine, and 5 µm troglitazone (all purchased from Sigma) in DMEM with 10% FBS. Cells were treated with differentiation cocktail for 2 days, after which cells were grown in DMEM containing 10% FBS and 5 µg/ml insulin with media replaced every 2 days. 3T3-L1 fibroblasts differentiated into adipocytes, and treated with GlcNAc for 24 h prior to the assay, were seeded in a 96-well plate for the QBT fatty acid uptake assay performed according to the manufacturer s instructions outlined in the kit. The kinetic reading monitoring the uptake of nonesterified long-chain fatty acid analog, using fluorescent BODIPY-FA, was quantified as Area Under the Curve (AUC) for Relative Fluorescence Units (RFU) Statistical Analysis All data are expressed as mean ± SEM values for experiments, including numbers of mice as indicated. Statistical significance was determined using unpaired, two-tailed Student s t-tests to compare differences between two group means (two samples and equal variance), or one-way 108

140 ANOVA with Dunnett s multiple comparison test using Microsoft s Excel or GraphPad Prism Software). In all experiments, a p-value of 0.05 or less was considered to be statistically significant. Metaboanalyst ( a comprehensive online software suite for metabolomic data analysis was used to generate the PCA scatter plot (Xia & Wishart, 2011). 3.4 Results Oral GlcNAc Supplementation Alters Liver Metabolism To identify dietary conditions where a modifying effect of GlcNAc supplementation on body-weight could be detected, three groups of young C57BL/6 male mice were maintained on diets containing 4%, 9% and 22% fat content. As expected, dietary fat content correlated with increased body-weight and with decreased RER (Figure 3.1A-C). Rates of weight-gain differed significantly in mice on different fat diets, with near cessation of growth on 4% diet, while 9% and 22% showing continued weight-gain. The 4% and 9% fat diets were selected for further experiments, as both left capacity for GlcNAc-dependent weight-gain, and represent diverse baseline dietary conditions. To determine if oral GlcNAc readily enters the bloodstream and accumulates in tissue, an oral gavage in mice with a bolus administration of heavy-glcnac ( 13 C6-GlcNAc) was performed. Blood samples were collected at different time intervals over a 3 hour period. Heavy-GlcNAc in blood peaked at 30 minutes and then gradually started to be cleared from the circulation, suggesting that it was being taken up by tissue (Figure 3.1D). At 180 minutes the blood level of heavy- GlcNAc has returned to normal, and heavy-glcnac was taken up by tissues and assimilated into UDP-GlcNAc via salvage HBP (Figure 3.1F). Heavy-GlcNAc was also co-administered with 109

141 heavy-glucose (Glc-d7). As expected, the uptake of heavy-glc peaked earlier than heavy-glcnac (Figure 3.1E), but the similarity displayed by the two curves suggests that a specific transporter might be responsible for GlcNAc uptake from the gut and subsequent release into circulation, where it is made available to the tissues. GlcNAc is commercially available as a dietary supplement, and oral GlcNAc in rats has shown no overt toxicity (Lee et al, 2004; Takahashi et al, 2009). A clinical study in children with severe treatment-resistant inflammatory bowel disease showed clinical improvement in a majority of cases following treatment with 3 6 g/day (~ mg/kg/day) of oral GlcNAc as an adjunct therapy (Salvatore et al, 2000). Oral GlcNAc at 0.25 mg/ml in drinking water, estimated to be ~40 mg/kg/day, suppressed spontaneous autoimmune diabetes in non-obese diabetic mice when initiated prior to disease onset (Grigorian et al, 2007). Importantly, oral GlcNAc increased UDP- GlcNAc supply and Mgat5-modified β1,6-glcnac-branched N-glycans on cell surface glycoproteins of T-cells in vivo (Grigorian et al, 2007). To explore dosage, GlcNAc at 0.5, 5.0 and 15 mg/ml (~80-2,500 mg/kg/day) was continuously provided in drinking water to weight and agematched male mice on 4% fat diet starting at weaning. Body-weight was measured weekly, and hepatic metabolites were analyzed by liquid-chromatography tandem mass-spectrometry (LC- MS/MS) 90 days following the beginning of GlcNAc treatment. Body-weight during this period of rapid growth increased with 5.0 and 15 mg/ml GlcNAc, on 4% fat diet (Figure 3.2A). GlcNAc at 0.5 mg/ml or greater in the drinking water increased hepatic GlcNAc-P and UDP-GlcNAc by 20%, while ATP remained unchanged (Figure 3.2B). Moreover, principal component analysis (PCA), a visualization technique that reduces the dimensionality of data by maintaining as much variance as possible, performed with the entire metabolite data set revealed a directional trend with GlcNAc dosage, moving away from the cluster of untreated controls 110

142 (Figure 3.2C). Intermediates in hepatic glycolysis and gluconeogenesis were increased with oral GlcNAc: including Glc-6P, Fru-6P, Fru-1,6BP, phosphoglycerate (3- & 2-PG), phosphophenolpyruvic acid (PEP), lactate, glycerol, and glycerol-3p (Figure 3.2D). Oral GlcNAc also increased hepatic levels of certain TCA cycle metabolites (Figure 3.2E), as well as the glucogenic polar amino acid Gln, and decreased essential amino acids Thr, Trp, Phe, and Iso/Leu (Figure 3.2F). Pyridine nucleotides soluble coenzymes, present as oxidized and reduced nicotinamide adenine dinucleotides: NAD+, NADH and NADPH were increased; as were the reduced and oxidized forms of the antioxidant glutathione (GSH and GSSG respectively) (Figure 3.2G). The GSH to GSSG ratio however was unaffected, while the ratio of NADH to NAD+ was elevated, consistent with catabolism supplying oxidative phosphorylation (Figure 3.2G) Oral GlcNAc Increases Body-Weight without Increasing Food Consumption I estimate that 0.5 mg/ml of GlcNAc (translating into ~40-80 mg/kg per day) was equivalent to less than 0.1% of total weight of daily food intake per mouse. Hence, even if GlcNAc was catabolized it would be an insignificant source of calories, suggesting that conversion to UDP- GlcNAc may regulate metabolism through protein glycosylation. A GlcNAc dose of 0.5 mg/ml was used to test for interaction with fat-enriched diet in mice. Oral GlcNAc was initiated at 14 weeks of age in weight-matched wild-type C57BL/6 male mice maintained on 4% and 9% fat diets (low fat and high fat, respectively), and continued for 30 weeks. Mice were also treated with glucosamine (GlcN), an amino-sugar dietary supplement used extensively in cell culture experiments to increase UDP-GlcNAc levels, primarily in O-GlcNAcylation studies (Hardiville & Hart, 2014). GlcN is taken up efficiently by cultured cells via Glut2 transporter (Uldry et al, 2002), and converted to GlcN-6P by hexokinases followed by two possible fates, either N-acetylation by 111

