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DAIEDUE DAIEDUE DATEDUE 6/07 p:/ClRC/DateDue.indd-p.1 DIABETES, LEPTIN AND THE STRESS AXIS Kimberly Ann Clark A Dissertation Submitted to Michigan State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Neuroscience Program 2007 Abstract Diabetes, Leptin and the Stress Axis By Kimberly Ann Clark Diabetes Mellitus is a chronic endocrine disease associated with a number of central and neuroendocrine dysfunctions due to lack of insulin, insulin action or both. A myriad of complications arise from the disease including retinopathy, nephropathy, neuropathy and cardiovascular dysfunction. Additionally, diabetes causes many neuroendocrine abnormalities including hyperphagia and polydypsia and hyperactivation of the hypothalamo-pituitary-adrenal (HPA) axis. The mechanisms, by which these neuroendocrine dysfunctions occur, however, are not known. It has been shown that diabetes causes a significant increase in norepinephrine (NE) levels in the paraventricular nucleus (PVN) of the hypothalamus. Moreover, circulating leptin levels decrease significantly with the disease. The goal of the research comprised in this dissertation was to investigate whether this decrease in leptin could be one of the reasons for the central and neuroendocrine changes associated with the disease, including activation of the hypothalamo-pituitary-adrenal (HPA) axis. The findings in this research suggest that leptin administration affects monoamine concentrations in the hypothalamus and serum corticosterone. Specifically, leptin administration was found to decrease NE concentration in the PVN and simultaneously decrease serum corticosterone. The dynamic changes in NE in the PVN due to leptin administration were assessed. It was found that peripheral leptin administration decreases noradrenergic activity in the PVN while simultaneously causing a decrease in HPA axis activity, as evidenced‘by decreased serum corticosterone. This effect was also observed in a subsequent study using a diabetic model, suggesting that leptin may play a modulatory role in the hyperactivation of the HPA axis seen in diabetes. The final experiment described in this dissertation shows that gene transfer using a lentiviral vector encoding leptin (0b) cDNA can ameliorate many of the neuroendocrine dysfunctions seen in diabetes including: hypoleptinemia, hypoinsulinemia, hyperglycemia, hyperphagia, polydipsia and diabetes-induced weight loss. Additionally gene transfer reduced the NE concentration in the PVN as well as serum corticosterone. Together these results suggest that leptin plays a modulatory role in many central and neuroendocrine actions and is likely involved in the neuroendocrine dysfunctions seen in diabetes. Acknowledgements I would like to thank my advisors, Drs. Sheba MohanKumar and Puliyur MohanKumar for the years of education, training, guidance and mentorship that they have provided. I cannot begin to put into words the impact that they have had on me both professionally and personally. Never before have I met two professors as dedicated to their students’ learning and training as them. They were never too busy or too occupied to set aside what they were doing to help. Their love for science, teaching and research are clearly visible in every thing they do, and I cannot begin to thank them for the lessons they have taught me. Thank you! I would also like to thank my other committee members: Drs. Robert Bowker, Peter Cobbett and Antonio Nunez for their time, guidance and suggestions over the years. I truly appreciate it. Lastly, I would like to thank the members of the lab, Madhu Siriveluprabhakar and Andrew Shin. I certainly couldn’t have finished this with out your unending help and support. You are both wonderful and I thank you for all you have done! iv Table of Contents List of Tables ................................................................... x List of Figures .................................................................. xi—xiv List of Abbreviations ......................................................... xv-xvii Chapter 1. Introduction ...................................................... 34 A. Statement of Purpose ................................................. 1-2 B. The Hypothalamo-pituitary-adrenal (HPA) Axis and its Regulation ................................................................... 2-9 1. The HPA Axis ................................................. 2 2. Cell Body Terminal Distribution of CRH Neurons ...... 3 3. Catecholamine Influence on CRH Neurons ................ 4 4. Noradrenergic Cell Groups ................................... 5 5. Adrenergic Cell Groups ...................................... 6 6. HPA Axis Regulation by Other Neurotransmitters ........ 6 C. Leptin and Its Effects on the HPA Axis ........................... 9-11 1. The Obese Gene Product, Leptin ............................ 9 2. Leptin and the HPA Axis ...................................... 10 D. Leptin and Mechanisms of Action ................................. 11-24 1. Leptin Signaling ................................................ ll 2. Leptin Receptors ................................................ 13 3. Leptin Receptors in the Hypothalamus ...................... 15 4. Leptin Receptors in the Brain Stem .......................... 16 5. Peripheral Leptin Receptors ................................... 17 6. Leptin and Energy Balance .................................... l8 7. Leptin Resistance .............................................. l9 8. Leptin and the Catecholamines ............................... 21 9. Leptin and Neuropeptide Y (N PY) ........................... 22 10. Leptin, Insulin and Glucose ................................. 23 E. Changes in Leptin & HPA Activity in Diabetes ................. 24-28 1. Diabetes Mellitus ............................................... 24 2. Insulin Dependent Diabetes Mellitus (IDDM) .............. 24 3. Non-Insulin Dependent Diabetes Mellitus (NIDDM). 25 4. Diabetes and Leptin ............................................ 26 5. Diabetes and the Brain Monoamines ......................... 28 F. Gene Therapy .......................................................... 29-33 1. Basic Physiologic Mechanisms ............................... 29 2. Viral Vectors .................................................... 30 3. Adenoviral Vectors ............................................. 30 4. Adeno-Associated Viral Vectors .............................. 31 5. Herpes Simplex Viral Vectors ................................ 32 6. Retroviral Vectors (Including Lentiviral Vectors) ......... 32 G. Thesis Objective ....................................................... 34 Chapter 2. Materials and Methods .................................... 35-43 Animals ...................................................................... 35 1. Sprague-Dawley Rats .......................................... 35 2. Streptozotocin-treated Rats .................................... 35 vi B. Intracerebroventricular Cannulae and Drug Administration ............................................................ 35 1. Implantation of ICV Cannulae (Lateral Ventricle) ........ 35 2. Administration of Drugs ICV ................................ 36 C. Implantation of Cannulae in the Paraventricular Hypothalamic Nucleus ....................................................................... 36 D. Push-Pull Perfusion and Perfusate Collection .................. 37 E. Jugular Catheterization and Blood Sample Collection........ 38 F. Drugs ..................................................................... 38-39 1. Leptin ............................................................ 38 2. Streptozotocin: Induction of Diabetes ...................... 39 3. Clonidine ......................................................... 39 4. Isoproterenol .................................................... 39 G. Radioimmunoassay ................................................... 39-40 1. RIA: Corticosterone and Leptin .............................. 39 2. Protein Assay ................................................... 40 H. Insulin Enzyme-linked Immunosorbant Assay (ELISA) ...... 40 1. Insulin Assay ................................................... 40 I. High Performance Liquid Chromatography with Electrochemical Detection (HPLC-EC) ............................... 40 J. Brain Microdissection ................................................ 41 K. Plasmid Construction ................................................ 42 L. Production of Leptin Lentiviral Particles ........................ 42 vii M. Statistical Analysis ................................................... 43 Chapter 3. The Effects of Central and Systemic Administration of Leptin on Neurotransmitter Concentrations in Specific Areas of the Hypothalamus ...................................................... 44-63 A. Introduction ................................................... 44 B. Rationale ...................................................... 45-46 C. Experimental Design ........................................ 47 D. Results ......................................................... 48-57 E. Discussion ..................................................... 58-62 F. Summary ...................................................... 63 Chapter 4. Leptin Decreases Serum Corticosterone by Decreasing Norepinephrine Release in the PVN: Reversal by Alpha Adrenergic Agonist ........................................... 64-84 A. Introduction ................................................... 64 B. Rationale ...................................................... 65-66 C. Experimental Design ....................................... 67 D. Results ......................................................... 68-78 E Discussion ..................................................... 79-83 F. Summary ...................................................... 84 Chapter 5. Leptin Suppresses Noradrenergic Activity in the PVN and HPA Axis Activity in Diabetic Rats ........................ 85-118 A. Introduction ................................................... 85 B. Rationale ...................................................... 86-87 viii F. Experimental Design ........................................ 88 Results ......................................................... 89-111 Discussion ..................................................... 112-117 Summary ....................................................... 118 Chapter 6. Neuroendocrine Dysfunction in STZ—induced Diabetes is Ameliorated by Leptin Lentiviral Vector Transfection ................................................................ 119-173 A. Introduction ................................................... 119 B. Rationale ....................................................... 120 C. Experimental Design ......................................... 121-122 D. Results .......................................................... 123-166 E. Discussion ...................................................... 167-172 F. Summary ....................................................... 173 Chapter 7. Summary and Conclusions .................................... 174-177 References ........................................................................ 178-202 ix List of Tables Table # Title Page 3-1 Changes in norepinephrine, dopamine and serotonin ......... 59 in the dorsomedial nucleus (DMD), medial preoptic area (MPA) and median eminence (ME) after leptin administration 6-1 Group distribution for gene transfer study ...................... 124 List of Figures Figure # Title Page 3-1 Changes in serum leptin and corticosterone following ........ 51 Central and peripheral leptin administration 3-2 Changes in monoamine concentrations in the PVN ........... 53 Following central and peripheral leptin administration 3-3 Changes in monoamine concentrations in the AN ............. 55 Following central and peripheral leptin administration 3-4 Changes in monoamine concentrations in the VMH .......... 57 Following central and peripheral leptin administration 4-1 NE release in the PVN following leptin administration ....... 72 4-2 NE release in the PVN following clonidine ..................... 73 Administration 4-3 NE release in the PVN following isoproterenol ................. 74 Administration 4-4 Serum corticosterone in animals treated with low ............. 77 And high dose leptin 4-5 Serum corticosterone in animals treated with .................. 78 Clonidine 4-6 Serum corticosterone in animals treated with .................. 79 Isoproterenol 4-7 Serum corticosterone in animals treated low and ............... 80 High dose leptin, clonidine or isoproterenol 5-1 Change in body weight following induction of ................ 93 Diabetes 5-2 Change in food intake following induction of .................. 95 Diabetes 5-3 Change in water intake following induction of ................ 97 Diabetes 5-4 NE release for all groups .......................................... 101 xi 5-5 5-6A 5-6B 5-6C 5-7 5-8A 5-8B 5-10A 5-lOB 5-11 5-12 5-13 5-14 6-1 6-2 NE release in the PVN ofdiabetic animals treated ............ 102 With leptin Average pre and post-treatment NE release in .................. 103 Non-diabetic controls Average pre and post-treatment NE release in .................. 103 Diabetic controls Average pre and post-treatment NE release in .................. 104 Diabetic animals treated with leptin NE release in the PVN of diabetic animals treated ............. 105 With clonidine Average pre and post-treatment NE release in .................. 106 Diabetic animals treated with clonidine Average pre and post-treatment NE release in .................. 106 Diabetic animals treated with leptin + clonidine NE release in the PW of diabetic animals treated ............. 107 With isoptoterenol Average pre and post-treatment NE release in .................. 108 Diabetic animals treated with isoproterenol Average pre and post-treatment NE release in .................. 108 Diabetic animals treated with leptin + isoproterenol Serum corticosterone in diabetic animals treated .............. 111 With leptin Serum corticosterone in diabetic animals treated ............... 112 With clonidine Serum corticosterone in diabetic animals treated .............. 113 With isoproterenol Serum corticosterone in diabetic animals treated ............... 1 14 With leptin, clonidine or isoproterenol Gene transfer study design ........................................ 125 Pre-treatment daily food intake for gene transfer ............... 128 xii 6-3 6-4 6-5 6-6 6-12 6-13 6-14 6-15 Study Pre-treatment average daily food intake for gene .............. 129 Transfer study. Daily food intake following transfection with .................. 130 Lentiviral vector containing GFP or (Ob) cDNA Average daily food intake following transfection .............. 131 With lentiviral vector containing GF P or (Ob) cDNA Daily food intake following induction of diabetes ............. 132 In animals transfected with lentiviral vector containing Either GFP or (Ob) cDNA Average daily food intake following induction of ............. 133 Diabetes Pre-treatment daily water intake for gene transfer ............. 136 Study Pre-treatment average daily water intake for gene ............. 137 Transfer study Daily water intake following transfection with ................ 138 Lentiviral vector containing GFP or (Ob) cDNA Average daily water intake following transfection ............ 139 With lentiviral vector containing GFP or (Ob) cDNA Daily water intake following induction of diabetes in ........ 140 Animals transfected with lentiviral vector containing Either GF P of (Ob) cDNA Average daily water intake following induction of ............. 141 Diabetes in animals transfected with lentiviral vector Containing either GF P or (Ob) cDNA. Pre-treatment daily % change in body weight .................. 144 For gene transfer study Average pre-treatment daily % body weight ................... 145 Change for gene transfer study xiii 6-16 6-17 6-19 6-20 6-21 6-22 6-23 6-24 6-25 6-26 6-27 Daily % body weight change following transfection .......... 146 With lentiviral vector containing GFP or (Ob) cDNA Average daily % body weight change following ............... 147 Transfection with lentiviral vector containing GFP Or (Ob) cDNA Daily % body weight change following induction of ......... 148 Diabetes in animals transfected with lentiviral vector Containing either GFP or (Ob) cDNA Average daily % body weight change following ............... 149 Induction of diabetes in animals transfected with Lentiviral vector containing either GF P or (Ob) cDNA NE concentration in the PVN of diabetic animals ............. 151 Transfected with lentiviral vector containing either GFP or (Ob) cDNA NE concentration in the DMD of diabetic animals ............ 153 Transfected with lentiviral vector containing either GFP or (Ob) cDNA NE concentration in the LH of diabetic animals ............... 155 Transfected with lentiviral vector containing either GF P or (Ob) cDNA NE concentration in the Hippocampus of diabetic ............. 157 Animals transfected with lentiviral vector containing Either GFP or (Ob) cDNA Serum corticosterone in diabetic animals transfected ......... 159 With lentiviral vector containing either GFP or (Ob) cDNA Blood glucose levels in diabetic animals transfected .......... 161 With lentiviral vector containing either GFP or (Ob) cDNA Serum leptin levels in diabetic animals transfected ............ 163 With lentiviral vector containing either GF P or (Ob) cDNA Insulin levels in diabetic animals transfected with ............. 165 Lentiviral vector containing either GF P or (Ob) cDNA xiv S-HT 5-HIAA a-MSH AAV ACSF ACTH AGRP AN BAT BBB BMI CART Clon CRH DA DM DMD DMEM DOPAC EAA ELISA Epi List of Abbreviations 5-hydroxy tryptamine 5-hydroxyindoleacetic acid a-melanocyte stimulating hormone Adeno-associated viruses Artificial cerebrospinal fluid Adrenocorticotropic hormone Agouti-related protein Arcuate nucleus Brown-adipose tissue Blood-brain barrier Body mass index Cocaine—and amphetamine-regulated transcript Clonidine Corticotrophin releasing hormone Dopamine Diabetes mellitus Dorsomedial nucleus Dulbecco’s modified Eagle’s medium Dihydroxyphenylacetic acid Excitatory amino acids Enzyme-linked immunosorbant assay Epinephrine XV GABA GAD GF P GnRH HEchB HIV-1 HSV-l HPA HPLC-EC i.c.v. IDDM IFG IGT i.c.v. i.p. Iso JAK LC Lep LR-IR MCH ME MPA Gamma-amino butyric acid Glutamic acid decarboxylase Green fluorescence protein Gonadotrophin releasing hormone Human embryonic kidney cells Human immunodeficiency virus type 1 Herpes Simplex Virus-1 Hypothalamo-pituitary-adrenal High performance liquid chromatography with electrochemical detection Intracerebroventricular Insulin-dependent diabetes mellitus Impaired fasting glucose Impaired glucose tolerance Intracerebroventricular Intraperitoneal Isoproterenol Janus kinase Locus coeruleus Leptin Leptin receptor immunoreactive Melanin-concentrating hormone Median Eminence Medial Preoptic Area xvi NE NIDDM NPY PVN POMC RIA STAT STZ VMH VSV-G Norepinephrine Non-insulin-dependent diabetes mellitus Neuropeptide Y Paraventricular hypothalamic nucleus Proopiomelanocortin Radioimmunoassay Signal transducers and activators of transcription Streptozotocin Ventromedial hypothalamus G envelope of vesicular stomatitis virus xvii Chapter 1. General Introduction A. Statement of Purpose Diabetes mellitus is a group of metabolic diseases characterized by high levels of blood glucose resulting from either a defect in insulin production or insulin action, or both (1). There are many secondary dysfunctions that accompany the disease including heart disease, stroke, high blood pressure, blindness, kidney damage and nervous system damage (2, 3). Type I diabetes, or insulin-dependent diabetes mellitus (IDDM) can be classified as either autoimmune/immune-mediated or idiopathic disease with pancreatic beta cell destruction (1). This form of diabetes usually has an early onset and requires multiple daily injections of insulin or a subcutaneous insulin pump for survival and is characterized by several neurological complications including activation of the stress axis, however, the mechanisms underlying this activation are not clear. It has been shown that diabetes causes a significant increase in norepinephrine (NE) levels in the paraventricular nucleus (PVN) of the hypothalamus (4-6), while circulating leptin levels decrease significantly (7-9). This decrease in leptin could be one of the reasons for the central and neuroendocrine changes associated with the disease, which results in the activation of the hypothalamo-pituitary-adrenal (HPA) axis. The overall aim of this dissertation research is to investigate the mechanisms by which the stress axis is elevated in Type I diabetes and to elucidate leptin’s role in controlling the level of norepinephrine in the hypothalamus. The following research uses a mechanistic approach to test the hypothesis that leptin decreases the level of NE in the PVN, thereby inhibiting CRH secretion and the HPA axis in a normal, non- diabetic animal. This research could provide a greater understanding of the neuroendocrine regulation of the HPA axis and the dysfunction that occurs in diabetes; possibly leading to alternative or adjunctive treatment for this life-long debilitating disease. B. The Hypothalamo-pituitary-adrenal (HPA) Axis and Its Regulation The HPA Axis Corticotrophin releasing hormone (CRH) is a 41-amino acid peptide hormone that is produced by a specific neuronal population (10). CRH neurons are found in a wide variety of areas within the central nervous system including the hypothalamus. The paraventricular nucleus (PVN) of the hypothalamus contains a high concentration of CRH cell bodies and these project to the median eminence (11). The PVN also has connections with the brain stem and spinal cord (12). When stimulated, the CRH neurons that project to the median eminence secrete CRH, which in turn causes the release of ACTH from the anterior pituitary. ACTH then, acts on the adrenal cortex to cause an increase in corticosteroids. This pathway is known as the Hypothalamo- pituitary-adrenal (HPA) axis or stress axis. Activation of the stress axis leading to elevated plasma glucocorticoids can serve as a beneficial mechanism in times of acute stress. In addition to serving as a feedback mechanism to suppress further HPA activity, glucocorticoids facilitate glucose and free fatty acid mobilization from the liver and adipocytes, respectively. It has been shown, however, that chronic HPA activation has various deleterious effects including hippocampal neuronal cell death and immune system suppression (13, 14). In fact, glucocorticoids have been found to inhibit the function of virtually all inflammatory cytokines by inhibiting the synthesis of pro-inflammatory substances such as leukotrienes and prostaglandins (15), which mediate numerous immune functions. Cell Body Terminal Distribution ofCRH Neurons Corticotropin-releasing hormone is a widely distributed peptide recognized for its intricate involvement in regulation of the stress response primarily via the HPA axis. It is, however, involved in a number of other endocrine functions including reproduction (16), feeding behavior and energy expenditure (17, 18) and inflammatory responses (Vamvakopoulous, NC 1994). The initiation of the hypothalamo-pituitary-adrenal axis begins when the parvocellular neurosecretory neurons in the PVN secrete CRH into the hypophyseal-portal system ultimately leading to an increase in plasma ACTH and corticosterone. However, CRH neurons project to sites other than the neurohypophysis including extrahypothalamic areas such as the thalamus, amygdala, hippocampus, cerebral cortex, striatum, midbrain, pons, medulla, cerebellum and the PVN itself (19-22). An ultra short positive feedback loop controlling the release of CRH in acute stress has been described (23). Histochemical studies of the PVN CRH projections have found that these fibers terminate on the parvocellular CRH cell bodies within the PVN itself (24). Additionally, Champagne et a1. performed retrograde tracing studies to determine the origin of CRH innervation to the PVN and found CRH-immunoreactive neurons in the perifomical hypothalamic nucleus, the bed nucleus of the stria terminalis, laterodorsal tegmental nucleus, the parabrachial nucleus and the dorsal raphe (25). It has also been found that lipopolysaccharide-induced stress or direct CRH administration to the PVN causes an increase in CRH receptor mRNA in the PVN and this increase was not seen in other brain areas (26). Additionally, the PVN sends CRH projections to the brain stem and spinal cord (12) targeting areas such as the locus ceruleus (27). In addition to the PVN, the locus ceruleus, receives CRH innervation from the central nucleus of the amygdala, Barrington’s nucleus and the brainstem nuclei paragigantocellularis (27). It has been shown that intracerebroventricular or direct C RH administration to the LC causes an increase in LC neuronal firing and NE release at target sites including the PVN (27-29) and that this effect is blocked by the CRH antagonist, alpha-helical CRF (30). These data together, suggest that it is likely that hypothalamic CRH may modulate its own activity under stress situations as well as modulate other neuroendocrine functions. Catecholamine Influence on CRH Neurons Direct adrenergie and noradrenergic input from the brainstem to the parvocellular PVN have been described (31) and is believed to have a stimulatory effect on CRH neurons and thus, the HPA axis (32). The PVN of the hypothalamus has a large number of CRH cell bodies and receives rich noradrenergic innervation from the brain stem. Sawchenko and Swanson performed retrograde tracer- immunoflurescent studies and found three specific brainstem areas provide noradrenergic input to the PVN, the A1 region of the ventral medulla, the A2 region of the dorsal vagal complex and A6 (the locus ceruleus) (33). These NE containing fibers are known to synapse with CRH cell bodies in the hypothalamic paraventricular nucleus (34). Szafarczyk et al., have shown that administration of NE into the PVN stimulates C RH secretion and that pharmacologic destruction of the ventral noradrenergic bundle significantly decreases CRH release (32, 35). Leibowitz et al., have shown that NE injected into the PVN causes a significant dose-dependent increase in circulating corticosterone. Then, in a mapping study, they showed that this NE stimulatory effect was localized with the highest rise in corticosterone levels following NE injection into the PVN compared to other areas in the brain (3 6). It has also been shown that adrenalin (Epi) has stimulatory affects on the HPA axis. Both i.c.v. and i.p. injection of adrenalin causes a dose-dependent increase in serum corticosterone in adult male rats and that this effect is blocked by pretreatment with prazosin, an alpha 1 adrenergie antagonist (37). Thus, it is clear that NE and Epi play a stimulatory role in CRH secretion from the PVN and this ultimately leads to an increase in corticosterone. Noradrenergic Cell Groups The noradrenergic neurons of the brainstem are organized into two columns, one dorsal and one ventral. In the medulla, the ventral column contains the A1 group or nucleus ambiguus. The dorsal column is comprised of the nucleus tractus solitarius and