published_or_final_version / Pharmacology and Pharmacy / Doctoral / Doctor of Philosophy

Not available / text

No description available.

No description available.

Male Sprague-Dawley rats were used to determine whether insulin resistance develops between 1.5-4.0 months of age and whether it is related to aging or the development of obesity. Animals were randomly placed into a single 1.5 months old group (1.5 CN) or raised in one of three 4.0 month old groups; exercise trained (ET), pairfed (PF), or sedentary control (4.0 CN). The ET group was fed ad Iibitum and had free access to a spontaneous exercise wheel, while the PF group was fed to maintain equal body weight with the ET group. The young group was sacrificed with nembutal injection (45 mg/kg body weight) at 1.5 months while the other three groups were sacrificed at 4.0 months of age. Epididymal fat pads were removed, digested with collagenase (5 mg/ml) and the isolated cells sized and assayed for 2-deoxyglucose transport over a range of insulin concentrations (0-1000 µU/ml). Body composition (percent fat, bone and muscle) was performed on the carcasses of these animals at a later date. The 2-deoxyglucose transport of the 1.5 CN group was significantly greater than the 4.0 CN group at insulin concentrations of 50, 250 and 1000 uU/ml and significantly greater than all 4.0 months groups at 1000 11U/ml- The adipocyte size was significantly smaller in the 1.5 CN group followed in ascending order by the ET, PF and 4.0 CN group. The body compositions demonstrated the expected trends as the 1.5 CN group had the highest percent bone and muscle while demonstrating the lowest percent fat. The ET group was most able to maintain the body composition of the 1.5 CN group, while the PF and 4.0 CN groups were least able to maintain this composition respectively.The results indicate that: 1) Adipocyte insulin resistance develops in the rat between 1.5 and 4.0 months of age. 2) This development of insulin resistance is related to obesity and not to aging. 3) Exercise may prevent the development of insulin resistance by preventing adipocyte hypertrophy. 4) Exercise helps maintain optimal body composition. These results should be of interest to type II diabetics as an exercise program may decrease their adipocyte size, enhance body composition and decrease insulin resistance.

The polycystic ovary syndrome (PCOS) is a heterogeneous disorder in women characterised by chronic ovulatory failure, hyperandrogenaemia, and insulin resistance. Some women are completely asymptomatic and others present with extreme menstrual disturbance, severe hirsutism, infertility and recurrent miscarriage. The pathophysiology of PCOS is not completely understood, but it is thought that insulin resistance plays a central role. In normal subjects, non-diabetic obese patients and patients with non-insulin dependent diabetes, insulin resistance is associated with elevated plasminogen activator inhibitor-1 (PAI-1) levels. PAI-1 is a glycoprotein, which inhibits the formation of plasmin (a proteolytic enzyme). Plasmin aids fibrinolysis and extracellular proteolysis. High PAI-1 and low plasmin levels increase the risk of thrombosis and impair extracellular proteolysis required in ovarian follicle growth, ovulation and embryo implantation. This study was designed to determine whether elevated plasminogen activator inhibitor-1 (PAI-1) was associated with the insulin resistance present in PCOS, investigate its possible role in the causation of anovulation and recurrent pregnancy loss in these women and ascertain whether it was an additional thrombotic risk factor so that clinicians and patients could take appropriate measures to reduce this risk In a pilot study, systemic PAI-1 activity was significantly elevated in oligomenorrhoiec women with PCOS. A larger study supported these findings, but demonstrated that obesity was a significant confounding factor, as the increase in PAI-1activity disappeared when standardised for weight. Activated Protein-C (APC) resistance was subsequently tested in these women because of the unexpected finding of an increased prevalence of a positive family history of thrombosis in women with PCOS compared with controls, but there was no increase in the prevalence of APC-resistance in PCOS. In another project, the cellular distribution of PAI-1 protein in human ovaries was described for the first time using immunohistochemistry. It was localised to the granulosa and theca cell compartments in both polycystic and normal ovaries, however there was no significant difference in the intensity of PAI-l staining between both groups on image analysis. PAI-1 messenger RNA expression was also evaluated in these biopsies by in-situ hybridisation, but no signal was detected suggesting that there was either a low overall RNA preservation in the tissues, or an insufficient sensitivity of the cocktail of oligonucleotide probes used. This study did not support the hypothesis that elevated PAI-1 was a feature of PCOS, however the in-situ location of PAI-1 protein was demonstrated for the first time in the human ovary and consistent with a previously suspected role in ovulation. The results did not support a role for PAI-1 in anovulation, recurrent miscarriage or increased thrombosis in PCOS.

