REGULATION OF GUANINE - NUCLEOTIDE-BINDING PROTEINS IN ISLET SUBCELLULAR-FRACTIONS BY PHOSPHOLIPASE-DERIVED LIPID MEDIATORS OF INSULIN-SECRETION

In the accompanying article (Kowluru, A., Rabaglia, M.E., Mose, K.E. and Metz, S.A. (1994) Biochim. Biophys. Acta 1222, 348-359) we identified three specific GTPase activities in islet subcellular fractions; most notably, two of these were enriched in the secretory granules. In the present study, we describe the regulation of GTPase activity in subcellular fractions of normal rat and human islets by insulinotropic lipids with a similar rank order as their insulin-releasing capacity. Arachidonic acid (AA), lysophosphatidylcholine (LPC), or phosphatidic acid (PA) inhibited the GTPase activities significantly (by 60-80%) in isIet homogenates; each also selectively inhibited certain GTPases in specific individual fractions. Less insulinotropic fatty acids, such as linoleic acid and oleic acid, inhibited GTPase to a lesser degree, whereas lysophosphatidic acid (LPA), phosphatidylcholine (PC) or palmitic acid, which do not acutely promote secretion, were ineffective. Similar inhibitory effects of these lipids were also demonstrable in fractions of human islets as well as those of transformed beta-cells (HIT cells). The effects of lipids were not attributable to their detergent properties (since several detergents failed to mimic lipid effects) or to inhibition of GTP binding (since they actually increased GTP gamma S binding modestly, and moreover, in reconstituted fractions, they potentiated GDP/GTP exchange activity up to 2-fold). These data indicate that the insulinotropic nature of the lipids might be due, in part, to their ability to maintain G-proteins in their GTP-bound (active) configuration by increasing GTP binding and decreasing its hydrolysis. These studies comprise the first evidence for the regulation by biologically active lipids of endocrine cell G-proteins at a locus distal to plasma membrane events (i.e., on endocrine secretory granules), and provide thereby a possible novel mechanism whereby the activation of islet endogenous phospholipases might culminate in insulin exocytosis.