Doctoral Dissertations

Date of Award

8-2002

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Biochemistry and Cellular and Molecular Biology

Major Professor

John W. Koontz

Committee Members

Wesley D. Wicks, Naima Moustaid-Moussa, Jeffery Becker

Abstract

Non-insulin dependent diabetes mellitus (NIDDM) is a heterogeneous disease arising from metabolic defects in insulin secretion and insulin action. The inability of insulin to trigger full metabolic responses results in impaired glucose transport, which elicits compensatory hyperinsulinemia with increased lipogenesis. In order to identify the molecular defects responsible for insulin resistance, we have investigated three different model systems: the ability of sulfonylurea receptor (SUR) ligands to modify insulin regulated events in cultured rodent adipocytes; the ability of agouti and/or agouti related polypeptide (AGRP) to modify insulin regulated events; and the regulation of the activity and amount of the HNF-1a transcription factor, mutations of which result in a subset of NIDDM, by variations in glucose concentration. Because the results of animal studies are often obscured by the interactions between multiple tissues, we have used model cell lines in culture to investigate these systems.

In obese Zucker rats, diazoxide decreases hyperinsulinemia and lipogenesis while improving the insulin sensitivity of glucose transport in adipose tissue. The opposing effects on glucose transport and lipogenesis suggest that diazoxide might have extrapancreatic effects that modify insulin signaling in adipose tissues. Accordingly, we have investigated the effect of diazoxide and glibenclamide (another SUR ligand) on glucose transport and insulin signaling in cultured 3T3-L1 adipocytes. Our results suggest that diazoxide has no direct effect on insulin signaling and glucose transport. In contrast, glibenclamide exhibited dual effects on glucose uptake. Acute exposure to glibenclamide resulted in inhibition of glucose uptake, while long-term incubation with glibenclamide enhanced glucose uptake independent of insulin action. Diazoxide was unable to antagonize either effect of glibenclamide, suggesting that the effects of glibenclamide were not mediated by the sulfonylurea receptor.

In the yellow mouse obesity syndromes, ectopic expression of the Agouti gene results in late-onset obesity and type II diabetes. Agouti overexpressed in hypothalamus mimics the natural action of AGRP, which antagonizes a-Melanocyte-Stimulating Hormone (a-MSH) at the Melanocortin-4-recptor (MC-4-R) that controls feeding behavior and metabolism. Although this antagonism in the central nervous system is sufficient to cause the agouti phenotype, a calcium-dependent mechanism for agouti’s actions in peripheral tissues has been proposed to account for enhanced lipogenesis and insulin secretion. To better understand the molecular mechanisms utilized by agouti to modify insulin sensitivity, we have investigated the effects of agouti and AGRP on insulin signaling and its biological responses in cultured 3T3-L1 adipocytes. We observed no effects of either agouti or AGRP on glucose uptake or FAS activity. In contrast, insulin stimulates glucose uptake in a dose-dependent manner and induces fatty acid synthase (FAS) activity as previously reported. In addition, the insulin-stimulated protein tyrosine phosphorylation and activation of downstream kinases were not affected by either agouti or AGRP.

Mutations in HNF-1 genes result in MODY3 (a subtype of NIDDM) that are characterized by pancreatic b-cell dysfunction. The diminished insulin storage and secretion are consistent with the reduced HNF-1 activity that regulates the transcription of insulin genes and genes involved in glycolysis and glucose transport. Reduction in HNF-1 activity and protein has been observed in the livers of type I diabetic animals. Restoring euglycemia without affecting insulin levels rescued hepatic HNF-1 activity and protein in these animals. Accordingly, we investigated whether hyperglycemia also suppresses HNF-1 activity in the type II diabetic animals. Examination of hepatonuclear extracts prepared from the obese Zucker rats revealed that HNF-1 binding activity was reduced. However, the reduction is not due to diminished HNF-1 protein levels as previously reported in the type I diabetic animals. Incubating Fao rat hepatoma cells with high concentrations of glucose resulted in a similar decrease in HNF-1 binding activity, with no changes in HNF-1 protein levels. The results with the hepatoma cells reflect the observation made with the obese Zucker rats and point to nutritional regulation of HNF-1 activity in the liver.

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