143 GNPNAT1/GNA1 to enter HBP, or deamination by GNPDA1 to Fru-6P and use in glycolysis (Wolosker et al, 1998). GlcNAc treated mice on 9% fat diet displayed significantly increased weight-gain compared to 9% fat diet alone, while GlcN had a negligible effect, suggesting that it did not follow the same path as GlcNAc (Figure 3.3A). After 20 weeks of continuous GlcNAc treatment, the mice had on average 13% and 19% increase in body-weight on 4% and 9% fat diet respectively, without any discernible increase in daily calorie intake (Figure 3.3B). In fact, after adjusting for body-weight, GlcNAc treated mice on 9% fat diet consumed fewer calories than control mice (Figure 3.3C). This suggests that exogenous GlcNAc promotes more efficient uptake and/or utilization of nutrients, as measured by conversion to biomass. These results were confirmed in five independent cohorts of mice and apply to both genders Oral GlcNAc Increases Lipid Accumulation and Catabolism At the time of sacrifice, GlcNAc treated mice weighed 10% more on 4% fat diet, and 16% more on 9% fat diet, than their control counterparts (Figure 3.3D). GlcNAc treated mice had similar muscle mass, but displayed increased fat content on both diets, as determined by DEXA (Figure 3.3E). The epidydymal fat-pads were increased by GlcNAc treatment by 46% and 12%, on the 4% and 9% fat diets respectively (Figure 3.3F). GlcNAc treatment with 9% fat diet resulted in hepatomegaly, an overall 42% increase in total liver weight, or a 22% increase after correcting for body-weight (Figure 3.3F). Thus, GlcNAc dependent weight-gain in C57BL/6 mice at 10 months of age was largely due to excess lipid accumulation in multiple sites. Serum free fatty acids were increased, whereas the level of triglycerides (TG) was the same in GlcNAc treated mice (Figure 3.3G-H). Consistent with increased fatty acid turnover, serum levels of glycerol and 112

144 glyceraldehyde were elevated; whereas, pyruvate, glycerol-3-phosphate, phosphoglyceric acid, propionate, and ketone-body 3-ketobutyrate were decreased (Figure 3.3I). The hepatomegaly observed in mice on GlcNAc with 9% fat diet suggests metabolic stress that might be revealed by nutrient-sensing signaling pathways. Notably, mtor is inhibited in lownutrient or stressed conditions, which promotes autophagy, hydrolysis of macromolecules and salvage of the constituents to promote cell survival (Efeyan et al, 2013; Rabinowitz & White, 2010). Western blot analysis of liver lysates revealed a dramatic reduction in the level of ribosomal protein p-s6, a marker of mtorc1 pathway activity, in GlcNAc treated mice on 9% fat diet (Figure 3.3J). Since hepatic mtorc1 activity negatively regulates autophagy and production of ketone bodies for peripheral tissues to use as an energy source, this observation suggests increased autophagy and macrolipophagy in these mice (Singh et al, 2009). No significant change in AMPactivated protein kinase (p-ampk) or Ac-CoA carboxylase (p-acc) was observed with GlcNAc, indicators of energy charge and fatty acid synthesis and oxidation (Hagiwara et al, 2012; Hardie, 2012) Oral GlcNAc Does Not Alter Physical Activity or Energy Expenditure Open circuit indirect calorimetry was used to estimate whole-body O2 consumption and CO2 production, while the activity of mice was measured by infrared photocells (Figure 3.4A-C). The RER was calculated from the volumes of O2 consumed and CO2 produced, and provides a measure of nutrients oxidized that ranges from 0.7 for oxidation of pure fats, to 1.0 for oxidation of pure carbohydrates. GlcNAc treated mice on 9% fat diet displayed increased oxidation of fat compared to mice on 9% fat diet alone (Figure 3.4D-E). GlcNAc treated and untreated mice on 9% fat diet were indistinguishable in total activity (ambulatory, grooming and rearing on hind 113

145 legs), and energy expenditure per mouse (Figure 3.4A and 3.4F). GlcNAc treated mice also consumed less food and water, but stored more fat, and relied more on oxidation of fat than their control counterparts (Figure 3.3C and 3.4D-H). Oral GlcNAc increased hepatic free fatty acids in mice on the 4% fat diet, either fasted or fed, and to a lesser degree in mice on 9% fat diet, where free fatty acids in controls were already high (Figure 3.4I). Hepatic TG levels in fasted and fed mice were increased by GlcNAc, but only on the 9% fat diet (Figure 3.4J). The livers of GlcNAc treated mice were larger, heavier and appeared pale, suggesting hepatic steatosis. Histology revealed extensive lipid deposition and confirmed that TG and neutral lipids were increased by GlcNAc treatment over levels observed with 9% fat diet alone (Figure 3.4K). However serum alanine aminotransferase (ALT), a nonspecific marker of liver damage was unchanged (Table 3.1). Blood plasma TG and electrolytes on 9% fat diet, in both fasted and fed mice, were not different in GlcNAc and respective control groups (Table 3.1) Oral GlcNAc Reprograms Fasting Metabolism Insulin receptor signaling phosphorylates/activates Akt, which leads to dephosphorylation/activation of glycogen synthase (GS), resulting in glycogen synthesis (Patel et al, 2004). Upon fasting, hepatic glycogen content, p-akt, and fatty acid synthase (FASN) were increased in GlcNAc treated mice, while phosphorylated/inactivated GS (p-gs) was decreased, relative to fasted GlcN treated and untreated controls (Figure 5A-D). Collectively, these findings suggest that GlcNAc supplementation enhanced the efficiency of nutrient uptake, and/or provided extra support for hepatic anabolic metabolism from muscle and adipose tissue, to delay depletion of glycogen during fasting. Alternatively, the increased reservoir of lipid and the greater 114

