This project is directed at developing transgenic mouse models in the nonobese diabetic (NOD) strain, for the purpose of elucidating the molecular mechanisms involved in aberrant glucose and fatty acid metabolism In the setting of insulin dependent diabetes mellitus (IDDM). The pathogenesis of IDDM is very complex. The primary acute clinical features include hyperglycemia and ketoacidosis. Specifically, hyperglycemia results from both decreased glucose uptake by insulin sensitive tissues and stimulated gluconeogenesis. Among the many mechanisms that are altered, transcriptional control of the rate limiting step of gluconeogenesis at phosphoenolpyruvate carboxykinase (PEPCK) has been shown to be a key component since it is markedly stimulated in IDDM. Likewise, excessive ketogenesis results from a flood of fatty acid substrates into the liver from adipose stores simultaneous with stimulated gene expression of the enzymes involved in fatty acid oxidation. We hypothesize that: (i) the elevated activity of the rate limiting enzymes of gluconeogenesis (PEPCK) and fatty acid oxidation (e.g., medium-chain acyl-CoA dehydrogenase-MCAD) necessary for ketogenesis during acute IDDM, results from increased stimulation of gene expression by a shared transcription factor, peroxisomal proliferator activated receptor (PPAR), important in both PEPCK and MCAD gene regulation. (ii) This results from the low insulin/high glucagon conditions that promote excessive lipolysis in this disease, and it is the elevated free fatty acids via PPAR, rather than a lack of insulin alone, that provides the major drive for elevated expression of gluconeogenesis and fatty acid oxidation exhibited by PEPCK and MCAD, respectively. If this is the case, one can envision improved treatment strategies in diabetic patients based on this mechanism, e.g., antagonists that act via PPAR. To test this hypothesis, we will accomplish the following SPECIFIC AIMS: (1) Further characterize the NOD mouse model by demonstrating the effects that IDDM has on liver gene expression of the important enzyme genes of gluconeogenesis and fatty acid oxidation important in development of hyperglycemia and ketogenesis, (2) Make a series of transgenic models using previously studied regulatory elements from the human medium-chain acyl-CoA dehydrogenase (hMCAD) gene. We will examine, specifically, the role of the nuclear receptor response element (NRRE- l) found at nucleotides -331 through -311 as an important control point in the diabetic mouse, as it is in the normal mouse. -This will allow us to examine expression of at least one pivotal gene of the fatty acid oxidation pathway in the physiologic setting of the actual disease state. (3) Make NOD transgenic mouse models with a transgene construct from the regulatory elements of PEPCK as a pivotal gene for controlling gluconeogenesis. This will include regulatory regions (nt.-460 - -360) that share similar transcription factors as those interacting with the NRRE-l described for MCAD. Using these models, it will be possible to isolate deranged physiologic processes and gain a much better understanding of the molecular mechanisms of aberrant metabolism in IDDM.
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