the dorsal motor vagal nucleus or A2 group. Both of these cell groups send ascending projections to the hypothalamus to contribute to endocrine and cardiovascular control (Kandel, 2000). The nucleus tractus solitarius receives visceral input from cranial nerves VII, IX and X and then modulates autonomic reflexes such as vagal motor control of the stomach and heart rate as well as regulation of blood flow to various vascular beds. Additionally, this nucleus is involved in the behavioral response to taste and other visceral stimuli (38). At the level of the pens, the A5 and A7 cell groups contribute to the ventral column and primarily project to the spinal cord to modulate nociception and autonomic reflexes (39). The largest group of noradrenergic neurons is located in the periaqueductal gray matter in the pens, the A6 group, or locus ceruleus. It projects extensively to the brain and spinal cord including areas such as the cerebral cortex, thalamus, hypothalamus and cerebellum. The locus ceruleus is primarily involved in maintaining arousal and the body’s response to new stimuli (40, 41). Adrenergic Cell Groups In the rostral ventrolateral medulla, the C1 adrenergie group, which is located near the nucleus ambiguus, sends ascending adrenergie input to the hypothalamus to regulate endocrine and cardiovascular responses. Additionally, the C1 group sends descending projections to the spinal cord to provide tonic excitatory input to vasomotor neurons (Kandel, 2000). Part of the nucleus of the solitary tract, the C2 adrenergie group, sends ascending projections to the parabrachial nucleus, which may be involved in transmission of gastrointestinal information. The C3 adrenergie group, located in the medulla, along with neurons in the C1 group also provide input to the noradrenergic A6 (locus ceruleus) (42). HPA Axis Regulation by Other Neurotransmitters The HPA axis is modulated by neurotransmitters and neuropeptides other than the brainstem noradrenergic system. There have been many studies investigating both the stimulatory and inhibitory modulators of the stress axis and it is now clear that the regulation of the HPA axis is intricately controlled by a myriad of substances. It has been found that the excitatory amino acid, glutamate, elicits a strong stimulatory response on ACTH and thus corticosterone when infused into the third ventricle of rats (43, 44). This was supported by additional studies showing that N- methyl-D-aspartic acid and kainic acid, excitatory amino acid (EAA) agonists, also produced a stimulatory response on ACTH and corticosterone (44-46). Studies have also shown that CRF-containing neurons that project from the PVN to the median eminence receive serotonergic (5-HT) input originating from the midbrain raphe nuclei (47), and that this input is stimulatory to the hypothalamic CRH neurons (48). Saphier and Feldman have used electrophysiological studies to show that electrical stimulation of the dorsal raphe leads to excitation of CRH-containing neurons in the PVN (49). Additionally, in vitro experiments have shown that S-HT stimulates the release of CRH from isolated rat hypothalami (48). Moreover, inflammatory mediators including cytokines such as tumor necrosis factor-a, interleukin-1 and interleukin-6, activate the HPA axis by stimulating hypothalamic CRH release and thus, ACTH and corticosterone (50-53). The glucocorticoids, then act to suppress these inflammatory mediators by inhibiting further cytokine production (15). Besides excitatory amino acids and inflammatory mediators, acetylcholine has also been found to be stimulatory to the HPA axis. Using an in vitro model, Suda et al., found that acetylcholine (ACh) stimulates CRH release from isolated hypothalami in a concentration-dependent manner and that this effect is blocked by atropine (54). These findings were supported by Calogero et al. who used a rat hypothalamic organ culture system to also show that ACh stimulates CRH in a concentration-dependent manner (55). The substances listed above are stimulatory to the CRH secretion. However, there are several substances that are inhibitory to hypothalamic CRH and the HPA axis. Some of these include GABA, substance P and the proopiomelanocortin (POMC)-derived peptides. There have been studies showing that much of the synaptic input to the PVN originates from local sources (56) and that approximately 50% of the synaptic input to the PVN is GABAergic (57). These arises from cells intrinsic to the hypothalamus (58). Additionally, it has been found that GABA is co- localized in CRH containing neurons in the PVN (59). Electrophysiological studies have shown that both magnocellular and parvocellular neurons in the PVN receive this inhibitory GABAergic input (60). The GABAergic neurons that immediately surround and project to the PVN have been shown to be stress-responsive by showing upregulation of glutamic acid decarboxylase (GAD), the GABA synthesizing enzyme, and c-Fos induction following acute stress (61, 62). Substance P, another transmitter widely distributed throughout the central nervous system, is believed to be inhibitory to the stress axis. Intracerebroventricular administration of substance P causes a decrease in circulating ACTH and corticosterone in rats (63), and it has been found to cause a concentration-dependent inhibition of CIUI secretion from isolated rat hypothalami, an effect that was reversed by a substance P receptor antagonist (64). Additionally, chronic inflammatory stress has been found to increase substance P levels in the PVN, AN and ME in rats, and an i.p. injection of a substance P receptor antagonist caused a significant increase in plasma ACTH and corticosterone (65) supporting the notion that substance P is an inhibitor of CRH secretion and the stress response. POMC-derived peptides are generally known to inhibit stress axis function. The paraventricular hypothalamic CRH neurons project to and innervate proopiomelanocortin (POMC) neurons in the arcuate nucleus as well as neurons in the pain control centers of the brainstem and spinal cord (66). Activation of the stress axis leads to CRH-induced POMC-derived and opioid peptides (67, 68), which are believed to mediate analgesia. These peptides, then inhibit the stress axis by suppressing CRH and NE (69) thereby limiting the stress response. This was further supported by Calogero et a1 (1988), who used an in vitro model to show that B- endorphin and a-melanocyte stimulating hormone (a-MSH) inhibited neurotransmitter-induced CRH release from isolated hypothalami. Together, these data support the notion that the HPA axis is modulated by many neurotransmitters and peptides in addition to the well known brain stem noradrenergic system. It is evident that each modulatory substance plays an intricate role in the regulation and function of the HPA axis. C. Leptin and Its Effects on the HPA Axis The Obese Gene Product, Leptin The obese gene product, leptin, was first isolated in 1994 by Zhang et al.(70). The adipocyte-derived hormone consists of 146 amino acids and is believed to play a role in metabolic homeostasis by serving as a signaling molecule to the central nervous system. It is believed that the primary role of leptin, as a signal of nutritional status, is to provide information regarding the amount of body fat to the hypothalamus, thereby modulating central nervous system functions that regulate food intake and energy expenditure (71). Leptin has been shown to produce a variety of central and neuroendocrine effects including inhibition of food intake (72, 73), increase energy expenditure (74, 75) and inhibition of the stress axis (76, 77). The mechanisms by which leptin produces these effects, however, are not clear. Leptin and the HPA Axis It has been shown that leptin deficiency in rodents is characterized by elevated glucocorticoid levels (78). Additionally, leptin injection decreases the elevated corticosterone in ob/ob mice even before significant body weight reduction occurs, indicating that leptin is able to acutely regulate the HPA axis (78). Mutations resulting in defective leptin signaling including, ob/ob mice, db/db mice and fa/fa rats are characterized by obesity, hyperinsulinemia and hypercorticosteronemia (79, 80). Chronic leptin replacement in ob/ob mice (lacking leptin entirely), but not db/db mice (lacking leptin receptor) corrects the hypercorticosteronemia (81). Moreover, fasting 10 in wild-type animals leads to a surge in plasma ACTH and corticosterone and administration of leptin blunts this effect (82). The mechanisms by which leptin is able to suppress the HPA axis is not known, however, there are strong implications to suggest leptin exerts its effects at the level of the hypothalamus via inhibition of CRH release. Support for this comes from the fact that leptin receptors have been identified in nuclei that are rich in CRH neurons such as the PVN and the nucleus tractus solitarius (83). However, this inhibitory effects of leptin appear to be specific to the CRH neurons associated with the stress axis and not to other CRH neurons. It has been shown that leptin treatment causes down-regulation of CRH expression in the PVN but not in the amygdala nor bed nucleus of the stria terminalis (84). This suggests leptin’s actions are specific to CRH neurons regulating the HPA axis. Moreover, in transgenic ob/ob mice, which are deficient in leptin, there is activation of the HPA axis. Chronic treatment with leptin suppresses HPA axis activation in these animals that is reflected by decreased CRH mRNA levels and serum corticosterone (77). Restraint stress also causes activation of the HPA axis and is ameliorated with the administration of exogenous leptin. In addition, it has been shown that glucose deprivation causes an increase in hypothalamic CRH release in in vitro studies. The administration of leptin blocks this effect suggesting that leptin suppresses stress-induced activation of CRH neurons. It has also been shown that leptin does not directly inhibit ACTH release from rat pituitary cells (76). This supports the hypothesis that leptin is able to suppress the HPA axis at the level of the hypothalamus, however, at this time it is not clear how leptin induces these effects. 11 D. Leptin and Mechanisms of Action Leptin Signaling The main receptor that is involved in leptin signaling is OB-R. It is a single transmembrane spanning receptor that belongs to the class I cytokine receptor superfamily (85). Several splice variants of the receptor have been identified and grouped into short and long forms. The OB-Rb has a long cytoplasmic domain and is primarily expressed in the hypothalamus while the short forms are ubiquitous. Binding of leptin to its receptor activates janus kinase (JAK), a class of cytoplasmic tyrosine kinases (86) and leads to phosphorylation of the intracellular domain of the receptor and a transcription factor, signal transducers and activators of transcription (STAT). Phosphorylation of STAT causes dimerization and translocation into the nucleus, thus activation of gene transcription (87). Leptin’s neuroendocrine effects are most likely to be mediated by JAK-STAT signaling via the OB-R in the hypothalamus. Studies have shown that it is the long form of the receptor (OB-Rb) that is capable of activating STAT proteins while the short forms cannot (88, 89). There has been much work done showing the association between changes in leptin levels and/or signaling and diabetes. Plasma leptin levels have been shown to decrease dramatically in untreated streptozotocin-induced diabetic rats (7) and this reduction is reversed with insulin (8). In addition, leptin has been shown to enhance insulin action on peripheral glucose uptake and hepatic glucose production (90). Conversely, dysregulation in leptin synthesis or action results in diabetes in rats and 12 mice. The Zucker (fa/fa) rats that have high serum leptin levels but are deficient in the leptin receptor. Additionally, two mutant mice strains have been extensively studied to better understand leptin and its receptor. The obese (ob/ob) and the diabetes (db/db) mice both result from a single gene mutation on mouse chromosome 6 and 4 respectively (91., 92) and result in extreme obesity. Coleman performed parabiosis studies in the 1970’s to elucidate the mechanisms behind these two mutant mice strains. When ob/ob mice were paired with wild-type controls, their body weight was normalized suggesting that these mutants were lacking the circulating factor regulating feeding and body weight (93). Cross-circulation did not lead to weight reduction in the db/db mice, however, suggesting that these animals were unresponsive to the signal (94). Many years later Zhang et al., used positional cloning to isolate the ob gene and its product, leptin (70). Since that time, it has been shown that the ob/ob mouse is completely unable to produce leptin. This deficiency results in profound obesity, increased food intake, decreased energy expenditure and reproductive dysfunction. Administration of exogenous leptin to these animals reverses these effects (72, 74, 75). The OB receptor is abnormally spliced in db/db mice resulting in dysfunctional leptin signaling and absence of the long form of the receptor (95), these animals are therefore, unresponsive to leptin administration. Leptin Receptors The obese gene product, leptin, serves as a signaling molecule regulating energy homeostasis. Leptin, produced in the periphery, is believed to circulate bound to plasma proteins and cross the blood-brain barrier to reach its receptors in the brain. 13 Leptin binds to the long form of the receptor (Ob-Rb) and activates janus kinase- signal transducer and activator of transcription (JAK-STAT) pathways (89). Once ligand binding occurs, a number of phosphorylation steps take place including autophosphorylation of the associated JAK kinase. This activates JAK proteins, which then phosphorylate specific tyrosine residues on the intracellular domain of the receptor, which then serve as docking sites for the STAT family of transcription factors (86). The JAKs then phosphorylate and activate the STAT proteins, which dimerize and translocate to the nucleus to alter gene transcription. Leptin, the product of the obese gene, is an important signaling molecule for the regulation of several central and neuroendocrine effects. In order to produce these effects, leptin must first bind with its receptors. The adipocyte-derived hormone, which is produced in the periphery, must enter the brain before it can produce its effects. Banks et al. showed that leptin was, in fact, able to cross the blood-brain barrier via a saturable transport mechanism (96). Since then, much work has been done to localize leptin receptors. The first leptin receptor was isolated from mouse choroid plexus by expression cloning (Tartaglia et al., 1995). Tartaglia, however, showed that multiple forms of the leptin receptor had to exist since this receptor was present in db/db mice (85). Several splice variants of the leptin receptor have been identified (97, 98) and can be grouped into short form (OB-Rs) or long form (OB-Rb) based on their intracellular domain. White and Tartaglia have shown that the single membrane-spanning receptor belongs to a family of class I cytokine receptors and all isoforms contain identical extracellular domains. The intracellular domains, however, are of different length and amino acid sequence (97). It is the long form of the 14 receptor that is believed to mediate leptin’s signal, while the exact function of the short isoforms remain to be determined. It has been suggested that the short forms of the receptor act as a transporting mechanism for leptin to enter the central nervous system where it can reach its hypothalamic targets. In fact, Hileman et al., have used a cell model to show that Ob-Ra is preferentially located on the apical cell membrane in canine kidney cells and that leptin transport occurs in a unidirectional manner in the apical to basolateral direction (99). These findings lend support to the hypothesis that the short—form of the leptin receptor serves as a transcellular transporter of leptin from the periphery into the brain. Ghilardi et al. has shown that the short form is expressed ubiquitously with the highest concentration in the lung, uterus, and lymph nodes, however, the long form accounts for only 3-5% OB-R mRNA in these tissues (89). This was not the case in the hypothalamus where the long form comprised 30-40% of the OB-R mRNA suggesting a greater physiologic function in the hypothalamus (89). These findings lend support to the belief that Leptin’s neuroendocrine effects are mediated via hypothalamic OB-Rb. Leptin Receptors in the Hypothalamus In the brain, immunohistochemistry has been employed to localize leptin receptor immunoreactive (LR-IR) cells in the rat. Receptors have been identified in a variety of areas including the choroid plexus, cerebral cortex, hippocampus, thalamus and hypothalamus, with the highest concentration in the hypothalamus (85, 100). Although a number of leptin receptor splice-variants exist, it is the variant with a long 15 cytoplasmic domain is primarily expressed in the hypothalamus (101). Strong LR-IR neurons were present in the paraventricular nucleus (PVN), periventricular nucleus, arcuate nucleus (AN), the ventromedial hypothalamus (VMH) and the dorsomedial hypothalamus (DMD), all areas believed to be involved in body weight regulation (100). Additionally, LR-lR neurons were found in the lateral and medial preoptic nuclei, supraoptic nucleus (SON), suprachiasmatic nucleus and the tuberomammillary nucleus (101). Hakansson, et. al., have shown that in the magnocellular neurons of the SON and PVN, leptin receptor-like imrnunoreactivity was found in both vasopressin and oxytocin-contairring neurons (1998) and in the CRH-containing neurons of the PVN (101). Within the AN, leptin receptor mRNA- expressing neurons have been identified and shown to contain neuropeptide Y mRNA (100, 102, 103) and these NPY neurons have been shown to project to the PVN (104). In addition to the NPY neurons in the AN, POMC-containing neurons in the AN have LR-IR (102) and project to the PVN (105) as well. F unahashi has shown that at the ultrastructural level, LR-IR was found to be concentrated predominantly in perikarya and dendrites of neurons in these areas (106). Since leptin receptors are found in a variety of hypothalamic nuclei and specifically on neurons that contain peptides known to be involved in feeding behavior and energy homeostasis, it is likely that leptin exerts a number of functions by modulating hypothalamic neuronal activity. Leptin Receptors in the Brain Stem The obese gene product, leptin, serves as a signaling molecule regulating energy homeostasis. Leptin, produced in the periphery, is believed to circulate bound to 16 plasma proteins and cross the blood-brain barrier to reach its receptors in the brain. Leptin binds to the long form of the receptor (Ob-Rb) and activates janus kinase- signal transducer and activator of transcription (JAK—STAT) pathways (89). It has recently been shown that in addition to the hypothalamus, the physiologically functional leptin receptor is in the brain stem as well (107). It had previously been shown that that the short-form of the receptor (Ob-Ra) is expressed in the brainstem in addition to many other areas (F ei et al., 1997; (100). Although its exact function is not known, it has been hypothesized that the Ob-Ra facilitates leptin’s transport across the BBB (99) where it may act to regulate autonomic as well as neuroendocrine functions. The mechanisms by which leptin experts its effects are not known, although the hypothalamic catecholarnines are likely candidates. Recently, it has been shown that brainstem catecholaminergic neurons were co-localized with leptin receptor immunoreactivity in noradrenergic Al , A2, A6, A7 and adrenergie C1, C2 and C3 cell groups (108). Peripheral Leptin Receptors In addition to the brain and brain stem, leptin receptors have been identified in a variety of peripheral tissues including skeletal muscle, liver, adipose tissue, pancreatic B-cells (109, 110), ovaries (111), testes (112) and rat and human adrenal cortex (113, 114). Therefore, it is possible that leptin may exert a variety of functions outside of the central nervous system as well. In muscle tissue, leptin has been shown to stimulate lipid oxidation, thus improving insulin sensitivity (110, 115). Additionally, leptin has been shown to directly regulate insulin secretion by 17 pancreatic B-cells (116). Peripheral leptin receptors on the gonadal organs, suggests that, in addition to leptin’s central affects on the hypothalamo-pituitary—gonadal (HPG) system, it likely exerts some of its effects directly. Studies have shown that leptin excess, deficiency and/or resistance may be associated with altered reproductive function (117). It is also possible that leptin has direct actions on glucocorticoid secretion from the adrenal cortex. Whether this action is stimulatory or inhibitory remains controversial. Some in vitro studies have found that leptin stimulates both aldosterone and corticosterone from adrenocortical cells (1 18). While others have shown that in both rhodents and humans, leptin inhibits basal and ACTH- stimulated corticosterone secretion (119). Malendowicz et. al., investigated this further by studying the effect of leptin fragments on adrenocortical cells (2003). They found that some leptin fragments had no effect on either aldosterone or corticosterone, some fragment decreased corticosterone with no effect on aldosterone, and yet other fragments stimulated both (120). Together these findings suggest that leptin may modulate endocrine functions by acting in the periphery in addition to the central nervous system. Leptin and Energy Balance Metabolic homeostasis is achieved via an intricate interaction of a variety of orexigenic and anorexigenic neuropeptides. Among them, the adipocyte-derived hormone leptin, plays a major role. The discovery of the ob gene product has filled in many of the gaps in our understanding of the feedback loop between the periphery and central nervous system controlling body weight homeostasis. It is proposed that 18 leptin not only acts as an “adipostat” signaling body fat mass in the periphery to the brain, but also as a short term sensor of energy balance. Leptin expression has been. shown to directly correlate with body fat mass (121, 122) in addition to changes in nutritional status. Both ob protein in the serum and ob mRNA in adipose tissue are markedly increased in obesity. Serum leptin levels are 5- 10—fold higher in db/db mice and undetectable in ob/ob mice (122). In addition, animals with both of these mutations show uncontrolled hyperphagia and weight gain. Food deprivation, however, leads to a rapid and dramatic decrease in leptin gene expression (122, 123). Normal animals food restricted for 1-3 days leads to a significant decrease in ob mRNA expression and this is reversed with re-feeding (122). Administration of recombinant leptin to rats causes a dramatic decrease in food intake and weight loss (72, 74, 75). Injection of 60 pmol (intraperitoneal) leptin lowered food intake within 30 minutes in both lean and ob/ob mice. Lean and ob/ob mice treated with leptin consumed 40 and 60% less food, respectively, than controls (Anahita et al., 1997). Campfield et al. has shown that a single i.c.v injection of leptin lowers food intake within 30 minutes and that this persists for over 24 hours (72). These acute changes reflect leptin’s ability to signal not only body fat mass but also dynamic changes in energy balance. The mechanism by which leptin produces these effects is not clear, however, many of leptin’s effects on the control of food intake and energy expenditure are believed to be mediated centrally since leptin injected directly into the lateral or third ventricle dramatically reduces food intake independent of body weight loss (72, 75). It has been suggested that neuropeptide Y 19 (NPY) may play a role in mediating leptin’s effects on food intake and metabolic homeostasis as well (124). Leptin Resistance Plasma levels of leptin are highly correlated with adipose tissue mass and decline in both humans and mice after weight loss (125). Plasma leptin levels are increased in several genetically induced rodent models as well as obese humans. Although leptin mRNA and protein levels were found to be significantly elevated in obese rodents, the rise in leptin was not able to suppress feed intake or weight gain (122, 125, 126). Since obesity is characterized by hyperleptinemia and not leptin deficiency, it is suggested that dysregulation in leptin signaling exists in obesity. Diet-induced obesity in humans is associated with increased plasma leptin levels with reduced sensitivity to leptin treatment (127). This may be indicative of some form of leptin resistance similar to what is seen in type II diabetes with respect to insulin. Many type 11 diabetic patients exhibit insulin resistance while secreting elevated insulin levels (128). It has been postulated that defective transport of leptin from the periphery to its targets in the brain may be responsible. Leptin may be produced in large amounts by the adipocytes, but with impaired transport, it may never reach its central target to exert its regulatory effects. Banks et al., has found there to be impaired transport of leptin across the blood-brain barrier (BBB) in obese rodents. They have shown that the transport rate of leptin into the brain is reduced by approximately 2/3 in obese mice and that the reduction in transport rate was not simply due to saturation of transporter but to a decreased capacity of the blood-brain barrier to transport leptin (129). It is also possible that errors in leptin signaling beyond BBB transport exist. Perhaps leptin receptors themselves are decreased in number or are dysfunctional. It may be hypothesized that leptin in circulation is altered as well. Several studies have shown that leptin circulates bound to plasma proteins in rodents and humans, and that these proteins appear to be saturated with obesity (130, 131). To date these binding proteins have not been cloned but it has been speculated that the soluble form of the leptin receptor (Ob-Ra) may serve as a serum leptin binding protein (130). Additionally, downstream leptin targets may be decreased or altered. Bergen et a1. (1999) has shown that levels of hypothalamic neuropeptides differ between diet- induced obese versus diet-resistant mice strains in response to a high fat diet. The orexigenic neuropeptide, NPY, is increased in mice prone to diet-induced obesity, while it is decreased in diet-resistant animals. In addition, POMC mRNA, the precursor to the anorexic neuropeptide a-MSH, significantly increased as body weight increased in obesity-resistant animals but not in diet-induced obese mice (132). It is not clear whether or not differences in these hypothalamic neuropeptides contribute to the altered leptin signaling seen in obesity. Leptin and the C atecholamines Leptin, the adipocyte-derived satiety factor is known to mediate a number of central and neuroendocrine effects including modulation of the stress axis, decrease feeding behavior and increase energy expenditure (133). Many of these actions are 21 mediated by catecholamines. It has been shown in an in vitro model that leptin suppresses NE efflux from