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 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the dissertation of ______________________________________________________ Zheng Sun candidate for the Ph.D. degree *. Henri Brunengraber (signed)_______________________________________________ (chair of the committee) Bryan Roth ________________________________________________ Laura Nagy ________________________________________________ Jonathan Whittaker ________________________________________________ Paul Ernsberger ________________________________________________ ________________________________________________ (date) _______________________09/07/2006 *We also certify that written approval has been obtained for any proprietary material contained therein. 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 I1-imidazoline receptors 14 Molecular identity of I1-imidazoline receptor 17 PC12 pheochromocytoma cells as a model system 30 I1-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 1 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 [125I]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 2 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 I1-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 I1-R Activation 156 Acute I1R Activation 158 Clinical significance 159 Chapter 6. Future Studies 161 Bibliography 164 3 List of Figures Figure 1. Working model of the I1R 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 95 125 Figure 7. Saturation kinetics of [ I]-p-iodoclonidine binding to I1R in PC12 98 Figure 8. Effect of antisense treatment on IRAS protein expression 99 Figure 9. IRAS antisense inhibits I1R 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 4 Figure 16. Dose-response curve for insulin activation of [3H]-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 5 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 6 Abbreviations Used 2-DG 2-deoxy-D-glucose α2AR alpha-2 adrenergic receptor ERK Extracellular Regulated Kinase I1-R I1-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 7 Abstract 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 I1- imidazoline receptor, antisense oligo-nucleotides directly against the initiation site of IRAS sequence were designed and transfected into PC12 cells. Antisense transfection for 48h reduced specific imidazoline radioligand binding to plasma membrane fractions by about 50%, with parallel drops in IRAS protein expression as detected by Western blot. Furthermore, transfection with antisense caused functional impairment of I1-imidazoline receptor signaling. Imidazoline agonist induced ERK1/2 activation was significantly inhibited with antisense transfection without affecting basal ERK level or ERK activation by growth factors. These findings strongly suggested that IRAS encodes an I1-imidazoline receptor or at least an important subunit of it. The mechanism of insulin sensitizing effect from imidazolines was studied in the SHROB rat, an animal model for human metabolic syndrome. Insulin induced Akt activation was found to be severely impaired in isolated adipocytes from SHROB compared to their lean SHR littermates. In addition, insulin induced glucose uptake in these cells from SHROB were also similarly resistant to stimulation by insulin. Chronic treatment of SHROB with the imidazoline agonist moxonidine partially restored both Akt activation and glucose uptake 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 Akt may contribute to this defect. Chronic oral treatment with I1- imidazoline 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 greatly preceded the concept of imidazoline receptors and can be traced back to Switzerland in 1939 (Hartmann & Isler, 1939). The first two imidazoline drugs were tolazoline, an α-adrenoceptor antagonist possessing vasodilating properties, and naphazoline, an α2- adrenoceptor/I1-imidazoline receptor agonist, still in daily use as an over the counter medication for topical application to relieve nasal congestion. Another milestone in the history of imidazoline drugs was the discovery of clonidine in 1962, initially named St155, which was first developed as a nasal decongestant but serendipitously was found to lower blood pressure, and subsequently became the prototype for centrally acting antihypertensive drugs (Hoefke and Kobinger, 1966;Ernsberger et al., 1987). Clonidine is still in use, mainly in the form of a patch to be worn on the skin for continuous control of blood pressure (Klein et al., 1985). Clonidine is also widely used for certain psychiatric disorders (Hieble et al., 1991), including posttraumatic stress disorder (Harmon and Riggs, 1996), Tourette’s syndrome (Leckman et al., 1991), autism (Jaselskis et al., 1992;Posey and McDougle, 2001), attention deficit disorder (Olfson, 2004) and opiate withdrawal (Agren, 1986). Clonidine may also have cognitive enhancing actions (Jackson and Buccafusco, 1991). Clonidine has also been used