PKC-dependent stimulation of exocytosis by sulfonylureas in pancreatic beta cells.

Hypoglycemic sulfonylureas represent a group of clinically useful antidiabetic compounds that stimulate insulin secretion from pancreatic beta cells. The molecular mechanisms involved are not fully understood but are believed to involve inhibition of potassium channels sensitive to adenosine triphosphate (KATP channels) in the beta cell membrane, causing membrane depolarization, calcium influx, and activation of the secretory machinery. In addition to these effects, sulfonylureas also promoted exocytosis by direct interaction with the secretory machinery not involving closure of the plasma membrane KATP channels. This effect was dependent on protein kinase C (PKC) and was observed at therapeutic concentrations of sulfonylureas, which suggests that it contributes to their hypoglycemic action in diabetics.

Ca2+ responses to interleukin 1 and tumor necrosis factor in cultured human skin fibroblasts. Possible implications for Reye syndrome.

Elevated concentrations of cytokines were found in the plasma of patients acutely ill with Reye syndrome (RS) but not in control subjects or recovered RS patients. To determine whether this disorder involves a genetically determined abnormal response to cytokines, the effects of tumor necrosis factor (TNF) and IL-1 on intracellular free Ca2+ were compared in cultured skin fibroblasts from control subjects and patients with RS. IL-1 and TNF caused rapid, transient, and concentration-dependent increases in cytosolic free Ca2+. The peak cytosolic free Ca2+ was greater and occurred at higher concentrations of IL-1 and TNF in patient cells than in cells from age-matched controls. In control cells, the Ca2+ transient diminished sharply with increasing amounts of IL-1 or TNF above the maximum stimulatory concentration. In contrast, in patient fibroblast this bell-shaped curve of concentration dependency was much less apparent. Bradykinin-stimulated Ca2+ transients were similar in the two groups and did not exhibit the bell-shaped concentration dependency. Thus, plasma cytokine levels are elevated in RS patients and the Ca2+ response to cytokines is increased in cells derived from these patients. We propose that the increased response reflects a genetic defect in cytokine receptor-modulated signal transduction.

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.

Morphological and biochemical characterization of possible intracellular precursors of milk lipid globules.

Within milk secreting mammary epithelial cells, immediate precursors of milk lipid globules existed as relatively large (diameter greater than 1 micron) cytoplasmic lipid droplets. The periphery of these lipid droplets was characterized by an electron-dense, granular material which lacked unit membrane character. Cytoplasmic lipid droplets which retained surface coat material were isolated from lactating cow and rat mammary glands. Surface coat material on isolated droplets was composed of polar lipids and proteins. In enzyme activities, this surface material was distinct from plasma membrane and endomembranes. In polar lipid composition, surface material on cytoplasmic lipid droplets was similar to milk lipid globule membrane or was intermediate between endoplasmic reticulum and milk lipid globule membranes. A previously unrecognized group of small structures (diameter less than 0.5 micron), which resembled cytoplasmic lipid droplets in matrix and surface coat appearance, was observed and characterized. These structures were isolated and found to contain large amounts of triacylglycerols, which closely resembled triacyl/glycerols of cytoplasmic lipid droplets in fatty acid composition. Surface coat materials of these small structures and of cytoplasmic lipid droplets were similar in enzymatic, polypeptide and polar lipid composition. Morphological evidence that these small structures may fuse with cytoplasmic lipid droplets was obtained. These small structures, for which we propose- the name micro lipid droplets, may provide triacylglycerols to support growth of cytoplasmic lipid droplets.

Microlipid droplets in milk secreting mammary epithelial cells: evidence that they originate from endoplasmic reticulum and are precursors of milk lipid globules.