isolated hypothalami (134). Barber et al., has found that STZ-induced diabetic rats show a marked increase in NE concentration in the PVN and subcutaneous leptin administration completely normalized this effect. They also showed that diabetic animals had significantly higher NE, DA and 5-1 IT levels in the arcuate nucleus and leptin administration reversed this effect (4). Additionally, a microdialysis study showed that leptin was able to decrease basal as well as feeding- evoked dopamine (DA) release from the arcuate nucleus in rats (135). The adipocyte- derived hormone is known to increase sympathetic nervous activity thus, energy expenditure as well (133). It has been found that central administration of leptin significantly increases plasma catecholamines in rats (136). Satoh et al., showed that a single i.c.v. or i.p. injection of leptin caused a significant increase in plasma NE and Epi concentrations in rats (137). Additionally, leptin has been found to increase NE turnover in thermogenic brown-adipose tissue (BAT) (138). Leptin is involved in many central and neuroendocrine effects and these data suggest that it is likely that the catecholamines may mediate many of these effects. Leptin and Neuropeptide Y flVP Y) Neuropeptide Y (NPY) is a potent stimulator of food intake and inhibitor of thermogenesis (139). It is synthesized in the arcuate nucleus of the hypothalamus and released in the paraventricular nucleus. There, NPY binds to receptors and elicits feeding behavior (140). NPY has been shown to induce a rapid and significant increase in both food intake and body weight while causing an increase in the hypothalamo-pituitary-adrenal axis activity, leading to an increase in plasma 22 corticosterone levels (141). Leptin has the opposite effects. Injection of recombinant leptin either centrally or peripherally has been shown to decrease feed intake and reduce body weight in rodents. An i.c.v. injection of leptin resulted in an almost immediate decrease in feed intake which persisted for over 6 hours following administration (75). Additionally, Campfield has shown that a single iv injection of leptin resulted in a significant reduction in food intake (72). With much research, it has become clear that leptin decreases feeding and body weight, however, the exact mechanisms behind this phenomenon are not entirely clear. One possible factor involved is NPY. The expression of NPY in the hypothalamus is increased in ob/ob mice and in fasted rats (142, 143). It has been shown, however, that leptin administration lowers NPY levels in ob/ob mice before body weight reduction (103). In addition, central administration of leptin to fasted rats causes a significant decrease in NPY mRNA in the arcuate nucleus (103). It has also been shown that leptin administration directly suppresses NPY release from perfused rat hypothalami of normal animals (81). Due to Erickson’s knockout experiments, it has become evident that NPY is not the only mediator of leptin’s actions. Central administration of leptin to these NPY knockout animals resulted in a greater reduction in feed intake and body weight compared to wild-type controls. This suggests that food intake can be regulated by mechanisms other than simply NPY (144). Mice that lack NPY are able to control their food intake and body weight within normal limits, in addition, they have nomral food intake response to administration of leptin (144). In addition to NPY, many other neuropeptides have been shown to mediate leptin’s effects on food intake and energy 23 expenditure. A number of studies have shown that a number of neurons also present in the hypothalamus are targets of brain leptin including POMC (proopimelanocortin) and CART (cocaine-and amphetaminc-regulated transcript) (145-147), MCH (rnelanin-concentrating homrone) and AGRP (agouti-related protein) in the hypothalamus (148, I49). Leptin, Insulin and Glucose It is widely accepted that insulin serves as an important mediator of metabolic homeostasis. In the brain, one of the main targets of action appears to be the hypothalamus where a high concentration of insulin receptors have been identified (150, 151). As one of insulin’s central effects is to reduce feed intake, it is not surprising that a correlation between leptin and insulin exists in the maintenance of energy balance. It has been shown that insulin stimulates leptin gene expression in adipose tissue and plasma leptin concentrations (7, 152, 153). Leptin concentration in the blood exhibits a diurnal pattern in both humans and rodents (154). In humans, there is a nocturnal rise in plasma leptin, which is believed to be due to a delayed effect of previously released insulin (131). In rodents, peak levels of leptin occur at night with the initiation of feeding and this is blocked by fasting (122, 154). Saladin has shown, however, that leptin levels are normalized with feeding or a single injection of insulin (154). Glucose is also likely involved in the regulation of leptin signaling. Havel has shown that leptin messenger RNA increases after glucose administration in mice and that inhibitors of glucose transport and/or metabolism cause a decrease in leptin release from cultured rat adipocytes even in the presence of 24 high concentrations of insulin (155). Together, these studies suggest that indeed leptin, insulin and glucose may interact to regulate feeding behavior. E. Changes in Leptin & HPA Axis in Diabetes Diabetes Mellitus Diabetes is a metabolic disorder that is known to produce a wide variety of dysfunctions in the body. Currently there are 20.8 million people, or 7% of the US. Population living with the disease. Complications include heart disease, stroke, high blood pressure, blindness, kidney disease, nervous system disease, amputations, gum and dental disease and complications of pregnancy (1). The total cost of diabetes in the United States in 2002 was estimated at $132 billion per year (156). Diabetes mellitus is a group of diseases characterized by high levels of blood glucose resulting from either a defect in insulin production, insulin action, or both. There are many secondary dysfunctions that accompany the disease but there are steps that can be taken to lower the risk of these complications (1). Insulin—dependent Diabetes Mellitus (IDDM) Type I diabetes, or insulin-dependent diabetes mellitus (IDDM) is now classified as either an autoimmune disease that develops when the body’s immune system destroys pancreatic beta cells or from a non-immune mediated mechanism (1). The beta cells of the Islets of Langerhom are the only cells in the body that make the hormone insulin, which regulates blood glucose. This form of diabetes usually has an early onset and requires the injection of insulin several times a day or a subcutaneous insulin pump for survival (157). Exogenous insulin is currently the major treatment for IDDM patients. The goal of insulin therapy is to eliminate the clinical symptoms and prevent long term complications associated with the metabolic alterations including repeated hyper/hypoglycernia, neuroendocrine and central nervous system dysfunction, diabetic ketoacidosis and hyperosmolar coma (158). Additionally, therapy aims to ameliorate complications most likely related to diminished vascular supply such as hypertension, retinopathy, nephropathy and peripheral neuropathy (3). Type I diabetes accounts for 5 to 10 percent of all cases of diabetes and often results in life-threatening complications for many people. Non-insulin-dependent Diabetes Mellitus OVIDDM) Type II diabetes, or non-insulin-dependent diabetes mellitus (NIDDM) accounts for 90-95% of diabetes. The number of people living with the disease has dramatically increased worldwide over the past few decades and continues to climb. More than 150 million people worldwide have diabetes and this number is projected to double by 2025 (159). Narayan reports that the typical American born today has a one in three chance of developing type II diabetes during his or her lifetime; and that for Hispanics and African-Americans, the risk is nearly one in two (160). Much of this has been attributed to changes in diet and decreased physical activity. The list of risk factors, currently known as the metabolic syndrome include the following: age greater than 45 years, overweight (Body mass index (BMI) greater than 25 kg/m2), First-degree relative with diabetes, habitual physical inactivity, member of a high-risk ethnic population (e. g. African-American, Latino, Native American, Asian-American, Pacific Islander), previously identified as impaired fasting glucose (IFG) or impaired glucose tolerance (IGT), history of gestational diabetes, hypertensive (>140/90 mmHg), HDL cholesterol <35 mg/dL or triglyceride level >250 mg/dL, polycystic ovary syndrome or a history of vascular disease. Currently approximately 24% of American Adults have the metabolic syndrome and this number increases to 44% over age 60 (161). This form of diabetes usually has a later onset and is associated with obesity, family history, race/ethnicity and physical inactivity. The obesity associated with the disease leads to or exacerbates insulin resistance in these patients. Treatment of NIDDM generally includes: lifestyle change, including diet modification, weight loss and the addition of regular exercise. Currently, pharmacological treatment options include the sulfonylureas, which stimulate endogenous insulin release, the biguanides (oral hypoglycemics), the glitazones, which increase insulin sensitivity, and the alpha-glucosidase inhibitors, which delay sugar hydrolysis and glucose absorption lowering postprandial hyperglycemia. Diabetes and Leptin It has been found that circulating leptin concentrations correlate with adiposity in both humans and rodents (125, 162) and decrease with food restriction or weight reduction (74, 82). The rise in leptin levels with increasing fat mass is thought to act via a negative feedback mechanism to inhibit feed intake and increase energy expenditure (72, 74, 75), thus maintaining normal body weight homeostasis. This feedback mechanism may be dysregulated in diabetes as there is a dramatic decrease 27 in adiposity and an increase in feeding behavior. Plasma leptin concentrations are correlated with plasma insulin levels (163), and it has been shown that insulin stimulates leptin gene expression in adipose tissue (7, 152, 153), and that ob mRNA is upregulated in adipose tissue after insulin infusion in normal rats (123). It has also been shown that a single insulin injection to fasted rats increased serum leptin levels to that of fed controls (154), thus it is likely that decreased insulin levels in diabetes leads to decreased leptin synthesis and/or action. In fact, it has been shown that adipocyte ob gene expression in decreased in insulin-deficient diabetic animals (7, 9), as well as circulating plasma leptin levels (8). Havel et al., showed that streptozotocin-induced diabetic animals showed a dramatic decrease in serum leptin levels within 24 hours, which was reversed with insulin treatment (8). In certain types of diabetes, especially type I, where there is a decrease in insulin levels, there is a loss of adipose tissue and decreased leptin levels (8). However, in other types of diabetes such as type II, where there is loss of insulin sensitivity, there is neither reduction in insulin levels nor decrease in adiposity. These patients remain obese but are also diabetic. In these patients, leptin levels also remain high, however, there is a decrease in leptin sensitivity (164). This could result in hyperphagia and aggravate the existing obesity. In the rat model, streptozotocin-induced diabetes causes a reduction in leptin levels and is associated with hyperphagia, hyperglycemia and rapid weight loss. This is alleviated, to a certain extent, by leptin treatment (4) 28 Diabetes and the Brain rllonoamines Diabetes is characterized by a number of neuroendocrine dysfunctions including metabolic abnormalities, hyperphagia and polydipsia and since hypothalamic monoamines mediate many of these, it is likely that they play a role in the disorder. A variety of studies have been conducted to investigate the possible interaction between the brain monoamines and diabetes. Reports have been published showing alterations in monoamine content in various brain nuclei in experirnentally-induced diabetic rats, including an increase in NE content in the hypothalamus (4, 5, 165) and that this was reversed by parenteral injection of insulin (166). Studies have also shown that along with an increase in NE levels, there is a concomitant increase in alpha-adrenergie receptors in specific brain areas of diabetic (db/db) mice and STZ- induced male rats (167, 168) and an altered binding affinity of alpha-2 adrenergic receptors, but no change in receptor number, in the brain stem of STZ-induced diabetic rats (169). It has also been found that the mRNA for tyrosine hydroxylase, the rate limiting enzyme in catecholamine synthesis, and the norepinephrine transporter mRNA are both upregulated in the LC of diabetic rats (170). These studies suggest that the brain monoamines are likely involved in the central and neuroendocrine dysregulation seen in diabetes. 29 F. Gene Therapy Basic Physiologic Mechanisms Researchers have long been searching for methods to insert genetic material into cells and ultimately organisms with the intent of curing disease. This process, theoretically would be most efficacious in diseases caused by a single gene mutation (171) such as hemophilia, cystic fibrosis or sickle cell disease, whereby gene therapy would replace the nonfunctional gene restoring normal function. Most gene therapy techniques thus far have been developed with the intent of “adding” a functional copy of a gene to a cell that contains a nonfunctional or mutated gene (172). In 1990, history was made when a four-year-old girl with severe combined immunodeficiency (SCID) was the first human to receive gene therapy. Her rare immune disorder, the result of a single gene defect, was a prime candidate for the experimental procedure (173). The disease would almost certainly result in death before adulthood and the only cause was a single enzyme deficiency. Doctors removed the patient’s white blood cells and let them grow in the laboratory. They then treated her defective cells by inserting the enzyme she lacked and re-infused the genetically modified blood back into her body with the hope that they would then produce the enzyme she lacked (174). The treatment was safe, although the effectiveness remains questionable. The cells did produce the missing enzyme, and the patient’s immune function improved. However, the cells did not give rise to fully functional new cells. The white blood cells were found to produce the enzyme for only a few months, the process, then had 30 to be repeated at regular intervals. Although this experiment was not a complete success, it was the impetus for a whole new generation ofclinical gene therapy. Viral Vectors Most gene therapy studies aim to introduce a “normal” gene into a cell to replace an “abnormal” disease-causing gene. This process is usually facilitated by some form of “carrier” or vector. Today, the most commonly employed vectors are viruses. Scientists have tried to capitalize on the modes of transmission used by common viruses to infect human hosts, while eliminating the pathogenicity of the parasite. Genetic modification is used to remove disease-causing genes, while incorporating therapeutic ones. These viral vectors may have a tropism for a particular cell type where they will seek to inject their genetic material containing the therapeutic gene of interest. It is then hoped that a functional protein product will be produced restoring normal function to the animal. Many different viruses have been investigated for therapeutic gene delivery, but as of now, four main types are primarily used. These include: adenoviruses, adeno-associated viruses, herpes simplex virus type 1, and retroviruses, including the lentiviral vectors. Adenoviral Vectors The adenoviruses, known for causing mild respiratory tract infections in humans, are non-enveloped viruses, which consist of a linear double stranded DNA genome. The adenoviral genome is approximately 35 kb and up to 30 kb can be replaced with foreign DNA for gene transfer (175). Adenoviral vectors have a few known 31 advantages including the fact that the life cycle does not normally involve integration into the host genome, which reduces the risk of insertional mutagenesis (175), however, this then means that the therapeutic gene is only transiently active and must be repeatedly administered. They can, however, be produced in high titers, which would allow maximal expression of the gene of interest. Unfortunately, there are also known draw-backs to using adenoviral vectors for gene delivery in humans. Researchers have found that the transgene expression is only temporary in vivo (176) and that approximately 90% of intravenously administered vector is degraded in the liver by non-irnrnune mechanisms (177). An immune response is then elicited and the remaining virus infected cells are likely destroyed (178). It is also important to note that an antibody-mediated response may quickly destroy the viral vector since many people have had previous exposure to this common respiratory pathogen. Adeno-Associated Viral Vectors Adeno-associated viruses (AAV) are non-pathogenic human parvoviruses, which are defective viruses, dependant on a helper virus for replication. The helper virus is usually the adenovirus, thus the name adeno-associated virus. The AAV is a single stranded DNA virus which has been shown to have a few great advantages over other viral vectors. First, they have been found to infect a wide range of cells including both dividing and non-dividing cells (179) additionally, they have been found to elicit little or no immune response (175). A few disadvantages of these viral vectors include the difficulty in manufacturing high titer production (180) and the possibility of causing insertional mutagenesis. Probably the most limiting feature of using AAV b) to as viral vectors is the small genetic carrying capacity. The total length of genetic insert cannot exceed 4.7 kb (175). Herpes Simplex Viral Vectors Herpes Simplex Virus-l (IISV-l) has a tropism for neurons and after infection can either enter a lytic life cycle or proceed to a latent state. Latently infected neurons appear to function normally and do not seem to elicit an immune response (181), however, the lytic cell cycle has been found to cause an immune response and ultimately leads to cell death. The HSV-l genome is a linear double stranded DNA molecule of 152 kb. Deletion of the non-essential genes allows approximately 40-50 kb of foreign DNA to be inserted for therapeutic transfer (182). HSV-l virus is a promising tool for neurologic disorders due to its neurotropism, large gene carrying capacity and latent stage. Its use, however, has been limited due to its ability to invoke a strong immune response (183), cause neurotoxicity (181) and non-neuronal cell infection and cell death (184). Retroviral Vectors (Including Lentiviral Vectors) Retroviruses consist of enveloped single stranded RNA viruses. Upon infection, the viral genome is reverse transcribed into double stranded DNA, which then integrates into the host genome and is expressed as proteins (175). The viral genome is approximately 10 kb and with deletion of non-essential material, can be used to transfer up to a 7.5 kb transgene (185). The envelope of the retrovirus determines which of the host’s cells will be infected. This has proven to be useful in 33 manipulating virus tropism. By replacing the env gene (which codes for the viral envelope protein), with that of another virus, the host cell range can be selected (186), this is known as pseudotyping. The biggest problem with the use of retroviruses for gene transfer is that they require target cell division for viral gene integration and expression. This would necessitate ex I’lI‘O transfection or simply limit in vivo application. Additionally, retroviruses have been found to be inactivated by the immune system (187). One solution to the limited use is a type of retrovirus, the lerrtivirus, including the human immunodeficiency virus type 1 (HIV-1). The biggest advantage of lentiviral vectors is the fact that they have the ability to infect both dividing and nondividing cells. They are able to replicate in non-mitotic cells because their preintegration complex, comprised of the viral genome, structural proteins, and the enzymes needed for reverse transcription and integration, take over the cell nuclear import machinery (188). Another major advance was pseudotyping lentiviral vectors with the envelope of other viruses (186). One of the most commonly used is the G envelope of vesicular stomatitis virus (VSV-G), which has been shown to greatly broaden the range of target tissues. Schlegel et a1. has found that VSV-G interacts with a ubiquitous cellular protein (189) that may be involved in post-binding viral entry (190). Although lentiviral vectors overcome some of the major obstacles in gene therapy, safety issues remain a pronounced concern. Besides the possibility of insertional mutagenesis, recombination to form replication- competent retroviruses (RCR) is of major concern. The use of an envelope of an unrelated virus lowers, but does not eliminate the risk that recombination may 34 originate a new type of virus (191). With resolution of some safety issues, lentiviral vectors could contribute significant progress in the field of gene transfer. 35 G. Thesis Objective Since diabetes is characterized by activation of the HPA axis, decreased leptin levels and increased NE in the PVN, the studies described in the following chapters were designed to test the hypothesis that leptin decreases the level of NE in the PVN, thereby inhibiting CRH secretion and the HPA axis. The experiments were designed to test the following hypothesis: 1) central and/or peripheral administration of leptin causes a decrease in NE levels in the PVN, 2) peripheral administration of leptin causes a decrease in NE release in the PVN while simultaneously decreasing serum corticosterone and treatment with a NE agonist will block this effect, 3) the leptin- mediated normalization of the neuroendocrine changes in diabetes are mediated through hypothalamic catecholamines, 4) leptin gene transfer can normalize the neuroendocrine dysfunctions seen in diabetes, including the HPA axis. A greater understanding of the mechanisms involved in the neuroendocrine dysregulation in diabetes could lead to the development of alternative treatment of the disease. 36 Chapter 2. Materials and Methods A. Animals Sprague-Dawley Rats Adult male Sprague-Dawley rats (3-4 months old) weighing approximately 350 g were obtained from Harlan Sprague-Dawley Inc., Indianapolis, IN. They were housed in light-controlled (light on from 0700-1900 h), air-conditioned (23i2° C) animal quarters and were fed rat chow and water ad libitum. Animals were used in the experiments in accordance with the NIH guide for the care and use of laboratory animals and were approved by the Institutional animal care and use committee. Streptozotocin-treated Rats Adult male Sprague-Dawley rats were treated with an i.p. injection of a 2% solution of Streptozotocin (STZ) (Sigma Chemical Co., MO) in cold 0.1 M citrate buffer, pH 4.5 i.p.). The day following STZ treatment, animals were anesthetized using halothane and blood from the tail vein was used to measure blood glucose levels using a glucometer (Accucheck, Boehringer-Mannheim, Indianapolis, IN, USA). Animals with blood glucose levels of 150 mg/dL or higher were considered diabetic. 37 B. Intracerebroventricular Cannulae and Drug Administration Implantation ofICVCannulae (Lateral Ventricle) Rats were anesthetized with sodium pentobarbital (50 mg/ Kg BW, i.p.). They were implanted with a stainless steel cannula (22-G) in the right lateral ventricle stereotaxically using the following coordinates with reference to Bregma (Paxinos and Watson, 1986): lateral ventricle: 0.8 mm posterior, 1.6 mm lateral, 3.7 mm ventral as described before (192). After securing the cannula with dental cement, a dummy inner cannula made of 30-G stainless steel tubing was used to plug the guide cannula to avoid the blockage due to gliosis. After recovering from the surgery, animals were given at least one week of rest before used in the treatment protocol. Administration of Drugs ICV At the time of experimentation, the dummy inner cannula made of 30 G steel tubing was removed. For infusion, the inner cannula was replaced with a 30 G inner cannula that was connected to a Hamilton syringe with polyethylene tubing which delivered the substance of interest at a low and steady rate. C. Implantation of Cannulae in the Paraventricular Hypothalamic Nucleus Implantation of Cannulae in the PVN Rats were anesthetized with sodium pentobarbital (50 mg/Kg BW, i.p.). They were implanted with a stainless steel guide cannula (22-G) in the PVN stereotaxically 38 using the following coordinates with reference to Bregma (Paxinos and Watson, 1986): PVN: 1.8 mm posterior, 0.2 rrrrn lateral, 8.47 mm ventral as described before (192). After securing the guide cannula with dental cement, an inner cannula made of 30-G stainless steel tubing was used to plug the guide cannula to avoid blockage due to gliosis. After recovering from surgery, the animals were given at least one week of rest before being used in the treatment protocol. D. Push-Pull Perfusion & Perfusate Collection The construction of the push-pull cannula has been described before. Briefly, the inner cannula was made out of the tip of a tuberculin syringe cut at the 0.05 ml mark. Two 29 G steel tubes of unequal lengths were placed in the inner cannula and held in place by epoxy resin. Care was taken to ensure that the longer (push) tube extended 0.5mm beyond the tip of the guide cannula. The push and pull tubes were connected to PE-20 tubings which, in turn, were connected to two channels of a P-3 peristaltic pump (Pharmaeia, Uppsala,Sweden). . The animals were ready for push—pull perfusion one week after stereotaxic surgery. Artificial cerebrospinal fluid (ACSF) containing CaCl2 (0.087 g/L), NaCl (7.188 g/L), KCL (0.358 g/L), MgSO4 (0.296 g/L) and NaHPO4 (1.703 g/L) in water, pH 7.3 was used as the perfusion medium. The PE-20 tubings were filled with the ACSF and the flow rate was adjusted to 10-12 ul/min. Animals were left in the perfusion chambers for an hour before beginning perfusion. The inner cannula attached to the peristaltic pump was inserted into the guide cannula. The perfusion 39 medium was pushed through the push tube and collected from the pull tube. Samples were collected from 1000 h until 1600 h and stored at -70 °C after the addition of 0.5 M HCLO4 at the ratio of 25:1 v/v. Perfusate samples were collected at 30-minute intervals. The perfusates were analyzed for neurotransmitter content using HPLC- EC. The location of the push-pull cannula was verified by examining stained coronal brain sections under a microscope. Only those animals whose cannulas were in the PVN were included in the study. E. Jugular Catheterization and Blood Sample Collection A 0.75” long incision was made on the ventral surface of the neck under halothane anesthesia. The jugular vein was isolated by blunt dissection and punctured using a sterile 20G needle. A catheter made of silastic tubing (Dow Corning, Midland, MI) was inserted into the vein and held in position by two sutures. The free end of the catheter was passed under the skin and extemalized at the base of the skull. The catheter was flushed with lock flush heparin (100 U/ml) and sealed with a piece of blunt steel tubing. During experimentation, blood samples were collected, samples were centrifuged at about 800 x g and plasma was collected and frozen at -20° C until they were analyzed for corticosterone by radioimmunoassay (RIA). The blood cells were re-suspended in heparinized saline and re-introduced into the animal. 