imidazoline receptors in insulin signaling and metabolic regulation

IMIDAZOLINE RECEPTORS IN INSULIN SIGNALING AND METABOLIC REGULATION by Zheng Sun Submitted in partial fulfillment of the requirements for the Degree of Doctor of Philosophy Thesis Advisor: Paul Ernsberger, Ph.D. Department of Nutrition CASE WESTERN RESERVE UNIVERSITY January 2007 UMI Number: 3226719 3226719 2006 UMI Microform Copyright All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. ProQuest Information and Learning Company 300 North Zeeb Road P.O. Box 1346 Ann Arbor, MI 48106-1346 by ProQuest Information and Learning Company. CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the dissertation of ______________________________________________________ candidate for the Ph.D. degree *. (signed)_______________________________________________ (chair of the committee) ________________________________________________ ________________________________________________ ________________________________________________ ________________________________________________ ________________________________________________ (date) _______________________ *We also certify that written approval has been obtained for any proprietary material contained therein. Zheng Sun Henri Brunengraber Bryan Roth Laura Nagy Jonathan Whittaker Paul Ernsberger 0 9/07/2006 1 Table of Contents Table of Contents 1 List of Figures 4 Acknowledgements 6 Abbreviations Used 7 Abstract 8 Chapter 1. Literature review Imidazoline ligands 10 Imidazoline receptors 11 Identification of imidazoline receptor subtypes 12 Cellular mechanisms of I 1 -imidazoline receptors 14 Molecular identity of I 1 -imidazoline receptor 17 PC12 pheochromocytoma cells as a model system 30 I 1 -imidazoline receptor agonists as therapeutic agents for the insulin resistance syndrome 31 Insulin and Akt (PKB) cell signaling 33 Metabolic Syndrome X 37 SHROB as animal model for Metabolic Syndrome 40 Phenotypic features of the SHROB model 43 Obesity 43 Hypertension 44 Hyperlipidemia 45 Retinal abnormalities 47 2 Glucose metabolism 47 Insulin and insulin signaling 48 Conclusion 50 Summary 50 Chapter 2. Research design Introduction 52 Specific aims 53 Chapter 3. Materials and methods Materials 57 Plasma membrane isolation for binding assays 58 [ 125 I]p-Iodoclonidine radioligand binding assays 58 Cell culture and transfection 59 Cell experiments 60 Akt activation assay in PC12 cells 62 Ruthenium red staining 63 Animals 63 Chronic drug treatment 64 Adipocyte isolation and insulin application 64 Western blot procedure 65 Glucose uptake assay 66 Statistical methods 68 Chapter 4. Manuscripts from the implementation of the research plan Manuscript 1. 69 3 Manuscript 2. 105 Chapter 5. Discussion and significance The significance of the present study on IRAS 133 Antisense and imidazoline binding studies 134 Antisense and I 1 -imidazoline cell signaling study 137 Possibility of IRAS as a subunit of imidazoline receptor 143 IRAS could also act as a scaffolding protein 145 Comparison of adipocyte glucose uptake results to previous studies 147 Impact of insulin concentration 147 SHR as control for SHROB 149 Insulin degradation in the experiments 151 SHROB as an animal model for human syndrome X 152 Akt activation studies 154 Chronic I 1 -R Activation 156 Acute I 1 R Activation 158 Clinical significance 159 Chapter 6. Future Studies 161 Bibliography 164 4 List of Figures Figure 1. Working model of the I 1 R signaling pathway 16 Figure 2. Functional domains illustration of human IRAS protein 21 Figure 3. Sequence alignment of EST106158 and human IRAS 28 Figure 4. Sequence alignment of EST106158 and predicted rat IRAS 29 Figure 5. A simplified illustration of Akt(PKB) cellular function 36 Figure 6. Domain map of the rat IRAS gene and sequence comparison to human and mouse 95 Figure 7. Saturation kinetics of [ 125 I]-p-iodoclonidine binding to I 1 R in PC12 98 Figure 8. Effect of antisense treatment on IRAS protein expression 99 Figure 9. IRAS antisense inhibits I 1 R signaling 101 Figure 10. Effect of antisense treatment on basal activation of ERK1/2 103 Figure 11. Insulin induced ERK activation in PC12 cells 104 Figure 12. Representative Western blot showing phosphorylated and total Akt immunoreactivity 125 Figure 13. Time course of insulin activation of Akt in lean SHR, SHROB and in SHROB treated with moxonidine for 21d in vivo 126 Figure 14. Dose response curves for insulin activation of Akt in lean SHR, SHROB and in SHROB treated with moxonidine for 21d in vivo 127 Figure 15. Basal Akt activation is not affected by phenotype or pharmacotherapy 128 5 Figure 16. Dose-response curve for insulin activation of [ 3 H]-2-deoxy-D-glucose uptake 129 Figure 17. Treatment with moxonidine alone in vitro does not affect Akt activation 130 Figure 18. Representative blot showing the time course of Akt activation by insulin with and without moxonidine pretreatment 131 Figure 19. Effect of in vitro moxonidine treatment on insulin activation of Akt in adipocytes from SHR and SHROB 132 6 Acknowledgements Helpful Lab Colleagues • Paul Ernsberger, Ph.D (Advisor) • Janean Johnson • Anna Saal • Ryan Strachan Collaborators • Laura Nagy, Ph.D • Becky Sebastian • Chung-Ho Chang, Ph.D Thesis Committee • Henri Brunengraber, M.D. • Laura Nagy, Ph.D • Jonathan Whittaker, Ph.D. • Bryan Roth, M.D., Ph.D. • Paul Ernsberger, Ph.D Personal support • Jing Han, Ph.D. Candidate 7 Abbreviations Used 2-DG 2-deoxy-D-glucose α 2 AR alpha-2 adrenergic receptor ERK Extracellular Regulated Kinase I 1 -R I 1 -imidazoline receptor IRAS Imidazoline Receptor Antisera-Selected IRBP Imidazoline Receptor Binding Peptide IRS Insulin Receptor Substrate (4 subtypes) JNK cJun N-Terminal Kinase MEK MAPK Kinase (phosphorylates ERK) NGF Nerve Growth Factor PAK p21-activated Kinase PKB protein kinase B PI3-K phosphoinositol-3-kinase PIX PAK-interacting exchange factor Rac Rho-family protein SHR spontaneously hypertensive rat SHROB spontaneously hypertensive rat obese strain [...]