Microlipid droplets, structures with diameters less than 0.5 micron, resemble larger cytoplasmic lipid droplets of milk secreting mammary epithelial cells in triacylglycerol core and surface coat composition. Previously, evidence was obtained that microlipid droplets fuse with and support growth of cytoplasmic lipid droplets, which are immediate precursors of large milk lipid globules. Morphological observations suggested that microlipid droplets may also be secreted directly from mammary epithelial cells, yielding the very small lipid globules of milk. The secretion mechanism, which involves envelopment of triacylglycerol droplets in apical plasma membrane, appeared to be the same for microlipid droplets as for larger cytoplasmic lipid droplets. Microlipid droplets appeared to originate by blebbing from cisternae of endoplasmic reticulum. By immunogold cytochemical localization and by immunological identification of electrophoretically separated polypeptides, endoplasmic reticulum, micro- and cytoplasmic lipid droplets, and milk lipid globules had a number of common polypeptides. Kinetics of incorporation of radiolabeled palmitate or glycerol into triacylglycerols and phospholipids were consistent with a possible endoplasmic reticulum origin of microlipid droplets and with the view that microlipid droplets may be secreted directly from the cell or may fuse with cytoplasmic lipid droplets.

Neomycin: a specific drug to study the inositol-phospholipid signalling system?

Neomycin, an antibiotic previously thought to interact specifically with inositol-containing phospholipids, was found to inhibit IP3-mediated Ca2+ release from the intracellular stores of permeabilized insulinoma and liver cells. This inhibition could be relieved by increasing the IP3 concentration. Radiolabelled IP3 was found to bind tightly to columns prepared from neomycin covalently attached to glass beads. ATP was also bound by these columns. It is concluded that neomycin acts in biological systems as a weak anion exchanger and is therefore unsuitable for use as a specific tool to study the role of inositol phospholipids in intracellular signalling.

Metabolic control of beta-cell function.

Glucose-induced insulin secretion is pulsatile. Glucose metabolism generates oscillations in the ATP/ADP ratio which lead to opening and closing of ATP-sensitive K(+)-channels producing subsequent oscillations in membrane potential, cytoplasmic calcium and insulin release. Metabolic signals derived from glucose can also stimulate insulin release independent of their effects on ATP-sensitive K(+)-channels. The ATP/ADP ratio may mediate both ATP-sensitive K(+)-channel-dependent and -independent pathways of secretion. Glucose metabolism also results in an increase in long-chain acyl-CoA, which is proposed to act as an effector molecule in the beta -cell. Long-chain acyl-CoA has a variety of effects in the beta -cell that may effect insulin secretion including opening ATP-sensitive K(+)-channels, activating endoplasmic reticulum Ca(2+)-ATPases and stimulating classical protein kinase C activity. In addition to stimulating insulin release, nutrients also effect gene expression, protein synthesis and beta -cell proliferation. Gene expression is effected by nutrient induction of a variety of immediate early response genes. Glucose stimulates proinsulin biosynthesis both at the translational and transcriptional level. beta -cell proliferation, as a result of insulin-like growth factor and growth hormone mitogenic pathways, is also glucose dependent. Thus, many beta -cell functions in addition to secretion are controlled by nutrient metabolism.

Cytosolic free calcium in adipocytes. Distinct mechanisms of regulation and effects on insulin action.

It has been proposed that an elevation in cytosolic free Ca2+ may play a role in either mediating or antagonizing the ability of insulin to stimulate glucose uptake in adipocytes. This question has been addressed in the present studies using isolated fura-2-loaded rat adipocytes stimulated with a variety of agonists. The effects of insulin, oxytocin, norepinephrine, ATP, and ionomycin on cytosolic free Ca2+ levels were assessed and compared with their effects on transport-limited glucose oxidation. Oxytocin and ionomycin at concentrations which caused 3-5-fold increases in cytosolic Ca2+, by releasing Ca2+ from internal stores, had no effect on insulin-stimulated glucose oxidation. ATP and norepinephrine which caused more modest increases in Ca2+, by mechanisms at least partially dependent on external stores, inhibited insulin-stimulated glucose oxidation. Insulin had no effect on basal Ca2+ levels nor did it modulate the Ca2+ elevation caused by other agonists. These data suggest that insulin-stimulated glucose transport is not associated with an increase in cytosolic Ca2+. In addition, although there appears to be a correlation between inhibition of insulin-stimulated glucose transport and the effect of certain agonists to promote Ca2+ influx, there is not a general obligatory relationship between an elevation in cytosolic Ca2+ and antagonism of this insulin action.

ATP-dependent control of steady-state cytosolic calcium in cultured gastric smooth muscle.