146 dependence of GlcNAc treated mice on fatty acid oxidation may reduce glucose consumption and depletion of glycogen. Indeed, p-ampk-α and its downstream target p-acc were increased in livers of fasted animals on GlcNAc or GlcN (Figure 3.5C-D). p-s6 levels were also elevated in GlcNAc and GlcN treated mice compared to controls upon 18 h of fasting, possibly a reflection of both reduced metabolic stress and a delayed fasting response. In this measure, GlcN has a similar effect to GlcNAc, consistent with some conversion of GlcN to UDP-GlcNAc. The increased phosphorylation of Akt and S6, and decreased phosphorylation of GS, along with FASN level, hormonal profile, and hepatic glycogen content suggest delayed fasting; while increased phosphorylation of AMPK-α and ACC is consistent with fasting and/or stress. Thus, GlcNAc treated mice show a delay in signaling that triggers glycogen breakdown and autophagy, along with enhanced AMPK and ACC signaling for promotion of mitochondrial fatty acid oxidation. Normally, blood glucose, insulin and leptin levels decrease during fasting, while glucagon increases. However, GlcNAc treated mice fasted for 18 h also displayed higher blood glucose and insulin levels, lower glucagon levels, and elevated hepatic glycogen and p-akt, comparable to that of GlcNAc treated fed mice (Table 3.1, Figure 3.5A-D). The ratio of circulating insulin to glucagon was indistinguishable between fasted and fed mice treated with GlcNAc, while the ratio changed 12-fold in untreated mice (Table 3.1). Circulating serum leptin was increased during both fasted and fed states in GlcNAc treated wild-type mice on 9% fat diet (Table 3.1 and Figure 9J), which implies increased fat depots, satiety and reduced appetite, and is consistent with the lowered food-intake observed in GlcNAc treated mice discussed above (Figure 3.3C and 3.4G). Serum leptin and insulin levels positively correlate with body-weight and energy stores in adipose tissue, while adiponectin inversely correlates with the same (Ye & Scherer, 2013). Adiponectin levels were lower in GlcNAc treated mice on 9% fat diet in the fed state (Table 3.1). 115

147 Hepatic HBP and Mgat5 activity have recently been shown to regulate the sensitivity of glucagon receptor to glucagon (Johswich et al, 2014). After 5 months of oral GlcNAc treatment, sensitivity to an injection of glucagon was increased, as indicated by elevated release and lower disposal rate of hepatic glucose, while glucose tolerance was not affected (Figure 3.5F-G). The hypersensitivity of glucagon receptor should place a higher demand on insulin to clear the excess hepatic glucose. In addition to lactate, amino acids are a major source of carbon for gluconeogenesis. After 7 months of oral GlcNAc, metabolite analysis in 18 h fasted mice revealed elevated serum levels of gluconeogenic precursors lactate, Gln, Phe and Ile (Figure 3.5E). These amino acids, along with Tyr and Leu are associated with human obesity and a high risk of diabetes (Kim et al, 2010; Newgard et al, 2009). Other metabolites associated with obesity, diabetes and metabolic syndrome were also elevated in GlcNAc treated mice, including sorbitol (Brownlee, 2001), aminoadipic acid (Wang et al, 2013), and uric acid (Johnson et al, 2013) (Figure 3.5E) Oral GlcNAc Increases Complex N-Glycan Branching in Liver Glycoproteins Glycopeptides were prepared from liver tissue of control and GlcNAc treated mice, and differentially labelled with light and heavy stable isotope dimethyl reagents respectively. Light and heavy labelled glycopeptides were mixed prior to mass spectrometry (MS) analysis. Intact glycopeptides and their deglycosylated form, obtained by treatment with PNGase F, were analyzed by LC MS/MS (Chen et al, 2014). The combined analyses identified glycosites and composition of N-glycosylation on glycoproteins. Specific glycosites in liver glycoproteins were compared for GlcNAc content in control and GlcNAc treated mice. Mice supplemented with GlcNAc on 4% and 9% fat diets, in both fed and fasted conditions, displayed significantly increased global GlcNAc 116

148 content in N-glycan structures of liver glycoproteins, which must be attributed primarily to increased N-glycan branching (Table 3.2). As a specific example, glycopeptides with site-specific N-glycosylation from the single transmembrane pass glycoprotein carcinoembryonic antigen related cell adhesion molecule 1 (CEACAM1 or CD66a) were analyzed in detail. The glycosite Asn89 on CEACAM1 was identified with high confidence, and the composition of the N-glycan structure was characterized as tri-antennary (Figure 3.6A and 3.6C). By normalizing with the ratio of deglycosylated peptides, this site-specific N-glycosylation was found to be 13-fold more abundant in livers from GlcNAc treated mice (Figure 3.6B and 3.6D). Structures for hybrid bi-antennary N-glycan with unsubstituted terminal mannose residues, and complex bi-antennary N-glycan were 0.72 and 0.70, respectively, suggesting that they are 1.4 times less abundant in livers from GlcNAc treated mice (Figure 3.6E-H). This result is consistent with GlcNAc incorporation into HBP and stimulation of the N-glycan branching pathway by UDP-GlcNAc (Abdel Rahman et al, 2014). Interestingly, CEACAM1 is found abundantly in the liver, where it regulates insulin clearance and hepatic lipogenesis (Najjar & Russo, 2014). CEACAM1 contains multiple potential N-glycosylation sites, and its glycans interact with lectins, including galectin-3 (Feuk-Lagerstedt et al, 1999) GlcNAc Increases Nutrient Uptake and Lipid Accumulation in Cultured Cells To explore the possibility that GlcNAc promotes increased nutrient uptake, AML12 - an immortal mouse hepatocyte cell line was cultured in medium supplemented with GlcNAc for 20 h, which markedly increased GlcNAc-P and UDP-GlcNAc levels, over 3-fold and 6-fold respectively, in LC-MS/MS analysis (Figure 3.7A). In a dose dependent manner, GlcNAc treatment increased L-PHA lectin binding, a probe for Mgat5-modified complex-type tri- and tetra- 117

