Type 2 diabetes mellitus (T2DM) is rapidly emerging as one of the greatest global health issues of the 21st century. Insulin-resistance is a condition associated with T2DM and in the cell it is defined as the inadequate strength of insulin signalling from the insulin receptor downstream to the final substrates of insulin action involved in multiple metabolic, gene expression, and mitogenic aspects of cellular function. To investigate the potential mechanisms involved in the development of insulin-resistance, two in vitro liver cell models were established using palmitate or a combination of insulin and fructose as inducers. The development of insulin-resistance was determined via the capacity of the hepatocytes to maintain normal glucose metabolism functionality by measuring hepatic gluconeogenesis and glycogenolysis. It was established that the treatments induced the development of insulinresistance after 24 hours chronic exposure. Previous studies have investigated the potential of Sutherlandia frutescens extracts as therapeutic agents for insulin-resistance. The aim of this study was thus to investigate the ability of a hot aqueous extract of S. frutescens to reverse the insulin-resistant state, via measuring gluconeogenesis and glycogenolysis, the associated changes in cellular physiology (lipid accumulation, oxidative stress, and acetyl- CoA levels), and changes in mRNA expression. The results showed that S. frutescens had a significant effect on reversing the insulin-resistant state in both models of insulin-resistance. Furthermore, S. frutescens was capable of reducing lipid accumulation in the form of triacylglycerol in the high insulin/fructose model, while this was unaffected in the palmitate model. However, S. frutescens did reduce the accumulation of diacylglycerol in the palmitate model. Oxidative stress, seen to be associated with the insulin-resistant state, was successfully treated using the extract, as indicated by a reduction in reactive oxygen species. However no change was seen in the nitric oxide levels, in either model. Interestingly, although S. frutescens had no effect on the level of acetyl-CoA in the insulin/fructose model, it was found to increase this in the palmitate model. It is suggested that this may be due to increased β-oxidation and metabolic activity induced by the extract. The analysis of mRNA expression gave some insight into possible mechanisms by which insulin-resistance develops, although the results were inconclusive due to high variability in samples and the possibility of the RNA being compromised. Future studies will address this issue. The results of this study reflect different proposed clinical causes of insulin-resistance through the responses seen in the two cell models. These indicate that liver steatosis and insulin-resistance are induced by high palmitate as well as high insulin and fructose levels, and reversed by S. frutescens. Therefore the potential of S. frutescens to be used as a therapeutic agent in the treatment of insulin-resistance is indicated by this study.

The effect of varying concentrations of glucose or the gastrointestinal hormones, gastric inhibitory polypeptide (GIP) and somatostatin (SS-14), on the in vitro immunoreactive insulin (IRI) response to the parasympathetic neurotransmitter, acetylcholine (ACh) was investigated. The isolated, vascularly perfused rat pancreas was used in all experiments. Acetylcholine (1.0 µM) did not stimulate IRI secretion in the presence of 2.2 mM glucose. However, in the presence of 4.4, 6.6, or 8.9 mM glucose, ACh (1.0 µM) potently stimulated IRI secretion (approximately fourfold). At a higher glucose concentration (17.8 mM), the IRI response to ACh was reduced. GIP also potentiated the IRI response to 1.0 µM ACh. This potentiation was most marked in the presence of 1.0 nM GIP, whereas the effect of concomitant infusion of 0.2 nM GIP and 1.0 µM ACh was only slightly greater than additive. SS-14 potently inhibited ACh-stimulated IRI secretion. These results demonstrated the glucose dependency of cholinergically stimulated IRI secretion, and that physiological levels of glucose and GIP increased B-cell sensitivity to cholinergic stimulation. It was suggested that the parasympathetic stimulation of IRI secretion associated with food intake could be affected by postprandial increases in glucose, GIP, and SS-14. The idea that endogenously released somatostatin may have influenced glucose or GIP-stimulated IRI secretion was not supported by the present experiments, since neither glucose (8.9 mM) nor GIP (2.0 nM) were found to have a significant effect on the release of pancreatic somatostatin-like immunoreactivity (SLI). Both atropine (1.0 µM) and hexamethonlum (100 µM) inhibited the IRI response to ACh. This suggested that the parasympathetic stimulation of IRI secretion was mediated not only by muscarinic receptors on the B-cell, but also by nicotinic receptors on intrapancreatic ganglia. Neither atropine nor hexamethonlum had a significant effect on glucose- or GIP-stimulated IRI secretion, indicating that the IRI response to these stimuli was not mediated by cholinergic receptors. Both SS-14 and the synthetic somatostatin analog SMS 201-995 (SMS; Sandostatin®) inhibited IRI secretion stimulated by 8.9 mM glucose, 2.0 nM GIP, or 1;.0 µM ACh, but not 17.8 mM glucose. The most potent inhibition by both SS-14 and SMS was observed in the presence of the weakest IRI stimuli (8.9 mM glucose and 1.0 µM ACh). These results suggested that the inhibitory effects of somatostatin on the B-cell could be overcome by the presence of strong stimuli. In addition, the inhibitory effects of the native hormone and the analog were found to be approximately equipotent (weight basis), indicating that the increased potency of SMS previously observed in vivo was due to its longer half-life in plasma, and not due to a more potent direct effect on the B-cell. / Medicine, Faculty of / Cellular and Physiological Sciences, Department of / Graduate