The regulation of intracellular calcium uptake and release in cultured gastric smooth muscle cells was studied in saponin-permeabilized cells derived from the rabbit antrum. Cells were studied in an ATP-regenerating medium in which the value of the ATP-to-ADP ratio was fixed by variation of the relative concentrations of creatine and creatine phosphate in the presence of a constant concentration of adenine nucleotides and creatine kinase. Free calcium in the medium was measured through the use of the fluorescent probe fura-2. As the ratio of ATP/ADP was increased (8.5, 55.0, and 155.0), the rate of calcium sequestration was increased, resulting in a decrease of steady-state free calcium (275.2, 178.4, and 98.1 nM, respectively). The addition of glucose (5 mM) and hexokinase (15 U/ml), which results in an increase of ADP due to the phosphorylation of glucose in the medium, caused an increase of free calcium concentration to a new set point of approximately 400 nM. Mitochondrial blockade with antimycin A before permeabilization had no effect on calcium sequestration or the resultant free calcium concentration, indicating that under physiological conditions calcium is sequestered predominantly into nonmitochondrial storage sites. Specific variation of ATP/ADP had no effect on the concentration dependence of inositol trisphosphate-induced calcium efflux, suggesting the functional independence of intracellular calcium influx and efflux pathways. These results indicate a significant role for cytoplasmic ATP/ADP in the control of intracellular calcium sequestration and the regulation of steady-state calcium concentration in cultured gastrointestinal smooth muscle cells.

Role of the isocitrate dehydrogenases and other Krebs cycle enzymes in lactating bovine mammary gland.

The role of the isocitrate dehydrogenases and other Krebs cycle enzymes in bovine mammary metabolism was studied by investigation of their distribution between cytosol and mitochondria. Citrate synthase was used as a marker for mitochondrial disruption, and distributions were normalized to this enzyme. Aconitase, fumarase, and NAD+:malate dehydrogenase were distributed between the mitochondria and the cytosol; evidence for the possible involvement of an aspartate:malate shuttle was also found. The NADP+:isocitrate dehydrogenase is predominantly cytosolic with a small but significant amount of mitochondrial component. Using the dye dichlorophenol-indophenol, a low level of NAD+:isocitrate dehydrogenase activity was observed in bovine mammary tissues. This assay also allows for detection of the enzyme in fresh mitochondria from a variety of other bovine tissues (heart, liver, kidney, and brain). Activities of the isocitrate dehydrogenases were also examined as a function of gestation and lactation. The NAD+:isocitrate dehydrogenase is apparently depressed during gestation with the NADP+ form of the enzyme (cytosolic) elevated postpartum. These results indicate that a substantial portion of Krebs cycle activity may become extramitochondrial in bovine mammary gland at the onset of lactation.

Acyl-CoA esters modulate intracellular Ca2+ handling by permeabilized clonal pancreatic beta-cells.

Cytosolic free Ca2+ rises in pancreatic beta-cells in response to glucose stimulation and is part of the coupling to insulin secretion. This study evaluates a possible role for cytosolic long chain acyl-CoA esters in modulating Ca2+ handling by clonal beta-cells (HIT). Intact cells incubated with 20 microM free palmitic acid exhibited a 40% decrease in basal cytosolic free Ca2+. In contrast, acyl-CoA esters, up to a chain length of 16, but not the corresponding fatty acids, significantly lowered the Ca2+ set point maintained by cells permeabilized with saponin. The maximum response to the various acyl-CoA esters increased with increasing chain length, with no differences in the half-maximally effective concentration of 0.5 microM. Long chain acyl-CoA esters caused a 40-50% increase in 45Ca2+ influx into a non-mitochondrial pool in the permeabilized HIT cells, consistent with a stimulatory effect on the endoplasmic reticulum Ca(2+)-ATPase activity, but did not affect inositol 1,4,5-trisphosphate-induced Ca(2+)-efflux. Thapsigargin, an inhibitor of endoplasmic reticulum Ca(2+)-ATPase activity, blocked the decrease in the Ca2+ set point caused by acyl-CoA esters. The ability of acyl-CoA esters to lower the Ca2+ set point depended on the ATP/ADP ratio (or free ADP); the Ca2+ set point was lowered by 36 +/- 3.6% at an ATP/ADP ratio of 90 and by 14 +/- 1.9% at an ATP/ADP ratio of 7. Depletion of cellular protein kinase C did not prevent the acyl-CoA-induced lowering of the Ca2+ set point. These findings suggest that the increases in long chain acyl-CoA esters may play a role in restoring cytosolic free Ca2+ through activation of Ca(2+)-ATPases.