149 antennary branched N-glycans (Figure 3.7B). Staining with ConA, a lectin that binds oligomannose- and hybrid-type N-glycans, was unchanged (Figure 3.7C). This indicates that N- glycosylation and early processing were not altered by increasing cellular UDP-GlcNAc. Immunoblots for O-GlcNAcylated proteins did not reveal differences with GlcNAc supplementation (Figure 3.7D). FASN level and lipid content in lipid droplets increased with GlcNAc treatment, in a dose dependent manner (Figure 3.7E-F). Metabolites involved in fat metabolism: citrate, Ac-CoA, malonyl-coa, carnitine and glycerol-3p increased, while glycerol, the immediate precursor for glycerol-3p, was depleted (Figure 3.7G). The uptake of fluorescent glucose analog (2-NBD-Glc) and dual-isotope-labelled glutamine ( 15 N2-Gln) was increased with GlcNAc treatment in AML12 cells (Figure 3.7H-I). Swainsonine (SW), an inhibitor of Golgi α-mannosidase II, blocks N-glycan-mediated branching by Mgat2, Mgat4 and Mgat5 (Lau et al, 2007). SW reduced the GlcNAc-dependent increase in complex-type N-glycan branching, and importantly, SW blocked the GlcNAc-induced increase in lipid droplet accumulation in AML12 cells (Figure 3.7J-K). GlcNAc also enhanced 15 N2-Gln uptake in the immortal epithelial HeLa cells, along with promoting lipid accumulation, and increasing levels of UDP-GlcNAc and L-PHA (Figure 3.8A-E). 3T3-L1 fibroblasts induced to differentiate into adipocytes exhibited a GlcNAc dose dependent increase in fatty acid uptake, lipid accumulation, and Mgat5-modified N-glycans (Figure 3.8F-H). These results show that GlcNAc induces a cell autonomous enhancement of Glc, Gln and fatty acid uptake. Thus, using different cell types, representative of distinct lineages with physiologically relevant metabolic functions, I show that exogenous GlcNAc elevates cellular UDP-GlcNAc level and promotes increase in Mgat5-modified tri- and tetra-antennary complex-type N-glycan branching, nutrient uptake, and lipid storage. Combined, these results suggest a model for GlcNAc dependent HBP 118

150 remodeling of cell surface N-glycan branching and reprogramming of cellular metabolism to enhance lipid accumulation (Figure 3.8I) Oral GlcNAc Partially Restores Anabolic Metabolism in Mgat5 -/- Mice GlcNAc supplementation in Mgat5 -/- mammary tumor cells has been shown to rescue a deficiency in retention of TGF-β and EGF cell surface receptors and their signaling (Lau et al, 2007; Mendelsohn et al, 2007). The rescue is due to compensating increases in the activity of the remaining Mgat branching enzymes driven by increased UDP-GlcNAc. The N-acetyllactosamine branches of structurally related N-glycans are redundant and compensate functionally by rescuing affinities for galectin association with glycoproteins' N-glycan branches at the cell surface (Lau et al, 2007). Mgat5 -/- mice display reduced body-weight and fat content (Lau et al, 2007), a phenotype opposite to that observed with GlcNAc supplementation in wild-type mice. Therefore, an attempt to rescue body-weight and metabolic insufficiency in Mgat5 -/- mice with oral GlcNAc on 9% fat diet was performed (Figure 3.9A). The Mgat5 -/- mice on GlcNAc had increased fat-tissue by 53%, compared to 26% increase in Mgat5 +/+ mice (Figure 3.9B). The increase in fat-tissue mass in Mgat5 -/- mice was offset by an 11% decrease in lean-tissue mass (Figure 3.9B). Similar results for body-weight and tissue composition were observed in female Mgat5 +/+ and Mgat5 -/- mice on 9% fat diet (Figure 3.9G-I). GlcNAc supplementation lowered RER in both genotypes, implying increased oxidation of fat relative to untreated mice (Figure 3.9C). The HBP metabolites GlcNAc- P and UDP-GlcNAc were elevated in liver by GlcNAc treatment in both genotypes (Figure 3.9D). Primary hepatocytes from young Mgat5 +/+ and Mgat5 -/- mice were cultured overnight in the presence of GlcNAc. Lipid droplet content was 40% lower in Mgat5 -/- hepatocytes, but increased with GlcNAc treatment, reaching levels similar to untreated Mgat5 +/+ hepatocytes (Figure 3.9E). 119

151 Liver histology revealed increased lipid accumulation in GlcNAc supplemented mice of both genotypes (Figure 3.9F). Collectively, GlcNAc treatment increased fat accumulation and fatty acid oxidation in Mgat5 -/- mice, consistent with the model of functional redundancy in N-glycan branches. However, it appears that Mgat5 is required for normal balance of lean to fat body tissue composition that cannot be compensated by increase in UDP-GlcNAc alone. 3.5 Discussion GlcNAc is a commercially available dietary supplement used by people and domestic animals, with unsubstantiated health claims (Schnaar & Freeze, 2008). Acute intravenous GlcNAc administration in humans does not seem to affect blood glucose homeostasis or insulin secretion (Gaulden & Keating, 1964), and insulin had no obvious effect on the disappearance rate of circulating GlcNAc (Levin et al, 1961). GlcNAc taken up by cells is largely converted into UDP- GlcNAc (Dennis et al, 2009). A previous publication has shown that C57/BL6 mice treated with 0.25 mg/ml GlcNAc in their drinking water for seven days displayed increased GlcNAc serum concentrations (Grigorian et al, 2011). This result strongly suggests that GlcNAc taken orally is absorbed in the small intestines and transferred into bloodstream. Using a time-course experiment I demonstrate that heavy-glcnac administered orally is rapidly absorbed in the small intestine and efficiently transferred into bloodstream. Furthermore, 3 hours following oral gavage heavy- GlcNAc is incorporated into UDP-GlcNAc in tissue examined, demonstrating that GlcNAc is systematically taken up from the circulation. UDP-GlcNAc is potentially an ideal sensor of cellular metabolic status (Hardiville & Hart, 2014; Love & Hanover, 2005; Marshall, 2006). GlcNAc supplementation might model surfeit or nutrient abundance in glucose, nitrogen and fatty-acid metabolism. Here I examined the effects of 120