... treatment with I 1imidazoline receptor agonists such as moxonidine significantly normalizes insulin resistance in adipose tissue of SHROB in both insulin cell signaling and glucose metabolism These changes in adipose tissue very likely contribute to the overall insulin sensitizing effect of imidazoline ligands on SHROB 9 CHAPTER 1 LITERATURE REVIEW Imidazoline Ligands The discovery of imidazoline compounds... through I1 -imidazoline receptors( Velliquette and Ernsberger, 2003b;Velliquette and Ernsberger, 2003a) Thus imidazoline agents could be new targets for drug development against insulin resistance and even metabolic syndrome Investigations on the mechanisms of insulin sensitizing effects from these agents would significantly facilitate the applications of them Insulin and Akt (PKB) Cell Signaling Insulin is... The I1 -imidazoline receptor is a novel target of drug development for hypertension and insulin resistance This thesis focused on the molecular basis for I1 -imidazoline binding and cell signaling and the mechanisms linking this signaling protein to regulation glucose metabolism IRAS is a gene candidate for the I1 -imidazoline receptor To investigate the possibility that IRAS is the I 1imidazoline receptor,... getting too high, the pancreas must produce additional insulin as compensation Many people with insulin resistance have high levels of blood glucose (hyperglycemia) and high levels of insulin (hyperinsulinemia) circulating in their blood at the same time If this situation continues, insulin resistance may lead to type 2 diabetes (Liashko and Dreval', 1973;Livingstone and Gould, 1995) The causes of insulin. .. hormone to stimulate the uptake and storage of carbohydrates, fatty acids, and amino acids into glycogen, fat, and protein, respectively Insulin binding induces tyrosine 32 autophosphorylation of the insulin receptor and further catalyzes the tyrosine phosphorylation of insulin receptor substrates (IRS-1 to IRS-4) as well as several other intracellular substrates including GAB-1 and Shc (Holgado-Madruga et... which bind to insulin receptors (Shoelson et al., 1986) By the same principal, antibodies against the imidazoline ligand idazoxan can be used to generate antiidiotype antibodies that bind to imidazoline receptors (Bennai et al., 1996) One theory is that the amino acid sequence forming the binding pocket in the idiotype region of an antibody will have some homology with the amino acids forming the binding... N-terminus): PX = phox homology domain, PI3P = phosphatidyinositol 3-phosphate, LRR = leucine rich repeat The PX domain 21 usually anchors to plasma and endoplasmic membranes through an interaction with PI3P The potential imidazoline binding region and ruthenium red binding region are possibly overlapping within the coiled coil domain The integrin α5 binding site also mediates interactions with rac and. .. adhesion and cellular trafficking The exact function of the LRRS in IRAS is still not clear However, in most cases LRRs are implicated in proteinprotein interactions (Kobe and Kajava, 2001) LRRs occur in a subfamily of Gprotein coupled receptors (Hsu et al., 2000) and in insulin and growth factor receptors (Edwards et al., 2001) These suggest that the LRRs in IRAS could possibly mediate protein-protein interactions... stimulated 8 by insulin in isolated abdominal adipocytes without affecting basal Akt activation level However, acute in vitro moxonidine administration did not yield similar effects, nor did moxonidine affect basal Akt level in adipocytes from either SHROB or SHR These results implicate adipose tissue as a locus of insulin resistance in this model of metabolic syndrome, and impairment of insulin signaling through... and PAK The insulin receptor substrate (IRS) proteins binding site resides at the C-terminal region of IRAS Within proteins that contain them, PX domains usually consist of 100-130 amino acids The main cellular function of PX domains is to mediate lipid-protein interaction with phosphorylated derivatives of phosphoinositides (PIP’s) Most proteins that contain a PX domain are sorting nexins (SNXs) that . I 1 -imidazoline binding and cell signaling and the mechanisms linking this signaling protein to regulation glucose metabolism. IRAS is a gene candidate for the I 1 -imidazoline receptor. To investigate. of SHROB in both insulin cell signaling and glucose metabolism. These changes in adipose tissue very likely contribute to the overall insulin sensitizing effect of imidazoline ligands on SHROB IMIDAZOLINE RECEPTORS IN INSULIN SIGNALING AND METABOLIC REGULATION by Zheng Sun Submitted in partial fulfillment of the requirements for