Regulation of steady-state free Ca2+ levels by the ATP/ADP ratio and orthophosphate in permeabilized RINm5F insulinoma cells.

Stimulation of insulin secretion in the pancreatic beta-cell by a fuel such as glucose requires the metabolism of the fuel and is accompanied by increases in oxygen consumption and intracellular free Ca2+. A very early signal for these events could be a decrease in the cytosolic ATP/ADP ratio due to fuel phosphorylation. To test this hypothesis the regulation of free Ca2+ was evaluated in permeabilized RINm5F insulinoma cells that sequester Ca2+ and maintain a low medium free Ca2+ concentration (set point), between 100 and 200 nM, in the presence of Mg2+ and ATP. ATP, creatine, creatine phosphate, and creatine phosphokinase were added to the media to achieve various constant ratios of ATP/ADP. Free Ca2 was monitored using fura-2. The results demonstrated that the steady-state free Ca2+ concentration varied inversely with the ATP/ADP ratio and orthophosphate (Pi) levels. In contrast, no correlation between free Ca2+ and the phosphorylation potential (ATP/ADP.Pi) was found. Regulation of the Ca2+ set point by the ATP/ADP ratio was observed at ratios between 5 and 50 and at Pi concentrations between 1 and 7 mM, irrespective of whether mitochondria were participating in Ca2+ sequestration or were inhibited. Increasing the ATP/ADP ratio stimulated Ca2+ uptake by the nonmitochondrial pool but did not modify Ca2+ efflux. Glucose 6-phosphate (1 mM) had no effect on the Ca2+ set point. The data suggest that variations in the cytosolic ATP/ADP ratio induced by fuel stimuli may regulate Ca2+ cycling across nonmitochondrial compartments and the plasma membrane by modulating the activity of Ca2+ -ATPases. A mechanism linking fuel metabolism and cytosolic ATP/ADP ratio to activation of the Ca2+ messenger system in pancreatic beta-cells is proposed.

Glucose-induced oscillatory insulin secretion in perifused rat pancreatic islets and clonal beta-cells (HIT).

Normal insulin secretion is oscillatory in vivo and from groups of perifused islets. Stimulation of rat islets with different glucose concentrations gave insulin oscillations of similar period (5-8 min) but increasing amplitude. It has been assumed that oscillatory secretion is due to oscillations in intracellular free Ca2+, as seen in single islets and single pancreatic beta-cells. However, when islets were perifused with diazoxide and high KCl to maintain high intracellular free Ca2+, insulin oscillations of similar amplitude and period still occurred on glucose stimulation, although superimposed on elevated basal secretion. Several likely possibilities for a diffusible synchronizing factor were tested, including pyruvate, lactate, ATP, and insulin itself; nevertheless, perifusion with high concentrations of these did not prevent insulin oscillations. Clonal pancreatic beta-cells (HIT) and dissociated islets also exhibited oscillatory insulin secretion, but with the 5- to 8-min period oscillations superimposed on 15- to 20-min period oscillations. These results indicate that the mechanisms for generating and synchronizing insulin oscillations reside in the beta-cell, although the structure of the islet may modulate the oscillation pattern.

The role of long-chain fatty acyl-CoA esters in beta-cell signal transduction.

