To gain insight into the regulation of pancreatic beta-cell mitochondrial metabolism, the direct effects on respiration of different mitochondrial substrates, variations in the ATP/ADP ratio and free Ca2+ were examined using isolated mitochondria and permeabilized clonal pancreatic beta-cells (HIT). Respiration from pyruvate was high and not influenced by Ca2+ in State 3 or under various redox states and fixed values of the ATP/ADP ratio; nevertheless, high Ca2+ elevated pyridine nucleotide fluorescence, indicating activation of pyruvate dehydrogenase by Ca2+. Furthermore, in the presence of pyruvate, elevated Ca2+ stimulated CO2 production from pyruvate, increased citrate production and efflux from the mitochondria and inhibited CO2 production from palmitate. The latter observation suggests that beta-cell fatty acid oxidation is not regulated exclusively by malonyl-CoA but also by the mitochondrial redox state. alpha-Glycerophosphate (alpha-GP) oxidation was Ca(2+)-dependent with a half-maximal rate observed at around 300 nM Ca2+. We have recently demonstrated that increases in respiration precede increases in Ca2+ in glucose-stimulated clonal pancreatic beta-cells (HIT), indicating that Ca2+ is not responsible for the initial stimulation of respiration [Civelek, Deeney, Kubik, Schultz, Tornheim and Corkey (1996) Biochem. J. 315, 1015-1019]. It is suggested that respiration is stimulated by increased substrate (alpha-GP and pyruvate) supply together with oscillatory increases in ADP [Nilsson, Schultz, Berggren, Corkey and Tornheim (1996) Biochem. J. 314, 91-94]. The rise in Ca2+, which in itself may not significantly increase net respiration, could have the important functions of (1) activating the alpha-GP shuttle, to maintain an oxidized cytosol and high glycolytic flux; (2) activating pyruvate dehydrogenase, and indirectly pyruvate carboxylase, to sustain production of citrate and hence the putative signal coupling factors, malonyl-CoA and acyl-CoA; and (3) increasing mitochondrial redox state to implement the switch from fatty acid to pyruvate oxidation.

The main function of white adipose tissue is to store nutrient energy in the form of triglycerides. The mechanism by which free fatty acids (FFA) move into and out of the adipocyte has not been resolved. We show here that changes in intracellular pH (pHj) (6).FFA are stored and released from adipose tissue in response to nutrient need and hormonal stimulation of the tissue. The increase in fuel storage that results from exposure of adipocytes to insulin is mediated by a series of signaling events, which also lead to the regulation of protein synthesis and gene transcription (7). The immediate consequences of some of the transducing mechanisms that have been defined to date are not entirely clear (7). For example, exposure of adipose tissue (8), muscle (9), and liver (10) to insulin results in an increase in intracellular pH (pHi). It has been suggested that this effect is mediated by activation of Na+/HI exchange (8). Alternatively, the effect of insulin on pHi also could arise from the removal of cytosolic FFA by esterification. The resulting alkalinization, by whatever mechanism, could function as a vital component of the tissue response to insulin, much as pH changes are involved in the regulation of fertilization, proliferation, and metabolism in other cellular contexts (11).The purpose of the studies presented here was to determine the mechanism by which FFA move into adipocytes (the major functional goal of insulin action) and out of adipocytes (the major functional goal of lipolysis). Our data indicate a passive diffusion mechanism as opposed to a carrier-mediated process.

Glucagon-like peptide 1 (GLP-1) is the most potent physiological incretin for insulin secretion from the pancreatic ␤-cell, but its mechanism of action has not been established. It interacts with specific cell-surface receptors, generates cAMP, and thereby activates protein kinase A (PKA). Many changes in pancreatic ␤-cell function have been attributed to PKA activation, but the contribution of each one to the secretory response is unknown. We show here for the first time that GLP-1 rapidly released free fatty acids (FFAs) from cellular stores, thereby lowering intracellular pH (pH i ) and stimulating FFA oxidation in clonal ␤-cells (HIT). Similar changes were observed with forskolin, suggesting that stimulation of lipolysis was a function of PKA activation in ␤-cells. G lucagon-like peptide 1 (GLP-1) is the most potent potentiator of glucose-induced insulin secretion that has been described (1,2). This peptide causes the elevation of cAMP and the activation of protein kinase A (PKA) (3); however, it releases insulin only in the presence of stimulatory glucose (4) and thus serves as an incretin rather than a secretagogue (5). Activation of PKA leads to phosphorylation of multiple ␤-cell proteins, many of which have been hypothesized to play a role in insulin secretion (6-8). The nature of the endogenous substrates for PKA that may potentiate insulin secretion is unknown. Because the islet contains large stores of triglycerides (9), particularly in diabetes (10), another possible role of the normal rise in cAMP could be to stimulate lipolysis (via lipase activation), thereby providing the cell with free fatty acids (FFAs). Recent research on hormone-sensitive lipase (HSL) in ␤-cells yielded results consistent with that notion (11). The released FFAs may directly effect secretion, or they may do so indirectly via generation of other lipids, including the putative long-chain acyl CoA (LC-CoA) signal, diacylglycerol (DAG), and phosphatidic acid (PA) (12). The acute addition of exogenous FFAs is also known to enhance glucose-stimulated secretion (9,13-15).We have shown in previous studies that added FFAs cause acidification in ␤-cells (16) and fat cells (17) as a consequence of the flip-flop mechanism of diffusion across the plasma membrane (16,17). Furthermore, we have shown in adipocytes that the elevation of cAMP stimulates lipolysis, with a resulting decrease in the intracellular pH (pH i ) caused by the release of FFAs, which become partially ionized (17). Therefore, in this study we assessed whether GLP-1 has a similar effect on lipolysis in ␤-cells. Our data show a decrease in pH i and a release of FFAs by agents that increase cAMP, presumably via activation of HSL. Furthermore, the incretin effect was largely diminished by a lipase inhibitor, whereas glucose-stimulated secretion was less affected. These findings indicate that cAMP-mediated lipolysis may play an important role in ␤-cell signal transduction and the incretin effect of GLP-1. RESEARCH DESIGN AND METHODSGrowth and incubation of cells. Clonal pancr...