Oscillations in oxygen consumption by permeabilized clonal pancreatic beta-cells (HIT) incubated in an oscillatory glycolyzing muscle extract: roles of free Ca2+, substrates, and the ATP/ADP ratio.

To determine whether oscillations in glycolysis could underlie the oscillations in O2 consumption observed in intact islets, we evaluated the capacity of an islet extract to exhibit spontaneous oscillations in glycolysis. When a cell-free extract obtained from approximately 1,000 islets was supplied with glucose and glycolytic cofactors, oscillations in NADH fluorescence were obtained. After this demonstration of spontaneous oscillations in islet extracts, we bathed permeabilized clonal beta-cells in the more plentiful spontaneously oscillating glycolytic muscle extract that generates pulses of alpha-glycerophosphate and pyruvate and induces oscillations in free Ca2+ and the ATP/ADP ratio. This preparation was used to investigate whether changes in Ca2+ and possibly alpha-glycerophosphate or pyruvate supply could underlie observed oscillations in O2 consumption and explain coordination between cytosolic and mitochondrial metabolism. We found that oscillations of O2 consumption and Ca2+ of a similar period were induced. Removal of medium Ca2+ with EGTA did not prevent the oscillations in O2 consumption nor were they greatly affected by the substantial rise in medium Ca2+ on treatment with thapsigargin to inhibit sequestration into the endoplasmic reticulum. The 02 oscillations were also not eliminated by the addition of relatively high concentrations of pyruvate or alpha-glycerophosphate. However, they were lost on addition of fructose-2,6-P2 at concentrations that prevent oscillations of glycolysis and the ATP/ADP ratio. Addition of a high concentration of ADP increased 02 consumption and also prevented 02 oscillations. These results suggest that the changes in respiration reflected in the 02 oscillations occur in response to the oscillations in the ATP/ADP ratio or ADP concentration and that this parameter is a primary regulator of 02 consumption in the pancreatic beta-cell.

Glucagon-like peptide 1 stimulates lipolysis in clonal pancreatic beta-cells (HIT).

Glucagon-like peptide 1 (GLP-1) is the most potent physiological incretin for insulin secretion from the pancreatic beta-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 beta-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 (pHi) and stimulating FFA oxidation in clonal beta-cells (HIT). Similar changes were observed with forskolin, suggesting that stimulation of lipolysis was a function of PKA activation in beta-cells. Triacsin C, which inhibits the conversion of FFAs to long-chain acyl CoA (LC-CoA), enhanced basal FFA efflux as well as GLP-1-induced acidification and efflux of FFAs from the cell. Increasing the concentration of the lipase inhibitor orlistat progressively and largely diminished the increment in secretion caused by forskolin. However, glucose-stimulated secretion was less inhibited by orlistat and only at the highest concentration tested. Because the acute addition of FFAs also increases glucose-stimulated insulin secretion, these data suggest that the incretin function of GLP-1 may involve a major role for lipolysis in cAMP-mediated potentiation of secretion.

Changes in internal pH caused by movement of fatty acids into and out of clonal pancreatic beta-cells (HIT).

Cells require a constant influx of free fatty acids for lipid resynthesis and metabolic energy. Fatty acids also act as second messengers and modulate channel activities. In the pancreatic beta-cell, fatty acids have both acute and chronic effects on insulin secretion. We show that the addition of fatty acid to pancreatic beta-cells in vitro produces a persistent decrease in intracellular pH, which begins immediately after the addition of fatty acid and has an exponential time course with t1/2 approximately 60 s. The pH drop can be largely reversed by the addition of albumin. The observed pH effect can be explained by passive diffusion ("flip-flop") of un-ionized fatty acid across the plasma membrane. Acidification by a fatty acid dimer and alkalinization by an alkylamine also favor the flip-flop mechanism of transport rather than a protein-mediated mechanism. Our method provides for the first time a real-time measurement of fatty acid import into cells. The significant pH change may be important in mediating some of the regulatory effects of fatty acid, such as inhibition of glycolysis.

Intracellular pH in adipocytes: effects of free fatty acid diffusion across the plasma membrane, lipolytic agonists, and insulin.

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 (pH1) in adipocytes correlate with the movement of FFA across cellular membranes as predicted by the Kamp and Hamilton model of passive diffusion of FFA. Exposure of fat cells to lipolytic agents or external FFA results is a rapid intracellular acidification that is reversed by metabolism of the FFA or its removal by albumin. In contrast, insulin causes an alkalinization of the cell, consistent with its main function to promote esterification. Inhibition of Na+/H+ exchange in adipocytes does not prevent the changes in pHi caused by FFA, lipolytic agents, or insulin. A fatty acid dimer, which diffuses into the cell but is not metabolized, causes an irreversible acidification. Taken together, the data suggest that changes in pHi occur in adipocytes in response to the passive diffusion of un-ionized FFA (flip-flop) into and out of the cell and in response to their metabolism and production within the cell. These changes in pHi may, in turn, modulate hormonal signaling and metabolism with significant impact on cell function.