Insulin resistance is a major metabolic defect associated with obesity and type 2 diabetes. The incidences of both are increasing at an alarming rate. Excessive consumption of nutrient rich foods have been implicated in the pathogenesis of insulin resistance. However, the mechanisms involved in the onset of insulin resistance in skeletal muscle caused by acute nutrient oversupply in vivo have not been fully elucidated. The broad aim of this thesis was to examine the mechanisms associated with the onset of skeletal muscle insulin resistance in models of acute nutrient oversupply. The effect of glucose oversupply was investigated in the first study, which resulted in insulin resistance at the whole body and skeletal muscle level following 5h of glucose infusion, but not after 3h. There was no change in markers of oxidative stress over the same time course during which insulin resistance developed. Furthermore, co-infusion of the antioxidant taurine had no effect on the decreased glucose uptake in skeletal muscle from glucose infused animals. There was no evidence of activation of inflammatory/stress signalling pathways or defects in the phosphorylation state of multiple insulin signalling intermediates over the same time course. In isolated soleus strips taken from control, 1h, or 5h glucose infused animals, insulin stimulated 2-deoxyglucose transport was similar. Although, insulin-stimulated glycogen synthesis was significantly reduced after 5h of glucose infusion, in the presence of significantly increased glycogen content. The reduced flux through the glycogen synthesis pathway and a reduced content of glucose-6-phosphate suggests in this model that the rate limiting step has shifted from glucose transport to glucose phosphorylation by hexokinase (HK). In an acute lipid and insulin infusion model, the onset of insulin resistance was similar to that observed in the glucose infusion model. The mechanisms for the insulin resistance in skeletal muscle in this model was not associated with defects in the phosphorylation of key insulin signalling intermediates or activation of inflammatory/stress signalling pathways. Furthermore, there was no change in markers of oxidative stress and the co-infusion of taurine had no effect on the onset of insulin resistance. There was an increased exposure of long chain acyl-CoA (LCACoA), although there was no change in the content of other lipid intermediates such as DAG or ceramides. Interestingly, muscle pyruvate dehydrogenase (PDH) kinase 4 (PDHK4) protein content was significantly decreased in hyperinsulinaemic glycerol infused rats after 3 and 5h, and this decrease was blunted in muscle from hyperinsulinaemic 3 and 5h lipid infused rats. These findings suggest that lipid infusion may reduce glucose metabolism by inhibition of the glucose phosphorylation due to LCACoA inhibition of HK and mitochondrial substrate competition regulated by increased PDHK4. In conclusion, the current studies demonstrate that the insulin resistance associated with nutrient oversupply was not associated with significant changes in phosphorylation of key insulin signalling intermediates, activation of inflammatory and stress signalling pathways, or a change in markers of oxidative stress. Overall, the studies in this thesis suggest that the initial onset of insulin resistance due to glucose and lipid oversupply (in the presence of high insulin) is associated with metabolic feedback regulation, which is likely to be a protective mechanism of the skeletal muscle to limit any further insult by the excess nutrients.

