The majority of low molecular weight G proteins undergoes a series of post-translational modification steps, e.g., isoprenylation, at their C-terminal cysteine, which seem to be critical for the transport of the modified proteins to the membrane sites for interaction with their respective effector proteins. Using lovastatin, an inhibitor of mevalonic acid, and hence, isoprenoid biosynthesis, we demonstrated previously that protein isoprenylation is critical for physiological insulin secretion from normal rat islets. Herein, we used more selective synthetic inhibitors of protein prenylation to examine their effects on glucose-and calcium-mediated insulin secretion from ␤TC3 cells. Both 3-allyl-and vinylfarnesols, which inhibit and/or modulate protein farnesyl transferases, significantly (80 -95%) inhibited glucoseand KCl-stimulated insulin secretion from these cells. In a similar manner, the allyl and vinyl forms of geranylgeraniol, reagents targeted toward protein geranylation, attenuated insulin secretion elicited by glucose and KCl. Furthermore, manumycin A, a natural inhibitor of protein farnesylation, and geranylgeranyl transferase inhibitor-2147 (GGTI-2147), a peptidomimetic inhibitor of protein geranylgeranylation, also inhibited glucoseand KCl-induced insulin secretion to comparable degrees. Treatment of ␤TC3 cells with either 3-vinylfarnesol or 3-vinyl geranylgeraniol resulted in accumulation of unprenylated proteins in the cytosolic fraction. These data further support our original formulation that inhibition of isoprenylation of small molecular weight G proteins might impede their interaction with their putative effectors, which may be required for physiological insulin secretion.

Mastoparan, a tetradecapeptide from wasp venom, stimulates insulin secretion from the islet beta-cells, presumably via activation of trimeric G proteins. Herein, we used Clostridial toxins, which selectively modify and inactivate the Rho subfamily of G proteins, to examine whether mastoparan-induced insulin secretion also involves activation of these signaling proteins. Mastoparan, but not mastoparan 17 (an inactive analog of mastoparan), significantly stimulated insulin secretion from betaTC3 and INS-1 cells. Preincubation of betaTC3 cells with either Clostridium difficille toxin B, which inactivates Rho, Cdc42, and Rac, or Clostridium sordellii toxin, which inactivates Ras, Rap, and Rac, markedly attenuated the mastoparan-induced insulin secretion, implicating Rac in this phenomenon. Mastoparan-stimulated insulin secretion was resistant to GGTI-2147, a specific inhibitor of geranylgeranylation of Rho G proteins (e.g. Rac), suggesting that mastoparan induces direct activation of Rac via GTP/GDP exchange. This was confirmed by a pull-down assay that quantifies the binding of activated (i.e. GTP-bound) Rac to p21-activated kinase. However, glucose-induced insulin secretion from these cells was abolished by toxin B or GGTI-2147, suggesting that the geranylgeranylation step is critical for glucose-stimulated secretion. Mastoparan significantly increased the translocation of cytosolic Rac and Cdc42 to the membrane fraction. Confocal light microscopy revealed a substantial degree of colocalization of Rac (and, to a lesser degree, Cdc42) with insulin in beta-cells exposed to mastoparan. Further, stable expression of a dominant negative (N17Rac) form of Rac into INS-1 cells resulted in a significant reduction in mastoparan-stimulated insulin secretion from these cells. Taken together, our findings implicate Rho G proteins, specifically Rac, in mastoparan-induced insulin release.

Acetyl-CoA carboxylase (ACC) catalyzes the formation of malonyl-CoA, a precursor in the biosynthesis of longchain fatty acids, which have been implicated in physiological insulin secretion. The catalytic function of ACC is regulated by phosphorylation (inactive)؊de-phosphorylation (active). In this study we investigated whether similar regulatory mechanisms exist for ACC in the pancreatic islet ␤-cell. ACC was quantitated in normal rat islets, human islets, and clonal ␤-cells (HIT-15 or INS-1) using a [ 14 C]bicarbonate fixation assay. In the ␤-cell lysates, ACC was stimulated by magnesium in a concentration-dependent manner. Of all the dicarboxylic acids tested, only glutamate, albeit ineffective by itself, significantly potentiated magnesium-activated ACC in a concentration-dependent manner. ACC stimulation by glutamate and magnesium was maximally demonstrable in the cytosolic fraction; it was markedly reduced by okadaic acid (OKA) in concentrations (<50 nmol/l) that inhibited protein phosphatase 2A (PP2A). Furthermore, pretreatment of the cytosolic fraction with anti-PP2A serum attenuated the glutamate-and magnesium-mediated activation of ACC, thereby suggesting that ACC may be regulated by an OKA-sensitive PP2A-like enzyme. Streptavidin-agarose chromatography studies have indicated that glutamate-and magnesium-mediated effects on ACC are attributable to activation of ACC's dephosphorylation; this suggests that the stimulatory effects of glutamate and magnesium on ACC might involve activation of an OKA-sensitive PP2A-like enzyme that dephosphorylates and activates ACC. In our study, 5-amino-imidazolecarboxamide (AICA) riboside, a stimulator of AMP kinase, significantly inhibited glucosemediated activation of ACC and insulin secretion from isolated ␤-cells. Together, our data provide evidence for a unique regulatory mechanism for the activation of ACC in the pancreatic ␤-cell, leading to the generation of physiological signals that may be relevant for physiological insulin secretion. Diabetes 50:1580 -1587, 2001