title: Effects of a phorbol ester and clomiphene on protein phosphorylation and insulin secretion in rat pancreatic islets.
abstract: The potentiation of glucose-stimulated insulin release induced by 100 nM-12-O-tetradecanoylphorbol 13-acetate (TPA) was inhibited by clomiphene, an inhibitor of protein kinase C (PK C), in a dose-dependent manner. Clomiphene at concentrations up to 50 microM had a modest inhibitory action (27%) on insulin release stimulated by 10 mM-glucose alone, but had no effect on the potentiation of insulin release induced by forskolin. Islet PK C activity, associated with a particulate fraction, was stimulated maximally by 100 nM-TPA. This stimulation was blocked by clomiphene in a dose-dependent manner, with 50% inhibition at 30 microM. Incubation of intact islets with TPA after preincubation with [32P]Pi and 10 mM-glucose to label intracellular ATP resulted primarily in enhanced phosphorylation of a 37 kDa protein (mean value, +/- S.E.M., 36,700 +/- 600 Da; n = 7). This increased phosphorylation was blocked by the simultaneous inclusion of clomiphene. Subcellular fractionation revealed the presence of the 37 kDa phosphoprotein in a 24,000 g particulate fraction of islet homogenates. Neither clomiphene nor TPA affected the rate of glucose oxidation by islets. These results show that the phosphorylation state of a 37 kDa membrane protein parallels the modulation of insulin release induced by TPA and clomiphene and support a role for PK C in the insulin-secretory mechanism.
authors.affiliation: Nuffield Department of Clinical Biochemistry, John Radcliffe Hospital, Headington, Oxford, U.K.
authors.forename: S J
authors.initials: SJ
authors.lastname: Hughes
authors.forename: S J
authors.initials: SJ
authors.lastname: Ashcroft
mesh_terms: D000818:Animals; D002996:Clomiphene / Q000494:pharmacology*; D005947:Glucose / Q000378:metabolism; D066298:In Vitro Techniques; D007328:Insulin / Q000378:metabolism*; D000078790:Insulin Secretion; D007515:Islets of Langerhans / Q000187:drug effects / Q000378:metabolism*; D008297:Male; D010084:Oxidation-Reduction; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism; D011506:Proteins / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*
chemical_list: D007328:Insulin; D011506:Proteins; D002996:Clomiphene; D011493:Protein Kinase C; D005947:Glucose; D013755:Tetradecanoylphorbol Acetate