archives@tulane.edu / Androgen excess predisposes females to type 2 diabetes. Using mouse models, our lab reported that androgen excess causes insulin resistance via activation of the androgen receptor (AR) in the brain. Neurons of the arcuate nucleus of the hypothalamus (ARC) regulate hepatic glucose production (HGP). Thus, I hypothesized that in female mice, androgen excess in neurons of the ARC causes hepatic insulin resistance by increasing HGP. To test this, I injected AaV-Cre-GFP or AaV-GFP into the ARC of ARlox/lox female mice to generate ARC-specific AR knockout (ARC-ARKO) and control mice, respectively. When exposed to a Western diet, control female mice chronically treated with dihydrotestosterone (DHT) developed insulin resistance and fasting hyperglycemia compared to vehicle-treated control mice. In contrast, DHT-treated ARC-ARKO mice remained insulin sensitive and normoglycemic compared to vehicle-treated ARC-ARKO mice. During a hyperinsulinemic-euglycemic clamp, insulin’s ability to suppress HGP was blunted in DHT-treated control mice. In contrast, insulin was still able to suppress HGP in DHT-treated ARC-ARKO females. Additionally, during the clamp, DHT-treated control mice showed no alteration in hepatic activation of AKT, a marker of hepatocyte insulin sensitivity, but exhibited reduced activation of hepatic STAT3, a marker of hypothalamic insulin sensitivity. In contrast, in DHT-treated ARC-ARKO mice activation of hepatic STAT3 was increased. In a parallel study, estradiol treatment improved insulin sensitivity in control ovariectomized (OVX) mice. In contrast, in DHT-treated OVX mice, estradiol treatment did not improve insulin sensitivity. Together these results suggest that in female mice exposed to a Western diet, androgen excess causes hypothalamic estrogen resistance and insulin resistance in ARC neurons via action at the AR leading to impairments in the brain-IL6-pSTAT3 pathway which results in unsuppressed HGP. / 1 / Jamie Morford

M.Sc. (Med.), Faculty of Health Sciences, University of the Witwatersrand, 2009

The study utilizes the yeast two-hybrid system to try and unravel the molecular link between the G protein-coupled receptors (GPCR) and the receptor tyrosine kinases (RTK). The fourth melanocortin receptor (MC4R) and the angiotensin receptor AT1 are both GPCRs while the insulin receptor (IR) and the epidermal growth factor receptor subtype-2 (ErbB2) belong to the RTK family. Alteration in the functioning of MC4R receptor can cause obesity. Development of insulin resistance and diabetes is a risk factor associated with obesity. Overexpression of the ErbB2 receptor is seen in a number of breast cancers. The interaction between the AT1 and ErbB2 receptors were studied based on previous studies that have shown an interaction between the epidermal and angiotensin receptors. Thus, Interactions between the MC4R and the IR, and that between the AT1 and ErbB2 receptors were studied for their possible roles in metabolic syndrome and cancer.

No description available.

