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Feeding State, Vitamin a Status and Atypical Protein Kinase C Modulate the Insulin-Regulated Gene Expression in Rat Hepatocytes

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Feeding State, Vitamin a Status and Atypical Protein Kinase C Modulate the Insulin-Regulated Gene Expression in Rat Hepatocytes University of Tennessee, Knoxville TRACE: Tennessee Research and Creative Exchange Doctoral Dissertations Graduate School 8-2014 Feeding State, Vitamin A Status and Atypical Protein Kinase C Modulate the Insulin-Regulated Gene Expression in Rat Hepatocytes Wei Chen University of Tennessee - Knoxville, [email protected] Follow this and additional works at: https://trace.tennessee.edu/utk_graddiss Part of the Molecular, Genetic, and Biochemical Nutrition Commons Recommended Citation Chen, Wei, "Feeding State, Vitamin A Status and Atypical Protein Kinase C Modulate the Insulin-Regulated Gene Expression in Rat Hepatocytes. " PhD diss., University of Tennessee, 2014. https://trace.tennessee.edu/utk_graddiss/2886 This Dissertation is brought to you for free and open access by the Graduate School at TRACE: Tennessee Research and Creative Exchange. It has been accepted for inclusion in Doctoral Dissertations by an authorized administrator of TRACE: Tennessee Research and Creative Exchange. For more information, please contact [email protected]. To the Graduate Council: I am submitting herewith a dissertation written by Wei Chen entitled "Feeding State, Vitamin A Status and Atypical Protein Kinase C Modulate the Insulin-Regulated Gene Expression in Rat Hepatocytes." I have examined the final electronic copy of this dissertation for form and content and recommend that it be accepted in partial fulfillment of the equirr ements for the degree of Doctor of Philosophy, with a major in Nutrition. Guoxun Chen, Major Professor We have read this dissertation and recommend its acceptance: Jay Whelan, Ling Zhao, Michael Karlstad Accepted for the Council: Carolyn R. Hodges Vice Provost and Dean of the Graduate School (Original signatures are on file with official studentecor r ds.) Feeding State, Vitamin A Status and Atypical Protein Kinase C Modulate the Insulin- Regulated Gene Expression in Rat Hepatocytes A Dissertation Presented for the Doctor of Philosophy Degree The University of Tennessee, Knoxville Wei Chen August 2014 Copyright © 2014 by Wei Chen All rights reserved ! ii To my beloved mother and father C.X. Li and Q.J. Chen To my endearing family and friends X. C., H. W., K. L., P. J. C. L.; Y. W.; H. L.; J. X. L. W.; Y. L. Y. H.; B. H.; M. Y.; H. Z.; X. G.; G. F. Y. D.; M. P.; Y. X.; D. G.; M. N.; J. B.; C. H. ! iii ACKNOWLEDGEMENTS The rewarding experience at the University of Tennessee in the past years has transformed me with a combination of intellectual guidance, hardworking, perseverance, team support, friendship, celebration and reflection. I have gained much more than I initially anticipated through my journey of becoming a person who seeks truth in the science world with a touch of curiosity and skepticism. Through the course of my study at UTK, I am blessed to come across people from all walks of life, to whom I am thankful for your unrelenting encouragements and supports. I wish to extend my gratitude to each and every one of you. Without your support, I could not have achieved what I present in this dissertation. I would like to express special thanks to my major professor, Dr. Guoxun Chen, for his academic guidance over the years. He provided precious research opportunities and taught many technical and academic skills that are indispensible in my development as a scientist. I would like to specifically thank Dr. Jay Whelan, Dr. Ling Zhao, Dr. Michael Karlstad and Dr. Jason Collier for their critical comments and advice regarding my research work. Their encouragements have been invaluable for making my graduate research productive and fruitful. My special thanks also go to my dear colleagues and lab mates over the years – Meredith Howell, Rui Li, Yang Li, Yi Zhao and Pan Hu. I very much enjoyed their company inside and outside the lab. And I am grateful that they were beside me when I needed assistance in my research. I would also like to send a final special thank you message to the Department of Nutrition at the University of Tennessee, and the China Scholarship Council, for all the financial supports that bolstered my study and research. ! iv ABSTRACT Hormonal and nutritional stimuli coordinately regulate the glucose and lipid metabolism in the liver. Dysregulated hormonal balance and nutrient metabolism not only disrupt the energy homeostasis, but also predispose individuals to the development of obesity and its related metabolic diseases. Research presented in this dissertation investigated the effects of a nutritional factor, vitamin A (VA), and a potential component of insulin signaling cascade, atypical protein kinase C (aPKC), on the glucose and lipid metabolic genes transcription