152 extended oral GlcNAc treatment on C57BL/6 mouse physiology and metabolism. In the postweaning rapid-growth phase, mice on a 4% fat diet showed more weight-gain with oral GlcNAc, as well as increased hepatic levels of glycolytic, gluconeogenic and TCA metabolites, and reduced levels of some amino acids, including branched-chain amino acids leucine/isoleucine. With prolonged oral GlcNAc weight-gain was enhanced, with greater effect on 9% compared to 4% fat diet, evidence of a clear interaction between GlcNAc and calorie enriched diet. GlcN at a similar dosage did not significantly increase body-weight, suggesting a less potent contribution to UDP- GlcNAc and down-stream effectors. Oral GlcNAc intake in rats for 13 weeks on 6% fat diet, has also been shown to increase body-weight without increase in food intake (Takahashi et al, 2009). Parameters that were not significantly altered by oral GlcNAc included food intake, total activity, and energy expenditure per mouse, as determined through indirect calorimetry, by measuring O2 consumption and CO2 production. Thus GlcNAc treated mice are not indolent or lethargic, but rather appear to utilize equivalent calories more efficiently, as measured by conversion to bodymass and fat content. It should be noted that in this experiment food intake and energy expenditure were measured only during a short time interval, a week for food intake and 24 hours for energy expenditure. Furthermore, by the time food intake and energy expenditure measurements were taken the mice have already diverged in body-weight. Ideally, food intake and energy expenditure should have been monitored throughout the experiment, since it is difficult to draw conclusions regarding energy balance over long time intervals from data obtained during a short time window, which is typically the case for calorimetry-based measures (Tschop et al, 2012). Food intake was monitored when mice were housed individually, as well as when they were caged together, and in both instances GlcNAc treatment decreased their food intake. Although food intake measurements 121

153 are often considered to be easy and trivial, they are in fact fraught with difficulty, and vary greatly in accuracy, reproducibility and practicality (Tschop et al, 2012). Not all ingested calories are introduced into metabolism, and there is even the possibility that mice might potentially consume their own faces, giving any residual nutrients a second chance as they transit through the digestive system. Thus, it is possible that food intake and/or energy expenditure measures employed may have been insufficiently sensitive or accurate. Interestingly, in a study using mannose supplementation, mice consuming mannose in drinking water weighted less and had decreased fat mass compared to untreated mice, but showed no difference in energy expenditure or physical activity, and an actual increase in calorie intake of 20% (Hudson Freeze personal communication). At first glance this result appears to violate the first law of thermodynamics, i.e. the law of conservation of energy which states that energy can be transformed from one form to another, but cannot be created or destroyed. However upon closer inspection a bomb calorimeter analysis revealed a significantly greater energy content in feces from mice supplemented with mannose, as compared to untreated controls, suggesting decreased nutrient absorption (Hudson Freeze personal communication). This result might be a cautionary tale and be relevant to my own findings with GlcNAc, which have also been suggested to violate the first law of thermodynamics, since I observe a body-weight increase with GlcNAc supplementation, without difference in energy expenditure or activity, and an actual decrease in food intake. I propose greater efficiency of nutrient uptake as the explanation for increased bodyweight and adiposity observed in GlcNAc treated mice. However at this point the energy content of their feces has not been analyzed to test for such a difference. An additional parameter that plays a role in body-weight regulation but was not accounted for in my experiments is adaptive thermogenesis, used to maintain core body temperature. Reduction of core body temperature by 122

154 0.3 to 0.5 C in male transgenic mice resulted in significant body-weight increase without affecting food intake or physical activity (Conti et al, 2006). Mass spectrometry analysis revealed that oral GlcNAc increases hepatic UDP-GlcNAc and GlcNAc content in glycoprotein N-glycans, consistent with more highly branched N-glycans, as demonstrated with CEACAM1. GlcNAc treatment was associated with elevated levels of leptin and insulin. In control mice on the 9% fat diet, the ratio of circulating insulin to glucagon (I/G) decreased 12-fold between fed and fasted conditions. The I/G ratio was abnormally low in fed and high in fasted GlcNAc-treated mice; with no net change between fed and fasted. With elevated levels of serum glucose, lactate and Gln, oral GlcNAc alters or delays the normal fasting response on 9% fat diet. The atypical response to fasting, as revealed by hormonal profile, serum metabolites, hepatic glycogen content, and AMPK and mtor signaling suggests a continued abundance of nutrients and/or hyperinsulinemia, possibly as a result of autophagy in fed GlcNAc treated mice associated with metabolic and ER stress induced by hepatic steatosis (Singh et al, 2009). My results suggest that HBP and the N-glycan branching pathway interact, descriptively as a thrifty genotype/phenotype (O'Rourke R, 2014). Mgat5 null mice are hypoglycemic, have reduced fat deposits, are resistant to weight-gain on a 9% fat diet, and display hypersensitivity to fasting manifested as faster glycogen depletion (Cheung et al, 2007); the opposite of GlcNAc fed mice in the present study. GlcNAc treatment partially restored fat accumulation in Mgat5 -/- mice and primary hepatocytes, consistent with the model of redundancy, where increasing UDP- GlcNAc drives compensating increases in N-glycan branching by the other Mgat enzymes (Dennis & Brewer, 2013; Lau et al, 2007). However, Mgat5 -/- mice treated with GlcNAc did not recover normal body-weight, and in fact displayed lower lean tissue mass, suggesting an important role for 123

