A reduction in the sensitivity of tissue to insulin is termed insulin resistance. In the clinic this condition is associated with obesity and inactivity and often leads to the development of type 2 diabetes. A major focus of antidiabetic therapy is to develop novel interventions to alleviate insulin resistance. However, the initial physiological and molecular defects in the development of insulin resistance remain elusive. This knowledge would greatly aid the development of novel and more effective insulin sensitisers.In an effort to improve the understanding of insulin resistance this thesis establishes that culturing liver cells in sera from obese diabetic patients reduces the ability of insulin to repress the key gluconeogenic gene, phosphoenolpyruvatecarboxykinase (PEPCK). Cells cultured in serum from obese diabetic human subjects exhibited defective PEPCK mRNA suppression by 0.1 and 0.5 nM insulin compared to cells cultured in control serum (p<0.0001), representing a shift to the right of the insulin dose response curve. Classification of human sera, using the response of the cell model following incubation with the sera, was actually more reliable than any single clinical biomarker at establishing whether the serum came from a volunteer with insulin resistance. This suggests that the cell model could be developed as a means to classify insulin resistance in the human population more reliably than simply measuring fasting glucose.The system was developed and optimised as a cell based humanised model of insulin resistance to aid the search for a biomarker for the development of obesity related insulin resistance. However, there was no linear relationship between any single biomarker and the resistance causing ability of the sera. Interestingly, cells cultured chronically in the presence of fetal calf serum supplemented with 5 pM insulin (the average increase in insulin between cases and controls) also exhibited reduced suppression of PEPCK by 0.1 and 0.5 nM insulin compared to controls (p=0.03 and 0.01 respectively). This has major implications for the understanding of how insulin resistance may develop. It suggests that minor increases in insulin release from beta cells, or minor loss of insulin clearance in the liver that elevate plasma insulin are potential initiating mechanisms for insulin resistance (at least in liver). Of course there may be many ways to initiate insulin resistance in vivo, but establishing the relative importance of the beta cell and the liver as an initial site for the development of insulin resistance is clearly important for effective intervention. Subsequent to the generation of insulin resistance in culture I could not detect significant differences in the response of the major post-receptor insulin signalling pathway components, between cells cultured under standard conditions and those cultured chronically in 5 pM insulin. Therefore the mechanism underlying this reduced insulin action on PEPCK gene transcription remains unclear.I then went on to develop reporter cell lines both for use in the study of the regulation of hepatic gene transcription by insulin and also as a potential screen for effective insulin sensitisers. Unfortunately the reporter cell lines did not turn out to be useful as hoped, as the reporter genes did not develop insulin resistance in response to chronic exposure to 5 pM insulin. In addition there were some differences between the reporter genes and endogenous genes in response to specific signalling inhibitors. This questions their suitability for the purposes proposed.Finally, I examined the signalling connections between the class of insulin sensitiser known as biguanides, and DNA repair mechanisms, as an initial characterisation of molecular links between diabetes and cancer. I established that inhibiting the DNA repair enzyme ATM reduces the phosphorylation of the biguanide target, AMPK in response to these drugs. However, although inhibition of ATM reduced biguanide suppression of G6Pase it had little effect on the regulation of PEPCK gene transcription by the drugs. This is consistent with AMPK not being the key mediator of biguanide regulation of PEPCK gene transcription and suggests that biguanide regulation of G6Pase and PEPCK gene transcription is mediated through distinct signalling pathways.In summary, I have developed a cell based model of insulin resistance that relies on factor (s) present in serum from humans with diabesity, and thus should be useful as a screen for more effective insulin sensitisers targeted at the population that donates the serum. It is likely that one of the factors responsible for generation of resistance is insulin itself as chronic exposure to low levels (albeit higher than background), of insulin reduces insulin sensitivity of the cells. The molecular details of the development of insulin resistance remain elusive as none of the major signalling pathways appear to be defective in the cells that have developed reduced insulin regulation of PEPCK. However, the data raise the intriguing possibility that chronic but mild hyperinsulinemia due to defective insulin secretion or clearance is an initial step in the development of insulin resistance. Hence, reducing insulin secretion (as opposed to current strategies of inducing insulin secretion) may be a more effective therapy for prevention of the development of insulin resistance. Finally, elements of the DNA repair pathways such as ATM may impinge on pathways that affect insulin sensitivity, including the biguanide target AMPK.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:578827 |
| Date | January 2011 |
| Creators | Schofield, Christopher James |
| Contributors | Sutherland, Calum |
| Publisher | University of Dundee |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | https://discovery.dundee.ac.uk/en/studentTheses/a5052794-9d39-4e09-90b5-dc88e02e309c |
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