Lipodystrophy: Metabolic and Clinical Aspects. Resource Room Slide Series

Transcription

1 Lipodystrophy: Metabolic and Clinical Aspects Resource Room Slide Series

2 Cellular Pathology of Insulin Resistance in Lipodystrophy Robert R. Henry, MD Professor of Medicine University of California, San Diego VA San Diego Healthcare System San Diego, CA Theodore P. Ciaraldi, PhD Project Scientist, UCSD La Jolla, CA

3 Disclosures Dr. Henry: Grants: AstraZeneca, Bristol-Myers Squibb, Eli Lilly, Medtronic, and Sanofi Consulting: Boehringer Ingelheim, Eli Lilly, Gilead, Intarcia, Isis, Novo Nordisk, Roche/Genentech, and Sanofi Advisory board: Amgen, AstraZeneca, Boehringer Ingelheim, Bristol-Myers Squibb, Daiichi Sankyo, Elcelyx, Eli Lilly, Gilead, Intarcia, Johnson & Johnson/Janssen, Merck, Novo Nordisk, Roche/Genentech, Sanofi Dr. Ciaraldi There are no relevant financial relationships to disclose.

4 Objectives Understand the role of ectopic fat storage in the development of insulin resistance Understand the nature of bi-directional signaling that occurs between adipose tissue and other organs: muscle, liver, and pancreas Recognize how post-translational modifications (PTMs) affect both signaling pathways and gene regulation

5 Overview 1. Fat storage Where and how? Fat metabolism in skeletal muscle Disordered fat metabolism 2. Insulin signaling Target tissues and responses Pathways PTMs Disordered insulin signaling 3. Adipose tissue-skeletal muscle communication Activation of stress kinases Altered phosphorylation of insulin receptor substrate 1 (IRS-1): consequences Altered PTMs of transcription factors: consequences

6 Body Fat Distribution Healthy Subcutaneous obesity Visceral obesity Lipodystrophy

7 Definitions Ectopic: Occurring in an abnormal position or unusual manner or form* Ectopic Fat: Abnormal fat accumulation other than in adipose tissue (eg, skeletal muscle, heart, liver, pancreas) * Merriam-Webster dictionary

8 Fat Distribution Between Tissues Adipose Tissue Compartment Plasma Muscle Compartment Lean Subcutaneous Obesity Visceral Obesity Lipodystrophy Unger RH. Trends Endocrinol Metab. 2003;14:

9 Insulin Sensitivity (clamp log 10 mol/l) (mg/min kg FFM ) Intramyocellular Lipid Relationship to Insulin Sensitivity r = P< Current Opinion in Pharmacology Skeletal Muscle-Associated Triglyceride (mmol/g wet weight of tissue) Nawrocki AR, Scherer PE. Curr Opin Pharmacol. 2004;4: Kelley DE, Goodpaster BH. Diabetes Care. 2001;24:

10 Lipid Droplets (LDs) Multi-Faceted Organelles for Lipid Storage and Mobilization Outer phospholipid monolayer Neutral lipid core: triglycerides, cholesterol esters Monolayer studded with LD proteins: Lipases and activators Adipocyte triglyceride lipase (ATGL) Hormone sensitive lipase (HSL) Comparative gene identification-58 (CGI58), activator of ATGL Perilipins (PLINs) 1-5: regulate access of lipases to lipid tissue-specific expression LDs permit organization and regulation of lipid storage and mobilization

11 Lipid Storage in Cells Adipocyte Skeletal muscle LD LD LD LD - HSL - ATGL - CGI58 - PLIN 1 - PLIN 2 - PLIN 5

12 Sources and Fates of Lipids and Fats Tissue Specificity Tissue Lipid Source Fate Adipose tissue Skeletal muscle Liver Internal stores Circulation Synthesis Circulation Internal stores (Limited) Circulation Internal stores (Limited) Synthesis Lipolysis and release to circulation Structural Oxidation Structural Oxidation Lipoprotein production Structural Pancreas Circulation Oxidation

13 Fat Metabolism in Muscle Healthy FFA Hyperlipidemia FFA FATP1 FAT FABPpm FATP1 FAT FABPpm CO 2 Ceramide CO 2 Ceramide FFA mito DAG FFA DAG LCFACoA LD LCFACoA Diffusion Protein-mediated transport and trafficking

14 Fats and Metabolites in Muscle Change in Lipodystrophy Intramyocellular lipid Triglyceride (TG) Diacylglycerol (DAG) * Long Chain Fatty Acyl CoA (LCFA-CoA) Ceramide Acylcarnatines CO 2 * Degree of fatty acid (FA) saturation altered in insulin-resistant conditions