40 F. Drugs Leptin Recombinant rat leptin (R & D Systems, Minneapolis, MN, USA) was dissolved in citrate buffer (pH 4.5) and administered i.p. or i.c.v. Streptozotocin: Induction ofDiabetes Diabetes was induced by administration of a 2% solution of streptozotocin (STZ) (Sigma, St. Louis, MO, USA) in cold 0.1M citrate buffer, pH 4.5 at a dose of 65 mg/kg BW given intraperitoneally. Control animals received vehicle (0.1 M citrate buffer i.p.). The following day, all animals were anesthetized using halothane and blood from the tail vein was used to measure blood glucose levels using a glucometer (Accucheck, Boehringer-Mannheim, Indianapolis, IN, USA). Animals with blood glucose levels of 150 mg/dL were considered diabetic. Clonidine Clonidine, an a2-adrenergic agonist, (Sigma, St. Louis, MO, USA) was dissolved in nanopure water and administered intraperitoneally at a dose of 0.3mg/kg BW. Isoproterenol Isoproterenol, a B-adrenergic agonist, (Sigma, St. Louis, MO, USA) was dissolved in nanopure water and administered intraperitoneally at a dose of 0.2 mg/kg BW. 41 G. Radioimmunoassay RIA: C orticosterone and Leptin Commercial RIA kits were used to measure plasma levels of corticosterone (Diagnostic Products Corp. Los Angeles, CA, USA) and leptin (Linco Research, St. Charles, MO, USA). The samples were assayed in duplicates according to the manufacturer’s instructions. The sensitivity of the corticosterone assay was 0.2 ng/ml and that ofleptin was 0.5 ng/ml. Protein Assay A IOul aliquot of the tissue homogenate was used in duplicate in the protein assay. Protein levels were determined using a microplate bicinehoninie acid assay (Pierce, Rockford, IL). Absorbanee at 562 nM was obtained using an ELX 800 microplate reader (Biotek Instruments, Winooski, VT). Neurotransmitter concentrations were expressed as pictogram per microgram protein. H. Insulin Enzyme-linked Immunosorbant Assay (ELISA) Insulin Assay A commercial ELISA kit was used to measure plasma levels of insulin (Linco Research, St. Charles, MO, USA). The samples were assayed in duplicates according to the manufacturer’s instructions. The sensitivity of the assay was 0.2 ng/ml insulin when using a 10 ul sample size. 42 I. High-performance Liquid Chromatography with Electrochemical Detection (HPLC-EC) The HPLC—EC system consisted ofa LC-4C amperometric detector (Bioanalytical Systems, West Lafayette, IN, USA), a phase II, 5 pm ODS reverse phase, C-18 column (Phenomenex, Torrance, CA, USA), a glassy carbon electrode (Bioanalytical Supplies, West Lafayette, IN) a CTO-IO AT/V P column oven, and a LC-lO AT VP pump (Shirnadzu, Columbia, MD, USA). The composition of the mobile phase was as follows: monochloroacetic acid (14.14 g/L), sodium hydroxide (4.675 g/L), octanesulfonic acid disodium salt (0.3 g/L), ethylenediaminetetraacetic acid (0.25 g/L), acetonitrile (3.5%), and tetrahydrofuran (1.4%). The mobile phase was made in pyrogen-free water and then filtered and degassed through a Milli-Q purification system (Millipore, Bedford, MA, USA) and pumped at a flow rate of 1.8 ml/min. The range of the detector is 1 nA full scale, and the potential of the working electrode is 0.65 V. At the time of HPLC analysis, the perfusate samples were thawed at 60°C for one min. A mixture of 50p] of the perfusate and 25 ul of the internal standard (0.45mM dihydroxybenzamine) was injected into the HPLC system. J. Brain Microdissection After treatment, the animals were sacrificed and their brains were removed quickly and frozen on dry ice. Trunk blood was collected and serum was separated and stored at -20 °C until analyzed by RIA. Serial sections (300 um thick) of the 43 brain were obtained using a cryostat maintained at -10 OC. The sections were transferred to cover slips, which were placed on a cold stage set at -10 °C. The nuclei were identified with the help of a stereotaxic atlas and microdissected using the Palkovits’ rnicrodisscction technique. Tissue samples were obtained bilaterally and included all subdivisions of the nuclei and stored at -70 OC. They were analyzed for neurotransmitter concentrations by HPLC. K. Plasmid Construction A 531 bp rat leptin PCR product was amplified using forward (AGACGCGGATCCGCGATGTGCTGGAGACCCCTGT) and reverse primers (AGACGCGTCGACTCAGCATTCAGGGCTAAG) using rat DNA as a template. The restriction enzymes BamH I and Sal I were added to forward and reverse primers, respectively. The plasmid pRleptin.lenti was derived from pCMV- EGFP.lenti by substituting EGF P fragment with rat leptin PCR product. L. Production of Leptin Lentiviral Particles Human embryonic kidney 293T (HEK) cells (3x105) were plated on 100 mm cell culture plates and transfected the following day with 3 plasmids: 10ug of pRleptin.lenti or pGFPlenti (the reporter gene green fluorescent protein, GFP, which produces green fluorescence under ultraviolet light), 3.5 pg of pVSV-G and 6.5 pg of pGag-Pol by calcium phosphate method. Infected 293T cells were maintained in 44 Dulbecco’s modified Eagle’s medium (DMEM) (Gibco Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (Gibco) and 1% antibiotic (10,000 IU/ml penicillin base and 10,000 ug/ml streptomycin base, Gibco) at 37°C with 5% C02. Conditioned medium was harvested 48 hr and 72 hr post transfection, cleared of debris by centrifuging at low speed then filtered through 0.45 pm filters and stored at -80 °C. Concentrated vector stock was prepared by centrifugation at 2000xg for 30 min in ultra centrifugal filter devices (Amicon Inc., Beverly, MA, USA) the flow through was discarded and the concentrate was collected in the filter and stored at -80 °C. HEK cells were transduced with either rat leptin lentiviral particles or GFP lentiviral particles for expression. The transduced media was tested for leptin 0r GFP production in HEK cells. M. Statistical Analysis Results from neurotransmitter release experiments were analyzed using two- way repeated measures ANOVA followed by F isher’s LSD test. Results from hormone assays were analyzed by one-way ANOVA followed by Fisher’s LSD test. Changes in average daily food intake, body weight and water intake were analyzed by repeated measures ANOVA followed by Fisher’s LSD test. 45 Chapter 3. The Effects of Central and Systemic Administration of Leptin on Neurotransmitter Concentrations in Specific Areas of the Hypothalamus A. Introduction Leptin is believed to help maintain metabolic homeostasis by modulating neurotransmitter systems that regulate food intake, energy expenditure and the HPA axis, although the mechanisms by which leptin exerts these central and neuroendocrine effects are not fully understood. There is evidence, however, that leptin modulates various neuropeptides and neurotransmitters, specifically the brain monoamines: NE, DA and 5-HT. Because the hypothalamic monamines are intricately involved in the regulation of these functions, it is hypothesized that leptin may produce its effects by altering the activity of these neurotransmitters. The study designed in this chapter tested this hypothesis by administering peripheral and central leptin to adult male rats then assessed neurotransmitter concentrations in specific hypothalamic nuclei. Additionally, serum corticosterone and leptin levels were measured by RIA. If this hypothesis is correct then: A) Central and systemic administration of leptin will affect hypothalamic monamines in a region-specific manner; and B) this change in monoamine concentration may cause a change in the level of serum corticosterone as well. 46 B. Rationale Leptin plays a critical role in metabolic homeostasis by serving as a signaling molecule to the brain (72). It is believed that the primary role of leptin is to act as a signal of nutritional status to the hypothalamus, thereby modulating neurotransmitter systems that regulate food intake and energy expenditure (71, 72). Additionally, leptin regulates several central and neuroendocrine functions including the reproductive system (193) and the stress axis (98). The mechanisms by which leptin produces these central and neuroendocrine effects are not clear. There is evidence to indicate that leptin modulates various neuropeptides and neurornodulators. Neuropeptide Y (NPY) is one such substance that is involved in the regulation of certain neuroendocrine effects associated with leptin (81, 109, 142). Apart from NPY, other brain neurotransmitters, specifically, the hypothalamic monoamines, norepinephrine (NE), dopamine (DA) and serotonin (5-HT) could play a critical role in many of leptin’s central and neuroendocrine effects. Hypothalamic monoamines are intricately involved in the regulation of feeding behavior, sympathetic outflow and alterations in the HPA and hypothalamo- pituitary-gonadal (HPG) axes as well as other neuroendocrine functions, all of which are also affected by leptin (194). The hypothalamic PVN receives direct monoaminergic input from the brain and brainstem (31) and is believed to have a stimulatory effect on C RH neurons and thus, the HPA axis (32). The PVN of the hypothalamus has a large number of CRH cell bodies and receives rich noradrenergic innervation from the brain stem. Deafferentation of the hypothalamus that severs connection from these nuclei can 47 disrupt the normal functioning of the HPA axis (35) feeding, etc. (195). Taken together, these studies indicate that monoaminergic activity in the hypothalamus is critical for the regulation of the HPA axis. Therefore, it is clear that monoamines could play an important role in mediating many of leptin’s central and neuroendocrine effects. However, the effects of exogenous leptin on hypothalamic monoamines have not been studied previously. The aim of this study therefore was to investigate the effects of peripheral and central administration of leptin on hypothalamic areas that are involved in the central and neuroendocrine effects associated with leptin. This study will help map the changes in hypothalamic monoamines in a number of different nuclei simultaneously, which will be helpful in understanding the complex effects of leptin on the neuroendocrine system. 48 C. Experimental Design To determine if central and/or peripheral administration of leptin affects monoamine concentrations in specific areas of the hypothalamus and serum corticosterone, adult male rats were treated as follows: The animals without cannulae were randomly divided into three groups of 7 rats/group. On the day of the experiment, animals were brought into the laboratory at least two hours before treatment. They were given an i.p. injection of 0 (control), 100 or 500 pg of rat recombinant leptin (n=7; R&D systems, Minneapolis, MN) in 250 pl of saline. Animals with cannula in the lateral ventricle received an i.c.v. infusion (n=4). At the time of treatment, the stylet was replaced with a 30G inner cannula that was connected to a Hamilton syringe. Leptin (5 pg in 5 pl) or the vehicle (saline, 5 ul) was infused slowly into the lateral ventricle over 5 minutes. Animals were sacrificed 5 h later and their brain was removed quickly and frozen using dry ice. Trunk blood was collected and the serum was separated and stored at -20°C until RIA analysis. The paraventricular nucleus (PVN), arcuate nucleus (AN), ventromedial hypothalamus (VMH), dorsomedial nucleus (DMD), median eminence (ME) and medial preoptic area (MPA) tissue samples were obtained using Palkovits’ microdissection technique. Monoamine concentrations in these areas were determined using HPLC-EC. 49 D. Results Serum Leptin Leptin levels (meaniSE; ng/ml) in control animals and animals administered with i.p. or i.c.v. of leptin are shown in Fig. 3-1. In control animals that were treated with the vehicle (i.p.) leptin level was 4.7i1.0. Intraperitoneal administration of 100 pg of leptin increased serum leptin concentration to 8.45zt1.3 (p<0.05). Treatment (i.p.) with 500 pg of leptin increased this further to 30.1i5.8 (p<0.05). In contrast, i.c.v. administration of leptin did not elevate serum leptin levels. Serum C orticosterone Serum corticosterone level (meaniSE; ng/ml; Fig. 3-lB) in control animals that were treated with the vehicle (i.p.) was 1 18.8il 7.1. Treatment with either 100 pg or 500 pg of leptin (i.p.) decreased corticosterone concentrations to 75.9i8.68 and 55.6i12.3 respectively (p<0.01). Similarly, corticosterone levels in animals that were given 5.0 pg ofleptin i.c.v. decreased by 75% (39.4i10.6) compared to the respective control group (125.3i27.6; p<0.05). 50 > Leptin (rig/ml) Corticosterone (nglml) II I IOCIVI 4o 40 30 30 20 20 * 10 M—j.::_.w 10 737mm I V ’ "nausea”... "am-.. .. Control 100 ug Control 5 ug Control 5 ug '/ I 500 ug Fig. 3-1. Changes in serum leptin (A) and corticosterone (B) levels afier i.p. administration of vehicle for leptin, 100 pg or 500 pg of leptin and after i.c.v. administration of vehicle for leptin or 5 pg of leptin. Animals (n=4-8) were sacrificed 5 hours after administration of vehicle or leptin. *p<0.05 compared to respective control groups. 51 Monoamine C oncentrations in the Parm'entriczrlar r\"ucleus (P VN) Changes in monoamine concentrations (meaniSE; pg/pg protein) in the PVN are shown in Fig. 3-2 after i.p. (3-2A) or i.c.v. (3-2B) administration of leptin. NE concentration in the PVN in animals that were given the vehicle (i.p.) was 61 .6i10.2. While administration of low dose of leptin did not have any effect on NE concentration, treatment with 500 pg of leptin decreased it by 50% (30.51240; p<0.05). Similarly, treatment with the higher dose of leptin also decreased DA and 5- HT concentrations significantly in the PVN (p<0.05). Intracerebroventricular administration of leptin decreased the levels of NE from 43.2 i 6.2 in the control group to 22.1 :t 5.3 in the leptin-treated group. Additionally, a decrease in DA levels (3.8 :l: 1.7) and 5-HT (4.4 i 2.3) compared to control groups (22.1 :t 5.3 and 13.1 :t 3.1, respectively; p<0.05). 52 Paraventricular Nucleus A 7////4 Control 100ug - 500ug pglug protein 60‘ 40“ 20‘% pglug protein * T. / A * 7T * E DA 5-HT Fig. 3-2. Changes in monoamine concentrations in the paraventricular nucleus after i.p. (A) administration of vehicle for leptin, 100 pg or 500 pg of leptin and after i.c.v. (B) administration of vehicle for leptin or 5 pg of leptin. Animals (n=4-8) were sacrificed 5 hours after administration of vehicle or leptin. Brains were removed quickly and frozen immediately. Monoamine concentrations were measured as described in the methods section. *p<0.05 compared to respective control groups. 53 Monoamine Concentrations in the Arcuate Nucleus (A N) Monoamine levels (MeaniSE.; pg/pg protein) in the AN are shown in Fig. 3-3 after i.p. (3-3A) or i.c.v. (3-3B) administration of leptin. NE level in control animals was 45i3.2 and decreased significantly with both 100 and 500 pg of leptin treatment (29.2il.9 and 32.54.35 respectively). A similar decrease was observed with i.c.v. administration of leptin (23.5il.4) compared to the control group (44.23238). However, leptin treatment (both i.p. and i.c.v.) did not affect DA or 5-HT levels in the AN. 54 A Arcuate Nucleus Control 1221 100 pg - 500 pg 80‘ 60‘ 40“ pglpg protein 4 NE DA 5-HT Control 5pg 80 - 60 r 40* pglpg protein 20‘ "' as 4- s § Fig 3-3. Changes in monoamine concentrations in the arcuate nucleus after i.p. administration of vehicle for leptin, 100 pg or 500 pg of leptin (A) and after i.c.v. administration of vehicle for leptin or 5 pg of leptin (B). Animals (n=4-8) were sacrificed 5 hours after administration of vehicle or leptin. Brains were removed quickly and frozen immediately. Monoamine concentrations were measured as described in the methods section. *p<0.05 compared to respective control groups. 55 rlvlonoamine Concentration in the lr’entromedial I‘ljpothalamus ( V til/H) Similar to what was observed in the PVN, only the highest dose of leptin (500pg) was effective in suppressing NE levels in the VMH (5.2i0.9 compared to 12.6i1.2 in the control; Fig 3-4A). A similar decrease was observed after i.c.v. administration of leptin (5.4i1.5 compared to 11.1i1.4 in the control; Fig 3-4B). Leptin treatment did not affect DA concentrations. Ilowever, i.c.v. administration of leptin decreased 5-HT levels in this nucleus (5.1:t0.9 compared to 13.5:l:2.1 in the control). 56 A Ventromedial Hypothalamic nucleus W Control 100 pg - 500 pg so - .5 60 ‘ 3 e a. 40 4 3 2 a 20 * o 2% B NE DA 5-HT Control 5pg 80 - .E 60 ‘ 2 2 °- 40 — CI E- 51 °- 20 - * * 0 -Wm NE DA 5-HT Fig.3-4 Changes in monoamine concentrations in the ventromedial hypothalamic nucleus after i.p. administration of vehicle for leptin, 100 pg or 500 pg of leptin (A) and after i.c.v. administration of vehicle for leptin or 5 pg of leptin (B). Animals (n=4-8) were sacrificed 5 hours after administration of vehicle or leptin. Brains were removed quickly and frozen immediately. Monoamine concentrations were measured as described in the methods section. *p<0.05 compared to respective control groups. 57 Monoamine Concentrations in Other Areas of the H)pothalamus Monoamine concentrations in the dorsomedial nucleus (DMD), median eminence (ME) and medial preoptic area (MPA) are shown in table 3-1. Intraperitoneal treatment with leptin did not produce any effect on monoamine concentrations in the DMD. In contrast, i.c.v. administration of leptin decreased NE significantly in the DMD from 40.2:t4.3 in the control group to 27.6:t4.l in the leptin-treated group (p<0.05). Neither i.p nor i.c.v. treatment produced any change in monoamine concentrations in the MPA and ME. 58 Table 3-1. Changes in norepinephrine, dopamine and serotonin in the dorsomedial nucleus (DMD), medial preoptic area (MPA) and median eminence (ME) after leptin administration. Area Treatment NE DA S-HT (pg/rig) (pg/rig) (pg/pg) DMD Control 40.1i4.3 6.3il.8 15.8i4.6 100 pg 45.5i8.1 6.8:12 19.2i2.6 i.p. leptin 500 pg 43.2i3.1 4.9i0.6 12.1:t1.4 leptin i-C-V- Control 40.2i4.3 7.1i2.4 13.7i3.1 5 pg leptin 27.6i4.1* 5.7il.3 15.9i3.8 MPA Control 49.1i9.2 11.1i1 .7 18.5322 100 pg 41.0i9.8 8.0i1.1 10.9i3.8 i.p. leptin 500 pg 40.6i5.4 ll.4i4.0 23617.7 leptin i-C-V- Control 43.4i212.8 12.0:t0.8 12.3i2.0 5 pg leptin 40.5i3.8 14.4i1.2 12.73214 ME Control 45.7i6.3 107.9i25 20.8:I23.6 100 pg 35.4i5.4 53.1110 l8.2i1.6 i.p. leptin 500 pg 41.7i5.8 102.3:l:30 25.9i7.9 leptin i.c.v. Control 46.2i6.3 84.9i6.8 24.1166 5 pg leptin 30713.4 65.7120 15.7i6.8 Changes in norepinephrine (NE), dopamine (DA) and serotonin (5-HT) in the dorsomedial nucleus (DMD), medial preoptic area (MPA) and median eminence (ME) after intraperitoneal (i.p.) administration of vehicle (control), 100 pg or 500 pg of leptin and after intracerebroventricular (i.c.v.) administration of vehicle (control) or 5 pg of leptin. * p<0.05 compared to the respective control group. 59 Illr E. Discussion Leptin plays a significant role in energy homeostasis by acting as a signaling molecule to the brain and by doing so also affects several central and neuroendocrine functions (70, 196). However, the mechanism by which leptin produces many of its effects are not clear. Results described in this chapter indicate that leptin can cause region-specific changes in neurotransmitter levels in the hypothalamus when given i.p. or i.c.v. and also reduces serum corticosterone levels significantly. Leptin-induced changes observed in hypothalamic monoamines could play an important role in bringing about its various neuroendocrine effects. The effect of leptin on hypothalamic monoamines has not been studied in detail. A few published in vitro studies involving leptin's effects on hypothalamic monoamines have had contradictory findings. A recent report by Hastings et al., showed that leptin had no significant effect on hypothalamic NE or S-HT overflow (197). Contrary to these findings, another in vitro study found that leptin inhibits depolarization-induced NE and DA release from rat hypothalamic neuronal endings without modifying basal levels (198). Recent in vitro studies provide evidence that leptin decreases NE efflux from the hypothalamus in a dose-dependant manner and that this decrease may be mediated through GABA (134). It is important to note that the study described in this chapter is the first systemic study as opposed to previous work, which was done in vitro. In the present study the effects of leptin on hypothalamic monoamines were studied simultaneously in multiple nuclei. This may provide information on how leptin affects monoaminergic temrinals differentially to modulate its many central and neuroendocrine effects. 60 The effect of leptin on the [IPA axis has been controversial. Earlier studies indicated that leptin is capable of increasing CRH mRNA levels in the PVN (81). This was attributed to the anorexic actions of CRH and its ability to increase energy expenditure (199). Van Dijk et al demonstrated that i.c.v. administration of leptin potentiates plasma leptin and increases plasma corticosterone at the onset of the dark phase (200). Other studies have reported increases in plasma ACTH and corticosterone in rats after i.c.v. administration of leptin (201). On the contrary, other studies indicate that leptin is capable of decreasing serum corticosterone by acting directly on the adrenal gland (202, 203). It is conceivable that leptin can decrease serum corticosterone because in conditions such as diabetes, where there is a well- documented decrease in leptin levels, there is a concurrent increase in HPA activity (4, 204). This indicates that a reduction in leptin levels causes activation of the HPA axis and vice versa. Additionally, it has been shown that rats with defective leptin receptors have constantly elevated HPA activity (205). Taken together, these studies support the findings presented in this chapter, that leptin treatment can indeed decrease serum corticosterone levels. In addition to the decrease observed in serum corticosterone level in the present study, both i.p. and i.c.v. leptin treatment decreased NE level in the PVN. The PVN has many CRH cell bodies and receives rich noradrenergic innervation from the brain stem (33, 34). Direct administration ofNE into the PVN is known to stimulate CRH synthesis (206). Moreover, lesioning of the ventral noradrenergic bundle that carries the majority of the noradrenergic innervation to the hypothalamus results in a dramatic suppression of the stress axis (207). Thus, it is evident that NE 61 levels in the PVN play a stimulatory role in CRH secretion. Since both i.c.v. and i.p. administration of leptin decreased NE levels in the PVN it is possible that this could be responsible for the decrease in plasma corticosterone seen with leptin treatment. Besides being a crucial part of the stress axis, the PVN is also involved in feeding behavior. NE levels in the PVN are also associated with feeding (208). NE injections into the PVN and treatment with (1-2 adrenergie agonists such as clonidine have suggested that NE levels in the PVN are critical for feeding (18, 209, 210). Since leptin suppresses feeding. the reduction in NE levels observed in the PVN correlate well with this behavior. Besides the PVN, the VMH is also known to be important in feeding behavior (211). NE levels in the VMH are known to stimulate feed intake (212). In the present study, leptin treatment produced a dose-dependent decrease in NE levels in the VMH indicating that this could be another possible target for leptin's actions. While the reduction in NE levels in the PVN and VMH could be related to leptin's effects on feeding and the stress axis, the significance of decreased NE levels in the AN is not clear. It does not correlate with any of the known neuroendocrine functions of the AN such as prolactin or growth hormone secretion. However, the AN contains cell bodies for a number of other neuropeptides such as NPY, GALP (213) and it is possible that these may be influenced by NE. Besides NE, DA and 5-IIT are also important in neuroendocrine functions associated with leptin. In the present study, DA level in the PVN decreased after leptin treatment but was not affected in any other hypothalamic area. DA is believed to be inhibitory to thyrotropin hormone secretion (Mueller and Nistico, 1989). The It In decrease in DA levels observed in the PVN could therefore be related to the stimulatory effect that leptin has on TRH neurons (214). Serotonin on the other hand increases with feeding (213). Moreover, serotonergic neurons that project to the hypothalamus express leptin receptor mRNA (108, 215). Therefore, the significance of the reduction in serotonin observed with leptin treatment in the present study is unclear. The DMII acts a nodal point receiving afferents from the VMH and the lateral hypothalamus and relays information to the PVN. It is rich in NPY and may participate in C RH secretion by the PVN (216). Therefore, the reduction in NE levels observed in the DMII could be related to the decrease in CRH secretion from the PVN. The mechanism by which leptin affects the hypothalamus is still unclear. Leptin has been shown to cross the blood-brain barrier via a saturable transport mechanism (96) and its receptors have been identified in several parts of the brain including the hypothalamus (103, 113). Functional leptin receptors have been found in the brainstem (108, 217) where leptin may act to modulate the neuroendocrine control of food intake and energy expenditure. Hosoi et al. have shown that noradrenergic fibers arising from the brainstem (A1, A2 and A6), are rich in leptin receptors (217) and, it is known that these regions project to the PVN and VMH in the hypothalamus (Mueller and Nistico, 1989). Leptin may therefore elicit its inhibitory actions on the hypothalamic monoamines by acting at the level of the brainstem. Leptin may also act directly at the level of the hypothalamus since leptin receptors are found in abundance in hypothalamic nuclei, particularly the PVN and 63 1161' 311'. VMH (100). Besides monoamines, leptin may affect a wide range of neurotransmitters and neuropeptides such as GABA, histamine and other anorexigenic peptides, the complex interaction of which could mediate many of its central and neuroendocrine effects (134, 199, 218-221). 