More than 10 million people are affected by type-II non-insulin dependent diabetes (NIDDM) . In the development of NIDDM, intracellular anomalies in glucose utilization precede the overt symptoms of NIDDM. These anomalies reflect a defect in the expression of proteins instrumental to glucose metabolism. Recently a thiazolidinedione derivative, pioglitazone (PIOG), was found to induce Glut 4 mRNA levels in the adipose and muscle of NIDDM rodents. This was associated with a decrease in blood glucose and insulin. The PIOG response is independent of insulin receptor binding, but is closely tied to IGF-1 actions. IGF-1, not insulin, is the stimulus for fat cell differentiation. The function of PIOG is to shift the dose-response for IGF-1 leftward. An examination of genes expressed during early stages of adipoblast differentiation revealed that PIOG amplified the IGF-1 induction of adipose fatty acid binding protein (a-FABP), Glut 4, and LPL. However only a-FABP mRNA was induced by PIOG alone, and the rise in a-FABP preceded that of Glut or LPL by 24-36 hrs. We hypothesize that the PIOG improvement of NIDDM is related to the amplification of the IGF-1 stimulus, where PIOG interacts with the nuclear IGF-1 signals to stimulate expression of proteins instrumental to glucose metabolism, and in this way enhances the capacity of the adipoblast and adipocyte to metabolize glucose following insulin stimulation. The outcome is a perceived increase in insulin responsivity. Using the a-FABP gene model, we have found that cis-acting elements for IGF-1 and PIOG reside in the 5'-flanking region of -7200 to +21. The cis-acting elements within this 5'-region that are responsive to IGF-1 and PIOG will be functionally mapped using 3T3-Ll preadipocytes stably transformed with pFABP/CAT constructs combined with an examination of the putative cis-elements to drive in vitro transcription initiation assays. DNase protection, band shift, and methylation interference assays will be applied to characterize nuclear proteins which interact with PIOG/IGF-1 response elements, and to elucidate the mechanism by which PIOG and IGF-1 regulate these nuclear factors. The data will address our goal of defining the nuclear mechanism by which IGF-1 and PIOG interact to improve NIDDM, and will shed light onto the transcriptional mechanisms which come into play as causative factors in the onset of NIDDM.
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