Glucose- and GTP-dependent stimulation of the carboxyl methylation of CDC42 in rodent and human pancreatic islets and pure beta cells. Evidence for an essential role of GTP-binding proteins in nutrient-induced insulin secretion.

Abstract

Several GTP-binding proteins (G-proteins) undergo posttranslational modifications (isoprenylation and carboxyl methylation) in pancreatic ␤ cells. Herein, two of these were identified as CDC42 and rap 1, using Western blotting and immunoprecipitation. Confocal microscopic data indicated that CDC42 is localized only in islet endocrine cells but not in acinar cells of the pancreas. CDC42 undergoes a guanine nucleotide-specific membrane association and carboxyl methylation in normal rat islets, human islets, and pure ␤ (HIT or INS-1) cells. GTP ␥ S-dependent carboxyl methylation of a 23-kD protein was also demonstrable in secretory granule fractions from normal islets or ␤ cells. AFC (a specific inhibitor of prenyl-cysteine carboxyl methyl transferases) blocked the carboxyl methylation of CDC42 in five types of insulin-secreting cells, without blocking GTP ␥ S-induced translocation, implying that methylation is a consequence (not a cause) of transfer to membrane sites. High glucose (but not a depolarizing concentration of K ϩ ) induced the carboxyl methylation of CDC42 in intact cells, as assessed after specific immunoprecipitation. This effect was abrogated by GTP depletion using mycophenolic acid and was restored upon GTP repletion by coprovision of guanosine. In contrast, although rap 1 was also carboxyl methylated, it was not translocated to the particulate fraction by GTP ␥ S; furthermore, its methylation was also stimulated by 40 mM K ϩ (suggesting a role which is not specific to nutrient stimulation). AFC also impeded nutrient-induced (but not K ϩ -induced) insulin secretion from islets and ␤ cells under static or perifusion conditions, whereas an inactive structural analogue of AFC failed to inhibit insulin release. These effects were reproduced not only by S -adenosylhomocysteine (another methylation inhibitor), but also by GTP depletion. Thus, the glucose-and GTP-dependent carboxyl methylation of G-proteins such as CDC42 is an obligate step in the stimulus-secretion coupling of nutrient-induced insulin secretion, but not in the exocytotic event itself. Furthermore, AFC blocked glucose-activated phosphoinositide turnover, which may provide a partial biochemical explanation for its effect on secretion, and implies that certain G-proteins must be carboxyl methylated for their interaction with signaling effector molecules, a step which can be regulated by intracellular availability of GTP.

Dominant-Negative α-Subunit of Farnesyl- and Geranyltransferase Inhibits Glucose-Stimulated, but Not KCl-Stimulated, Insulin Secretion in INS 832/13 Cells

“…1B). It must be added that Cdc42, another geranylgeranylated protein, which we and others have implicated in GSIS (3,6,7,9,10,22), also accumulated in the cytosolic fraction in INS 832/13 cells after incubation with GGTI-2147 (Fig. 1A).…”

“…Original observations from multiple laboratories, including our own, demonstrated critical involvement of small G-proteins, such as Rac1, Cdc42, Rap1, and ARF6 (ADP-ribosylation factor 6) in GSIS from normal rat islets, human islets, and clonal ␤-cell preparations (3,(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23). Such conclusions were primarily based on data from experiments using 1) inhibitors of requisite posttranslational modifications of these G-proteins; 2) clostridial toxins, which monoglucosylate and inactivate specific G-proteins; and 3) gene depletion (5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23). Despite this compelling evidence, the precise biochemical mechanisms underlying glucose-mediated activation of these signaling proteins leading to GSIS remain only partially understood.…”

The atypical antipsychotic clozapine impairs insulin secretion by inhibiting glucose metabolism and distal steps in rat pancreatic islets

“…In the present study, clozapine did not affect mastoparan-induced insulin secretion at 3.3 mmol/l glucose, but did suppress its augmentation of glucose in a Ca 2+ -independent manner. Glucose augments posttranslational modifications of specific small G proteins in a GTP-sensitive manner [38,39], and glucose augmentation is extremely dependent on GTP, which increases in parallel with ATP on glucose stimulation [40]. Therefore, clozapine may affect the activation of a specific G protein by inhibiting ATP production, and this could contribute, at least in part, to the alteration in distal steps of insulin secretion.…”

Activation of Acetyl-CoA Carboxylase by a Glutamate- and Magnesium-Sensitive Protein Phosphatase in the Islet β-Cell

“…Human and rat pancreatic islets and clonal ␤ (HIT-T15 and INS-1) cells. Pancreatic islets were isolated from male SD rats (300 -400 g body wt) by the collagenase digestion method, as described in our earlier publications (7,17,18). Islets were manually picked twice under stereo microscopic control to avoid any contamination by exocrine tissue.…”

“…Isolation of subcellular fractions from ␤-cell homogenates. Subcellular fractions from insulin-secreting ␤-cells were isolated by a differential centrifugation procedure, as previously described (7,17,18). All procedures were carried out at 4°C unless stated otherwise.…”