64 F. Summary The experiment described in this chapter tested the hypothesis that leptin may produce its central and neuroendocrine effects by altering the activity of the brain monoamines. The results from this experiment indicate that central and systemic administration of leptin does, in fact, affect hypothalamic monamines in a region- specific manner and this change in monoamine concentrations may be responsible for the change in serum corticosterone levels as well. 65 Chapter 4. Leptin Decreases Serum Corticosterone by Decreasing NE Release in the PVN: Reversal by Alpha Adrenergic Agonist A. Introduction Leptin, the protein product of the ob gene, is known to regulate a variety of neuroendocrine functions, including the hypothalarno-pituitary-adrenal (HPA) axis activity in several animal models. The exact mechanism by which it does so, however, is not known. The PVN contains a large number ofCRH neurons and NE is known to stimulate these neurons and cause elevated H PA activity. We have shown previously that norepinephrine concentrations decrease in the PVN after both central and peripheral administration of leptin paralleling a decrease in serum corticosterone levels (Chapter 3). To study the exact time at which this occurs after leptin treatment and the duration of mechanism of leptin’s actions, we have designed a push-pull perfusion study with simultaneous blood sample collection. Additionally, we investigated the mechanism by which leptin may mediate its effects by administering alpha or beta adrenergic receptor agonists. 66 B. Rationale It is known that leptin plays a major role in metabolic homeostasis by serving as a signaling molecule to the brain. providing information on the amount of body fat to the hypothalamus. Leptin has been shown to decrease feed intake (72), increase energy expenditure (74, 75), and affect the reproductive axis (222). In addition, leptin has been shown to inhibit the stress axis (223). The effects on feeding behavior and energy balance is thought to be mediated through a variety of anorexigenic and orexigenic peptides in the brain, however, the mechanisms by which leptin affects the stress axis are not clear. Corticotrophin releasing hormone (C RH) neurons are found in a wide variety of areas within the central nervous system including the hypothalamus. The paraventricular nucleus (PVN) of the hypothalamus contains a high concentration of CRH cell bodies, and when stimulated, these neurons secrete CRH, which in turn causes the release of adrenocorticotrophic hormone (ACTH) from the anterior pituitary. This then, acts on the adrenal cortex to cause an increase in corticosteroids. Norepinephrine (NE) is known to play a critical role in stimulating CRH secretion (224). The PVN receives rich noradrenergic innervation from the A2 noradrenergic area of the brain stem and these NE fibers are known to synapse with CRH cell bodies in the PVN (225). Since leptin receptors have been identified in nuclei that are rich in CRH neurons such as the PVN (83), and in the brainstem (217), these are probable sites of leptin’s actions. Since leptin has been shown to decrease NE concentration in the PVN and it has been shown that leptin causes a decrease in serum corticosterone (Chapter 3), it is hypothesized that leptin decreases 67 noradrenergic activity in the PVN thereby, decreasing CRH secretion and the HPA axis. The aim ofthis study was to determine if peripheral administration of leptin is able to decrease noradrenergic activity in the PVN while causing a simultaneous decrease in HPA activity and if so, the exact time course and duration of leptin’s actions. Additionally, we wanted to investigate the mechanism by which leptin may mediate its effects by administering alpha or beta adrenergic receptor agonists. 68 C. Experimental Design The study described in this chapter was designed to investigate the tirne- course by which peripheral administration ofleptin causes a decrease in norepinephrine release in the PVN while simultaneously causing a decrease in serum corticosterone levels. To do this, we implanted adult male Sprague Dawley rats with both a push-pull cannulae in the PVN and a jugular catheter. On the day of the experiment, animals were brought into the lab at least two hours before treatment and were randomly divided into seven groups and treated with the vehicle for leptin (control), 100 or 500 pg of recombinant rat leptin i.p., an alpha adrenergie agonist, clonidine (0.6 mg/kg BW) i.p.. a combination ofclonidine and leptin, a beta adrenergie agonist, isoproterenol (0.2 mg/kg BW) i.p., or a combination of isoproterenol and leptin. Push-pull perfusion was performed for 6 hours. Perfusate samples were collected every 30 min and blood samples were collected every 60 min for the duration of the experiment. Adrenergic agonists were administered 30 min before leptin or saline. Perfusate samples were analyzed for neurotransmitter content using HPLC-EC and plasma was analyzed for corticosterone (CS) by RIA. 69 D. Results NE release in the PVN Profiles of NE release (mean i S.E; pg/min) in the PVN for the different groups are shown in Figures 4-1, 4-2 and 4-3. Pre-treatment NE release was not significantly different between groups. Fig 4-1 shows the NE release (mean t SE; pg/min) for the saline (control), low dose leptin (100 pg i.p.) and high dose leptin (500 pg i.p.) treated groups. The pre-treatment NE release for the control animals was 6.6 i 0.5 and remained at that level for the duration of the experiment. On the other hand low dose leptin caused a significant decrease in NE release from 7.7 i 2 to 2.6 i 0.6. Additionally, high dose leptin produced a significant reduction in NE release from 9.3 i 2 to 2.8 i 0.8 (p < 0.05). Fig 4—2 shows there were no significant differences in NE release (mean 3: SE; pg/min) in the PVN between animals treated with clonidine (0.3 mg/kg BW i.p.) then saline or clonidine then 500pg leptin (i.p). Pre—treatment NE release was 8.2 :1: 0.5 and remained at that level for the duration of the experiment for the saline + clonidine group and was not significantly different from the leptin + clonidine group, which had a pre-treatment NE release of 8.0 i 0.3 and did not change significantly by the end of the experiment. The NE release in the saline and 500pg leptin treated group, however, was significantly lower than the clonidine + saline and the clonidine + leptin treated groups (p<0.05). Fig. 4-3 shows the NE release in the PVN (mean t SE; pg/min) of animals treated with isoproterenol (0.2 rug/kg BW i.p.) followed by saline, isoproterenol 70 followed by 500pg leptin (i.p) or saline followed by 500 pg leptin (i.p). In the saline + isoproterenol group, pre-treatment NE release was 7.8 i 0.7, and remained at that level for the duration ofthe experiment. In the leptin + isoproterenol group, there was a significant difference between pre and post-treatment NE release, however, 9.3 :t 0.7 to 3.6 i 0.4 (p<0.05). In the saline + leptin group, there was a significant difference between pre and post-treatment NE release, 8.7 i 0.6 to 2.8 i 0.8 (p<0.05). Beginning 1.5 hours post-treatment, both the leptin + isoproterenol group and the saline + leptin group had significantly lower NE release compared to the saline + isoproterenol treated group (p<0.05). 71 + Control "'—- 100 ug —‘@"'— 500 ug Lep Lep N E release (pg/min) Fig 4-1. NE release in the PVN in saline (control), low dose leptin (100 pg i.p.) and high dose leptin (500 pg i.p.) treated animals. The pre-treatment NE release for the control animals was 6.6 i: 0.5 and remained at that level for the duration of the experiment. On the other hand, both low and high dose leptin caused a significant decrease in NE release (*p < 0.05) (N=6-8/group). The agonist was given at — 0.5 hrs (solid arrow) and the vehicle for leptin or leptin was given at 0 hrs (dashed arrow). 4?- Saline+ “V“ Leptin+ —9— 500 ug Clonidine Clonidine Leptin 20 — 16 — g, 12 - ' 7: l g T ‘\ 2 H,, \“~ — 2 8 ” ‘\\_/ ‘3“ . T "J v --+-— ——w z a 4 — l T o I 1 I l l l I l l l l l -1 0 1 2 3 4 Time(hts) Fig. 4-2. There were no significant differences in NE release in the PVN between animals treated with clonidine then saline or clonidine then 500pg leptin. However, the NE release in the saline and 500pg leptin group was significantly lower than the clonidine + saline and the clonidine + leptin treated groups (*p<0.05). The agonist was given at —— 0.5 hrs (solid arrow) and the vehicle for leptin or leptin was given at 0 hrs (dashed arrow) (N=4-5/group). 73 —9— Saline+ “*— Leptin+ + 500 ug Isoproterenol Isoproterenol Leptin 20 r 16 r E E, 12 z ‘o’ (D (B 2 2 8 “ Lu 2 l 4 __ 0 | l l l l l l l l -1 0 1 2 3 4 Time (hrs) Fig. 4-3. NE release in the PVN of animals treated with isoproterenol followed by saline, isoproterenol followed by 500pg leptin or saline followed by leptin are shown. There is no change between pre and post—treatment NE release in the saline + isoproterenol group. The leptin + isoproterenol group and the saline + leptin treated groups have significantly lower NE release compared to pretreatment and the saline + isoproterenol treated group beginning 1.5 hours post-treatment (*p < 0.05). The agonist was given at — 0.5 hrs (solid arrow) and the vehicle for leptin or leptin was given at 0 hrs (dashed arrow) (N=4—5/ group). 74 Serum Corticosterone Serum corticosterone levels for all groups for the duration of the experiment are shown in Figures 4-4 through 4-7. Pre-treatment corticosterone levels (mean t SE; ng/ml) were not significantly different among groups. Figure 4-4 shows serum corticosterone levels (mean :t 8.13.; ng/ml) for the control animals (N=10), the low dose leptin treated animals (100 pg i.p.; N=4) and the high dose leptin treated animals (500 pg i.p.; N=l 1). Pre-treatment corticosterone in the control group was 212.6 i 28 and remained at that level for the duration of the experiment. Both high and low dose leptin administration, however, caused a significant reduction in serum corticosterone compared to controls and pretreatment levels. High dose leptin administration caused a significant reduction in serum corticosterone by 3 hours post-treatment from 256.3 i 17.6 to 197 i 20 (p<0.05) and was significantly lower than controls and pre-treatment levels for the remainder of the experiment (p<0.05). By 4 hours post-treatment, low dose leptin administration caused a significant decrease in serum corticosterone (248.8 d: 13 to 155.5 :1: 55.1) compared to pre-treatment levels and controls (p < 0.05). Figure 4-5 shows serum corticosterone levels (mean 1 SE; ng/ml) for the control animals (N=10), high dose leptin (500 pg i.p.; N=1 1), clonidine (0.3mg/kg body weight i.p.; N=6) and clonidine + high dose leptin (N=4) treated animals. Pre- treatment corticosterone levels were not different between groups. Pre-treatment corticosterone in the control group was 212.6 i 28 and remained at that level for the duration of the experiment. High dose leptin administration caused a significant reduction in corticosterone compared to pre-treatment levels (p<0.05) and controls 75 COT ill. E15 311i and pre-treatment levels (p<0.05). Clonidine treated animals had pre-treatment levels of 250.7 d: 30 and did not change significantly during the experiment. Interestingly, treatment with leptin did not cause a decrease in corticosterone when given with clonidine; pre-treatment levels were 222.5 1: I 1.9 and remained at that level for the duration of the experiment. Figure 4-6 shows serum corticosterone levels (mean t SE; ng/ml) for the control animals (N=10), high dose leptin (500 pg i.p.; N=1 1), isoproterenol (0.2mg/kg body treatment levels were not different between groups. Pre-treatment corticosterone in the isoproterenol groups was 293.0 i 20 and did not change significantly throughout the experiment. On the other hand, treatment with high dose leptin + isoproterenol caused a significant reduction in corticosterone from 286.6 :t21 to 176.9 :1: 21(p<0.05). Figure 4-7 summarizes the serum corticosterone levels for all groups. Serum corticosterone did not change significantly throughout the experiment in the control, isoproterenol, clonidine nor clonidine + 500 pg leptin treated groups. On the other hand, both high and low dose leptin as well as isoproterenol + high dose leptin caused a significant reduction is corticosterone as compared to pre-treatment levels ( p<0.05) and pre-treatment and controls (p<0.05) (N=4-I l/group). 76 I\ l_..l A__._L=____‘____--____ I: Control m 100 pg 500 pg leptin leptin E400 " U) 5300 - “c’ 2200- # .. l 3 4. m * I§1oo~ ‘I: 8 0 / / / / / Time (h) Fig. 44. Serum corticosterone levels (mean :1: SE; ng/ml) for the control, low dose (100 pg) and the high dose leptin (500 pg) treated animals are shown above. Corticosterone in the control group was did not change significantly throughout the experiment, however, both high and low dose leptin administration caused a significant reduction in serum corticosterone compared to pre-treatment levels and to controls (*p<0.05). By 3 hours post-treatment, high dose leptin caused a significant reduction is corticosterone compared to pre-treatment (#p<0.05). By 4 hours post- treatment, low dose leptin caused a significant decrease in serum corticosterone compared to controls and pre-treatment levels (*p<0.05), which persisted for the remainder of the experiment (N=4-1 l/group). 77 1:1 Control 500 pg leptin 1:] Clonidine (CLO) E 400 - - CLo+5oo pg leptin U) f; 300 - C 'I 7 a CD 8 100 - A A é ? 5 t t r r 2 3 o / / a / 4 a 0 1 2 3 4 5 Time(h) Fig. 4-5. Serum corticosterone levels (mean t SE; ng/ml) for the control animals, high dose leptin (500 pg i.p.; N=11), clonidine (0.3mg/kg body weight i.p.) and clonidine + high dose leptin treated groups. Pre-treatment corticosterone levels were not different between groups. High dose leptin administration caused a significant reduction in corticosterone compared to pre-treatment levels (a p<0.05) and controls and pre-treatment levels (* p<0.05). Serum corticosterone did not change significantly in the clonidine treated animals during the experiment. Interestingly, treatment with leptin did not cause a decrease in corticosterone levels when given with clonidine (clo + 500 pg leptin group) (N=4-11/group). 78 l:l Control 500 pg leptin E Isoproteronol (ISO) 400 ‘ - ISO + 500 pg leptin 300 ” 200 7 l A o 1 2 3 4 5 Time (h) \ 100 \ Corticosterone (nglml) L\\\\\\\\\\V Fig. 4-6. Serum corticosterone levels (mean t SE; ng/ml) for the control, high dose leptin (500 pg i.p.), isoproterenol (0.2mg/kg body weight i.p.) and isoproterenol + high dose leptin treated groups. Pre-treatment levels were not different between groups. Treatment with 500 pg leptin caused a significant decrease in corticosterone as compared to pre-treatment levels (a p<0.05) and controls (* p<0.05). Pre- treatment corticosterone in the isoproterenol groups was not significantly different from post-treatment corticosterone. On the other hand, treatment with high dose leptin + isoproterenol caused a significant reduction in corticosterone from 286.6 3:21 to 176.9 i 21(*p<0.05). 79 7- Control % 100 pg leptin - it?” '9" ”tom onI ine - lcso roteronol IBO) 500 pg ep_tin 400 ‘ ICSLC + +500 pg leptin 300 ‘ Corticosterone (nglml) . 7 7 V? 7 A A A 7 20° t? r e r a r A r r r rt 2 r 100 / M / / r a r r t t! t t 0 a 24 a a 0 1 2 3 4 5 Time (h) Fig. 4-7. Semm corticosterone for all groups is shown above. Serum corticosterone did not change significantly throughout the experiment in the control, isoproterenol, clonidine nor clonidine + 500 pg leptin treated groups. On the other hand, both high and low dose leptin as well as isoproterenol + high dose leptin caused a significant reduction is corticosterone as compared to pre-treatment levels (a p<0.05) and pre- treatment and controls (* p<0.05) (N=4-11/group). 8O E. Discussion Results from this study indicate for the first time that systemic administration of leptin causes a decrease in norepinephrine release in the paraventricular nucleus while simultaneously decreasing serum corticosterone. Norepinephrine release in the PVN was decreased 30 min following leptin administration and remained decreased for the duration of the post-treatment period. An important aspect of this study is the concomitant measurements ofplasrna corticosterone. Leptin decreased serum CS levels at 240 and 300 min post-treatment. This effect was blocked by the a- adrenergic agonist clonidine and not by the B-adrenergic agonist isoproterenol. These results indicate that leptin decreases hypothalamic NE, thus IIPA activity and that this effect is most probably mediated via alpha-adrenergie receptors. Previous work from our laboratory has shown that leptin causes a dose-dependent decrease in NE concentration in the PVN. It has also been shown that leptin causes a decrease in serum corticosterone, although the mechanism by which it does this is not known (Chapter 3). This current study suggests that leptin decreases noradrenergic activity in the PVN thereby, decreasing CRH secretion and the HPA axis through alpha- adrenergic receptors. Since leptin’s identification in 1994, much work has been done to investigate its role as a regulator of metabolic homeostasis. Leptin has been shown to regulate a variety of orexigenic and anorexigenic peptides in the brain including, neuropeptide Y (NPY), melanin-concentrating hormone, agouti-related proteins, orexin, melanocortin and cocaine-anrphetamine related transcripts (72, 81, 226). Among its 81 many functions, leptin has also been shown to be inhibitory to the HPA axis (76, 223), however, the mechanism by which leptin affects the stress axis is not clear. There have been a variety of animal models used to show leptin’s inhibitory action on the stress axis. Genetically altered ob/ob mice are deficient in leptin. The HPA axis of these animals remains activated, however, chronic treatment with leptin suppresses this activation (1 Ialaas et al., 1995; Pellymounter et al., 1995; Campfield et al., 1995). In addition, leptin has been shown to inhibit restraint stress-induced activation of the HPA axis. There have also been in vitro studies showing that glucose deprivation causes an increase in CRI-I release from the hypothalamus, which is again blocked by leptin treatment (76). These studies su gest that leptin causes suppression of stress-induced activation of C RH neurons. A variety of neurochemicals are believed to regulate the HPA axis. Among them, the catecholamines, specifically norepinephrine, are thought to be of primary importance. The PVN of the hypothalamus has a high concentration of CRH cell bodies and receives rich noradrenergic innervation from the brain stem (33, 34). It has been shown that administration of NE into the PVN can stimulate CRH secretion, while a neurotoxic blockade of the ventral noradrenergic bundle, which supplies noradrenergic innervation to the hypothalamus from the brain stem, markedly reduces NE levels in addition to causing a significant reduction in CRH release (227). To produce its effects, leptin must first bind with its receptors. Leptin has been shown to cross the blood-brain barrier via a saturable transport mechanism (96). Several splice variants of the leptin receptor (OB-R) have been identified and are found in a variety of areas including the hypothalamus, hippocampus and several peripheral tissues. The “long form” (OB-Rb) ofthe receptor is highly expressed in the hypothalamus and is believed to play a major role in mediating leptin’s central and neuroendocrine effects (228). Many populations of neurons contain leptin receptors including, CRII neurons in the hypothalamus (229, 230), which may mediate some of leptin’s actions. The inhibitory effects of leptin are specific to the CRII neurons associated with the IIPA axis and not to other CRII neurons. It has been shown that leptin treatment causes a downregulation of C Rl-I expression in the PVN but an increase in the amygdala and bed nucleus of the stria terminalis (84). In the present study, it is shown that leptin causes a decrease in NE release in the PVN along with a decrease in serum corticosterone. This effect was blocked by the alpha-adrenergie agonist clonidine and not by the beta-adrenergie agonist isoproterenol. The exact mechanism by which clonidine is able to produce this effect is not known, however there are a few possibilities. C lonidine preferentially binds to a 2 — adrenergic receptors, which are G-protein coupled receptors that primarily mediate inhibitory effects of the catecholamines in the central and peripheral nervous systems (231). Located both pre- and post-synaptically, a z-ARs are known to mediate cardiovascular function, analgesia and memory (232). There are three sub- classes of a 2-ARs, or z-A, a 2-B and or z-C, which have been found in many areas of the brain and brainstem including: the hypothalamic PVN noradrenergic terminals (233), presynaptic GABA terminals in the PVN (234) and the brainstem noradrenergic neurons of Al, A2, A5, A7 and the locus coeruleus (233, 235), So, clonidine could have exerted its effects at any or all ofthcse locations. First, we must consider the dose ofclonidine used in this current study; 0.3 mg/kg BW. It has been 83 shown that in the brain clonidine acts preferentially at or 2-adrenergic receptors, when administered at systemic doses between 0.05-0.1 mg/kg, to decrease noradrenergic neuronal firing, NE release and NE tumover. But, at higher doses, appears to produce agonist effects at or .-adrenergic receptors counteracting the decreased noradrenergic activity (40). Thus. it is possible that the dose used in this current study produced noradrenergic agonist effects at or I ARs counteracting leptin’s suppressive effects on NE release and corticosterone. Additionally, it has been shown that both i.c.v. and i.v. injection of the nitric oxide donor 3-morpholino-sydnonimine (SIN-1) causes a significant increase in ACTI-I release in non-anesthetized freely moving rats (236). Additionally, i.c.v. injection of SIN-1 increases NE levels in the PVN and serum corticosterone (237). Rivier et. al., (2003) found that the SIN-1 -induced release of ACTH is blocked by the a 2-adrenergic agonist, clonidine but not by a 1 or B-adrenergic antagonists (236). The results suggest that although a 1 or B-adrenergic receptors may be involved in HPA axis activity, only a 2-adrenergic receptors are involved in mediating nitric oxides ability to release ACTH. These findings suggest that a 2-adrenergic receptors may in fact, modulate HPA activity in various contexts and may play a role in leptin’s ability to suppress HPA axis activity. Another possible explanation is that clonidine acted on presynaptic or 2- adrenergic receptors on GABA neurons in the PVN. In the PVN of the rat hypothalamus, Norepinephrine is known to increase or decrease GABAergic synaptic currents to the PVN neurons via at u I- or u 2-adrenergic receptors respectively (234). Chong, et al., has used slice patch recordings to show that guanfacine, an a 2-A 84 selective agonist, reduced inhibitory post-synaptic currents (IPSC) in magnocellular and parvocellular neurons of the PVN (238). This suggests that noradrenalin excites both magnocellular and parvocellular neurosecretory neurons of the PVN by decreasing GA BA input via a 3-adrenergic receptors in the PVN. Therefore, it is reasonable to assume that clonidine, a non—selective a 2-adrencrgic agonist could act at the pre-synaptic or 2-ARs on GABA neurons, thereby decreasing inhibitory input to the PVN, thus facilitating C RH release from the parvocellular neurons. This could explain how clonidine was able to reverse leptin’s suppressive effects on corticosterone in this current study. This does not explain why there was no change in NE release, however. Ifclonidine acted to dis—inhibit the CRH neurons in the PVN by decreasing GABA release, one would have expected to still see a decrease in NE release. However, this was not shown. It is possible that disinhibiting the CRH neurons in the PVN lead to increased noradrenergic input to the PVN. It has been shown that the CRH neurons in the PVN project to brainstem noradrenergic nuclei including the LC (239) and that CRH administration to these nuclei, including the LC, causes an increased neuronal firing and central nervous system (CNS) NE release in target sites including the PVN (30, 239). So, it is possible that clonidine acted to decrease GABA’s inhibitory input to the PVN, thereby increasing CRH release which lead to increased ACTH and corticosterone while also causing an increase in NE release in the PVN mediated by increasing C RH release in the brainstem. Additionally, we cannot rule-out the possibility that clonidine acted to reverse leptins suppressive effects on corticosterone by some as yet, urridcntilied means. However, the results from this study do suggest that leptin decreases HPA activity by 85 decreasing hypothalamic NE which is most likely mediated via alpha-adrenergie receptors. 86 F. Summary The study described in this chapter was designed to investigate the time- course by which peripheral administration of leptin causes a decrease in norepinephrine concentrations in the PVN while simultaneously causing a decrease in serum corticosterone levels. Norepinephrine release in the PVN was decreased 30 min following leptin administration and remained decreased for the duration of the post-treatment period. Leptin decreased serum corticosterone levels at beginning at 240 min post-treatment. This effect was blocked by the cit—adrenergie agonist clonidine and not by the B-adrcnergic agonist isoproterenol. These results indicate that leptin decreases hypothalamic NE, thus HPA activity and that this effect is most probably mediated via alpha-adrenergie receptors. 87 Chapter 5. Leptin Suppresses Noradrenergic Activity in the PVN and I-IPA Axis Activity in Diabetic Rats A. Introduction Diabetes is a metabolic disorder characterized by a variety of central nervous system dysfunctions including hyperphagia, polydypsia and activation of the IIPA axis (240-242). The mechanisms by which these neuroendocrine dysfunctions occur are not known. Metabolic imbalances may be sensed by the brain via signaling molecules such as glucose and insulin. In addition, leptin may serve as a metabolic regulator in diabetes mellitus. Iv-Iypothalamic monoamines are also likely to play a role as they are intricately involved in modulation of many of these functions. It is known that serum leptin levels decrease in diabetes (7, 8, 243), in addition, it has been shown that hypothalamic NE levels are elevated (4, 165, 166). This is associated with an increase in HPA activity in diabetes (244). Therefore, it is hypothesized that leptin decreases noradrenergic activity in the PVN, thereby inhibiting CRH secretion and HPA axis activity and that this regulatory mechanism is altered in diabetes. To test this hypothesis, we have designed a push-pull perfusion study, using STZ-induced diabetic rats, with simultaneous blood collection. Additionally, we investigated the mechanism by which leptin may mediate its effects by administering alpha or beta adrenergic receptor agonists. 