Glucose-induced insulin secretion is associated with inhibition of free fatty acid (FFA) oxidation, increased esterification and complex lipid formation by pancreatic beta-cells. Abundant evidence favors a role for cytosolic long-chain acyl-CoA (LC-CoA), including the rapid rise in malonyl CoA, the inhibitory effect of hydroxycitrate or acetyl CoA carboxylase knockout, both of which prevent malonyl CoA formation, and the stimulatory effect of exogenous FFA. On the other hand, some evidence opposes the concept, including the fall in total LC-CoA levels in response to glucose, the stimulatory effect of LC-CoA on K(ATP) channels and the lack of inhibition of glucose-stimulated secretion either by overexpression of malonyl CoA decarboxylase, which markedly lowers malonyl CoA levels, or by triacsin C, which blocks FFA conversion to LC-CoA. Alternative explanations for these data are presented. A revised model of nutrient-stimulated secretion involving two arms of signal transduction that occur simultaneously is proposed. One arm depends on modulation of the K(ATP) channel evoked by changes in the ATP/ADP ratio. The other arm depends upon anaplerotic input into the tricarboxylic acid cycle, generation of excess citrate, and increases in cytosolic malonyl-CoA. Input from this arm is increased LC-CoA. Signaling through both arms would be required for normal secretion. LC-CoA esters and products formed from them are potent regulators of enzymes and channels. It is hypothesized that their elevations directly modulate the activity of enzymes, genes and various beta-cell functions or modify the acylation state of key proteins involved in regulation of ion channels and exocytosis.

Activation of the ATP-sensitive K+ channel by long chain acyl-CoA. A role in modulation of pancreatic beta-cell glucose sensitivity.

Long-term exposure to elevated levels of long chain free fatty acids decreases glucose-induced insulin secretion from pancreatic islets and clonal pancreatic beta-cells. The mechanism for this loss of glucose sensitivity is at present not known. In this study, we evaluated the possibility that increases in long chain acyl-CoA esters (LC-CoA), the metabolically active form of free fatty acids, might mediate the loss of glucose sensitivity. We observed that cellular levels of LC-CoA increased more than 100% in response to overnight incubation with 0.5 mM palmitic acid complexed to albumin. In the same studies, the total CoA pool increased by about 40%. Patch-clamp studies demonstrated that saturated and unsaturated LC-CoA, but not malonyl-CoA or free CoASH, induced a rapid and slowly reversible opening of ATP-sensitive K+ channels. The effect was concentration-dependent between 10 nM and 1 microM. These findings indicate that the ATP-regulated K/ channels is a sensitive target for LC-CoA and suggest that high levels of LC-CoA, which accumulate in response to hyperglycemia or prolonged exposure to free fatty acids, may prevent channel closure and contribute to the development of beta-cell glucose insensitivity.

Stopped flow and steady state kinetic studies of the effects of metabolites on the soluble form of NADP+:isocitrate dehydrogenase.

The cytosolic form of NADP+:isocitrate dehydrogenase, a primary source of the NADPH required for de novo fatty acid synthesis in lactating bovine mammary gland, was studied to determine possible mechanisms of regulation by metabolites. Stopped flow kinetics showed a distinct lag time, followed by attainment of an apparently linear final velocity. Direct nonlinear regression analyses of the reaction progress curves allowed for the calculation of the rate constant (kappa) for the transition of the enzyme from an inactive to an active form; this transition is best catalyzed by its metal-substrate complex. Preincubation with metal-substrate or metal-citrate nearly abolished the lag by increasing kappa 10-fold. In steady state experiments, analyses of velocity versus metal-citrate complex as a binding isotherm, following the assumptions of Wyman's theory of thermodynamic linkage, showed that binding of metal-citrate complex could both activate and inhibit the enzyme. This analysis suggested: (a) activation by binding to sites with an average dissociation constant of 0.25 mM; (b) inhibition by binding to sites with an average dissociation constant of 3.83 mM; and (c) modulation (reactivation) by binding to sites with an average dissociation constant of 1.54 mM. Concentration ranges observed for these transitions are compatible with physiological conditions, suggesting that complexes of metal-citrate and metal-isocitrate serve to modulate the activity of NADP+:isocitrate dehydrogenase.

Oscillations in cytosolic free Ca2+, oxygen consumption, and insulin secretion in glucose-stimulated rat pancreatic islets.

