We have attempted to define the nature of insulin secretory defect(s) in aged animals. In these studies, pancreatic islets were isolated from 2- and 18-mo-old Fischer 344 rats. Margination of secretion vesicles during exocytosis was assessed by measuring the recruitment of somatostatin (SRIF) receptors to the surface membrane. Section vesicle lysis was studied by measuring insulin release into the incubation media. Submaximal and maximal glucose-induced insulin secretion was significantly greater in islets isolated from younger rats (P less than 0.01). SRIF receptor recruitment was stimulated by glucose in both younger and older Fischer 344 rats. However, an increase in SRIF receptor recruitment was reduced in islets isolated from older animals (from 2.14 +/- 0.4 to 4.6 +/- 0.4 fmol/10 islets) (P less than 0.01) as compared with islets from younger animals (from 2.6 +/- 0.2 to 6.2 +/- 0.4 fmol/10 islets). When secretion vesicle lysis was inhibited by the presence of sodium isethionate in the incubation media, glucose (300 mg/dl) failed to stimulate secretion vesicle margination to the plasma membrane. In contrast, glyburide (0.6 micrograms/ml) continued to stimulate directly secretion vesicle margination in islets from aged animals (from 2.1 +/- 0.3 to 6.0 +/- 0.3 fmol/10 islets). We conclude that glucose-induced margination of secretion vesicles at the plasma membrane is impaired by the aging process. This impairment results in lower submaximal and maximal insulin secretory response to glucose. The fact that glyburide is capable of stimulating secretion vesicle margination suggests that glucose signal recognition and/or stimulus-secretion coupling may be the locus of impairment in the process of insulin secretion in older animals.

Farnesylation of p21Ras by farnesyltransferase (FTase) is obligatory for anchoring p21Ras to the plasma membrane, where it can be activated by growth factors. Insulin significantly stimulates the phosphorylation of the alpha-subunit of FTase (4-fold) and the enzymatic activity of FTase in 3T3-L1 fibroblasts and adipocytes. FTase activity was assessed by the amount of [3H] mevalonate (a precursor of farnesyl) incorporated into p21Ras in vivo and by quantitating the amount of farnesylated p21Ras before and after insulin administration. Insulin-stimulated phosphorylation of the alpha-subunit of FTase in 3T3-L1 fibroblasts and adipocytes was blocked by the mitogen-activated protein/extracellular-signal regulated kinase-kinase inhibitor, PD98059, but not by wortmannin or bisindolylmaleimide. Additionally, PD98059 blocked insulin-stimulated [3H]mevalonic incorporation and farnesylation of unprocessed p21Ras in both cell lines. Furthermore, expression of the dominant negative mutant of p21Ras precluded insulin-stimulated phosphorylation of the FTase alpha-subunit and activation of its enzymatic activity. In contrast, 3T3-L1 fibroblasts, expressing the constitutively active Raf-1, exhibited enhanced phosphorylation of the FTase alpha-subunit. It seems that insulin's effect on the phosphorylation and activation of FTase in both fibroblasts and adipocytes is mediated via the Ras pathway, resulting in a positive feedback augmentation of the cellular pool of farnesylated p21Ras.

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