in primary hepatocytes from Zucker lean (ZL) and fatty (ZF) rats. To examine VA effects, we put ZL and ZF rats with different VA statuses on various feeding conditions. After the isolation of their primary hepatocytes, we analyzed the expressions of hepatic model genes in response to the treatments of insulin and retinoic acid (RA). We report that the insulin- and RA-regulated glucokinase (Gck), sterol regulatory element-binding protein-1c (Srebp-1c) and cytosolic form of phosphoenolpyruvate carboxykinase (Pck1) expressions are impaired in hepatocytes of ZF rats fed chow or a VA sufficient (VAS) diet ad libitum. The impairment is partially corrected when ZF rats are fed a VA deficient (VAD) diet ad libitum or pair-fed a VAS diet to the intake of their VAD counterparts in non-fasting conditions. Interestingly, in the pair-fed ZL and ZF rats, transient overeating on the last day of pair-feeding regimen changes the expression levels of some VA catabolic genes, and impairs the insulin- and RA-regulated gene expression in hepatocytes. To investigate the roles of aPKC, PKCζ(zeta) and PKCι/λ(iota/lambda), they were overexpressed in primary hepatocytes from ZL rats using recombinant adenoviruses. aPKC overexpression in primary hepatocytes significantly attenuated the insulin- regulated expressions of Gck, Srebp-1c and Pck1. Additionally, aPKC overexpression increased their basal transcript levels. The impairment is associated with reduced protein level of insulin receptor substrate 1, and the phosphorylation of protein kinase B in response to insulin treatment. ! v Taken together, the data demonstrate that feeding state, VA status, and aPKC expression regulate the hepatic glucose and lipid metabolism at the gene expression level, and provide interesting insights into the development of metabolic diseases. ! vi TABLE OF CONTENTS CHAPTER I. LITERATURE REVIEW ON INSULIN SIGNALING PATHWAY, VITAMIN A AND ITS ROLES IN THE REGULATION OF MACRONUTRIENT METABOLISM. ..................................................................................................................1 1. Metabolic diseases and metabolic syndromes .............................................................2 2. Insulin signaling, insulin resistance and atypical protein kinase C .............................3 3. VA, carotenoids and retinoids .....................................................................................7 4. Physical and chemical properties of VA ...................................................................11 5. The absorption, transport and metabolism of VA .....................................................12 6. VA Status assessment and VA dietary recommendation ..........................................19 7. VA, retinoid signaling pathway and gene transcription ............................................24 8. Major physiological functions of VA .......................................................................32 9. VA and metabolism ..................................................................................................38 10. Conclusion ...............................................................................................................47 CHAPTER II. VITAMIN A AND FEEDING STATUSES MODULATE THE INSULIN-REGULATED GENE EXPRESSION IN PRIMARY RAT HEPATOCYTES ..............................................................................................................48 Abstract ..........................................................................................................................49 1. Introduction ................................................................................................................50 2. Materials and methods ...............................................................................................51 3. Results ........................................................................................................................53 4. Discussion ..................................................................................................................78! CHAPTER III. OVEREXPRESSION OF ATYPICAL PROTEIN KINASE Cζ (PKCζ) OR ι/λ (PKCι/λ) ATTENUATES INSULIN-REGULATED GENE EXPRESSION IN PRIMARY RAT HEPATOCYTES .................................................................................82 Abstract ..........................................................................................................................83 1. Introduction ................................................................................................................84 2. Materials and methods ...............................................................................................86 3. Results ........................................................................................................................91 4. Discussion ................................................................................................................109
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