Peripheral insulin resistance is characterised by reduced insulin-stimulated glucose uptake in skeletal muscle and adipose tissue, and the condition represents one of the earliest hallmarks in the development of type 2 diabetes (T2D). In patients with T2D, protein expression of the insulin-stimulated glucose transporter, GLUT4, is reduced in adipose tissue, but preserved in skeletal muscle. Transgenic studies in rodents provide evidence that overexpression of GLUT4 selectively in either skeletal muscle or adipose tissue enhances whole-body insulin action. Since skeletal muscle accounts for the majority of insulin-stimulated glucose disposal, the effect of adipose tissue GLUT4 on insulin sensitivity is thought to be secondary to an altered secretion of adipokines which affect insulin action in muscle, in the context of a ‘metabolic crosstalk’ between insulin sensitive tissues. Increasing GLUT4 expression in skeletal muscle and adipose tissue could be an effective therapy in the treatment of insulin resistance and T2D. Exercise training increases GLUT4 protein expression in skeletal muscle of patients with T2D. This adaptation occurs in the face of enhanced insulin sensitivity, and results from the cumulative and transient increase in GLUT4 mRNA following each acute exercise bout. Less is known regarding the regulation of skeletal muscle GLUT4 expression by a single bout of exercise in patients with T2D, or the effect of exercise training on GLUT4 expression in adipose tissue. / The primary aim of the studies undertaken for this thesis was to enhance understanding of exercise-mediated GLUT4 expression in skeletal muscle and adipose tissue of patients with T2D. The first investigation determined the effect of a single bout of exercise on skeletal muscle GLUT4 mRNA, and the signalling pathways which regulate GLUT4 expression, in patients with T2D and healthy control volunteers, matched for age and BMI. Increased (p<0.05) expression of GLUT4 and PGC-1α mRNA, together with increased (p<0.05) phosphorylation of AMPK and p38 MAPK was observed following exercise in patients with T2D, to a similar extent as in age- and BMI-matched control subjects. These findings lead to the conclusion that exercise-mediated regulation of GLUT4 expression is normal in patients with T2D. The second investigation of this thesis sought to identify the effect of a 4 week exercise training program on skeletal muscle and adipose tissue GLUT4 expression in patients with T2D. It was found that exercise training increased (p<0.05) GLUT4 protein expression by ~36% and ~20% in adipose tissue and skeletal muscle, respectively. These adaptations occurred in the absence of changes in insulin sensitivity or plasma levels of adipokines, adiponectin and resistin. Accordingly, the third study of this thesis sought to identify novel adipokines that regulate peripheral glucose metabolism in an adipocyte model of GLUT4 overexpression. Amyloid precursor protein (APP) was reduced (p<0.05) in culture media of GLUT4 overexpressing adipocytes, and the APP cleavage product, amyloid-beta (Aβ), reduced (p<0.05) insulin-stimulated Akt phosphorylation in L6 myocytes in vitro. / These observations lead to the conclusion that increased adipose tissue GLUT4 expression may influence whole body glucose metabolism through reduced levels of Aβ. The primary aim of the final study undertaken was to identify novel changes in the abundance of proteins in skeletal muscle following exercise training in patients with T2D, including proteins of glucose metabolism, which may regulate of GLUT4 expression. Exercise training altered the abundance of several proteins involved in energy metabolism, as well as some novel proteins which may play a role in cytoskeleton interactions with mitochondria. In summary, this thesis demonstrated that skeletal muscle from patients with T2D responds normally to an acute exercise bout in terms of increased GLUT4 mRNA expression. In addition, it was shown that exercise training increased GLUT4 protein expression, not only in skeletal muscle, but also in adipose tissue of patients with T2D. This is significant because adipose tissue GLUT4 overexpression enhances insulin sensitivity. Data from this thesis suggest that improvements in insulin sensitivity may be secondary to altered secretion of Aβ from adipose tissue. Collectively, the findings provide a number of therapeutic targets for the treatment of insulin resistance and T2D.

The Ser/Thr kinase Akt plays an important role in many of insulin's actions including GLUT4 translocation to the plasma membrane (PM). However, there are several features of Akt's regulation of GLUT4 translocation that remain unclear. The goal of my thesis was to resolve some of the following questions: Is activation of Akt sufficient to stimulate GLUT4 translocation? What is the quantitative relationship in signal transmission between individual components within the Akt cascade? What is the role of Akt in insulin resistance? To determine if activation of Akt is sufficient to mediate GLUT4 translocation, I developed a drug-inducible heterodimerisation strategy to activate Akt2 independently of other potential insulin signalling pathways. These studies revealed that activation of Akt2 resulted in rapid stimulation of GLUT4 translocation to a similar extent with maximum insulin, indicating that Akt2 is sufficient for this event. It was previously observed that maximum effect of insulin on GLUT4 translocation was obtained with minimum activation of Akt. To resolve this discrepancy, the relationship between Akt signalling components was examined using a quantitative kinetic and dose response approach combined with hierarchical cluster analysis. Most notably I observed a strong relationship between Akt at the PM, but not Akt in the whole cell lysate, with its substrate phosphorylation. Active pools of phospho-Akt and -AS160, a major substrate involved in GLUT4 translocation, were found in the lipid raft, highlighting the importance of subcellular partitioning of key signalling components for achieving biological specificity. The involvement of Akt in insulin resistance was investigated using the heterodimerisation strategy. These studies revealed that insulin itself initiates a pathway that causes insulin resistance by converging on target(s) downstream of Akt. This inhibitory pathway emanates from PI3-kinase and is likely induced by a range of insults including chronic insulin and dexamethasone. In conclusion, Akt is a crucial element in the insulin action pathway that exhibits precise spatial regulation. While the role of this nanoregulation of Akt in disease remains to be evaluated, my studies suggest that the major defect contributing to insulin resistance occurs downstream of Akt. The elucidation of this target will have major implications for metabolic diseases.

Thesis (M.S.)--Auburn University, 2007. / Abstract. Vita. Includes bibliographic references (ℓ. 65-73)