ROLE OF PROTEIN PHOSPHATASES IN INSULIN SECRETION

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DESCRIPTION: (From applicant's abstract) While a majority of extant studies have focused since on the protein kinases, very little is known of the regulation and roles of protein phosphatases (PPases) in physiologic insulin secretion from the pancreatic beta cell. The catalytic activity of certain enzymes of intermediary metabolism is influenced by their phosphorylation status. For example, acetyl CoA carboxylase (ACC), a lipogenic enzyme, is modulated by phosphorylation (inactive)- dephosphorylation (active). Here in the investigators describe a novel cytosolic, okadaic acid-sensitive (PP2A-like) PPase that dephosphorylates and activates ACC in the beta cell. Activity of this PPase is augmented by methylation at its C-terminal leucine. Further, the methylated form is converted to its inactive, demethylated form by a cytosolic serine esterease-like enzyme, also localize in the beta cell. Physiologic concentrations of glutamate and magnesium stimulate the carboxyl methylation as well as the catalytic activity of this PPase and concomitantly increase ACC activity. Effects of glutamate and magnesium are specific for ACC-phosphatase, but not ACC-kinase. Insulinotropic concentrations of glucose or glutamine stimulate the carboxyl methylation of this PPase in intact beta cells. More importantly, activity of this ACC-PPase is abolished in islet derived from the GK rat, a model for NIDDM, compared to islets from the control Wistar rats. Using isolated rat islets and pure beta (HIT-T15 and INS-1) cells, the investigators propose to study the regulation of this PPase, and thereby ACC, at 3 cellular levels (i.e. subcellular fractions, intact islets, and purified proteins in reconstituted systems). The investigators will also purify this PPase using microcystin -sepharose affinity columns to determine changes in the (A, B and C) subunit composition of PP2A under various experimental conditions, to establish a link between the carboxyl methylation of the C-subunit and the assembly of PP2A haloenzyme. The investigators will characterize the methylating and demethylating enzymes of PP2 Ac and study the interaction of ACC with its putative PPase. Using similar approaches, the investigators will examine the abnormalities in ACC-phosphatase in the diabetic GK islet and investigate if such a defect is reversed by treatment of diabetic rats with insulin or phlorizin. The proposed studies should establish a link between ACC activation and physiologic insulin secretion from isolated beta cells. These studies will also identify the locus that is causal to the observed defect in ACC-phosphatase and ACC activation signaling cascade in the diabetic beta cell, leading to abnormal insulin secretion demonstrable in this model for NIDDM.