Background: Endothelial dysfunction is a key factor in the development of vascular complications in insulin resistance and diabetes and recent studies have established that endothelium-derived hyperpolarising factor (EDHF) plays an important physiological role in endothelium-derived relaxation responses, especially in small arteries and arterioles. Objective: This project aimed to identify the role of, and characterise, EDHF in animal models of insulin resistance, including the obese Zucker rat (OZR) as well as the fructose-fed (FF) Sprague-Dawley rat. Methods: Vascular function was studied in third-order mesenteric arteries from male and female Zucker rats using pressure myography, and in lobar arteries from male FF rats using wire myography. Endothelial function was determined by studying responses to the endothelium-dependent dilator acetylcholine (ACh) and the endothelium-independent dilator levcromakalim in the presence of a variety of inhibitors to study the roles of NO, EDHF and gap junctions. The effect of insulin resistance on gap junctions was further assessed by measuring the protein and mRNA expression of vascular connexins. Protein levels were determined by western blotting followed by semi-quantitative analysis of band intensity, whilst mRNA levels were quantified using real-time PCR, in which beta-actin was used as the housekeeping gene. Results: Metabolic parameter comparisons confirmed that male OZRs were type 2 diabetic, whilst female OZRs were insulin resistant. Responses to ACh were reduced in both the male and female OZRs compared with their gender controls, with the male OZR showing a greater degree of endothelial dysfunction. In all Zucker third-order mesenteric arteries, inhibition of NO had no effect; however inhibitors of EDHF abolished relaxation responses to ACh. Inhibitors of gap junctions associated with connexin 40 significantly (p less than 0.05, Student's t-test) attenuated the maximal response to ACh in the LZR, but had no effect in the OZR. Comparison of Western blot band intensity indicated that connexin 40 protein levels in mesenteric vascular homogenates in the OZR were significantly smaller (p less than 0.05, Student's t-test) than in the LZR, with no difference in connexin 43 protein levels. mRNA levels showed a significant (p less than 0.05, Student's t-test) decrease in connexin 40 expression in the OZR compar ed with the LZR, with no change in connexin 43 mRNA expression. Although FF rats did develop insulin resistance, responses to ACh were not altered in the FF rats as compared with their controls, and ACh responses were abolished by NO inhibitors. Conclusion: The findings presented in this thesis demonstrate that endothelial dysfunction is present in third-order mesenteric arteries from insulin-resistant female and type 2 diabetic male OZRs, and is associated with a defect in EDHF. However, endothelial function was not compromised in the insulin-resistant FF rats. Furthermore, the reduction in EDHF-mediated vasodilatation in the mesenteric arteries from female OZRs was associated with the functional absence of connexin 40-related gap junctions as well as a reduction in connexin 40 protein and mRNA levels. This novel finding suggests that gap junctions associated with connexin 40 may be a potential therapeutic target for diabetic vascular disease.

Includes bibliographical references (leaves 291-311) / xxiii, 313 leaves : ill. (chiefy col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Molecular Biosciences, 2002

Includes bibliographical references (43 leaves) This study shows that when LR3IGF-I is administered to animals in pharmacologically active doses, it may be present in either the free form or bound to IGF-binding protein(s) in the circulation. Age and nutrition which are factors that regulate synthesis of endogenous IGF-I and IGF-binding proteins, affect the in vivo formation of complexes between the analog and IGFBP(s). This study also suggests that IGFBP-1 inhibits the pharmacological activity of circulating LR3IGF-I on thymus whereas it appears to stimulate the pharmacological activity of LR3IGF-I in kidneys.

A novel reverse microemulsion (RM) mediated synthesis of glucose-responsive nanoparticles was developed for controlled insulin delivery. Nanoparticles were constructed using a model system comprised of dextran, poly(α-1,6 glucose), physically crosslinked with the tetrafunctional glucose-binding protein, Con A. A rapid-screening technique was used to quantify RM phase behavior in the presence of dextran, Con A and insulin. The extent of the RM existence region diminishes with increasing dextran and Con A concentrations and with increasing dextran molecular weight. Crosslinking efficiency between Con A and fluorescein isothiocyanate dextran (FITC-Dex) was found to depend on the total concentration of Con A as well as the ratio of Con A to FITC-Dex. Functionalizing dextran with higher affinity mannose ligands and increasing dextran molecular weight both improved crosslinking efficiency. The nanoparticles dissolved when dispersed in buffered saline solutions containing elevated glucose concentrations and were most responsive within the physiological range. Finally, insulin was encapsulated in select formulations and found to release preferentially at these elevated glucose concentrations. / Singapore-MIT Alliance (SMA)

