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Sorting Nexin 5 and Dopamine 1 Receptor Regulate Insulin Signaling and Metablism in Renal Proximal Tubule Cells

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SORTING NEXIN 5 AND DOPAMINE 1 RECEPTOR REGULATE INSULIN SIGNALING AND METABLISM IN RENAL PROXIMAL TUBULE CELLS A Dissertation submitted to the Faculty of the Graduate School of Arts and Sciences of Georgetown University in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Physiology and Biophysics By Fengmin Li, MS Washington, DC May 2, 2011 Copyright 2011 by Fengmin Li All Rights Reserved ii SORTING NEXIN 5 AND DOPAMINE 1 RECEPTOR REGULATE INSULIN SIGNALING AND METABLISM IN RENAL PROXIMAL TUBULE CELLS Fengmin Li , MS Thesis Advisor: Pedro A. Jose, MD, PhD ABSTRACT The mechanisms that regulate the renal degradation of insulin are unknown. This dissertation research tests the overall hypothesis that in the kidney, sorting nexin 5 (SNX5) and dopamine D1 receptor (D1R) regulate insulin receptor (IR) expression and insulin signaling pathway, as well as the insulin degrading enzyme (IDE). Two parallel objectives of this dissertation research are: 1) to investigate how SNX5 and D1R regulate the IR and insulin signaling pathway; and 2) to investigate how SNX5 and D1R regulate IDE and insulin degradation. SNX5 is one of the members of the sorting nexin family, which is composed of a diverse group of proteins that mediate trafficking of various receptors. The D1R is defective in individuals with essential hypertension and diabetes. In this work, we determined the roles of D1R and SNX5 in the regulation of the IR, IDE and insulin signaling pathway in human renal proximal tubule cells (hRPTCs). We studied the expression of these proteins and their interaction in hRPTCs, by laser scanning confocal microscopy and immunoprecipitation. Insulin promoted the co-localization of SNX5 and IRβ in the perinuclear area. SNX5 and IRβ did not co- immunoprecipitate in the basal state or after treatment with insulin. In contrast, D1R and IRβ co- localized and co-immunoprecipitated, and were increased by insulin. Moreover, IDE co- immunoprecipitated with both SNX5 and D1R in the basal state and was also increased by iii insulin. To silence stably the endogenous SNX5 and D1R expression, shRNA was transfected into hRPTCs. SNX5 or D1R depletion decreased IRβ and IDE expression, as well as IDE activity. SNX5 depletion also decreased the phosphorylation of IRS1 and Akt. In conclusion, SNX5 needed to interact with D1R to regulate IRβ expression, and the downregulation of SNX5 or D1R impaired the insulin signaling pathway. Moreover, both SNX5 and D1R were involved in the degradation of insulin by increasing IDE expression and activity. This work may help explain why up to 65% of individuals with diabetes also suffer from hypertension and 25–47% of individuals with hypertension have insulin resistance or impaired glucose tolerance. iv ACKNOWLEDGMENTS • Pedro A. Jose, MD, PhD, my research mentor and thesis advisor for inspiring this work and for his generous, brilliant, and dedicated commitment to my education. • Susan E. Mulroney, PhD, for her faith, support, encouragement, and critical help with preparation of the dissertation and document. • Ines Armando, PhD, for painstakingly reviewing the drafts of figures, manuscript and dissertation, for teaching me how to write, and her wholehearted help. • Kathryn Sandberg, PhD, for her willing hands-on assistance in developing my presentation skills and revising my research statement and for her true and valued friendship and advices. • Peter M. Andrews, PhD, for his support and advice on the kidney tissue images. • Van Anthony Villar, MD, PhD, and John E. Jones, PhD, for their enthusiastic support of the project, hands-on assistance with some experiments and for painstakingly reviewing the drafts. • Hewang Li, MD, PhD, for helping and teaching me how to do FRET microscopy and co- localization analyses. • Julie Jurgen, BS, for correcting my pronunciation, helping me practice my presentation, and revising my writing. • Yu Yang, MD, PhD, for teaching me how to do immunohistochemistry in kidney tissues. • Peiying Yu, MD, PhD, for helping and teaching me some basic cell biological techniques. • Xiaoyan Wang, MD, PhD, for sharing some antibodies and her expertise in protein expression studies. v • Laureano Asico, DVM, PhD, and Laura B Teitelbaum, BS, for helping me with all the documents and ordering of all the supplies. • Crisanto Escano, DVM, PhD, for helping me with the animal experiments. • All members of Dr. Jose’s laboratory at Children’s National Medical Center for their willing assistance and camaraderie. • And finally, I owe a special “thank you” to my husband and son, Bingchen Du and Allen Yu Du, my parents, and my sisters for their patient love and understanding during this challenging endeavor. vi The research and writing of this thesis are dedicated to everyone who helped along the way. Many thanks! vii TABLE OF CONTENTS Introduction ...................................................................................................................... 1 Hypertension ..................................................................................................................... 1 Diabetes mellitus .............................................................................................................. 2 Hypertension and diabetes ............................................................................................... 4 Metabolic syndrome......................................................................................................... 4 CHAPTER I Sorting nexin family ................................................................................... 6 1.1 Sorting nexin classification ........................................................................................ 6 1.2 Functions of SNXs .................................................................................................... 7 1.3 Retromer .................................................................................................................... 8 1.4 Sorting nexin 5 ........................................................................................................... 9 CHAPTER II Dopamine and dopamine receptor .......................................................... 11 2.1 Dopamine ................................................................................................................. 11 2.2 Renal dopamine D1 receptors ................................................................................... 12 2.3 Intracellular signaling .............................................................................................. 14 CHAPTER III Insulin receptor and insulin metabolism ................................................ 16 3.1 Insulin receptor ........................................................................................................ 16 3.1.1 Insulin receptor (IR) synthesis .............................................................................. 16 3.1.2 Insulin signaling pathways .................................................................................... 16 3.1.3 Insulin receptor substrate (IRS) ............................................................................ 19 3.1.4 Insulin receptor (IR) crossover and degradation ................................................... 20 viii 3.2 Insulin metabolism ................................................................................................... 20 3.2.1 Insulin ................................................................................................................... 20 3.2.2 Insulin degradation................................................................................................ 21 3.2.3 Insulin degrading enzyme (IDE) .......................................................................... 23 3.2.4 Cathepsin D (CD) ................................................................................................ 24 3.3 Insulin function in the kidney .................................................................................. 24 CHAPTER IV Hypotheses and specific aims ................................................................ 27 4.1 Hypothesis................................................................................................................ 27 4.2 Specific aim 1 .......................................................................................................... 27 4.3 Specific aim 2 .......................................................................................................... 27 4.4 Significance.............................................................................................................. 28 4.5 Novelty ..................................................................................................................... 29 4.6 Rationale .................................................................................................................. 29 CHAPTER V Materials and methods ............................................................................ 31 5.1 Cell lines .................................................................................................................. 31 5.2 Antibodies ................................................................................................................ 31 5.3 RNA preparation and reverse transcription PCR (RT-PCR) ................................... 31 5.4 Quantitative real-time PCR (qRT-PCR) .................................................................. 32 5.5 SNX5 and D1R shRNA stable transfection .............................................................
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