Temporal sequence of metabolic and ionic events in glucose-stimulated clonal pancreatic beta-cells (HIT).

Stimulation of insulin release by glucose requires increased metabolism of glucose and a rise in cytosolic free Ca2+ in the pancreatic beta-cell. It is accompanied by increases in respiratory rate, pyridine and flavin nucleotide reduction state, intracellular pH and the ATP/ADP ratio. To test alternative proposals of the regulatory relationships among free Ca2+, mitochondrial metabolism and cellular energy state, we determined the temporal sequence of these metabolic and ionic changes following addition of glucose to clonal pancreatic beta-cells (HIT). Combined measurements of the native fluorescence of reduced pyridine nucleotides and oxidized flavin, intracellular pH, and free Ca2+ were performed together with simultaneous measurement of O2 tension or removal of samples for assay of the ATP/ADP ratio. The initial changes were detected in three phases. First, decreases occurred in the ATP/ADP ratio (<3 s) and increases in pyridine (2 +/- 1 s) and flavin (2 +/- 1 s) nucleotide reduction. Next, increases in the O2 consumption rate (20 +/- 5 s), the ATP/ADP ratio (29 +/- 12 s) and internal pH (48 +/- 5 s) were observed. Finally, cytosolic free Ca2+ rose (114 +/- 10 s). Maximal changes in the ATP/ADP ratio, O2 consumption and pyridine and flavin nucleotide fluorescence preceded the beginning of the Ca2+ change. These relationships are consistent with a model in which phosphorylation of glucose is the initial event which generates the signals that lead to an increase in respiration, a rise in the ATP/ADP ratio and finally influx of Ca2+. Our results indicate that Ca2+ does not function as the initiator of increased mitochondrial respiration.

Glucose-stimulated increase in cytoplasmic pH precedes increase in free Ca2+ in pancreatic beta-cells. A possible role for pyruvate.

The temporal relationship of glucose-induced increases in cytoplasmic pH (pHi) and cytoplasmic free Ca2+ was studied in single mouse pancreatic beta-cells and suspensions of clonal beta-cells (HIT). In both preparations of cells the increase in pHi preceded the cytoplasmic free Ca2+ increase. Therefore the alkalinization cannot be a consequence of the Ca2+ influx. A potential metabolic mechanism for the increase in pHi, involving stimulation of pyruvate transport and oxidation, was demonstrated in a model system of liver mitochondria incubated with pyruvate, ATP, and hexokinase to which glucose was then added to initiate ATP use. The involvement of this mechanism in beta-cells is suggested by the observation that the alkalinization was prevented in most cells by incubation with 3-hydroxycyanocinnamate, a mitochondrial pyruvate transport inhibitor. On the other hand, the inhibited cells exhibited normal Ca2+ responses to glucose stimulation. This indicates that neither pyruvate metabolism nor the alkalinization is of critical importance for the Ca2+ signal, though pyruvate oxidation or its metabolites may be important in downstream regulation of secretion.

Regulation of pancreatic beta-cell mitochondrial metabolism: influence of Ca2+, substrate and ADP.

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.

Fat depot origin affects fatty acid handling in cultured rat and human preadipocytes.

Regional differences in free fatty acid (FFA) handling contribute to diseases associated with particular fat distributions. As cultured rat preadipocytes became differentiated, FFA transfer into preadipocytes increased and was more rapid in single perirenal than in epididymal cells matched for lipid content. Uptake by human omental preadipocytes was greater than uptake by abdominal subcutaneous preadipocytes. Adipose-specific fatty acid binding protein (aP2) and keratinocyte lipid binding protein abundance was higher in differentiated rat perirenal than in epididymal preadipocytes. This interdepot difference in preadipocyte aP2 expression was reflected in fat tissue in older animals. Carnitine palmitoyltransferase 1 activity increased during differentiation and was higher in perirenal than in epididymal preadipocytes, particularly the muscle isoform. Long-chain acyl-CoA levels were higher in perirenal than in epididymal preadipocytes and isolated fat cells. These data are consistent with interdepot differences in fatty acid flux ensuing from differences in fatty acid binding proteins and enzymes of fat metabolism. Heterogeneity among depots results, in part, from distinct intrinsic characteristics of adipose cells. Different depots are effectively separate miniorgans.