155 Mgat5 in balancing fat and lean tissue with aging. Early aging in Mgat5 -/- mice is associated with an imbalance in TGF-β and growth factor signaling, and premature loss of muscle satellite cells and osteoprogenitor bone-marrow cells (Cheung et al, 2007). In the absence of Mgat5, positive feedback between metabolism, HBP and N-glycan branching may be insufficient to maintain muscle satellite cells (Cheung et al, 2007). GlcNAc supplementation also rescued glucagon receptor sensitivity in Mgat5 -/- primary hepatocytes and in vivo in mice (Johswich et al, 2014), and in this study enhanced its sensitivity in wild-type mice. Treatment of glucose-starved hematopoietic cells with GlcNAc has been shown to promote IL-3 receptor surface expression and signaling in a manner dependent on Golgi remodeling of N-glycans, which in turn increased Gln uptake and utilization for energy production, lipid biosynthesis and essential amino acid uptake (Wellen et al, 2010). In cultured cells, GlcNAc increased Mgat5-dependent N-glycan branching, and enhanced uptake of Glc, Gln and fatty acid. Swainsonine (SW), an inhibitor of N-glycan branching, blocked the GlcNAc-dependent increase in lipid accumulation. Similarly, GlcNAc increased Gln uptake in an Mgat5-dependent manner in Hek293 cells (Abdel Rahman et al, 2014). Amino acid uptake and catabolism, coupled with glucagon-driven gluconeogenesis may play a major role in hepatic metabolic phenotypes observed in GlcNAc wild-type and Mgat5 -/- mice. Genes in the HBP have been implicated in lipid accumulation in different species, supporting its role in weight regulation. A polymorphism in GFAT is associated with obesity in men (Weigert et al, 2005), and GFAT overexpression in HepG2 liver cells increased transcript levels associated with ER stress and lipid accumulation (Sage et al, 2010). Genetic or pharmacological suppression of GFAT inhibited lipogenesis in HepG2 (Sage et al, 2010), murine 3T3-L1 adipocytes (Hsieh et al, 2012), and in human visceral adipocytes (O'Rourke et al, 2013). 124

156 Furthermore, transgenic mice overexpressing GFAT in the liver were heavier than non-transgenic littermates (Veerababu et al, 2000). In the red flour beetle, down-regulation of UDP-GlcNAc pyrophosphorylase (UAP1) results in depletion of fat-body (Arakane et al, 2011). Hepatic Slc35B4, a Golgi UDP-GlcNAc transporter, was identified as a quantitative trait locus associated with diet-induced obesity, insulin resistance and gluconeogenesis in mice (Yazbek et al, 2011). Slc35B4 is also a candidate gene for obesity in humans (Chen et al, 2013), where it could provide more effective transport of UDP-GlcNAc. Golgi UDP-GlcNAc transport activity controls UDP- GlcNAc supply to the N-glycan branching pathway, where Mgat5 is most sensitive to its concentration (Lau et al, 2007). As a practical consideration, the sources and amounts of GlcNAc in our diet are unknown. Dietary GlcNAc may interact with gene polymorphisms in the HBP and Golgi N-glycan pathway, playing a role in the obesity epidemic. The microbiomes of obese humans and mice have lower bacterial diversity and a significantly greater ratio of Firmicutes to Bacteriodetes (DeWeerdt, 2014). With a high abundance and variety of carbohydrate-active enzymes such as glycosidases in the microbiota (El Kaoutari et al, 2013), the microbiome associated with obesity may release more GlcNAc from chitin and other polysaccharides into the gut. Chitin is a long-chain polymer of GlcNAc found widely in nature, and used as a food and feed additive (Hirano, 1996; Shahidi & Abuzaytoun, 2005), which is obscured on labelling as carbohydrates or sugar. Oral GlcNAc may also provide a selective advantage to certain gut microbial communities, and modulate host metabolism and physiology. Further work will be necessary to elucidate the complex relationship between diet, gut microbiota and the host. 125

157 fasted 18 h fed ad libitum control GlcNAc control GlcNAc ALT (IU/L) ± ± ± ± Triglycerides ± ± ± ± (mmol/l) Cholesterol (mmol/l) ± ± * ± ± Chloride (mmol/l) ± ± ± ± Sodium/Potassium ± ± ± ± Ratio Glucose (mm) ± ± ** ± ± Insulin (pm) ± ± * ± ± Glucagon (pm) ± ± * ± ± Insulin to Glucagon ± ± * ± ± * Ratio HOMA2-IR ± ± * N/A N/A Leptin (pm) ± ± ** ± ± * Adiponectin (ng/ml) ± ± ± ± * Table 3.1 Phenotypic differences in serum biochemistry between GlcNAc treated and untreated mice on 9% fat diet, under fasted and fed conditions. At sacrifice mice were 10 months old (n=4-5 per group), and have been on 0.5 mg/ml oral GlcNAc supplementation for 30 weeks, or 7 months. Data are mean ±SEM. *p<0.05 versus fasted or fed ad libitum control mice on 9% fat diet (2-tailed, unpaired Student's t-test). ALT alanine aminotransferase, HOMA2-IR homeostatic model assessment-insulin resistance. 126

158 Diet GlcNAc content (GlcNAc treated/control) Glycopeptides with N-glycans p-values 4% fat < % fat, fasted < % fat < % fat, fasted Table 3.2 Global analysis of relative GlcNAc content in liver N-glycans. N-glycans from liver glycopeptides, from control and GlcNAc treated mice, were identified by differential labelling with stable light and heavy isotope dimethyl labelling followed by LC-MS/MS. Liver was isolated from mice on 4% and 9% fat diets, in fasted and fed conditions. Sign test with probability of 0.5 and two-tail p-value was performed. 127

