Zucker diabetic fatty (ZDF) rats, an animal model of human type 2 diabetes, have a blunted response to hyperglycemia with a failure to suppress net hepatic glucose production and increase glucose flux to glycogen, which are associated with a failure to stimulate glucose phosphorylation catalyzed by glucokinase (GK). We found impairments in GK regulation occur during the progression of diabetes in ZDF rats. During an early stage of diabetes (~10 weeks of age), characterized by excessive postprandial hyperglycemia and hyperinsulinemia, GK resides with its inhibitory protein (GKRP) in the nucleus and fails to translocate to the cytoplasm where its activity would normally promote glucose phosphorylation. During a middle stage of diabetes (~14 weeks of age), characterized by prolonged and marked hyperglycemia and hyperinsulinemia, glucose phosphorylation is impaired despite a continuous residency in the cytoplasm of GK. During a late stage of diabetes (~20 weeks of age), characterized by prolonged and extreme hyperglycemia and euinsulin- emia, GK expression was markedly decreased. Additionally, preventing large excursions of postprandial and prolonged postabsorptive hyperglycemia reversed the GK impairments observed in ZDF rats. To explore the mechanisms responsible for each type of altered GK regulation, we propose three aims. First, we propose to examine why GK fails to translocate from the nucleus to the cytoplasm in response to a rise in plasma glucose in 10-week old ZDF rats. We will assess whether the colocalization of GK and GKRP in the nucleus after a rise in glucose concentration is due to a failure of GK to dissociate from GKRP or to be retained in the cytoplasm. Second, we will determine how chronically increased postprandial hyperglycemia impairs GK translocation from the nucleus to the cytoplasm in response to a postprandial signal of hyperglycemia and hyperinsulinemia in 10-week old ZDF rats. We will address whether the ability of insulin to facilitate glucose action and/or the ability of glucose per se to signal hepatic GK translocation is impaired and whether GK translocation is prevented by the inability of GK to bind to a cytoplasmic protein partner, 6-phosphofructose-2- kinase/fructose-2,6-phosphatase. Thirdly, we will examine why chronic hyperglycemia decreases GK expression in 14 to 18 week old ZDF rats. We will assess whether chronic hyperglycemia decreases GK expression by directly affecting the liver or by decreasing insulin secretion, and whether maintaining GK expression by preventing hyperglycemia is associated with maintaining GK gene transcription or improved stability of GK protein. To execute these aims we will use isolated hepatocytes for Aim 1 and isotopic and chronic catheterization techniques to perform glucose and pancreatic clamps in conscious ZDF rats in Aim 2 and 3. Chronic hyperglycemia will be altered by a phlorizin analogue. These experiments directed towards understanding the mechanism by which chronic hyperglycemia impairs the action of glucose and/or insulin on the liver will provide the basis for improved management and therapeutic treatment of type 2 diabetes.
The liver is an important organ for the maintenance of normal blood glucose and an impairment of the liver to switch from a producer to a consumer of glucose contributes to the elevated blood glucose associated with many of the complications of type 2 diabetes. We found that inappropriate regulation within the liver of a key metabolic enzyme, glucokinase, is caused by elevated blood glucose, which can lead to a further elevation of blood glucose. We propose to determine in a diabetic animal model of type 2 diabetes the reasons for impaired glucokinase activity, and mechanism by which improved glycemic control reverses this impairment and the potential for diabetic complications.
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