88 B. Rationale Diabetes Mellitus is a chronic endocrine disease associated with a number of central and neuroendocrine dysfunctions due to lack of insulin, insulin action or both. A myriad ofcomplications arise from the disease including retinopathy, nephropathy, neuropathy and cardiovascular dysfunction. Diabetes causes neuroendocrine abnormalities including hyperphagia and polydypsia as well (240-242). It has also been shown that diabetes causes hyperactivation of the HPA axis in rodents (240, 245, 246) and humans (247, 248). The mechanisms by which these neuroendocrine dysfunctions occur, however, are not known. Streptozotocin (STZ) is used to induce experimental diabetes in animals as it is selectively toxic to the pancreatic beta cells, the only cells in the body known to produce insulin (249). In experimentally-induced diabetic rats, the HPA axis activation is shown by elevated plasma corticosterone and ACTH (250) as well as, increased CRH mRNA in the PVN (251). It has been shown that the elevated plasma corticosterone and ACTH levels in STZ-induced diabetic rats can be normalized with i.p. insulin administration (4, 251). In addition to insulin and glucose, leptin may serve as a metabolic regulator in diabetes mellitus. Leptin has been shown to cause several central and neuroendocrine effects including decreasing feed intake (72, 75) altering sympathetic outflow and increasing energy expenditure and thermogenesis (252, 253). Leptin has also been shown to modulate hypothalamic monoamine concentrations (chapter 3) and decrease noradrenergic activity in the PVN while simultaneously decreasing serum corticosterone (chapter 4). All of these effects are generally opposite of what is seen in diabetes, and since leptin levels decrease markedly in untreated STZ diabetic rats (7, 8) it is likely that it is this 89 decrease in leptin levels that are responsible for the neuroendocrine dysregulation including hyperactivation ofthe HPA axis seen in diabetes. Hypothalamic monoamines are also likely involved since they are known to modulate of many of these functions. In an in vitro model, it has been shown that leptin administration to isolated rat hypothalami causes a significant reduction in NE efl'lux (134), and we have shown in chapter 3, that a single i.p. or i.c.v. injection of leptin causes a significant reduction in NE content in the hypothalamus. Additionally, we have shown that peripheral administration ofleptin decreases noradrenergic activity in the hypothalamus of normal rats by decreasing NE release in the PVN (chapter 4). It has been shown that hypothalamic NE levels are elevated in diabetes (4, 5, I65) and that i.p. or i.c.v. administration ofleptin reverses this effect (4). So, since it is known that serum leptin levels decrease in diabetes, and that hypothalamic NE levels are elevated, it is possible that leptin decreases the levels of NE in the PVN, thereby inhibiting CRH secretion and HPA axis activity and that this regulatory mechanism is altered in diabetes. 90 C. Experimental Design The experiment described in this chapter was designed to investigate leptin’s role in hypothalamic noradrenergic activity and the IIPA axis dysregulation seen in STZ-induced diabetes. To do this, we implanted adult male Sprague Dawley rats with both a push—pull cannulae in the PVN and ajugular catheter. Animals were randomly divided into seven groups and treated as follows: Group I (n=4) served as the non-diabetic control and received the vehicle for STZ (i.p.) and the vehicle for leptin (i.p.). Group II (n=5) was the diabetic control and received STZ (65 mg/kg BW, i.p.) and the vehicle for leptin (i.p.). Group III (n=6) received STZ (65 mg/kg BW, i.p.) and recombinant rat leptin (500 pg i.p.). Group IV (n=5) received STZ (65 mg/kg BW, i.p.) and alpha-adrenergie agonist clonidine (0.3mg/kg BW). Group V (n=5) received STZ (65 mg/kg BW, i.p.), alpha-adrenergie agonist clonidine (0.3 mg/kg BW i.p.) and recombinant rat leptin (500 pg i.p.). Group VI (n=5) received STZ (65 mg/kg BW, i.p.) and beta-adrenergie agonist isoproterenol (0.2 mg/kg BW i.p.). Group VII (n=5) received STZ (65 mg/kg BW, i.p.), adrenergie agonist isoproterenol (0.2 mg/kg BW i.p.) and recombinant rat leptin (500 pg i.p.). Push-pull perfusion was performed for 6 hours. Perfusate samples were collected every 30 min and blood samples were collected every 60 min for the duration of the experiment. Adrenergic receptor agonists were administered 30 min before leptin or saline. Perfusate samples were analyzed for neurotransmitter content using HPLC-EC and plasma was analyzed for corticosterone (CS) by RIA. 91 D. Results Body Weight There were no significant differences in body weight between diabetic and non-diabetic groups at the beginning of the experiment. Body weight (mean i S.E.; g) of non-diabetic control animals on day 0 was 322.6 i 10.8 and increased sigrrilicantly to 365.1 i: 13.6 on day 14. On the other hand, body weight of diabetic animals on day 0 was 304.7 i 12.7 and decreased significantly to 255.1 i 16.1 on day 14(Fig 5—1). —‘3’— Control "V" Diabetes 3 E 27 d) 3 >4 '0 O m 250* 200 4 l 1 L1 ' l l l l l— 12 3 4 5 6 7 8 91011121314 Days Fig. 5-1. Changes in body weight after the induction of diabetes or saline administration. Body weight was measured daily as described above. N= 7/group; (* p<0.5) as measured by repeated measures ANOVA. 93 Food Intake Changes in food intake over the entire observation period are shown in Fig. 5-2. The daily food intake (mean t SE; g) of non-diabetic control animals on day 0 was 22.6 i 2.1 and remained at that level for the duration ofthe experiment. In contrast, induction ofdiabctcs caused a significant increase in food intake from 20.9 i 1.8 on day 0 to 45.2 i 2.3 on day 14 (p < 0.05). 94 —V— Control "V“ D'abetes 60 — T T/,/V m” ,V’ _ T T/ 40 X\\T T ,v a "" ID x S .E “O O 0 LI. V 20 __ 0 I 1 1 1 l l J I l l l 1 L I 1234567891011121314 Days Fig. 5-2. Changes in food intake after injection of STZ (65 mg/kg BW) (N = 7) or vehicle (N = 6) are shown above. Beginning at day 5, food intake of diabetic animals was significantly higher than controls (* p < 0.05 as compared by repeated measures ANOVA) 95 Water Intake Fig. 5-3 shows the changes in water intake among the diabetic and non-diabetic controls for the entire observation period. The daily water intake (mean i S.E.; ml) of non-diabetic control animals on day 1 was 35.7 i 4.4 and remained at that level for the duration of the experiment. In contrast, induction of diabetes caused a significant increase in water intake from 95.8 i 5.9 on day 1 to 202.5 4: 14.4 on day 14 (p < 0.05). 96 Water Intake (ml) 250 200 150 100 50 —V— Control "V“ D'abetes A 1 L L114 11 l l l 111 01234567891011121314 Days Fig. 5-3. Changes in water intake for diabetic and non-diabetic controls for the duration of the experiment. Day 1 represents the first day ofdiabetes or control (24 hours after administration of STZ (65mg/kg BW) or vehicle). Water intake was measured daily as described above. * p< 0.05 as compared by repeated measures ANOVA (N =6-7/ group). 97 Norepinephrine Release in the PVN Profiles ofNE release in the PVN for the different groups are shown in Figures 5- 4 through 5-10. Pre-trcatment NE release (before agonist or vehicle for agonist and leptin or vehicle for leptin administration) was significantly different between diabetic and non-diabetic animals only. There were no significant pre-treatment NE release differences between diabetic groups. Fig. 5-4 shows the NE release (mean 3: SE; pg/min) for all groups. Relevant group comparisons and statistical significance are shown in subsequent graphs. Fig. 5-5 shows: NE release (mean i S.E.; pg/min) in non-diabetic control animals was 13.3 i 1.6 prior to treatment and did not change significantly after injection of vehicle. NE release in diabetic control animals was 67.5 i 3.5 prior to treatment and did not change significantly after injection of vehicle. On the other hand, the diabetic + leptin group had a significant decrease in NE release from pre- treatment levels 0f71.0 i 2.8 to 51.1 i 2.8 (p<0.04). Fig. 5-6A shows the average NE release (mean :1: SE; pg/min) pre and post- treatment for the non-diabetic control animals. The average pre-treatment NE release was 13.6 i 1.3, which was not different from the average post-treatment NE release (12.8 d: 1.7). Fig. 5-6B shows the average NE release (mean :t SE; pg/min) pre and post-treatment for the diabetic control animals. The average pre-treatment NE release was 67.4 i 4.7, and was not different from the average post-treatment NE release (67.9 i 5.4). Fig. 5—6C shows the average NE release (mean i S.E.; pg/min) pre and post-treatment for the leptin treated animals (500 pg i.p.). The average post—treatment 98 NE release was 62.9 d: 2.8 which was significantly lower than the average pre- treatment NE release (72 i: 2.5) (p<0.04). Fig. 5-7 shows the NE release (mean i S.E.; pg/min) in the PVN ofdiabetic animals treated with clonidine (0.3 mg/kg BW) then saline, clonidine then 500pg leptin or saline then 500pg leptin. The leptin + clonidine treated group was significantly different than the saline + clonidine treated group (p<0.05), the leptin + saline treated group was significantly different than the leptin + clonidine treated group (p<0.05), and the leptin + saline treated group was significantly different than the saline + clonidine treated group (p<0.05) as shown. Fig. 5-8A shows the average NE release (mean i S.E.; pg/min) pre and post- treatrnent for the saline + clonidine (0.3 mg/kg BW) treated animals. The average pre-treatment NE release was 66.5 d: 1.2, which was not different from the average post-treatment NE release (69.8 i: 2.4). Fig. 5-8B shows the average NE release (mean :1: SE; pg/min) pre and post-treatment for the leptin (500 pg) + clonidine (0.3 mg/kg BW) treated animals. The average pre-treatment NE release was 56.2 :1: 1.9, and was not different from the average post-treatment NE release (58.4 :t 3.1). Fig. 5-9 shows the NE release (mean i S.E.; pg/min) in the PVN of diabetic animals treated with isoproterenol (0.2 mg/kg BW) followed by saline, isoproterenol then 500pg leptin or saline then 500pg leptin. In the leptin + isoproterenol treated group there was a trend showing decreased NE release after leptin administration, which was significantly different from the saline + isoproterenol group at 2.5 and 4 hours post-treatment (p<0.02) and significantly different than the saline + leptin 99 treated group as shown ( p<0.05). The saline + leptin treated group was significantly different than the isoproterenol + saline treated group as shown (p<0.05). Fig. 5-10A shows the average NE release (mean :t SE; pg/min) pre and post- treatment for the saline + isoproterenol (0.2 mg/kg BW) treated animals. The average pre-treatment NE release was 60.5 i 2.8, which was not significantly different from the average post-treatment NE release (62.3 i 3.1). Fig. 5-IOB shows the average NE release (mean :1: SE; pg/min) pre and post-treatment for the leptin (500 pg) + isoproterenol (0.2 rug/kg BW) treated animals. The average post-treatment NE release was 54.0 i 3.3, which was significantly lower than the average pre-treatment NE release (66.7 i 3.2) (p<0.04). 100 + ND Con 6— Diabetes +— Lep —e—- Iso + Lep + Iso —E1— Clon + Lep + Clon 90 r 80 r 70 r 60— 50” 40‘ N E Release (pg/min) 30b 20— -1 0 1.0 2.0 3.0 4.0 Time (hrs) Fig. 5-4. NE release (mean i S.E.; pg/min) for all groups. Relevant group comparisons and statistical significance are shown in subsequent graphs. The agonist or vehicle was given at — 0.5 hrs (solid arrow) and the vehicle for leptin or leptin was given at 0 hrs (dashed arrow). 101 + Control —9— Diabetes “1‘— Diabetes+ Leptin 70‘ 4o N E Release (pg/min) 20* 10? -1 0 1.0 2.0 3.0 4.0 Time (hrs) Fig. 5-5. NE release in the PVN of non-diabetic controls, diabetic controls and diabetic animals treated with 500pg leptin i.p. The agonist or vehicle was given at 0.5 hrs (solid arrow) and the vehicle for leptin or leptin was given at 0 hrs (dashed arrow). NE release was significantly different between non-diabetic and diabetic animals for the duration of the experiment ** p< 0.0001 and was significantly different between diabetic and diabetic + leptin animals beginning at 2.5 hours (* p<0.04). A ve. N E mlease (pglnln) A ve. N E mlease (pglmln) 1:1 70 ” Pre- treatment Pre- treatment T W Post- Post- treatment treatment 103 Fig. 5-6A. Average NE release (mean i S.E.; pg/min) pre and post saline-treatment for the non-diabetic control animals. The average pre-treatment NE release was not different from the average post-treatment NE release. Fig. 5-6B. Average NE release (mean i S.E.; pg/min) pre and post saline-treatment for the diabetic control animals. The average pre-treatment NE release was not different from the average post-treatment NE release. Ave. NE release Wain) 70 50 Pre- ”'7 “catnient ._ g - _. f i Post- treatment 104 Fig. 5-6C. Average NE release (mean i S.E.; pg/min) pre and post leptin—treatment. The average post-treatment NE release was significantly lower than the average pre-treatment NE release (*p<0.04). N E release (pg/min) 90 40 30 20 10 Saline + Clonidine I + Leptin + Clonidine + Leptin + Saline l l I l 1 l I I 1 l l g -1 0 1.0 2.0 3.0 4.0 Time (hrs) Fig. 5-7. NE release in the PVN of diabetic animals treated with clonidine then saline, clonidine then 500pg leptin or saline then leptin. The leptin + clonidine treated group was significantly different than the saline + clonidine treated group (* p<0.05), the leptin + saline treated group was significantly different than the leptin + clonidine treated group (a p<0.05), and the leptin + saline treated group was significantly different than the saline + clonidine treated group (b p<0.05) as shown. The agonist or vehicle was given at - 0.5 hrs (solid arrow) and the vehicle for leptin or leptin was given at 0 hrs (dashed arrow). 105 A vo. N E roloau (pgtmin) Ave. N E release (pglrrin) 70' 60' 50‘ 70 m Pre- Eg treatment Pre- treatment :1 Post- treatment ‘ Post- ; treatment 106 Fig. 5-8A. Average NE release (mean i S.E.; pg/min) pre and post-treatment for the saline + clonidine treated animals. The average pre-treatment NE release was not different from the average post-treatment NE release. Fig. 5-8B. Average NE release (mean i S.E.; pg/min) pre and post-treatment for the leptin + clonidine treated animals. The average pre-treatment NE release was not different from the average post-treatment NE release. N E release (pg/min) 40 30 20 10 Saline + lso + Leptin + Iso + Leptin + Saline 1.0 2.0 Time (hrs) 3.0 4.0 Fig. 5-9. NE release in the PVN of diabetic animals treated with isoproterenol followed by saline, isoproterenol then 500pg leptin or saline then leptin. In the leptin + isoproterenol treated group there was a trend showing decreased NE release after leptin administration, which was significantly different from the saline + isoproterenol group at 2.5 and 4 hours post-treatment (*p<0.02) and significantly different than the saline + leptin group (a p<0.05). The saline + leptin treated group was significantly different than the isoproterenol + saline group (b p<0.05). The agonist or vehicle was given at — 0.5 hrs (solid arrow) and the vehicle for leptin or leptin was given at 0 hrs (dashed arrow). 107 Ave. N E release (pglrrin) A ve. N E release (pglrrl'n) - P'e' t::-:.-:. P03" treatment treatment 70 r 65 ~ “T” was so 5’ I lift: 3:4 Fig. 5-10A. Average NE I release (mean :1: SE; pg/min) pre and post—treatment for the saline + isoproterenol treated 55 animals. The average pre- treatment NE release was not different from the average post- so treatment NE release. Chaps Pre- Post- :3 treatment :3 treatment 70- T Fig. 5-10B. Average NE release (mean :t SE; pg/min) pre and post-treatment for the leptin + isoproterenol treated animals. The average post- treatment NE release was significantly lower than the pre- treatment release (*p<0.04). 108 Serum Corticosterone Serum corticosterone levels (mean i 8.13.; ng/ml) for all groups at the time of sacrifice are shown in Figures 5-1 1 through 5-14. Fig. 5-1 1 shows non-diabetic animals had significantly lower corticosterone levels (220.5 :1: 5.5) compared to diabetic animals (365.4 :1: 58.4) (p<0.01) and neither group changed significantly throughout the experiment. On the other hand, 500 ug leptin administration (i.p.) to diabetic animals caused a significant reduction in corticosterone from 321.7 :t 15.2 pre-treatment to 92.9 i 17.8 by 5 h post-treatment). Additionally, leptin caused a significant reduction in corticosterone compared to diabetic controls beginning 1 h post-treatment, which persisted throughout the experiment (p<0.002). Fig. 5-12 shows non-diabetic animals had significantly lower corticosterone levels (220.5 d: 5.5) compared to diabetic animals (365.4 d: 58.4) (p<0.01) and neither group changed significantly throughout the experiment. Additionally, clonidine (0.3 mg/kg body weight, i.p.) did not cause a significant change in corticosterone. Pre-treatment levels were 404.1 i 22.9 and remained at that level for the duration of the experiment. On the other hand, 500 pg leptin administration (i.p.) to diabetic animals caused a significant reduction in corticosterone (p<0.02) but not when given with clonidine. Pre-treatment level of corticosterone in the clonidine + leptin group was 425.4 i 52.6 and did not change significantly throughout the experiment. Fig. 5-13 shows non-diabetic animals had significantly lower corticosterone levels compared to diabetic animals (p<0.01). Additionally, isoproterenol (0.2 mg/kg body weight, i.p.) did not cause a significant change in corticosterone. Pre-treatment levels 109 were 303.6 d: 8.8 and remained at that level for the duration of the experiment. On the other hand, 500 ug leptin administration (i.p.) to diabetic animals caused a significant reduction in corticosterone (p<0.02), which persisted when given with isoproterenol. Pre-treatment corticosterone level in the leptin + isoproterenol group was 374.6 :t 26.5 which decreased to 244.2 i 43.6. Figure 5-14 summarizes the serum corticosterone levels for all groups. Serum corticosterone did not change significantly throughout the experiment in the non- diabetic control, diabetic control, isoproterenol, clonidine nor clonidine + 500 ug leptin treated groups. On the other hand, both 500pg leptin as well as isoproterenol + leptin caused a significant reduction is corticosterone as compared to pre-treatment levels and controls (N=4-6/group). 110 ND Con DCon Lep - V////A 71/4 550‘ 8 a / a a a é ¢ 6 e a a 7 ° 5, ./ 2 a a, O 2 3 4 5 Time(h) Fig. 5-11. N on-diabetic animals had significantly lower corticosterone levels compared to diabetic animals (p<0.01), and neither group changed significantly throughout the experiment. On the other hand, 500 pg leptin administration (i.p.) to diabetic animals caused a significant reduction in corticosterone from 321.7 i 15.2 to 92.9 i 17.8 (N=4-6/group). Leptin caused a significant reduction in corticosterone compared to diabetic controls beginning 1 h post-treatment, which persisted throughout the experiment (*p<0.002). 111 2” D Con Lep 00" SIB; 0n - V/////A 7’11 :1 550 7 g: 425 I 7 '/ 5 z é ‘” / ¢ = / f e 300 4 /* g 9 w v 8 a a -° 175 — é! /* .. E W i .. 8 £5 £7 50 _ £4 £1 _ 0 1 2 3 4 5 Time lhl Fig. 5-12. Non-diabetic animals (ND Con) had significantly lower corticosterone levels compared to diabetic animals (p<0.01) and neither group changed significantly throughout the experiment. Clonidine administration (0.3 mg/kg body weight, i.p.) did not cause a significant change in corticosterone. On the other hand, 500 pg leptin administration (i.p.) to diabetic animals caused a significant reduction in corticosterone compared to diabetic controls (*p<0.02) but not when given with clonidine (N=4-6/group). 112 ND D Con Lep [so [so + Con Lep -’--l:] 550— 425 — 300 — k\\\\\\\\\\\\\l\\\\\\‘\\\\\‘l O L\\\\\\\\\\V 3(- 175 r X- a- X- C orticosterone (nglml) mm mm W W mm: m com J I“\\\\‘: 50‘ vom- Isis. Time (hl Fig. 5-13. Non-diabetic animals had significantly lower corticosterone levels compared to diabetic animals (p<0.01) and did not change throughout the experiment. Isoproterenol (0.2 mg/kg body weight, i.p.) did not cause a significant change in corticosterone. Pre-treatment levels were 303.6 i 8.8 and remained at that level for the duration of the experiment. On the other hand, 500 pg leptin administration to diabetic animals caused a significant reduction in corticosterone compared to diabetic controls (*p<0.02), which persisted when given with isoproterenol beginning at 3 h post-treatment (#p<0.002). Pre-treatment corticosterone level in the leptin + isoproterenol group was 374.6 i 26.5 which decreased to 244.2 i 43.6 by 5 h post- treatment. 113 - ND 7/////A D co" Lep - Clon [:100“ [3 [so I:] [so 550 +Lep +Lep € 3: 425 ‘ 5 3 2 300 ‘ ’2? 'J .. _ r o r 50 — ‘ 3 Time (h) Fig. 5-14. Serum corticosterone levels for all groups are shown above. Serum corticosterone did not change significantly throughout the experiment in the non- diabetic control, diabetic control, isoproterenol, clonidine nor clonidine + 500 pg leptin treated groups. On the other hand, both 500pg leptin as well as isoproterenol + leptin caused a significant reduction in corticosterone as compared to pre-treatment levels and controls (*p<0.02, #p<0.002 respectively) (N=4-6/group). 114 E. Discussion Diabetes Mellitus is a chronic metabolic disorder known to cause a variety of complications including kidney damage, neuropathy, retinopathy and cardiovascular dysfunction. In addition, many central and neuroendocrine effects are seen, including hyperactivation of the stress axis. Activation of the HPA axis results in increased secretion of hypothalamic C R11, which is secreted into the hypophyseal-portal circulation and is transported to the anterior pituitary where it causes synthesis and release of ACTH ultimately leading to secretion of corticosterone from the adrenal cortex. During times of acute stress, elevated plasma glucocorticoids serve beneficial functions including mobilization of energy stores and suppression of further HPA activity. However, chronically elevated glucocorticoid levels have deleterious effects including hippocampal neuronal cell death and immune system suppression (l3, l4). Normally, the stress response is turned off via glucocorticoid negative feedback; this mechanism, however, is dysregulated in diabetes as is evidenced by the sustained HPA activation (240, 246). The mechanism by which the stress axis is chronically elevated in diabetes is not known, however, it is likely that leptin’s regulation of hypothalamic NE is involved. The PVN of the hypothalamus has a large number ofCRH cell bodies and receives rich noradrenergic innervation from the brain stem, specifically, the Al region of the ventral medulla, the A2 region of the dorsal vagal complex and A6 (the locus ceruleus) (33). These NE containing fibers are known to synapse with CRH cell bodies in the hypothalamic PVN (34), and it has been shown that administration of NE into the PVN stimulates CRH section (35). In addition, pharmacologic 115 destruction of the ventral noradrenergic bundle significantly decreases CRH release (32, 35). Leibowitz ct al., has shown that NE injected into the PVN causes a significant dose-dependent increase in circulating corticosterone. And, in a mapping study, they showed that this NE stimulatory effect was localized with the hi ghest rise in corticosterone levels following NE injection into the PVN compared to other brain areas (36). Together, these studies clearly show that PVN noradrenergic activity stimulates CRH release and the HPA axis. The adipocyte-derived hormone, leptin may modulate this activity. Leptin administration to isolated rat hypothalami causes a dose-dependent decrease in NE release (134), and we have shown that both i.p. and i.c.v. administration of leptin caused a decrease in NE content in the PVN of male rats (chapter 3). We then showed that peripheral administration of leptin suppresses NE release in the PVN while concomitantly decreasing serum corticosterone (chapter 4) in non-anesthetized freely moving rats. Together, these results suggest that in fact, leptin is able to suppress noradrenergic activity in the PVN, thus HPA axis activity. We then wanted to explore this relationship in a diabetic model. It has been shown that in experimentally induced diabetes, there is an increase in NE content in the hypothalamus (4, 5, 165) and that this was reversed by parenteral injection of insulin (166) or leptin (4). Since it is known that serum leptin levels decrease in diabetes (7, 8) we wanted to test the hypothesis that this decrease in circulating leptin levels in diabetes contributes to the elevated noradrenergic activity in the PVN and thus the HPA axis activity. The results from this study are consistent with previous findings that diabetes causes an increase in serum corticosterone and hypothalamic noradrenergic activity. When a single i.p. injection of leptin was 116 administered to the diabetic animals, a significant reduction in NE release in the PVN was accompanied by a dramatic decrease in serum corticosterone. This suggests that noradrenergic activity in the PVN plays an important role in mediating leptin’s suppressive effects on the HPA axis activity, and since leptin levels are significantly reduced in diabetes, it may contribute to the hyperactivation of the stress axis. One observation to point out is that the decrease in NE release following leptin administration did not reach significance until 2.5 hours after administration while a significant decrease in serum corticosterone was observed 1 hour post-administration. It is quite possible that in this chronic diabetic state, there are many “stressors” contributing to the hyperactivation of the stress axis and leptin may be involved in modulating some of these via mechanisms exclusive ofNE release in the PVN. One such possibility involves arginine vasopressin. Vasopressin has been shown to stimulate ACTH secretion by itself and to act synergistically with CRH (254-256). Stimuli such as hypovolernia, hypotension and hyperosmolality cause the release of vasopressin (257). The animals in this study were uncontrolled diabetic rats with very high blood glucose levels. This hyperosmolar state may have caused an increase in vasopressin secretion contributing to the increased ACTH secretion and activation of the HPA axis. It has been shown that leptin plays a role in regulating glucose homeostasis (252, 258) and acts directly on a number of peripheral tissues including pancreatic B-cells, hepatocytes and skeletal muscle (259, 260). Therefore, it is probable that leptin acted at these peripheral sites to modulate glucose homeostatic mechanisms thereby ameliorating the hyperosmolality thus vasopressin contribution to HPA hyperactivation. This effect may have occurred prior to leptins affect on 117 hypothalamic NE release, thus the reason the decrease in corticosterone was observed before the decrease in NE in the PVN. It is also possible that leptin has direct actions on glucocorticoid secretion from the adrenal cortex, as leptin receptors have been identified here (113, 114). Whether this action is stimulatory or inhibitory remains controversial. Some in vitro studies have found, that leptin stimulates both aldosterone and corticosterone from adrenocortical cells (118). While others have shown that in both rhodents and humans, leptin inhibits basal and ACT H-stimulated corticosterone secretion (1 19). Malendowicz et. al., investigated this further by studying the effect of leptin fragments on adrenocortical cells (2003). They found that some leptin fragments had no effect on either aldosterone or corticosterone, some fragment decreased corticosterone with no effect on aldosterone, and yet other fragments stimulated both (120). Together these findings suggest that leptin may affect glucocorticoid secretion by mechanisms independent of hypothalamic NE, thus the reason leptin was shown to reduce corticosterone prior to NE in the PVN in this study. We also found that the alpha-adrenergie agonist, clonidine, was able to block leptin’s suppressive effects on corticosterone, whereas the beta-adrenergie agonist isoproterenol was not. These results suggest that leptin’s suppressive effects on the HPA axis may be mediated via org-adrenergie receptors. Further support for this comes from the fact that adrenalectomy causes a reduction in alpha 2 adrenoceptors and not alpha 1 adrenoceptors in the PVN (261). Thus, alpha 2 adrenoceptors could play a role in leptin’s effect as indicated in the present study. 