159 Figure 3.1 Oral GlcNAc is rapidly absorbed by gut to enter bloodstream and be taken up by tissue from circulation. (A) Change in body-weight for wild-type C57BL/6 male mice on diets containing different percentages of fat. Data shown are mean ± SEM, n=8, analyzed by one-way ANOVA followed by Dunnett s multiple comparison test compared with 4% fat diet, with significant differences indicated as #p<0.05 and ##p<0.01 versus 9% fat diet, and *p<0.05 and **p<0.01 versus 22% fat diet. (B) Respiratory exchange ratio (RER) of mice fed different percentage fat diets for 50 weeks, (C) quantification of data in (B) for night and day. Data shown are mean ±SEM, analyzed by one-way ANOVA followed by Tukey's multiple comparison test, with significant differences indicated as *p<0.05, **p<0.01, and ***p< (D) Time-course of relative abundance of 13 C6-GlcNAc in serum of mice orally gavaged with a bolus administration of 13 C6-GlcNAc at 20 µg/g of mouse. (E) Time-course of relative abundance of Glc-d7 in serum of mice orally gavaged with a bolus administration of Glc-d7 at 50 µg/g of mouse. (F) At 180 minutes following oral gavage with 20 µg/g 13 C6-GlcNAc, UDP- 13 C6-GlcNAc was detected as a strong peak in different mouse tissues. 128

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161 Figure 3.2 Oral GlcNAc increases UDP-GlcNAc level and promotes weight-gain in mice. (A) Analysis of change in body-weight of wild-type C57BL/6 male mice on low fat diet over 90 days supplemented with oral GlcNAc. (B) Relative abundance of liver metabolites in the distal portion of HBP measured by LC-MS/MS in 90 day GlcNAc treated mice on low fat diet. (C) Principle component analysis of all measured liver metabolites in mice on low fat diet and GlcNAc supplied in drinking water at 0.5, 5.0 and 15 mg/ml. Steady-state liver metabolites in the glycolysis and gluconeogenesis pathways (D), tricarboxylic acid (TCA) cycle (E), amino acids (F), and oxidized and reduced forms and ratios of glutathione and nicotinamide adenine dinucleotides (G). All metabolites measured by LC-MS/MS and expressed as fold change in 90 day GlcNAc treated mice on low fat diet. Relative levels of specific metabolites were normalized to liver weight. Metabolomic data shown are mean ±SEM, n=10, analyzed by oneway ANOVA followed by Dunnett s multiple comparison test compared with vehicle control of 0 mg/ml GlcNAc, with significant differences represented as *p<0.05, **p<0.01, and ***p<

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163 Figure 3.3 Oral GlcNAc promotes weight-gain and lipid accumulation. (A) Change in body-weight for wild-type C57BL/6 male mice on 4% and 9% fat diet over weeks of oral GlcNAc or GlcN supplied in drinking water at 0.5 mg/ml. Data shown are mean ± SEM, n=10, analyzed by 2-tailed unpaired Student's t-test, with significant differences indicated as #p<0.05 for 4% fat versus GlcNAc 4% fat, and *p<0.05 for 9% fat versus GlcNAc 9% fat. (B) Body-weight and (C) calorie intake per body-weight per day at 34 weeks of age, or following 20 weeks of GlcNAc treatment. Terminal body-weight (D), tissue composition (E), and epidydymalfat and liver weight normalized to body-weight (F) at sacrifice. Error bars represent ±SEM, n=10, *p<0.05 or **p<0.01 GlcNAc treated versus control, on either 4% or 9% fat diet (2-tailed, unpaired Student's t-test). Serum free fatty acids (FFA) (G), triglycerides (TG) (H), and significant steady-state metabolite changes (I) in mice on 9% fat diet and supplemented with 0.5 mg/ml oral GlcNAc for 90 days. Error bars represent ±SEM, n=5, *p<0.05, or **p<0.01 versus control (2-tailed, unpaired Student's t-test). (J) Western blot analysis of metabolic signaling pathways in liver lysates from mice maintained on 9% fat diet supplemented with GlcN or GlcNAc, probed for phosphorylated AMP-Activated Protein Kinase (AMPK-α), Acetyl-CoA Carboxylates Kinase (ACC) and ribosomal protein S6 (S6). Tubulin was used as a loading control. 132

164 Figure 3.4 Oral GlcNAc promotes fatty acid oxidation without affecting activity or energy expenditure. (A) Analysis of total activity, (B) oxygen consumption rate, (C) carbon dioxide emission rate, and (D) Respiratory Exchange Ratio (RER=VCO2/VO2) over 20 hour period, (E) quantification of data in (D) for night and day, (F) energy expenditure, and (G) food and (H) water intake. Liver free fatty acids (FFA) (I) and triglycerides (TG) (J) in mice on 9% fat diet and orally supplemented with 0.5 mg/ml GlcNAc. Error bars represent ±SEM, n=5, *p<0.05, or ** p<0.01 versus control (2-tailed, unpaired Student's t-test). (K) Representative images of liver histology sections stained with oil red O to detect TG and neutral lipids in mice on 9% fat diet, fed ad libitum or fasted for 18 h, and supplemented with 0.5 mg/ml GlcNAc in drinking water for 30 weeks. 133

165 Figure 3.5 Oral GlcNAc alters fasting liver metabolism. (A) Representative images of liver histology sections stained with periodic acid-schiff, with glycogen deposits detected as purple-magenta areas, obtained from mice on 9% fat diet mice, fed ad libitum or fasted for 18 h, and supplemented with 0.5 mg/ml GlcNAc in drinking water for 30 weeks. (B) Liver glycogen content. Error bars represent ±SEM, n=5, *p<0.05 versus control (2- tailed, unpaired Student's t-test). (C) Immunoblot analysis of metabolic signaling pathways with FASN, and phosphorylated versions of GS, Akt kinase, ribosomal protein S6, AMPK-α, and ACC in liver lysates of fasted mice maintained on 9% fat diet and treated with GlcN or GlcNAc. (D) Quantification of immunoblots using tubulin as loading control. (E) Steady-state relative abundance of metabolites in blood serum from mice maintained on 9% fat diet and GlcNAc for 30 weeks. (F) Intraperitoneal glucagon tolerance test, and (G) intraperitoneal glucose tolerance test, with Area Under the Curve (AUC) quantification in mice treated with 0.5 mg/ml oral GlcNAc for 23 weeks. Error bars represent ±SEM, n=10, analyzed with 2-tailed unpaired Student's t-test *p<0.05 versus control on the same diet. 134