Obesity can lead to Type 2 Diabetes, both conditions increase in association with physical inactivity and high-energy diets, resulting in elevated blood glucose, decreased insulin sensitivity and increased insulin resistance. Sutherlandia frutescens (S.frutescens), an anti-diabetic plant, reverses and prevents insulin resistance in a rat model and human cell culture model. Gene expression analysis in hepatocyte cultures, identified genes down regulated in insulin resistance and up regulated by S.frutescens. These included genes encoding vesicle transporter proteins, hypothesised to be linked to hepatic lipid accumulation and lipid droplet formation during insulin resistance. The aim of this study was to investigate critical genes involved in lipid droplet formation, vesicle assembly and transport in high fat diet (HFD)-induced insulin resistant rat liver tissue during the development of insulin resistance and the reversal of these changes by S.frutescens. Rats were fed a low fat diet (LFD) or HFD supplemented with S.frutescens for 2, 4 and 8 weeks. Rats fed a HFD for 12 weeks developed insulin resistance, confirmed by plasma glucose and insulin levels (compared to normal controls). Groups of these rats were gavaged with S. frutescens (50mg/kg BW), Metformin (13mg/kg BW) or water for a further 4 weeks and starved for 12 hours, anaesthetized and blood removed by heart puncture. Liver was stored in RNA-Later™ for qRT-PCR and snap-frozen in liquid nitrogen for western blotting and confocal microscopy analysis. Changes in expression of vesicle transporter genes VAMP3 and NSF were analysed by qRT-PCR and changes in the protein expression by western blotting analysis. Proteins were localised within the liver by confocal immunohistochemistry using ZEN lite™ software. Statistical analysis was performed using One-Way ANOVA and unpaired t-test. mRNA gene expression of vesicle transport components VAMP3, NSF and SNAP25 showed relatively moderate changes with considerable individual variation within control or experimental groups. Uncorrelated changes in mRNA and protein products were found and may be due to differential regulation by siRNA. Proteins also showed altered staining patterns in high fat diet rats that reverted towards normal on S. frutescens treatment, potentially reflecting functional changes associated with transport of lipid-filled vesicles.

Insulin resistance is the major metabolic defect in obesity and Type 2 diabetes. Increased lipid accumulation is strongly associated with insulin resistance. A significant component of insulin resistance is thought to be a reduced ability of insulin to activate the cascade of phosphorylation events that lead to the metabolic effects of this hormone. The broad aims of this thesis were to examine the effect of high-fat diets containing different fat subtypes on in vivo insulin signalling, under conditions normally used to detect whole body insulin resistance, and to compare the effects of acute and chronic lipid oversupply on insulin signalling in vivo. Time-course and dose-response effects of insulin stimulation on site-specific phosphorylation of key signalling proteins were studied in rat tissues in vivo, to establish an appropriate experimental system to examine the onset of activation of the insulin signalling pathway. It was determined that short insulin infusions with concurrent glucose infusion, similar to the beginning of a euglycaemic-hyperinsulinaemic clamp, significantly increased the phosphorylation of major intermediates of the insulin signalling pathway in important tissues of insulin action (skeletal muscle [RQ], liver [LIV] and white adipose tissue [EPI]). These experiments provided a platform to study insulin signalling under the same conditions used to study lipid-induced insulin resistance. The provision of diets enriched in polyunsaturated or saturated fatty acids (FA) resulted in the corresponding enrichment of these fat subtypes in rat plasma and tissues. However, the effects on insulin signalling were essentially the same. Both fat diets induced defects in sitespecific phosphorylation of insulin receptor substrate (IRS)-1 and protein kinase B (PKB) in RQ and LIV, but not EPI. This suggests that the amount of fat in the diet, rather than enrichment in a particular fat subtype, had a greater impact on the development of signalling defects and that the response to high-fat feeding was tissue-specific. A 3hr elevation of circulating FA (using a lipid/heparin infusion), to a level that is relevant in clinical Type 2 diabetes, impaired insulin-stimulated PKB phosphorylation with no significant effect on IRS-1 phosphorylation. This suggests that there may be differences in the way acute and chronic exposure to increased FA impair insulin signalling. The phosphorylation defects observed in both chronic and acute studies did not seem to be associated with activation of major stress signalling pathways (JNK and NFkB), which have been suggested to have a role in lipidinduced insulin resistance. In conclusion, these studies demonstrate that impaired IRS-1 and PKB phosphorylation do have a role in the reduced insulin action observed with lipid oversupply in vivo, because the changes were detected under similar conditions as those used to determine whole body insulin resistance.

Thesis (Ph. D.)--Ohio State University, 2006. / Title from first page of PDF file. Includes bibliographical references (p. 118-125).

published_or_final_version / Pharmacology and Pharmacy / Doctoral / Doctor of Philosophy