15 Fat Cell Functions Then Lipoprotein LPL FFA FFA FFA (in lipodystrophy) TG Glucose LD

16 Adipose Tissue Functions Now FAT Increased in lipodystrophy Decreased in Lipodystrophy Resistin Adiponectin Leptin Angiotensin II Visfatin CRP PAI-1 FFA IL-6 MCP-1 TNF-α

17 Summary, Part 1 In healthy individuals, lipid is stored predominately in adipose tissue, with subcutaneous adipose tissue > visceral adipose tissue. Lipid storage in muscle, liver, and pancreas is limited and present primarily to meet energetic and structural needs. Lipid storage in tissues is tightly regulated, primarily through the actions of proteins associated with LDs.

18 Summary, Part 1, cont. Loss of the storage capacity of adipose tissue, such as in lipodystrophy, leads to increased lipid stores in other tissues (ectopic fat). Lipid oversupply results in altered/incomplete FFA oxidation. Specific FA metabolites can have deleterious effects on cellular function, including secretion (eg, adipokines).

19 Insulin Target Tissues 1 Tissue Skeletal muscle Adipose tissue Liver Heart Response Glucose uptake Glycogen synthesis Lipolysis Lipogenesis Glucose production Lipogenesis Lipoprotein production Glucose oxidation FFA oxidation Hypertrophy

20 Insulin Target Tissues 2 Tissue Brain Endothelium Macrophage Response Appetite Sympathetic tone Vasoconstriction Vasodilation Leukocyte adhesion Low density lipoprotein uptake

21 Insulin Signaling Cast of Players IR Insulin Receptor GLUT4 Glucose Transporter 4 IRS (1-5) Insulin Receptor Substrate GSK3 Glycogen Synthase Kinase 3 PI3-K Phosphoinositide 3 Kinase (p85 & p110 kda Subunits) Grb2 Growth Factor Receptor Bound-2, Scaffold Protein PDK1 Phosphoinositide-Dependent Kinase SOS Son of Sevenless, Scaffold Protein Akt Also known as Protein Kinase B ERK Extracellular Regulated Kinase apkc Atyptical Protein Kinase C mtor Mammalian Target of Rapamycin AS160 Akt Substrate of 160 kda Shc Src homology 2 and collagen-like

22 Insulin Receptor subunit -S-S- Insulin Binding Plasma Membrane - Transmembrane Region ß subunit Y953 Y960 Y1146 Y1150 Y1151 Y1316 Y Juxtamembrane Region - Kinase Regulatory Region - C-Terminal Regulatory Region Adapted from Ciaraldi TP. In Principles of Diabetes Mellitus. 2 nd ed. Poretsky LO, ed. New York, Springer,2009, p

23 Principles of Insulin Signaling The IR can phosphorylate both itself and other proteins (eg, IRS) on the amino acid tyrosine. Phosphorylation creates recognition sites for IRS and other proteins to serve as scaffolds, recruiting other molecules into signaling complexes. Phosphorylation on other sites can oppose complex formation.

24 Principles of Insulin Signaling, cont. Intracellular localization of these multi-molecular signaling complexes plays an important role in the subsequent pathways activated. Phosphorylation is just one of a number of posttranslational modifications (PTMs) that can influence complex formation, sub-cellular localization, enzyme activity, and stability or degradation of the protein(s).

25 PTMs of Proteins Modification Enzyme Responsible Phosphorylation Kinase (eg, IR) Phosphatase P Phosphate Acetylation HAT HDAC Ac Acetyl group O-GlcNAcylation OGT OGA O-GlcNAC O-linked-Nacetylglucosamine Ubiquitination E1, E2, E3 DUB Ub Ub Ub Ubiquitin SUMOylation E1, E2, E3 SENP, DeSI SUMO Small ubiquitinrelated modifier

26 Phosphorylation of IRS-1 IR NH 2 Y 613 Y 623 PH PTB PI3K binding COOH IRS-1 has no intrinsic enzymatic activity PH pleckstrin homology domain, binds phosphoinositides PIP x PTB phosphotyrosine binding domain

27 IRS-1 as a Molecular Scaffold IR NH 2 Y 613 Y 623 PH PTB PI3K binding COOH p85 Grb-2

28 PDK1 Insulin Signaling: Metabolism Insulin PI(4,5)P 2 PI(3,4,5)P 3 Insulin Receptor IRS p85 p110 apkc? GSK3 _ Akt GS AS160 -ps -py GS Rab-GTP GLUT4 GLUT4 Vesicle Adapted from Fröjdö S, Vidal H, Pirola L. Biochim Biophys Acta. 2009;1792:83-92.