118 The exact mechanism by which clonidine is able to produce this effect is not known, however, it is consistent with previous work from our lab (chapter 4). A possibility to consider is the dose ofclonidine used in this current study; 0.3 mg/kg BW. It has been shown that in the brain clonidine acts preferentially at a 2-adrenergic receptors, when administered at systemic doses between 0.05-0.1 mg/kg, to decrease noradrenergic activity, but at higher doses, appears to produce agonist effects at or 1- adrcnergic receptors counteracting the decreased activity (40). Thus, it is possible that the dose we used produced noradrenergic agonist effects at or I ARs counteracting leptin’s suppressive effects on NE release and corticosterone. Another possible explanation is that clonidine acted on presynaptic 0r 2-adrenergic receptors on GABA neurons in the PVN, thereby decreasing inhibitory input to the PVN, thus facilitating CRH release from the parvocellular neurons. This could explain how clonidine was able to reverse leptin’s suppressive effects on corticosterone in this current study. The persistent elevation in HPA activity in diabetic rats may also indicate a failure in the negative feedback mechanism exerted by glucocorticoids. Glucocorticoid receptors are distributed in various parts of the brain including brainstem noradrenergic neurons (262, 263). Increased levels of glucocorticoids may therefore act to decrease noradrenergic levels in the PVN to suppress stress axis activity (264). On the other hand, CRH sensitive neurons have the ability to positively influence noradrenergic neurons in the brain stem (265). Stress paradigms that increase the mRNA levels of tyrosine hydroxylase (TH) in the locus coeruleus under acute circumstances, fail to decrease TH mRNA levels after chronic stress paradigms (263). However, it is not clear if glucocorticoids can affect other 119 noradrenergic regions besides the locus coeruleus. This could be another possible reason why higher levels ofcorticosterone in diabetic rats fail to decrease NE levels in the PVN. Besides acting on brain stem noradrenergic neurons, glucocorticoids can act directly on the PVN (266). Glucocorticoid receptor expression decreases in chronic stress models in the PVN. However, it is elevated in the central amygdala (267) that provides innervation to noradrenergic neurons in the brain stem. This could be an alternate route by which systemic glucocorticoids can increase the level of NE in the PVN in chronic conditions such as diabetes. Regardless of the route, the present study indicates that diabetes causes an increase in norepinephrine in the PVN and promotes stress axis activity. Leptin blocks this effect and this is most probably mediated via alpha adrenergic receptors. The ability of leptin to act on brain stem noradrenergic neurons to produce this effect has to be investigated. 120 II F. Summary The study in this chapter was designed to test the hypothesis that leptin decreases noradrenergic activity in the PVN, thereby inhibiting CRH secretion and HPA axis activity, and that this is dysregulated in diabetes. Additionally, we wanted to investigate the mechanism by which leptin mediates its effects by administering alpha or beta adrenergie agonists. We found that in STZ-induced diabetic rats, a single i.p. injection of leptin decreased NE release in the PVN while simultaneously causing a decrease in serum corticosterone levels and that this effect was blocked by the alpha-adrenergie agonist clonidine and not by the beta-adrenergie agonist isoproterenol. These results suggest that the decrease in serum leptin levels seen in diabetes contributes to the hyperactivation of the HPA axis and that this effect is mediated via alpha-adrenergie receptors. 121 Chapter 6. Neuroendocrine Dysfunction in STZ-induced Diabetes is Ameliorated by Leptin Lentiviral Vector Transfection A. Introduction Diabetes is characterized by several neuroendocrine complications including hyperglycemia, hyperphagia and activation of the stress axis. It has been shown that diabetes causes a significant increase in norepinephrine (NE) levels in the paraventricular nucleus (PVN) of the hypothalamus, while circulating leptin levels decrease dramatically. This decrease in leptin could be one of the reasons for the central and neuroendocrine changes associated with the disease, including activation of the hypothalamo-pituitary-adrenal (HPA) axis. Leptin in known to inhibit HPA activity in several animal models and we have previously shown that NE concentrations decrease in the hypothalamus paralleling a decrease in serum corticosterone following leptin administration (chapter 3), and that peripheral administration of leptin to normal (chapter 4) and diabetic (chapter 5) rats results in decreased NE release in the PVN while simultaneously decreasing serum corticosterone. However, we have found that peripheral administration of leptin to diabetic rats does not bring serum leptin levels to that of non-diabetic controls (4), thus one possible reason that we see only partial resolution of hyperphagia, polydipsia and hyperactivation of the HPA axis. The study in this chapter was designed to investigate whether or not transfection ofdiabetic rats with lentiviral vector containing leptin gene can normalize serum leptin levels and the neuroendocrine dysfunction seen in the disease. B. Rationale It is well established that there is a dramatic decrease in the level of leptin in diabetes (7, 8, 243), and as described previously, leptin has been shown to reduce feed intake (72, 75) alter sympathetic outflow, increase energy expenditure and thermogenesis (252, 253). All of these effects are generally opposite of what is seen in diabetes, and since leptin levels decrease markedly in untreated STZ diabetic rats, it is likely that it is this decrease in leptin levels that is responsible for the neuroendocrine dysregulation including hyperactivation of the HPA axis. We have shown that not only does leptin modulate hypothalamic monoamines, thus HPA axis activity (chapters 3 & 4), but that it can also ameliorate the HPA hyperactivation via decreasing hypothalamic noradrenergic activity and serum corticosterone in STZ- induced diabetes (chapter 5). Previous work from our lab has shown that exogenous administration of leptin can partially alleviate central and neuroendocrine changes during diabetes such as hyperphagia, polydipsia, body weight loss and hyperglycemia in addition to stress axis activation (4). However, leptin is known to have a short half-life (268, 269), and the dose used in previous studies (100 pg/kg BW) did not bring serum leptin levels to that of non—diabetic controls. For these reasons, we designed a gene transfer study to try to normalize serum leptin levels in STZ-induced diabetic rats with the hope of completely normalizing the neuroendocrine dysfunction. 123 C. Experimental Design The study described in this chapter was designed to investigate the neuroendocrine function of STZ-induced diabetic rats after transfection with leptin lentiviral vector. To do this, we constructed human immunodeficiency virus type 1 lentiviral vectors that encoded either leptin (01)) or control green fluorescence protein (OF P) genes and transfected adult male Sprague Dawley rats. The animals were randomly divided into three groups and administered vehicle for vector (control), lentiviral vector containing GFP gene or Lentiviral vector containing leptin (Ob) gene via intravenous infusion. Seven days later, animals were further divided and half were treated, with vehicle for streptozotocin or streptozotocin (65mg/kg body weight) (Table 6-1). Food and water intake and body weight were monitored daily for 21 days (Fig 6-1). Fourteen days following STZ or vehicle administration, animals were sacrificed, brains were quickly removed and frozen and blood samples were collected. Serum was assayed for leptin, and corticosterone (CS) using RIA and insulin using ELISA. Brains were microdissected and analyzed for neurotransmitter concentration by HPLC. Treatment Leptin Leptin OF P GFP STZ Vector Vector Vector Vector Only + ST Z + + STZ + Citrate Citrate Number N=7 N=5 N=5 N=7 N=4 of Animals Table 6-1. Experiment group distribution. Fig. 6-1. Experimental design to assess neuroendocrine function afier transfection with either leptin lentiviral vector, GF P lentiviral vector or no vector. Pre-treatment observation lasted 7 days; food and water intake and body weight was monitored daily. On the 8th day, animals were randomly divided into three groups and administered vehicle for vector (control), lentiviral vector containing GFP gene, Lentiviral vector containing leptin gene via intravenous infusion. Seven days later, animals were further divided and half were treated with vehicle for streptozotocin or streptozotocin (65mg/kg body weight). 125 D. Results Food [make Changes in food intake over the entire observation period are shown in Figs. 6-2 through 6-7. Fig 6-2 shows the daily food intake (mean 1: SE; g) ofall groups for the 7 day pre-treatment observation period. On day one, control animals daily food intake was 20.1 d: 2.7 and remained at that level for the duration of the observation period. There were no significant differences between groups for the pre-treatment period. Fig 6-3 shows the average food intake (mean i S.E.; g) for the 7 day pre- treatment observation period. There were no significant differences between groups. Fig 6-4 shows the daily food intake (mean i S.E.; g) for the 8 days following administration of either lentiviral vector containing GFP cDNA (GFva), lentiviral vector containing leptin (0b) cDNA (Lepvv) or no vector. On day one, control animals daily food intake was 19.9 :1: 2.3 and remained at that level for the duration of the observation period. There were no significant differences between groups during this time period. Fig 6-5 shows the average food intake (mean i 3.13.; g) for the 8 days following administration of either lentiviral vector containing GFP cDNA (GFva), lentiviral vector containing leptin (Ob) cDNA (Lepvv) or no vector. There were no significant differences between groups. Fig 6-6 shows the daily food intake (mean :1: 8.13.; g) for the 1 1 days following the induction ofdiabetes (administration of STZ; 65 mg/kg body weight) or control (citrate). The GFva + Citrate group had a daily food intake of 23.9 i 1.4 at the beginning of the observation period and remained at that level for the duration of the time period. The Lepvv + Citrate group had a daily food intake of 19.7 i 0.7 on day l and remained at that level for the duration of the experiment. On the other hand, the diabetic groups had significantly higher daily food intake for the duration of the time period. The GFva + STZ group had a daily food intake of 18.5 i 1.7 on day one and increased to 36.3 i 2.8 by the end of the time period. On day one, the Lepvv + STZ group had a daily food intake of 18.2 i: 1.1 and increased to 36.8 i 1.5 by the end of the observation period. The diabetic controls (ST Z group) increased the daily food intake from 17.7 d: 0.7 to 42.6 3: 2.2. Fig 6-7 shows the average food intake (mean 5: SE; g) for the 11 days following the induction of diabetes (STZ, 65mg/kg body weight), or control (citrate). The average daily food intake for the Lepvv + Citrate group (19.6 i 0.4) was significantly lower than the GFva + Citrate group 23.2 i 0.9) (p<0.03). The diabetic groups had significantly higher daily food intake than the non-diabetic groups, however, there were no significant differences between diabetic groups. GFPVV GFva Lepvv Lepvv STZ + Cit + STZ + Cit + STZ —~v— ——v—— —e— — e — —El—— 50 " 40 ” Food Intake (9) w 0 1 20— Fig. 6-2. Daily food intake (mean i S.E.; g) of all groups for the 7 day pre-treatment observation period. On day one, control animals daily food intake was 20.1 i 2.7 and remained at that level for the duration of the observation period. There were no significant differences between groups for the pre-treatment period (N=4-7/ group). GFva GFva Lepvv Lepvv STZ +Cit +STZ +Cit +STZ - V//////. :1 [:1 50 7 4o — 3'2 dl :5 g 30 — IL 6 > < fi— 7 - 20 — // 10 A /é Goups Fig. 6-3. Average food intake (mean :t SE; g) for the 7 day pre-treatment observation period. There were no significant differences between groups (N=4- 7/group). 129 GFva GFva Lepvv Lepvv STZ + Cit + STZ + Cit + STZ —v— ——v—— —e— —e— —B—— 50 — 4o — § 0) x g 30 — .5 O 0 LI. 20 - 10 l l L l l l l l 1 2 3 4 5 6 7 8 Days Fig. 6-4. Daily food intake (mean i S.E.; g) for the 8 days following administration of either lentiviral vector containing GFP cDNA (GFva), lentiviral vector containing leptin (Ob) cDNA (Lepvv) or no vector. There were no significant differences between groups (N=4-7/group). 130 GFva GFva Lepvv Lepvv STZ + Cit + STZ + Cit + STZ - V////A VIA 1:1 1: Ave. Food Intake (9) a O 1 10 Goups Fig. 6—5. Average food intake (mean :t SE; g) for the 8 days following administration of either lentiviral vector containing GF P cDNA (GFva), lentiviral vector containing leptin (0b) cDNA (Lepvv) or no vector. There were no significant differences between groups (N=4-7/group). GFva GFva Lepvv Lepvv STZ + Cit + STZ + Cit + STZ —V— ——v—— —9 ~ — e — —B—— 50 " T f 40 T i T ;/ i $71 :> T,/ "(la/4% / /‘ I a ,0 l // /.’ x/ I/ O 30 — ”/Z’Tr/ / / Food Intake (9) l l l l l l l I 10111 1234567891011 [hys Fig. 6-6. Daily food intake for the 11 days following the induction of diabetes or control. Food intake in the Lepvv + Citrate group was lower than the GFva + Citrate group, but did not reach significance. The STZ-treated groups had significantly higher daily food intake than the citrate treated groups (*p<0.0001). There was a trend showing that the Lepvv + STZ group had lower daily food intake than both the GFva + STZ group and the diabetic controls (STZ), however, this did not reach significance (N =4-7/group). 132 GFva GFPw Lepvv Lepvv STZ + Cit + STZ +Cit + STZ - V////A 1:1 I: 50 r * 4o - | I 1'7 g a 2 a a '§ 30 r n5 . > n: < . .3 20 " W7 Goups Fig. 6—7. Average food intake for the 11 days following the induction of diabetes (STZ) or control (cit). The average daily food intake for the Lepvv + Citrate group (19.6 i 0.4) was significantly lower than the GFva + Citrate group (23.2 i: 0.9) (* p<0.03). The STZ-treated groups had significantly higher daily food intake than the citrate-treated groups (a p<0.0001), however, there were no significant differences between STZ-treated groups (N=4-7/group). 133 .A‘i-‘J it Water Intake Changes in water intake over the entire observation period are shown in Figs. 6—8 through 6-13. Fig 6-8 shows the daily water intake (mean i 8.13.; ml) ofall groups for the 7 day pre-treatment observation period. On day one, control animals daily water intake was 38.8 :t 6.3 and remained at that level for the duration of the observation period. There were no significant differences between groups for the pre-treatment period. Fig 6—9 shows the average water intake (mean i 8.13.; ml) for the 7 day pre- treatment observation period. There were no significant differences between groups. Fig 6-10 shows the daily water intake (mean i S.E.; ml) for the 8 days following administration of either lentiviral vector containing OF P cDNA (GFPVV), lentiviral vector containing leptin (01)) cDNA (Lepvv) or no vector. On day one, control animals daily water intake was 30.0 :1: 5.0 and increased to 43.8 :1: 2.4 by the end of the observation period. Both Lepvv groups had lower daily water intake than the GFva and STZ treated groups (p<0.0005). Fig 6-11 shows the average water intake (mean t SE; ml) for the 8 days following administration of either lentiviral vector containing OF P cDNA (GFva), lentiviral vector containing leptin (Ob) cDNA (Lepvv) or no vector. The Lepvv + Citrate group had significantly lower daily water intake (32.9 i2) than the GFva + Citrate group (42.5 3:09) (p < 0.0001). In addition, daily water intake ofthe Lepvv + STZ group (33.4 i1.4) was significantly lower than the GFva + STZ group (38.8 :t 1.1; p < 0.005) and the diabetic control group (STZ, 39.5 i 1.5; p < 0.003). Fig 6-12 shows the daily water intake (mean :1: SE; ml) for the 11 days following the induction of diabetes (administration of STZ; 65 mg/kg body weight) or 134 control (citrate). There was a trend showing that the Lepvv + Citrate group had lower daily water intake than the GFva + Citrate group (34.0 i 2.4 vs. 46.4 i 2.4) but did not reach significance. Both groups remained at that level for the duration of the observation period. On the other hand, the diabetic (STZ-treated) groups had significantly higher daily water intake than non-diabetic (citrate-treated) animals for the duration ofthe observation period (p<0.0001). The GFva + STZ group had a daily water intake of98.3 i 8.3 on day one and increased to 215.0 :t 7.3 by the end of the time period and was not different than the diabetic controls (STZ group) (106.3 d: 5.5 to 217.5 :1: 10.3). The Lepvv + STZ treated group daily water intake increased from 97.1 :1: 4.1 to 192.9 d: 9.7 and was significantly lower than the GFva + STZ and STZ treated groups beginning on day 7 post-STZ administration (p<0.05). Fig 6-13 shows the average water intake (mean 1: SE; ml) for the 1 1 days following the induction of diabetes (STZ, 65mg/kg body weight), or control (citrate). There were no significant differences between non-diabetic groups, however, the average daily water intake of the Lepvv + STZ group (147.2 i 4.8) was significantly lower than the GFva + STZ group (164.9 :t 6.5; p < 0.005) and the STZ group ( 169.3 3: 4; p < 0.003). 135 GFva GFva LCPVV Lepvv STZ + Cit + STZ + Cit + STZ —v— ——v—— —€»-— — e — —El—- 250 " 225 r“ r 200 / 50 r I'\ Waterlntake (ml) .b O I Fig. 6-8. Daily water intake (mean :t 8.13.; ml) of all groups for the 7 day pre- treatment observation period. On day one, control animals daily water intake was 38.8 i: 6.3 and remained at that level for the duration of the observation period. There were no significant differences between groups for the pre-treatment period (N=4- 7/ group). 136 .1?“- GFva GFva Lepvv Lepvv STZ + Cit + srz + Cit + STZ - 7//////. E 1__J 250— 225 ” 200 "’— A ve. Water Intake (ml) 0'! O ‘r‘ 20” A 1O Fig. 6-9. Average water intake (mean t SE; ml) for the 7 day pre-treatment observation period. There were no significant differences between groups (N=4- 7/group). 137 GFva GFva Lepvv Lepvv STZ + Cit + STZ + Cit + STZ -—Ja—— ——v—— ——£%~— —-e-— ——EF— 250 " 225 — 200 4 Water Intake (m1) Fig. 6-10. Daily water intake for the 8 days following administration of either lentiviral vector containing GFP cDNA (GFva), lentiviral vector containing leptin (0b) cDNA (Lepvv) or no vector. On day one, control animals daily water intake was 30.0 i 5.0 and increased to 43.8 i 2.4 by the end of the observation period. Both Lepvv groups had lower daily water intake than the GFva and STZ treated groups (*p<0.0005) (N=4-7/group). 138 GFva GFva Lepvv Lepvv STZ + Cit + STZ + Cit + STZ - V////A VIA I: :1 250 " 225 ‘ 200 J‘ 50 Ave. Waterlntake (ml) Goups Fig. 6-11. Average water intake for the 8 days following administration of either lentiviral vector containing GF P cDNA (GFPW), lentiviral vector containing leptin (0b) cDNA (Lepw) or no vector (STZ). The Lepvv + Citrate group and the Lepvv + STZ group had significantly lower daily water intake than all other groups (* p < 0.007) and were not difi‘erent from each other (N=4-7/group). 139 G Fva GFva Lepvv Lepvv STZ + STZ + Cit + Cit --v—— —e-— — e — —&— T F-Vi’“ wax—r13“ 1/% x T T E [,1 /..— 4 I //V/ T / x/ .’ . / / /‘.f’ T / / i/ 7% __ / ' a: ;/-r / / 2/7 /0/ B / +Cit __V_. 250 r 200 r E 150 — a) x 53 E i// a: / *5 100 "‘ ’/ 3 50 — 0 l 234567891011 Days Fig. 6-12. Daily water intake for the 1 1 days following the induction of diabetes (STZ) or control (Cit). The diabetic (STZ-treated) groups had significantly higher daily water intake compared to non-diabetic (citrate-treated) groups (#p<0.0001). The Lepvv + STZ treated group had lower daily water intake than the GFva + STZ and the STZ treated groups (*p<0.05) as shown (N=4-7/group). 140 GFva GFva Lepvv Lepvv STZ + Cit + STZ + Cit + STZ - 7////A VIA 1:1 1:] 250 ” 190 ’ E e» x E h 130 “ 45 d > < 70 — 1O Fig. 6-13. Average water intake for the 11 days following the induction of diabetes (STZ), or control (Cit). The non-diabetic groups (GFva + Cit and Lepvv + Cit) had significantly lower water intake than the diabetic groups (STZ-treated) (*p<0.0001). There were no significant differences between non-diabetic groups, however, the average daily water intake of the Lepvv + STZ group was significantly lower than the GFva + STZ group (# p < 0.005) and the STZ group (@ p < 0.003). 141 Body I'll’eig/zt F igurcs 6-14 through 6-19 show the % body weight change (mean :1: SE; %) for the different groups for the entire experimental period. Fig 6-14 shows the 0/o body weight change (mean 2+: S.E.; 0/o) for all groups for the pre-treatment observation period. The GFva + citrate group % body weight change increased from 1.4 i 0.4 on day 1 to 9.1 i 0.6 on day 7 and was not significantly different from any other group. Fig 6-15 shows the average % body weight change (mean i S.E.; %) for all groups for the pre-treatment observation period. The GFva + citrate group average % body weight change for the entire pre-treatment observation period was 6.2 i 0.5 and was not significantly different from any other group. Fig 6—16 shows the 0/o body weight change (mean i: S.E.; %) for all groups for the 9 days following the administration of either lentiviral vector containing GFP cDNA (GFva) or lentiviral vector containing leptin (Ob) cDNA (Lepvv) or no vector (STZ). The GFva + citrate group % body weight change increased from 10.6 :1: 1.1 on day 1 to 20.0 i 1.5 on day 9 and was not significantly different from any other group, however, there was a trend showing the Lepvv groups % body weight change was lower (9.2 :1: 0.7 on day 1 to 18.1 i 0.6 on day 9) but did not reach significance. Fig 6-17 shows the average % body weight change (mean i S.E.; %) for all groups for the 9 days following the administration of either lentiviral vector containing GFP cDNA (GFva) or lentiviral vector containing leptin (Ob) cDNA (Lepvv) or no vector (STZ). The GFl’vv + citrate group average % body weight change for the 9 day period was 15.2 i 1.1 and was not significantly different from any other group. however, there was a trend showing the Lepvv groups average % body weight change was lower (13.3 i 0.5) but did not reach significance. Fig 6-18 shows the 0/o body weight change (mean :1: SE; °/o) for all groups for the 12 days following the administration ofeither STZ (65 mg/kg body weight) or vehicle (citrate). The GFva + citrate group % body weight change increased from 0.8 :1: 0.6 on day l to 8.4 i 1.0 on day 12 and was not significantly different from the Lepvv + citrate group. On the other hand, all diabetic groups (STZ treated) lost ,Fum; fl! weight. There was no significant difference between diabetic groups, however there was a trend showing the Lepvv + STZ group % body weight change (6.6 i 0.8 to 1 1 i 2.6) was less than both the GFva + STZ (7.9 :1: 0.9 to 15.0 i 2.4) and STZ (9.6 :1: 0.7 to 16.4 i 3.4) treated groups although significance was not reached. Fig 6-19 shows the average % body weight change (mean i S.E.; %) for all groups for the 12 days following the administration of either STZ (65 mg/kg body weight) or vehicle (citrate). The GFva + citrate group average % body weight change was 4.3 i 0.6 and was not significantly different from the Lepvv + citrate group (4.7 :t 0.7). All diabetic groups (STZ treated) lost weight, however, there was no significant difference between groups. Note, there was a trend showing the Lepvv + STZ group average % body weight change (9.3 a: 1.5) was less than both the GFva + STZ (1 1.8 :1: 1.6) and STZ (13.6 i 2.1) treated groups although significance was not reached. 143 G F va GFva Lepvv LCPVV STZ + Cit + srz + Cit + STZ —v—— ——v—— ——-e—~— — e — ——E}—— 20 — 15 — (D m C N .C 0 E I m >— g 10 //§ >5 1: o m o\° 5 .— 0 I l I 1 l l 1 2 3 4 5 6 Days Fig. 6-14. The % body weight change (mean i S.E.; %) for all groups for the pre- treatment observation period. The GFva + citrate group % body weight change increased from 1.4 i 0.4 on day 1 to 9.1 i 0.6 on day 7 and was not significantly different from any other group (N=4-7/ group). 144 GFva GFva Lerv Lepvv STZ +Cit +STZ +Cit +STZ - V//////. I: I: 20 _ 10 * Ave. % Body Weight C hange O Groups Fig. 6-15. The average % body weight change for the pre-treatment observation period for all groups. There were no significant differences between any group (N=4- 7/group). 145 "'1 #r-t ' GFPVV GFva Lepvv Lepvv STZ + Cit + STZ + Cit + STZ —v—— -—v—— ——~e-— — e — —E}—- °/o B ody Weight C hange a I Fig. 6-16. The% body weight change for all groups for the 9 days following the administration of either lentiviral vector containing OF P cDNA (GFva) or lentiviral vector containing leptin (Ob) cDNA (Lepvv) or no vector (STZ). There was no significant difference between groups, however note the trend showing Lepvv groups had lower % body weight change than the GFva and STZ groups (N=4-7/group). 146 GFva GFva Lepvv Lepvv STZ + Cit + STZ + Cit + STZ - V////A 1:: 1:1 .3 O l s \\\i1 Z Ave. % Body Weight C hange O L O I Goups Fig. 6-17. The average % body weight change for all groups for the 9 days following the administration of either lentiviral vector containing GFP cDNA (GFva) or lentiviral vector containing leptin (Ob) cDNA (Lepw) or no vector (STZ). There was no significant difference between groups, however note the trend showing Lepvv groups had lower average % body weight change than the GFva and STZ groups (N=4-7/group). 147 GF va GFva Lepvv Lepvv STZ + Cit + STZ + Cit + STZ ——v— ——v—— —e-— -— e — —-El—— 20 " 10 ’— OJ 5" C (U .C U H 5: g 0 >5 '5 o _7_ m i" “i \‘r‘ T °\° "T**\T*TTTI —r- \'\\ \.\ \ T -10 ”Rfl:§r\\ O~ —O— * l l \ \\ E T ‘.~ a—— —O \ K —‘—_ \ B‘\B\\;‘x;\‘\ _‘ T Fee‘s; i \Bxfl _zolllll|llllll 123456789101112 Days Fig. 6-18. The % body weight change for all groups for the 12 days following the administration of either STZ (65 mg/kg body weight) or vehicle (citrate). The GFva + Cit group % body weight change was not significantly different from the Lepvv + Cit group. All diabetic groups lost weight, but there was no significant difference between groups. There was, however, a trend showing the Lepvv + STZ group % body weight change was less than both the GFva + STZ and the STZ treated groups (N=4-7/group). 148 GFva GFva Lepvv Lepvv STZ + Cit + STZ + Cit + STZ - m 1: 1:1 207 10” Ave. % Body Weight C hange O Goups Fig. 6-19. The average % body weight change for all groups for the 12 days following the administration of either STZ (65 mg/kg body weight) or vehicle (citrate). The GFva + Cit group average % body weight change was not significantly different from the Lepvv + Cit group. There was no significant difference between STZ treated groups, however, there was a trend showing the Lepvv + STZ group average % body weight change was less than both the GFva + STZ and STZ treated groups (N =4-7/group). 