Metabolic homeostasis is orchestrated by the hypothalamus through the neuroendocrine and the autonomic nervous systems. The hypothalamic nuclei respond to the peptide leptin secreted from adipose tissue to suppress feeding and increase energy expenditure by promoting fat metabolism via sympathetic activity. Another important, but perhaps less appreciated function of central leptin signaling is to elevate peripheral insulin sensitivity. Environmental and genetic risk factors that affect hypothalamic leptin signaling can lead to obesity and type 2 diabetes mellitus (T2DM). Here, we discovered that LIM domain only 4, LMO4, is a novel protein participating in central leptin signaling. In a process strikingly similar to T2DM in humans, CaMKIIα-Cre;LMO4flox/flox mice, which have LMO4 knocked out in the postnatal brain including the hypothalamus, develop visceral adiposity, reduced insulin sensitivity, obesity and diabetes when fed with regular chow. Central leptin signaling was significantly lost in key hypothalamic nuclei of mutant mice. Caloric restriction prevents obesity but not insulin resistance in these mice. Taken together, our results suggest that LMO4 function in the brain is required for central leptin signaling to control fat metabolism and peripheral insulin sensitivity.

Atherosclerosis is a leading cause of morbidity and mortality worldwide and type 2 diabetes and obesity-associated metabolic syndrome, both characterized by insulin resistance, are potent risk factors. These conditions also increase the risk for restenosis after revascularization procedures used for treatment of atherosclerosis. Studies have shown that insulin and resveratrol (RSV), a red wine polyphenol, decrease neointimal growth after vessel injury in models of restenosis, demonstrating a protective effect on the vasculature. However, oral glucose and sucrose were used in insulin studies to maintain normoglycemia, and their effect on neointimal formation was not assessed. Several studies have shown that nitric oxide (NO) production is stimulated by insulin and RSV, and since NO can decrease neointimal growth, the objective of this thesis was to address the mechanism of action of insulin or RSV to protect against restenosis, and determine whether NO production mediates these effects. To examine this, we treated rats with insulin or RSV and performed arterial balloon injury. In Study 1, insulin reduced neointimal area after injury in rats receiving oral glucose but not oral sucrose. Oral glucose alone had no effect on neointimal formation or insulin sensitivity whereas oral sucrose increased neointimal growth and induced insulin resistance. In Study 2, insulin decreased neointimal area and cell migration, and increased re-endothelialization. These effects were abolished by nitric oxide synthase (NOS) inhibition. In addition, insulin increased eNOS protein expression in the vessel. In Study 3, RSV reduced neointimal growth, cell proliferation, and migration after injury, without affecting re-endothelialization. Most of these effects were abolished by NOS inhibition, except for the decrease in cell migration. Insulin sensitivity and systolic blood pressure were not affected by RSV. Together, the results demonstrate that insulin, independent of glycemic effects, and RSV have a protective effect on the vessel against restenosis, which is mediated by NO. Since both insulin and RSV decrease neointimal formation without negatively impacting re-endothelialization, insulin or RSV treatment could provide some advantage over anti-mitogenic agents currently used in drug-eluting stents, which delay re-endothelialization. These studies suggest that insulin or RSV may have clinical potential in the prevention of restenosis after angioplasty.

<p>Olanzapine is an atypical antipsychotic drug exhibiting a low incidence of extrapyramidal side effects. It is not only effective in treating positive symptoms of schizophrenia, but also more efficacious against negative and depressive symptoms than classical antipsychotics. Olanzapine has been recommended as the first-line drug for the treatment of schizophrenia. Unfortunately, a common side effect of olanzapine, namely weight gain, has also been observed. A comprehensive literature analysis revealed that olanzapine induced higher weight gain than most other antipsychotics, only second to clozapine. The incidence of olanzapine-induced weight gain and related diseases, such as diabetes and cardiovascular diseases, is higher than that of the general population. These unwanted side effects have decreased the adherence to treatment. Many clinical observations and preliminary animal studies have attempted to elucidate the possible mechanism involved. To date, the mechanism for olanzapine-induced weight gain remains unclear.</p><p>This research project utilizes an animal model to investigate the possible mechanism of olanzapine-induced weight gain. The specific research objectives include: 1) does olanzapine affect feeding behavior; 2) can olanzapine influence the levels of glucose and triglyceride; 3) are cytokines, such as insulin, leptin, and TNF-Ñ involved in olanzapine-induced weight gain; 4) how does olanzapine affect adipose tissue?</p><p> An olanzapine-induced weight gain animal model has been established in the present investigation. An increase in food and water intake and increase in fat deposition accompanied with weight gain after treatment were observed. No significant increase in levels of glucose and triglyceride was detected. The changes of insulin and leptin levels in blood suggest that olanzapine may affect the endocrine system. A dramatic morphological alteration of adipose tissue by olanzapine was serendipitously observed. Immunohistochemical staining revealed that olanzapine stimulated collagen VI expression and deposition in the extracellular matrix suggesting that adipocyte differentiation may be enhanced. The effect of olanzapine on fat deposition might play a critical role in olanzapine-induced weight gain. The data from adipose tissue have provided a new clue on future research in understanding the mechanism of olanzapine-induced weight gain. Due to limitation of small number of animals and relatively short term of treatment, a large variation in groups diminished the power of analysis regarding the effects of olanzapine related to weigh gain.