29 PDK1 Mitogenesis & Gene Expression Insulin PI(4,5)P 2 PI(3,4,5)P 3 Insulin Receptor Shc SOS Grb2 IRS p85 p110 Akt GSK3 Erk 1/2 p70s6k mtor Transcription Factors Nucleus Fröjdö S, Vidal H, Pirola L. Biochim Biophys Acta. 2009;1792:83-92.

30 Insulin Action in Lipodystrophy and Type 2 Diabetes Muscle Adipose Tissue IR Binding IR Kinase IRS Phosphorylation IRS1 PI-3K Activity Akt Phosphorylation apkc Activity AS160 Phosphorylation GLUT4 Abundance GLUT4 Translocation GSK3 Erk 1/2 Activity Change expressed relative to healthy individuals Type 2 Diabetes Lipodystrophy

31 PTMs of Transcription Factors: Regulation of Gene Expression Transcription Factor Modification Insulin Effect Effect on Activity Phosphorylation FOXO1 SREBP1c PPARγ NF-кB Acetylation O-GluNAcylation Phosphorylation Acetylation SUMOylation Phosphorylation SUMOylation Phosphorylation Acetylation Ubiquitination O-GluNAcylation

32 Summary, Part 2 Insulin is a pleotropic hormone, generating an array of responses in multiple tissues. Insulin signaling is initiated by binding to its receptor and stimulation of IR tyrosine kinase activity. Phosphorylation cascades regulate the assembly and subcellular localization of multi-molecular signaling complexes.

33 Summary, Part 2, cont. Different substrates and signaling complexes mediate insulin signaling to regulation of metabolism and gene expression/mitogenesis. Disruption of phosphorylation cascades, either by phosphorylation at alternative sites or other PTMs, can lead to insulin resistance.

34 Stress-Activated Kinases Kinase Substrates Activators Erk 1/2 (p44/42 Mitogen Activated Protein Kinase) p38 Mitogen Activated Protein Kinase (p38) IкB Kinase (IKK) c-jun NH 2 Terminal Kinase (JNK) C-Jun p53 IKKα Caspase 3/6 SP1 CEBP IkBα IRS-1 c-jun p53 Insulin Thrombin FFA Glucose Inflammatory cytokines Inflammatory cytokines FFA Inflammatory cytokines FFA

35 Phosphorylation of IRS-1 Positive Negative Mixed IR NH 2 Y 613 Y 623 PH PTB PI3K binding S 307 S 312 S 323 S 332 S 616 S 636 S 731 S 794 S 1101 COOH JNK mtor JNK IKKß PKC GSK3 PKC ERK mtor AMPK p70s6k PKC p70s6k

36 Phosphorylation of IRS-1, cont. Kinase Site Effect on Activity Akt S 522, S 629 JNK S 307, S 312 mtor S 307, S 731 IKKß S 312 PKCα S 24 PKCΘ S 312, S 1101 PKCζ S 323 p70s6k S 312 ERK S 636 GSK3 S 322 AMPK S 794

37 PTMs of Transcription Factors: Regulation of Gene Expression Transcription Factor PTM Effect on Activity FOXO1 SREBP1c PPARγ NF-кB Phosphorylation Acetylation Phosphorylation Acetylation SUMOylation Phosphorylation SUMOylation Phosphorylation (IкBα) Acetylation Ubiquitination

38 Summary, Part 3 Kinases activated by FFAs and inflammatory cytokines can phosphorylate IRS-1 on serine residues to impair downstream insulin signaling. Phosphorylation of transcription factors (TFs) can either stimulate or impair their activity, depending on the site modified. Phosphorylation and other PTMs of TFs can influence subcellular localization, protein stability, and/or transcriptional activity. Phosphorylation of IkB leads to its degradation, releasing NFkB to move into the nucleus and stimulate the transcription of proinflammatory genes, including cytokines and chemokines, maintaining a vicious circle.

39 Hyperlipidemia and Inflammation-mediated Insulin Resistance FFA p38 Cytokines DAG LCFACoA Ceramides JNK IKKß IRS IkB Degradation NFkB NFkB Proinflammatory Genes

40 A Downward Spiral Lipodystrophy to Insulin Resistance AT Ectopic Fat Circulating FFA Altered FA Metabolism Activation of Stress Kinases Circulating FFA Inflammatory Cytokines Phosphorylation of IRS-1, etc. Insulin Resistance Insulin Secretion