149 Norepi)rep/nine Concentration in the PVN Fig. 6-20 shows NE concentration (pg/pg protein; :t SE.) in the PVN following administration oflentiviral vector containing GFP cDNA (GFPW), leptin cDNA (Lepvv) or no vector, and either STZ (65 mg/kg BW) or vehicle (Citrate). The Lepvv + Citrate group (N = 5) had a NE concentration of 15.5 i 1.3, which was significantly lower than all other groups (p<0.03). The GFva + citrate treated group ( N = 7), which had a NE concentration of 29.9 i 2.9 was significantly different from the GFva + STZ, Lepvv + citrate and the STZ treated groups (p<0.03). The GFva + STZ treated group had a NE concentration of 45.6 :t 8.0 and was significantly different than the GFva + citrate, Lepvv + citrate and the Lepvv + STZ treated groups (p<0.02). The Lepvv + STZ group (N = 7) NE concentration was 21.1 i 2.9, which was significantly lower that the GFva + STZ group (N = 5) (45.6 :1: 8.0; p = 0.0003) and the STZ group (N = 4) (50.0 i 7.0; p = 0.0003). 150 GFva GFva Lepvv Lepvv srz +Cit +STZ +Cit +STZ - V////A 1:1 :1 60 r b 48 ” .15 3 g 36 7 a O) E V # 3 _ o 1.1.! 9: Z /_I_ 12— // 0 //a /A Groups Fig. 6-20 NE concentration the PVN following administration of lentiviral vector containing GFP cDNA (GFPW), leptin cDNA (Lepvv) or no vector, and either STZ or vehicle (Cit). The Lepvv + Cit group was significantly lower than all other groups (*p<0.03). The GFva + Cit treated group was significantly different from the GFva + STZ, Lepvv + Cit and the STZ treated groups (a p<0.03). The GFva + STZ treated group was significantly different than the GFva + Cit, Lepvv + Cit and the Lepvv + STZ treated groups (b p<0.02). The Lepvv + STZ group was significantly lower that the GFva + STZ group and the STZ group (# p = 0.0003). 151 Nm'epineplzrine Concentration in the DMD Fig. 6-21 shows NE concentration (pg/pg protein; i: SE.) in the DMD following administration of lentiviral vector containing GFP cDNA (GFPW), leptin cDNA (Lepvv) or no vector, and either STZ (65 mg/kg BW) or vehicle (Citrate). The Lepvv + Citrate group (N = 5) had a NE concentration of 7.9 i 1.0, which was significantly lower than the GFva + Citrate group (N = 7), which had a NE concentration of29.4 i 6.5 and the GFva + STZ treated group (p < 0.002). The Lepvv + STZ group (N = 7) had a NE concentration of 15.2 i 5.2, which was significantly lower than the GFva + Citrate treated group (p<0.02) and note the trend showing that the Lepvv + STZ treated group was lower than the GFva + STZ group (25.3 i 1.2; N=5) and the STZ group (19.6 i 1.3; N=4) but did not reach 5 ignificance. GFva GFva Lepvv Lepvv + Cit + srz + Cit + STZ STZ - 7////A :3 I: 45” 36" E E27— m 3 E 0 518- 0 m z * 9’ “‘1— 7 0 2/1 Goups Fig. 6-21. NE concentration in the DMD following administration of lentiviral vector containing GF P cDNA (GFva), leptin cDNA (Lepw) or no vector, and either STZ or vehicle (Cit). The Lepvv + Cit group was significantly lower than the GFva + Cit and the GFva + STZ treated groups (* p < 0.002). The Lepvv + STZ group was significantly lower than the GFva + Cit treated group (# p<0.02) also note the trend showing that the Lepvv + STZ treated group was lower than the GFva + STZ and STZ treated groups but did not reach significance. 153 Norepincplzrine Concentt'utirm in the U] Fig. 6-22 shows NE concentration (pg/pg protein; :1: SE.) in the LH following administration of lentiviral vector containing GFP cDNA (GFva), leptin cDNA (Lepvv) or no vector, and either STZ (65 mg/kg BW) or vehicle (Citrate). There were no significant differences between groups (N = 4-7/group). 154 GFva GFva Lepvv Lepvv STZ + Cit + STZ + Cit + STZ - m 1:] 1:1 30— 24* E ," l , an Fig. 6-22. NE concentration (pg/ pg protein; i SE.) in the LH following administration of lentiviral vector containing GFP cDNA (GFPW), leptin cDNA (Lepvv) or no vector, and either STZ (65 mg/kg BW) or vehicle (Citrate). There were no significant differences among groups (N = 4-7). 15 £11 Norepittephrine Concentration in the VMH Fig. 6-23 shows NE concentration (pg/pg protein; :t SE.) in the VMII following administration of lentiviral vector containing GFP cDNA (GFva), leptin cDNA (Lepvv) or no vector, and either STZ (65 mg/kg BW) or vehicle (Citrate). There were no significant differences between groups, although the Lepvv + Cit was lower than the GFva + Cit (20.5 i 4.6 compared to 22.6 i 2.9) and the Lepvv + STZ (26.2 i 4.4) was lower than the GFl’vv + STZ (34.4 i 5.6) and the STZ treated animals (28.8 i 1.6) (N = 4-7/group). GFva GFva Lepvv Lepvv srz + Cit + STZ + Cit + STZ - V////A [:1 1:] 50— 40- —— E a g 30- 01 i __ E 20— w 3 7% ”.1 z / 1o~ o A /fl Goups Fig. 6-23. NE concentration (pg/ pg protein; i SE.) in the VMH following administration of lentiviral vector containing GFP cDNA (GFPW), leptin cDNA (Lepvv) or no vector, and either STZ (65 mg/kg BW) or vehicle (Citrate). There were no significant differences between groups, although the Lepvv + Cit group was lower than the GFva + Cit group, and the Lepvv + STZ group was lower than the GFva + STZ and STZ treated animals (N = 4-7/group). I 7 kl! i\"orepinephrine (.‘oncentrution in the I lippocumpus Fig. 6-24 shows NE concentration (pg/pg protein; at SE.) in the Hippocampus following administration of lentiviral vector containing GFP cDNA (GFva), leptin cDNA (Lepvv) or no vector. and either STZ (65 mg/kg BW) or vehicle (Citrate). There were no significant differences between groups (N = 4-7). 158 GFva GFva Lepvv Lepvv STZ +Cit +STZ +Cit +STZ - V////A :1 [:1 9 _ 6 _ .II' 'I' ‘- ,A' 1.", ‘7‘ -.- ,II'. I -. r l‘hxr‘l r f4 . . ‘ 'v' '1' V ‘. .1. ‘ ‘1. .a . I: R“ c ‘. . er... .stttstifrjgxte '3 13;. '1.) .’ _I __.‘bQ :I _'. .1 ' ‘ 2* In: ' " Tc 'fi’f"‘.l. r - ..‘,'__¥o. ~32 s‘uu' - 'zlf'.,i’.-I'E.II~’J1:‘ 3'3-- - N E conc (pglug protein) % Fig. 6-24. NE concentration (pg/ pg protein; i SE.) in the Hippocampus following administration of lentiviral vector containing GFP cDNA (GFPW), leptin cDNA (Lepw) or no vector, and either STZ (65 mg/kg BW) or vehicle (Citrate) (N= 4-7). 159 Norepirrep/nine Concentration in the Cortex Fig. 6—25 shows NE concentration (pg/pg protein; :2 SE.) in the Cortex following administration of lentiviral vector containing GFP cDNA (GFva), leptin cDNA (Lepvv) or no vector, and either STZ (65 mg/kg BW) or vehicle (Citrate). There were no significant differences between groups (N = 4-7). 160 GFva GFva LePYV Lepvv srz + Cit + STZ + Cit + STZ - W 7]]; 1:1 1:1 10* ,- g __ a /—- 2— 7/ Goups Fig. 6-25. NE concentration (pg/ pg protein; i SE.) in the Cortex following administration of lentiviral vector containing GFP cDNA (GFPW), leptin cDNA (Lepvv) or no vector, and either STZ (65 mg/kg BW) or vehicle (Citrate). There were no significant differences between groups (N = 4-7). 161 Serum Corticosterone Serum corticosterone levels for all groups are shown in Fig 6-26. At the time of sacrifice, corticosterone levels (mean i S.E.; ng/ml) in the non-diabetic groups (Lepvv + Cit and GFva + Cit) were significantly lower than the diabetic (STZ treated) groups (p<0.0001). The Lepvv + Citrate group (22.2 i 4.2) was lower than the GFva + Citrate group (91.9 a: 15.1) but did not reach significance. A similar trend persisted in the diabetic groups. The Lepvv + STZ group (302.8 i: 54.8) was lower than the GFva + STZ group (367.3 i 45.8) and the diabetic controls (STZ; 403.6 i 42.2) although did not reach significance (p = 0.06). 162 GFPW GFPW Lepvv Lepvv STZ + Cit + STZ + Cit + STZ - V//////. f: 1:] 500 ' 400 — —— .345} E 300 ~— ii 2 f'. 2 .. i .0 200 ” E O o * 100 — * 0 A// / Groups Fig. 6-26. Serum corticosterone levels for all groups are shown. At the time of sacrifice, corticosterone levels in the in the non-diabetic groups (Lepvv + Cit and GFPW + Cit) were significantly lower than the diabetic (STZ treated) groups (*p<0.0001). The Lepw + Cit group was lower than the GFPW + Cit group but did not reach significance. A similar trend persisted in the diabetic groups. The Lepvv + STZ group was lower than the GFPW + STZ group and the diabetic controls (STZ) but did not reach significance (p = 0.06) (N = 4-7/group). 163 Blood Glucose Levels Mean blood glucose levels (mg/dl; i SE.) at the time of sacrifice are shown in Fig. 6-27. Non-diabetic groups, GFva + Citrate (N = 7) and Lepvv + Citrate (N = 5) had blood glucose levels of 98.6 :t 5.9 and 96.2 i 1.9, respectively, which were significantly lower than diabetic controls (STZ, N = 4; 499.3 i 6.0), Lepvv + STZ (N = 7; 414.0 i 13.3) and GFPW + STZ (N = 7; 513.0 J: 13.5) (p < 0.0001). The blood glucose level ofthe Lepvv + STZ treated group was significantly lower than the diabetic controls (STZ) and the GFPW + STZ treated animals (p < 0.0001). 164 GFva GFva Lepvv Lepvv STZ + Cit + STZ + Cit + STZ - V//////. I: 1:] 600 — 480 — g a» E. 360 — Q, 8 0 2 to '§ 240 - E 120 — # # 7% o a /// Groups Fig. 6-27. Mean blood glucose levels (mg/d1; :t SE.) at the time of sacrifice are shown. Non-diabetic groups (GFPW + Cit and Lepvv + Cit) had blood glucose levels significantly lower than diabetic controls (STZ), Lepvv + STZ and GFPW + STZ (# p < 0.0001). The blood glucose level of the Lepvv + STZ group was significantly lower than the diabetic controls (STZ) and GFva + STZ treated groups (* p < 0.0001) (N = 4-7/group). 165 Serum Leptin Levels Serum leptin levels (mean i S.E.; ng/ml), at the time of sacrifice, are shown in Fig. 6-28. Lepvv + Citrate treated animals (3.74 3: 0.44; N = 7) had significantly higher serum leptin levels than the GFPW + Citrate treated group (2.01 i 0.19; N = 7) (p< 0.001) and both groups were significantly higher than all STZ treated groups (p<0.001). Lepvv + STZ treated animals (0.63 d: 0.05; N = 7) had higher leptin levels than both the Diabetic controls (STZ; N = 4) and GFPW + STZ (N = 7) treated groups (undetectable levels), but did not reach significance. 166 GFva GFPW Lepf'v Lep“ STZ + Cit + STZ + Crt + STZ - m EEC] if % /._ Groups Fig. 6-28. Serum leptin levels (mean i S.E.; ng/ml), at the time of sacrifice, are shown. Lepvv + Citrate animals had significantly higher serum leptin levels than the GF PW + Citrate group (* p< 0.001) and both groups had significantly higher leptin levels than all STZ treated groups (# p<0.001). Lepvv + STZ animals had higher leptin levels than both the Diabetic controls (STZ) and GFva + STZ groups, but did not reach significance. (N=4-7). 167 Serum Insulin Levels Serum insulin levels (mean i 8.15.; ng/ml), at the time of sacrifice, are shown in Fig. 6-29. Lepvv + Citrate treated animals (0.84 i 0.06; N = 7) had significantly higher serum insulin levels than all other groups (p<0.0004). The GFPW + Citrate treated group (0.43 :1: 0.1 1; N = 7) was significantly different than the OFva + Citrate, Lepvv + Citrate and the STZ treated groups (p< 0.007). Lepvv + STZ animals (0.27 :L- 0.05; N = 7) had higher insulin levels than both the Diabetic controls (0.1 i 0.01; N = 4) and GFva + STZ (0.14 i 0.04; N = 7) groups, but did not reach significance. 168 GFPW GFPW Lepvv Lepvv STZ + Cit + srz + Cit + srz - W [:1 [:1 1.257 0.83 — Insulin (nglml) 0.42 0.00 f a Fig. 6-29. Serum insulin levels at the time of sacrifice. The Lepvv + Cit treated animals had significantly higher serum insulin levels than all other groups (*p<0.0004). The GFva + Cit treated group was significantly different than the GFPW + Cit, Lepvv + Cit and the STZ treated groups (#p< 0.007). Lepvv + STZ animals had higher insulin levels than both the STZ and GFPW + STZ treated groups, but did not reach significance (N=4-7/group). 1 O\ 9 E. Discussion The American Diabetes Association estimates that there are currently 20.8 million eo )le, or 7% of the US. 0 )ulation livin with diabetes (wwwdiabetesorg . P 1 P l g - Approximately 1-2 million of these are Type I, or insulin-dependent diabetes mellitus (IDDM) patients. Type I diabetes is thought to result from either an autoimmune disease that develops when the body’s immune system destroys pancreatic beta cells, the only cells in the body that make the hormone insulin or from unknown causes resulting in loss of pancreatic beta cell function. Currently, there are few treatment options available, with exogenous insulin being the major therapy for IDDM patients. Many complications arise from the disease including cardiovascular dysfunction, kidney damage, retinopathy and peripheral neuropathy. In addition, a number of neuroendocrine changes occur such as hyperphagia, polydipsia, hyperglycemia and hyperactivation of the HPA axis. Leptin is likely involved in the central and neuroendocrine dysregulation seen in diabetes as it has been shown to modulate many of these. Leptin in known to inhibit HPA activity in several animal models and we have shown that NE concentrations decrease in the hypothalamus paralleling a decrease in serum corticosterone following leptin administration (chapter 3), and that peripheral administration of leptin to normal (chapter 4) and diabetic (chapter 5) rats results in decreased NE release in the PVN while simultaneously decreasing serum corticosterone. We have found, however, that daily peripheral injections of leptin to diabetic rats does not bring serum leptin levels to that of non—diabetic controls (4), which is likely to be one reason that we see only partial normalization of 170 hyperphagia, pol ydipsia and HPA axis activity. In an attempt to overcome this obstacle, we designed this current study using gene transfer with HIV-1 lentiviral vector encoding rat leptin (Ob) cDNA. A number of other studies have used this approach in an attempt to increase leptin levels. Dhillon et al., showed that central administration of adeno-associated viral vector containing leptin cDNA to normal rats reduces age-related weight gain, adiposity and serum insulin levels (270). While others have shown that a single i.c.v. injection of adenoviral vector encoding leptin reduces body weight and feed intake in both normal and Zuckerfir/fa rats (271). Additionally, much work has been done on leptin gene transfer to diet-induced obese, aged-obese and non-insulin dependent diabetes mellitus (NIDDM) animal models (272-274), however, little work, has been done in insulin-dependent diabetes mellitus. In the current study we used a form of retrovirus, the lentivirus; human immunodeficiency virus type 1 (HIV-1). The biggest advantage of this vector is the fact that it can infect both dividing and nondividing cells. Another major advance is the fact that it can be pseudotyped with the envelope of other viruses such as the G envelope of vesicular stomatitis virus (VSV-G), which broadens the range of target tissues (189). Other viral vectors are used for gene transfer but have a number of disadvantages. Adenoviral vectors are advantageous because they can be produced in high titers and the life cycle does not normally involve integration into the host genome, which reduces the risk of insertional mutagenesis (175), however, this then means that the therapeutic gene is only transiently active and must be repeatedly administered (176). Additionally, researchers have found that approximately 90% of 171 intravenously administered vector is degraded in the liver by non-immune mechanisms (177). In fact, it has been shown that cerebrospinal fluid leptin levels decrease by 87% after 14 days in rats administered adenoviral vector containing leptin cDNA (271 ). Another vector commonly used is the adeno-associated viruses (AAV), which are non-pathogenic human parvoviruses that are dependant on a helper virus for replication; usually the adenovirus, They are capable of infecting both dividing and non-dividing cells (179), however, these viral vectors are difficult to manufacture in high titer production (180), but probably the most limiting feature of using AAV as viral vectors is the small genetic carrying capacity. In the present study, we hoped to overcome these obstacles by using a lentiviral vector. In the present study transfection of STZ-induced diabetic rats with lentiviral vector encoding leptin cDNA ameliorated many of the neuroendocrine abnormalities associated with diabetes. STZ-induced diabetes results in a significant increase in both feed and water intake with a concomitant decrease in body weight. In this study, Lepvv transfected animals showed decreased feed intake in both the citrate and STZ treated groups. In the citrate treated animals, Lepvv transfection decreased feed intake after 5 days but did not reach significance until 10 days post-transfection. In the STZ treated animals, Lepvv transfection decreased feed intake for the duration of the experiment, but did not reach significance. Lepvv transfection also significantly decreased water intake in both citrate treated and STZ treated animals beginning on day 4 post-transfection. This reduction in water intake was maintained following the induction ofdiabetes and persisted for the duration of the experiment. Lepvv transfection caused a slowing of body weight gain during the pre-diabetic period, 172 however, interestingly, Lepvv transfection was also able to ameliorate the body weight loss associated with STZ-induced diabetes although this did not reach significance. STZ—induced diabetes causes a dramatic decrease in body weight as the pancreatic B-cells are destroyed and the animals are unable to utilize glucose for energy and storage. The transfection with Lepvv was able to slow this wasting process possibly via increased serum insulin, which would allow greater glucose uptake and decrease the need for fatty acid oxidation and protein catabolisrn. Another possibility to consider is that leptin may be acting to maintain body weight within certain limits. It has been shown that with decreased caloric intake, there is a concomitant decrease in metabolic rate in humans and rodents (275, 276). Additionally, central administration of leptin has been shown to attenuate the expected fall in metabolic rate in rodents subject to caloric restriction (277). So, it is quite conceivable that leptin may play a role in resisting fluctuations in body weight due to changes in energy availability by adjusting metabolic rate. Diabetes is characterized by decreased serum insulin levels as well as increased blood glucose. In this study, transfection with lentiviral vector containing leptin cDNA was shown to partially normalize both of these. Lepvv transfection produced an increase in serum insulin levels in both the citrate and STZ treated animals. The Lepvv + citrate group had significantly higher serum insulin than the GFPW + citrate group. The Lepvv + STZ treated animals had higher serum insulin than both the GFva + STZ and the STZ treated groups, although significance was not reached. Lepvv was also shown to partially normalize the hyperglycemia which results from diabetes. At the time of sacrifice, Lepvv + STZ treated animals had 173 significantly lower blood glucose levels than both the GFPW + STZ and the STZ treated animals, however, animals were still hyperglycemic (approximately 400 mg/dl. compared to 500 mg/dl.) STZ-induced diabetes is also characterized by a number ofother endocrine abnormalities including increased NE concentration in the PVN ofthe hypothalamus along with hyperactivation of the HPA axis as shown by increased serum corticosterone. Lepvv transfection caused a significant reduction in NE concentration in the PVN of both citrate treated and STZ treated animals. Simultaneously, Lepvv transfection decreased serum corticosterone of both citrate and STZ treated animals (p=0.06). This reduction in HPA axis activity is ofgreat importance as chronic hyperactivation of the stress axis is known to produce a myriad of deleterious effects which may further compromise the health of diabetic patients. In this study, transfection with lentiviral vector containing leptin cDNA partially normalized all the central and endocrine abnormalities associated with STZ- induced diabetes. One possibility for only partial resolution of these is the fact that serum leptin levels of diabetic animals were not brought to that of non-diabetic animals. Transfection with Lepvv produced an increase in serum leptin levels of both citrate and STZ treated animals but significance was reached only in the citrate treated groups. It is possible that the decreased weight loss in the Lepvv + STZ treated animals maintained a greater percentage of adiposity, thus natural leptin producing ability, however, this was not able to keep up with the severe diabetes that resulted with the dose of STZ used (65 mg/kg body weight). Future studies may investigate the central and neuroendocrine normalizing effects of leptin lentiviral 174 gene transfer in a less severe form ofdiabetes. This might produce more clinically relevant results as most type 1 diabetic patients do not maintain blood glucose levels in the 4003 on a regular basis. Besides leptin, the lack of insulin in the STZ treated. animals could be another contributing factor for the failure of Lepvv to completely reverse the effects of STZ. Although Lepvv transfection did increase insulin levels. they did not reach the levels ofcontrol animals. Therefore. a combination of leptin and insulin gene transfer therapy may help to totally reverse the effects of diabetes. Leptin gene therapy could also reduce the dependence of diabetics on insulin. This needs further investigation. 175 F. Summary Diabetes produces a number ofcentral and endocrine abnormalities including hyperphagia, polydipsia, weight loss, decreased serum insulin and leptin, increased blood glucose, increased hypothalamic noradrenergic activity and hyperactivation of the HPA axis. The study in this chapter was designed to investigate whether or not transfection ofdiabetic rats with lentiviral vector containing leptin (01)) cDNA could normalize serum leptin levels and the neuroendocrine dysfunction seen in the disease. We have found that leptin lentiviral vector gene transfer to STZ-induced diabetic rats was able to partially normalize all central and neuroendocrine abnormalities studied and with further investigation may provide either an alternative or adjunctive treatment for the disease. 176 Chapter 7. Summary and Conclusions The American Diabetes Association estimated the total annual economic cost of diabetes in 2002 was over $132 billion dollars and represents greater than 1 1% of the US health care expenditure. They estimate that nearly 21 million people or 7% of the US population is currently living with the disease (wwwdiabctesorg). In addition to the life-long complications experienced by patients with diabetes, this is a huge economic burden and any research or further understanding may provide alternative therapy or cure for the chronic debilitating disease. The goal ofthe research described in this dissertation was to investigate the role the adipocyte-derivcd hormone, leptin, plays in regulating the hypothalamic monoamines, particularly NE, in the pathophysiology of the hyperactivation of the stress axis seen in diabetes. To do this, a variety of techniques were employed and in vivo experiments were conducted. To begin with, we wanted to determine if indeed leptin affects the hypothalamic monoamines, and possibly HPA activity. Chapter 3 describes the experiment whereby we showed that both central and peripheral administration of leptin produces significant changes in hypothalamic monoamines in a region specific manner, including a reduction in NE concentration in the PVN of the hypothalamus along with a simultaneous decrease in serum corticosterone. This study showed for the first time that systemic leptin administration is able to produce region-specific changes in brain monoamines, thus may mediate leptin’s central and endocrine effects including the suppression ofHPA axis activity. 177 Following the observation that leptin caused a significant decrease in NE concentration in the PVN. we wanted to study the exact time at which this occurs after leptin treatment and the duration of mechanism of leptin’s actions. To do this, we designed a push-pull perfusion study with simultaneous blood sample collection as described in chapter 4. Additionally, we investigated the mechanism by which leptin may mediate its effects by administering alpha and beta adrenergie agonists. Results from this study indicate that systemic administration of leptin causes a decrease in NE release in the PVN while simultaneously decreasing serum corticosterone. We then showed that this effect was blocked by the a-adrenergic agonist clonidine and not by the [3-adrenergic agonist isoproterenol. At this time. the mechanism by which clonidine was able to produce this effect is not clear. We have considered the possibility that the dose ofclonidine we used was high enough to produce agonist effects at a I adrenergic receptors, thus negating leptins suppressive effects on II PA axis activity. It is also possible that leptin acted at pre-synaptic a2 adrenergic receptors on GABA neurons, thereby, decreasing GABAergic inhibitory input to the hypothalamic CRH neurons. Further investigation will be needed to elucidate the mechanism by which clonidine was able to produce this effect. In summary, the results described in chapter 4 indicate that leptin decreases hypothalamic noradrenergic activity. thus HPA activity and that this effect is most probably mediated via alpha-adrenergie receptors. Our next step was to investigate leptins role in a diabetic model. It had been shown that in experimentally induced diabetes, there is an increase in NE content in the hypothalamus (4, 5, 165) and that this is reversed by parenteral injection ofleptin 178 (4). And, since it is known that serum leptin levels decrease in diabetes (7, 8) we wanted to test the hypothesis that this decrease in circulating leptin levels in diabetes contributes to the elevated noradrenergic activity in the PVN and thus the HPA axis activity. As described in chapter 5, we found that a single i.p. injection of leptin to STZ-induced diabetic animals caused a significant reduction in NE release in the PVN along with a dramatic decrease in serum corticosterone and this was reversed by the alpha adrenergie agonist clonidine but not the beta adrenergie agonist isoproterenol. This supported out hypothesis that noradrenergic activity in the PVN plays an important role in mediating leptin’s suppressive effects on IIPA axis activity, and since leptin levels are significantly reduced in diabetes, it is likely that it is responsible for the hyperactivation of the stress axis. Since we had found that not only are the hypothalamic monoamines affected by central and peripheral administration of leptin, but that NE in the PVN specifically is decreased by leptin administration. And that this decrease in NE is accompanied by a simultaneous decrease in serum corticosterone in both normal and diabetic animals, we wanted to conduct a gene transfer study to see if transfection with lentiviral vector containing leptin cDNA could normalize the many neuroendocrine abnormalities seen in diabetes including the elevated hypothalamic NE content and the hyperactivation of the HPA axis activity. Indeed we found that leptin gene transfer ameliorated all central and neuroendocrine abnormalities investigated including, hyperphagia, polydipsia, body weight loss, hyperglycemia, hypoinsulinemia, hypoleptinemia, increased hypothalamic NE concentration and hyperactivation of the stress axis. It is important to note that in this study these 179 abnormalities were not completely normalized to non-diabetic levels. One possible reason is the severe form of diabetes produced by the dose of STZ used. 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