Postprandial overproduction of apolipoprotein B48 (apoB48)-containing lipoproteins has been observed in states of insulin resistance and is important to the sequelae of cardiovascular disease, but little is understood about factors that regulate their secretion. The glucagon-like peptides (GLPs) are released from ileal enteroendocrine L-cells following lipid ingestion. I hypothesized that the GLPs could acutely affect the production of apoB48-containing triglyceride (TG)-rich lipoproteins (TRL) in the small intestine. Using the Syrian golden hamster, I first characterized the gross effects of the GLPs on TRL secretion in response to an oral fat load and then continued to dissect the mechanisms of these changes using primary intestinal cell cultures and a variety of knockout mouse models. An exogenous GLP-1 receptor (GLP-1R) agonist was found to acutely inhibit chylomicron secretion in both hamsters and mouse models, and extending the bioactivity of endogenously-secreted GLP-1 with a dipeptidyl peptidase-4 inhibitor had suppressive effects in insulin-resistant fructose-fed hamsters. The insulinotropic and delayed gastric emptying functions do not completely account for the hypolipidemic effect of GLP-1R agonism, and the effect of the GLP-1R agonist exendin-4 could be seen directly in the apoB48 secretion of primary enterocytes. In contrast, the sister peptide GLP-2 was a potent acute stimulator of chylomicron secretion in hamsters and mice. The hyperlipidemic effect of GLP-2 could be attributed to an increased rate of luminal FA uptake mediated by the posttranslational modification of the FA transporter CD36, and CD36-deficient mice were found to be refractory to the stimulatory effects of GLP-2. The activity of nitric oxide synthase was also found to be essential to the hyperlipidemic action of GLP-2. I identified a set of intercellular communications that could contribute in mediating the action of GLP-2, in which GLP-2 secreted from the enteroendocrine L-cell stimulates intestinal subepithelial myofibroblasts to release vascular endothelial growth factor, which directly activated the enterocyte to secrete apoB48. In summary, this thesis demonstrates that two co-secreted postprandial hormones have considerable but completely opposite influences on chylomicron production. Changing the balance of the GLPs’ actions in vivo could provide a therapeutic strategy to combat postprandial dyslipidemia.

The metabolic syndrome is a collection of pathologies including dyslipidemia, obesity and insulin resistance. A thorough understanding of the mechanisms behind metabolic syndrome development would help in the development of treatment and prevention strategies. Our lab has previously shown that cholesterol feeding exacerbates features of the metabolic syndrome in high fat-, high fructose-fed mice. The nuclear receptor Liver X Receptor (LXR), a master transcriptional regulator of cholesterol, fat and carbohydrate metabolism in the liver, is endogenously activated by oxysterols, metabolic derivatives of cholesterol. In order to determine whether cholesterol exerts its metabolic effects via LXR activation, parallel studies were conducted comparing chronic cholesterol supplementation with LXR activation in the hamster. Results showed that cholesterol feeding and LXR activation caused similar dyslipidemia, glucose intolerance and upregulation of target mRNA and proteins. These data support the hypothesis that the dyslipidemic effects of dietary cholesterol are mediated at least in part by LXR.

ChREBP has emerged as one of the key controllers of hepatic lipogenesis. While the function of ChREBP has been extensively investigated, mechanisms underlying its transcriptional regulation remain largely unknown. We located a conserved POU-binding site within mammalian ChREBP promoters, and demonstrated that the POU homeodomain protein Oct-1 binds to this site in the human HepG2 cell line. Oct-1 transfection significantly repressed ChREBP promoter activity 50-75%. Conversely, knockdown of Oct-1 expression with shRNA significantly increased ChREBP expression levels. Furthermore, insulin treatment resulted in a two-fold activation of ChREBP promoter activity, and stimulated endogenous ChREBP expression. We found that the stimulatory effect of insulin on the ChREBP promoter is at least partially dependent on the presence of the POU-binding site, and that insulin treatment reduced Oct-1 expression. Our observations identify Oct-1 as a transcriptional repressor of ChREBP, and suggest that insulin stimulates ChREBP expression via attenuating the repressive effect of Oct-1.