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167 Figure 3.6 Oral GlcNAc increases tri-antennary N-glycan structures on glycosite Asn89 of CEACAM1 hepatic transmembrane glycoprotein. (A) By matching the Y1 ion (peptide + GlcNAc) from the MS/MS spectrum to the list of deglycosylated peptides identified by Mascot database search with accurate molecular weight and retention time, the peptide sequence was identified as Asn89 of CEACAM1 transmembrane glycoprotein. (B) The extracted ion chromatogram peak area for control (light) and GlcNAc (heavy) labelled precursor from deglycosylated CEACAM1 peptides. With a ratio of GlcNAc to control of 1.68, no significant difference was found, which demonstrates that the increase in abundance of intact glycopeptides with tri-antennary complex structure was due to increased branching of complex N-glycans. (C) Annotated MS/MS spectrum of heavy-labelled intact glycopeptide identified with complex tri-antennary N-glycan structure. Terminal sialylation could be verified by the existence of oxonium ion with m/z 292 and 274. (D) The extracted ion chromatogram (XIC) of control (light) and GlcNAc (heavy) labelled peptide precursor from full MS scan indicates the abundance of tri-antennary glycopeptide being much higher in liver lysates from GlcNAc treated mice, with a ratio of 21 in GlcNAc to control. (E) Hybrid bi-antennary N- glycan structure with unsubstituted terminal mannose residues. (F) XIC from control and GlcNAc bi-antennary hybrid N-glycan with mannose, with a peak area ratio of GlcNAc to control of (G) Complex bi-antennary N-glycan structure. (H) XIC from control and GlcNAc complex biantennary N-glycan, with a peak area ratio of GlcNAc to control of

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169 Figure 3.7 GlcNAc increases UDP-GlcNAc, β1,6-glcnac branched N-glycans and lipid accumulation. (A) Fold changes of distal HBP metabolites upon GlcNAc treatment for 20 h. (B) Analysis of Mgat5-mediated β1,6-glcnac branched complex-type N-glycans on cell surface glycoproteins quantified with fluorophore conjugated lectin L-PHA. (C) Analysis of oligomannose-type and hybrid-type N-glycans on cell surface glycoproteins with fluorophore conjugated lectin ConA. (D) Immunoblot analysis of O-GlcNAcylation detected with monoclonal-antibody CTD (E) Protein expression level by western blot analysis of FASN enzyme and loading control tubulin, used for relative quantification of immunoblot. (F) Intracellular lipid droplets, quantified microscopically with lipophilic fluorescent probe BODIPY 493/503. (G) Fold change in specific metabolites involved in fat metabolism, normalized to cell number. (H) Glucose uptake in cells treated with GlcNAc for 20h, grown in the presence of fluorescent glucose analog 2-NBD-Glc for 1h, and quantified using flow cytometry as mean fluorescent intensity (MFI). (I) Analysis of heavy-isotope labelled 15 N2-Gln uptake in cells treated with GlcNAc for 20 h, pulsed with 15 N2- Gln for designated times, and quantified using mass spectrometry. (J) Tri- and tetra-antennary Mgat5-modified N-glycans, and (K) intracellular lipid accumulation in the absence and presence of GlcNAc and/or Swainsonine (SW), quantified microscopically with fluorophore-conjugated lectin L-PHA or BODIPY 493/503. Data shown are mean ±SEM, analyzed by one-way ANOVA followed by Dunnett s multiple comparison test compared with vehicle control (A-C, E-G, J-L), with significant differences represented as *p<0.05, **p<0.01, and ***p<

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171 Figure 3.8 HBP and N-glycan dependent regulation of cellular metabolism. (A) Analysis of 15 N2-Gln uptake and its downstream metabolites (B) glutamate ( 15 N-Glu) and (C) 15 N-Gln in HeLa cells treated with GlcNAc for 20 h, pulsed with 15 N2-Gln, and quantified using LC-MS/MS. (D) Analysis of intracellular lipid in HeLa cells. (E) Fold change (FC) in L- PHA and UDP-GlcNAc as a function of GlcNAc treatment in HeLa cells. (F) Kinetic reading monitoring uptake of non-esterified long-chain-fatty-acid-analog (BODIPY-FA) quantified as Area Under the Curve (AUC) for Relative Fluorescence Units (RFU). (G) Analysis of intracellular lipid and (H) complex-type N-glycans in 3T3-L1 adipocytes. Data are mean ±SEM, analyzed by t-test or one-way ANOVA followed by Dunnett s test, significant differences represented as *p<0.05, **p<0.01, and ***p< (I) GlcNAc salvaged by HBP increases UDP-GlcNAc, the substrate for N-acetylglucosaminyltransferase enzymes (Mgat1, 2, 4, 5) acting on glycoprotein acceptors trafficking through Golgi en route to the cell surface. Km values for UDP-GlcNAc decline from Mgat1, Mgat2, Mgat4 to Mgat5, making biosynthesis of tri- and tetra-antennary N-glycans sensitive to UDP-GlcNAc levels. N-glycan branching increases the affinity of glycoproteins for galectins, which cross-link and oppose loss of receptors and transporters to endocytosis. This increases cell surface residency of glucose (Glc), glutamine (Gln), and fatty-acid (FA) transporters (Glut, SLC and CD36 respectively), and other transmembrane glycoproteins such as CEACAM1. The improved cell surface retention allows more nutrients to enter the cell and contribute to increased lipid accumulation via FASN. A positive-feedback loop is formed by increasing uptake and flux of Glc, Gln and Ac-CoA through de novo HBP to UDP-GlcNAc and N-glycan branching on receptors and transporters. 140

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