There is considerable evidence to show that alterations in the pattern of insulin secretion from the beta cell of the endocrine pancreas may play a pathogenic role in type II diabetes. Normally, the pattern of insulin release in response to an increase in glucose concentration is a biphasic one. Recent work done by the principla investigator and his collaborators has led to the hypothesis that the two phases of insulin secretion are regulated by different biochemical branches within the calcium messenger system: the first branch operates via a rise in the calcium concentration of the cell cytosol activating calmodulin-dependent reactions; the second branch via a rise in diacylglycerol content activating C-kinase-dependent reactions. The purposes of the proposed studies are to establish this hypothesis more fully by studying the biochemical events which underline each phase of insulin secretion. First phase secretion will be induced by either glucose, the ionophore, A23187, or phospholipase A2. Examination of the effects of these agents on the following processes will be undertaken: a) the turnover of phosphoinositides and the production of diacylglycerol and inositol triphosphate; b) the concentration of Ca2+ in the cell cytosol; c) the phosphorylation of cytosolic proteins; and d) the production of arachidonic acid and its metabolism via the lypoxygenase and epoxygenase pathways. In addition, the products of these pathways will be employed to determine their effects on insulin release, islet cell calcium metabolism, and protein phosphorylation. Second phase secretion will be induced by either the phorbol ester, TPA, or the diglycerides 1-acetyl, 2-arachidonyl glycerol and 1-oley1 2-acetyl glycerol. Their effects on insulin secretion, cellular calcium metabolism, protein phosphorylation, and various aspects of lipid metabolism will all be studied. The basic method to be employed is the perifused rat pancreatic islet. Insulin will be measured by radioimmunoassay. The turnover of lipids is assessed by a variety of methods, but in particular, high pressure liquid chromatography. Protein phosphorylation will be examined using labeling with [32p]-HPO4= and separating protein by SDS-polyacrylamide slab gel electrophoresis either in one or two dimensions. Free calcium will be measured by the HOST-aequorin method of Borle and Snowdowne.

Agency
National Institute of Health (NIH)
Institute
National Institute of Arthritis, Diabetes, Digestive and Kidney Diseases (NIADDK)
Type
Research Project (R01)
Project #
5R01AM034381-02
Application #
3153153
Study Section
Metabolism Study Section (MET)
Project Start
1984-07-01
Project End
1987-06-30
Budget Start
1985-07-01
Budget End
1986-06-30
Support Year
2
Fiscal Year
1985
Total Cost
Indirect Cost
Name
Yale University
Department
Type
Schools of Nursing
DUNS #
082359691
City
New Haven
State
CT
Country
United States
Zip Code
Zawalich, W S; Diaz, V A; Zawalich, K C (1988) Role of phosphoinositide metabolism in induction of memory in isolated perifused rat islets. Am J Physiol 254:E609-16
Zawalich, W S (1988) Modulation of insulin secretion from beta-cells by phosphoinositide-derived second-messenger molecules. Diabetes 37:137-41
Zawalich, W S; Zawalich, K C (1988) Induction of memory in rat pancreatic islets by tolbutamide. Dependence on ambient glucose level, calcium, and phosphoinositide hydrolysis. Diabetes 37:816-23
Zawalich, W S; Zawalich, K C (1988) Phosphoinositide hydrolysis and insulin release from isolated perifused rat islets. Studies with glucose. Diabetes 37:1294-300
Zawalich, W S (1988) Synergistic impact of cholecystokinin and gastric inhibitory polypeptide on the regulation of insulin secretion. Metabolism 37:778-81
Zawalich, W; Takuwa, N; Takuwa, Y et al. (1987) Interactions of cholecystokinin and glucose in rat pancreatic islets. Diabetes 36:426-33
Rossetti, L; Shulman, G I; Zawalich, W S (1987) Physiological role of cholecystokinin in meal-induced insulin secretion in conscious rats. Studies with L 364718, a specific inhibitor of CCK-receptor binding. Diabetes 36:1212-5
Zawalich, W S; Diaz, V A; Zawalich, K C (1987) Cholecystokinin-induced alterations in beta-cell sensitivity. Duration, specificity, and involvement of phosphoinositide metabolism. Diabetes 36:1420-4
Zawalich, W S; Diaz, V A (1987) Prior cholecystokinin exposure sensitizes islets of Langerhans to glucose stimulation. Diabetes 36:118-22
Zawalich, W S; Diaz, V; Cote, S (1986) Influence of mebendazole on insulin secretion from isolated rat islets. J Pharmacol Exp Ther 237:826-9

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