THE SENSITIVITY OF THE LIVER TO GLUCAGON IS INCREASED DURING INSULIN-INDUCED HYPOGLYCEMIA. Noelia Rivera Gonzalez. Thesis

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1 THE SENSITIVITY OF THE LIVER TO GLUCAGON IS INCREASED DURING INSULIN-INDUCED HYPOGLYCEMIA By Noelia Rivera Gonzalez Thesis Submitted to the Faculty of the Graduate School of Vanderbilt University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE in Molecular Physiology and Biophysics August, 2008 Nashville, Tennessee Approved: Professor Owen McGuinness Professor Mary C. Moore Professor Alan D. Cherrington

2 To Mami, Papi, Bebo, Natalia, Jarvis, family and friends that have contribute throughout my educational and life journey ii

3 ACKNOWLEDGMENTS I am very grateful to each person that has assisted me in the accomplishment of this research project. It could not have been completed without the support, contribution, effort and sacrifice of my colleagues, friends and family. First, I would like to thank my mentor Alan D. Cherrington for trusting in me and truly believing in me as a student and human being. I m very grateful for the guidance, support and knowledge that I have received from him over these years. He never gave up on me and I will always be deeply appreciative for all his encouragement and unconditional support through the development of my professional career and life. I would like to thank my committee members, Drs. Owen McGuinness and Mary C. Moore, for willingly contributing their time, effort and insightful comments to this research project. I would like to thank all the past and present members of the Cherrington laboratory for their technical, intellectual and personal support during these years. I m very grateful to Doss Neal, Ben Farmer, Tiffany Farmer and Phil Williams for their surgical and technical expertise. Without their help, I could not have performed all these experiments. THANK YOU!!! Dale Edgerton and Genie Moore, thank you for always be willing to answer questions and for all of the unconditional help regarding science and life. Chris Ramnanan, thanks for all your help with the liver tissues and for always been there to listen and helping me stay positive. I would also like to thank to all the other members of the laboratory for making this experience an amazing process of learning and growing as a better scientist and a human being. Patsy, thanks so much for been my iii

4 second mom. You are an amazing woman and thank you for all your funny and inspiring words and more importantly your advice. In addition, I would like to thank to all the members of the Animal Resources Core and Hormone Core Laboratory for all of their effort and work for this project. I would like to thank Maria, Trenis, Nora, Veronica and Jarvis for being there unconditionally. Especially I would like to thank Jarvis, for all of his support and for truly believing in me. I am truly blessed to have you by my side. Lastly, I would like to thank MY FAMILY. I am who I am today because of them. Ever since I was a child, they always taught me the importance of a good education. No matter which path in life I have taken, they always supported me, stood by me and have believed in me. GRACIAS!! Los quiero con toda mi alma!. Finally, thank you to without whom none of this would have been possible, God, who leads me, guides me and walks besides me. Financially, this research was supported by the National Institutes of Health (NIH) grants: R37 DK18243, R37 DK18243S and P60 DK iv

5 TABLE OF CONTENTS Page DEDICATION...ii ACKNOWLEDGMENTS... iii LIST OF TABLES...vii LIST OF FIGURES...viii Chapter I. INTRODUCTION...1 Counterregulatory response to hypoglycemia...1 Defense against hypoglycemia...3 Hormone Action...4 Autoregulation and other factors...6 Glucagon action and signaling...9 Insulin action and signaling...16 Insulin and glucagon interaction...23 II. MATERIALS AND METHODS...25 Animal Care...25 Surgical Procedures...25 Experimental Procedure...28 Experimental Design...29 Collection and Processing of Samples...31 Sample Analysis...33 Plasma Glucose...33 Plasma [3-3 H] glucose...33 Plasma Free Fatty acids...35 Metabolites...36 Lactate...37 Alanine hydroxybutyrate...37 Glycerol...38 Hormones...38 Insulin...38 Glucagon...39 v

6 Cortisol...41 Catecholamines...41 Pancreatic Polypeptide...43 Blood Flow...43 Tissue Analysis...47 Calculations...50 Net Hepatic Substrate Balance and Fractional Extraction...50 Glucose Turnover...52 Hepatic Gluconeogenesis and Glycogenolysis...56 Statistical Analysis...59 III. THE SENSITIVITY OF THE LIVER TO GLUCAGON IS INCREASED DURING INSULIN-INDUCED HYPOGLYCEMIA...60 Aim...60 Results...60 Discussion...78 Summary and Conclusions...86 REFERENCES...90 vi

7 LIST OF TABLES Table Page 3.1 Pancreatic Polypeptide during control (-40 to 0 min) and experimental periods (0-180 min) of studies conducted on 18h fasted conscious dogs exposed to a controlled rise in glucagon in the presence of euglycemia and hypoglycemia Tracer determined glucose production, utilization and glucose clearance during control (-40 to 0 min) and experimental periods (0-180 min) of studies conducted on 18h fasted conscious dogs exposed to a controlled rise in glucagon in the presence of euglycemia and hypoglycemia Lactate and alanine blood levels and net hepatic balance during control (-40 to 0 min) and experimental periods (0-180 min) of studies conducted on 18h fasted conscious dogs exposed to a controlled rise in glucagon in the presence of euglycemia and hypoglycemia Glycerol and BOHB blood levels and net hepatic balance during control (-40 to 0 min) and experimental periods (0-180 min) of studies conducted on 18h fasted conscious dogs exposed to a controlled rise in glucagon in the presence of euglycemia and hypoglycemia Arterial plasma free fatty acids and net hepatic FFA balance during control (-40 to 0 min) and experimental periods (0-180 min) of studies conducted on 18h fasted conscious dogs exposed to a controlled rise in glucagon in the presence of euglycemia and hypoglycemia...77 vii

8 LIST OF FIGURES Figure Page 1.1 Glucagon receptor-signaling pathway Insulin receptor signaling pathway Experimental Design Arterial plasma insulin and glucagon during basal (-40 to 0 min) and experimental periods (0 to 180 min) in 18h fasted conscious dogs exposed to a controlled rise of glucagon in the presence or euglycemia and hypoglycemia Arterial plasma cortisol, epinephrine and norepinephrine during basal (-40 to 0 min) and experimental periods (0 to 180 min) in 18h fasted conscious dogs exposed to a controlled rise of glucagon in the presence or euglycemia and hypoglycemia Arterial plasma glucose and glucose infusion rate between 60 to 180 caused by glucagon during basal (-40 to 0 min) and experimental periods (0 to 180 min) in 18h fasted conscious dogs exposed to a controlled rise of glucagon in the presence or euglycemia and hypoglycemia Net Hepatic Glucose Balance and the Delta AUC: for the increase in NHGO between 60 to 180 caused by glucagon during basal (-40 to 0 min) and experimental periods (0 to 180 min) in 18h fasted conscious dogs exposed to a controlled rise of glucagon in the presence or euglycemia and hypoglycemia Net Hepatic gluconeogenic and glycogenolytic flux during basal (-40 to 0 min) and experimental periods (0 to 180 min) in 18h fasted conscious dogs exposed to a controlled rise of glucagon in the presence or euglycemia and hypoglycemia (A) Phosphorylation of Akt (Ser 473), GSK3- (Ser 9), FOXO1 (Ser 256), CREB(Ser 133) and PGC-1 and (B) relative gene expression of PEPCK and G-6-Pase of liver samples taken from 18h fasted conscious dogs exposed to a controlled rise of glucagon in the presence of euglycemia and hypoglycemia...72 viii