Type 2 diabetes is characterized by defects in insulin secretion, peripheral glucose utilization (PGU) and hepatic glucose production (HGP). The ability of insulin to stimulate PGU and repress HGP in patients with type 2 diabetes is reduced as a consequence of insulin resistance. In addition, in Type 1 diabetics, HGP can increase if circulating insulin levels are low, a particular problem when poor glycemic control has led to the development of insulin resistance. In both type 1 and type 2 diabetes this increased HGP was thought to be a consequence of an increased rate of gluconeogenesis, rather than glycogenolysis, but more recent data suggest that the latter may also be important. In either case, the final reaction in both metabolic pathways, the hydrolysis of glucose-6-phosphate to glucose, is catalyzed by glucose-6-phosphatase. In animal models of both type 1 and type 2 diabetes glucose-6-phosphatase catalytic subunit (G6Pase) gene expression is elevated. Moreover, hepatic overexpression of G6Pase is sufficient to induce an increased rate of HGP. These observations suggest that the suppression of G6Pase gene expression may represent a potential strategy for reducing HGP in diabetic patients. The rationale development of a pharmaceutical agent that suppresses G6Pase gene transcription will require a detailed knowledge of the cis-acting elements and trans-acting factors through which transcription of the gene is regulated. We have shown that the inhibitory action of insulin on basal G6Pase gene transcription requires two promoter regions designated A and B. Region A binds hepatocyte nuclear factor-1 (HNF-1) but does not directly mediate the action of insulin. Instead, HNF-1 enhances the action of insulin mediated through Region B. Region B contains three insulin response sequences (IRSs) designated IRS 1-3. IRS 1 and 2 bind the insulin-responsive transcription factor FKHR (FOXO1a) whereas IRS 3 binds an unidentified insulin-responsive factor.
Aim 1 of this grant application seeks to address several questions, namely (i) how does HNF-1 enhance the inhibitory action of insulin on G6Pase gene transcription? (ii) how does insulin inhibit glucocorticoid stimulated G6Pase gene transcription? (iii) how does insulin inhibit cAMP-stimulated G6Pase gene transcription? and (iv) what is the unidentified insulin-responsive factor binding IRS 3? In Aims 2 and 3 we propose characterizing the cis-acting elements and trans-acting factors that mediate the inhibitory effect of phorbol esters and the stimulatory effect of PGC-1 on G6Pase gene transcription, respectively. We have shown that the G6Pase promoter region between -484 and +66 is sufficient to confer maximal hormonally regulated G6Pase fusion gene transcription in situ.
In Aim 4 we propose generating transgenic mice to determine whether this same promoter region can confer a developmental, tissue-specific and hormonally regulated pattern of G6Pase fusion gene expression in vivo that mimics that of the endogenous gene. ? ?
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