No description available.

No description available.

The canonical PI3K/Akt pathway is activated downstream of numerous receptor tyrosine kinases, including the insulin and insulin-like growth factor receptors, and is a crucial regulator of growth and survival in metazoans. The deregulation of Akt is implicated in the pathogenesis of numerous diseases including cancer, making the identification of modifiers of its activity of high chemotherapeutic interest. In a transheterozygous genetic screen for modifiers of embryonic Akt function in Drosophila, in which the PI3K/Akt signaling pathway is conserved, we identified the A subunit of the vacuolar ATPase (Vha68-2) as a positive regulator of Dakt function. Our characterization of this genetic interaction in the larval stage of development revealed that Vha68-2 mutant phenotypes stereotypically mimicked the growth defects observed in mutants of the Drosophila insulin signaling pathway (ISP). The loss of Vha68-2 function, like Dakt-deficiency, was found to result in organismal and cell-autonomous growth defects, and consistent with its putative role as a positive regulator of Dakt function, both the mutational and pharmacological inhibition of its activity were found to downregulate Akt iv activation. Genetic epistasis experiments in somatic clones of Vha68-2/dPTEN double mutants demonstrated that the loss of Vha68-2 function suppressed the growth defects associated with dPTEN-deficiency, placing Vha68-2 activity downstream of dPTEN in the ISP, while the examination of PI3K activity and PH domain-dependent membrane recruitment in pharmacologically inhibited larval tissues further placed Vha68-2 function downstream of PI3K. These findings were recapitulated in cultured NIH-3T3 cells, whose treatment with bafilomycin A1, a potent and specific inhibitor of V-ATPase, resulted in the downregulation of Akt phosphorylation, particularly in non-cytoplasmic intracellular compartments. Furthermore, cellular subfractionation of bafilomycin-treated NIH-3T3 cells demonstrated a decrease in the localization of Akt to early endocytic structures, and a downregulation in the localization and activation of Akt in the nuclei of both Drosophila and mammalian cells. Finally, the pharmacotherapeutic relevance of V-ATPase inhibition was addressed in two tumor models – multiple myeloma and glioblastoma – and our preliminary findings in these cancers, which are often associated with ectopic PI3K/Akt signaling, showed significant cytotoxic efficacy in vitro, warranting its consideration as a tractable pharmacological option in the treatment of cancer.

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.

No description available.

No description available.

No description available.

No description available.

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.