digitonin and Insulinoma

After protein cross-linking by dimethyl suberimidate, tumoral insulin-producing cells of the RINm5F line were either exposed to digitonin for measurement of hexokinase activity in the resulting cell pellet and supernatant, or incubated in the presence of D-[5-3H]glucose, D-[U-14C]glucose or L-[U-14C]glutamine to assess the metabolism of these nutrients. After digitonin treatment, the activity of hexokinase recovered in the cell pellet was about 40% higher in cross-linked than control RINm5F cells. Although failing to affect the metabolism of L-[U-14C]glutamine, and severely decreasing the oxidation of D-[U-14C]glucose, the cross-linking of proteins accentuated the increase in D-[5-3H]glucose utilization and D-[U-14C]glucose conversion to acidic metabolites resulting from a rise in hexose concentration from 2.8 to 16.7 mM. The latter change represents a mirror image of that previously found in cross-linked pancreatic islets. Taking into account the vastly different participation of glucokinase to hexose phosphorylation in RINm5F cells and normal islet cells, the present findings further support, therefore, the regulatory role of protein-to-protein interaction in the control of glucokinase catalytic activity in these fuel-sensing cells.

Topics: Animals; Cross-Linking Reagents; Digitonin; Dimethyl Suberimidate; Fructosephosphates; Glucose; Glucose-6-Phosphate; Glutamine; Hexokinase; Insulinoma; Rats; Tumor Cells, Cultured

Mastoparan, a tetradecapeptide found in wasp venom that stimulates G-proteins, increases insulin secretion from beta-cells. In this study, we have examined the role of heterotrimeric G-proteins in mastoparan-induced insulin secretion from the insulin-secreting beta-cell line beta-TC3. Mastoparan stimulated insulin secretion in a dose-dependent manner from digitonin-permeabilized beta-TC3 cells. Active mastoparan analogues mastoparan 7, mastoparan 8, and mastoparan X also stimulated secretion. Mastoparan 17, an inactive analogue of mastoparan, did not increase insulin secretion from permeabilized beta-TC3 cells. Mastoparan-induced insulin secretion from permeabilized beta-TC3 cells was inhibited by pretreatment of the cells with pertussis toxin, suggesting that mastoparan-induced insulin secretion is mediated through a pertussis toxin-sensitive G-protein present distally in exocytosis. Enriched insulin secretory granules (ISG) were prepared by sucrose/nycodenz ultracentrifugation. Western immunoblotting performed on beta-TC3 homogenate and ISG demonstrated that G alpha i was dramatically enriched in ISG. Levels of G alpha o and G alpha q were comparable in homogenate and ISG. Mastoparan stimulated ISG GTPase activity in a pertussis toxin-sensitive manner. Mastoparan 7 and mastoparan 8 also stimulated GTPase activity in the ISG, while the inactive analogue mastoparan 17 had no effect. Selective localization of G alpha i to ISG was confirmed with electron microscopic immunocytochemistry in beta-TC3 cells and beta-cells from rat pancreas. In contrast to G alpha o and G alpha q, G alpha was clearly localized to the ISG. Together, these data suggest that mastoparan may act through the heterotrimeric G-protein G alpha i located in the ISG of beta-cells to stimulate insulin secretion.

Topics: Animals; Blotting, Western; Cell Membrane Permeability; Cytoplasmic Granules; Digitonin; Dose-Response Relationship, Drug; Exocytosis; GTP Phosphohydrolases; GTP-Binding Proteins; Insulin; Insulinoma; Intercellular Signaling Peptides and Proteins; Islets of Langerhans; Microscopy, Immunoelectron; Peptides; Pertussis Toxin; Rats; Tumor Cells, Cultured; Virulence Factors, Bordetella; Wasp Venoms