Insulin secretion in the intact organism, and by the perfused pancreas and groups of isolated perifused islets, is pulsatile. We have proposed a metabolic model of glucose-induced insulin secretion in which oscillations in the ATP/ADP ratio drive alterations in metabolic and electrical events that lead to insulin release. A key prediction of our model is that metabolically driven Ca2+ oscillations will also occur. Using the fluorescent Ca2+ probe, fura 2, digital image analysis, and sensitive O2 electrodes, we investigated cytosolic free Ca2+ responses and O2 consumption in perifused rat islets that had been maintained in culture for 1-4 days. We found that elevated ambient glucose increased the average cytosolic free Ca2+ level, the ATP/ADP ratio, and oxygen consumption, as previously found in freshly isolated islets. Oscillatory patterns were obtained for Ca2+, O2 consumption, and insulin secretion in the presence of 10 and 20 mM glucose. Very low amplitude oscillations in cytosolic free Ca2+ were observed at 3 mM nonstimulatory glucose levels. Evaluation of the Ca2+ responses of a large series of individual islets, monitored by digital image analysis and perifused at both 3 and 10 mM glucose, indicated that the rise in glucose concentration caused more than a doubling of the average cytosolic free Ca2+ value and a 4-fold increase in the amplitude of the oscillations with little change in period. The pattern of Ca2+ change within the islets was consistent with recruitment of responding cells. The coexistence of oscillations with similar periods in insulin secretion, oxygen consumption, and cytosolic free Ca2+ is consistent with the model of metabolically driven pulsatile insulin secretion.

Malonyl-CoA and long chain acyl-CoA esters as metabolic coupling factors in nutrient-induced insulin secretion.

Several approaches were used to test the hypothesis proposing a role for acyl-CoA esters in nutrient-induced insulin release (Prentki, M., and Matschinsky, F. M. (1987) Physiol. Rev. 67, 1185-1248; Corkey, B. E., Glennon, M. C., Chen, K. S., Deeney, J. T., Matschinsky, F. M., and Prentki, M. (1989) J. Biol. Chem. 264, 21608-21612). Exogenous saturated long chain fatty acids markedly potentiated glucose-induced insulin release and elevated long chain acyl-CoA esters in the clonal beta-cell line (HIT). The secretory action depended on the fatty acid chain length, occurred in the range 3-20 microM (free concentration of palmitate), and was reversible and inhibitable by the neuromodulator somatostatin. 2-Bromopalmitate, an inhibitor of carnitine palmitoyl transferase I, suppressed the oxidation of endogenous fatty acids and promoted release of insulin. Only the nutrients or the combination of nutrients that caused secretion elevated malonyl-CoA. The short-chain acyl-CoA profile of HIT cells stimulated by various nutrients was determined in the presence of the nonstimulatory fuel glutamine. Glucose and leucine each provoked similar changes in acyl-CoA compounds. Both secretagogues elevated malonyl-CoA 3-6-fold, whereas succinyl-CoA, free CoASH, acetyl-CoA, and the free CoASH to acetyl-CoA ratio remained unaltered. Furthermore, only when inhibition of fatty acid oxidation was associated with a rise in malonyl-CoA did the total (mitochondrial plus cytoplasmic) content of long chain acyl-CoA esters correlate inversely with insulin release promoted by various nutrients. The results are consistent with the concept that fuel stimuli cause a rise in malonyl-CoA which by inhibiting fatty acid oxidation increase cytosolic long chain acyl-CoA esters. These data provide further support for a model in which malonyl-CoA and long chain acyl-CoAs esters serve as metabolic coupling factors when pancreatic beta-cells are stimulated with glucose and other nutrient secretagogues.

A role for malonyl-CoA in glucose-stimulated insulin secretion from clonal pancreatic beta-cells.

To gain insight into the relationship between acyl coenzyme A (CoA) esters and glucose-induced insulin release, acyl-CoA profiles were determined in clonal pancreatic beta-cells (HIT). A high sensitivity high performance liquid chromatography method was used to measure malonyl, succinyl, beta-hydroxy beta-methylglutaryl and acetyl-CoA esters and free CoASH. Malonyl-CoA content increased more than 3-fold following exposure of HIT cells to 10 mM glucose. The rise in malonyl-CoA, which preceded insulin secretion, was evident 2 min after exposure to glucose and was sustained for at least 30 min. The increase in malonyl-CoA was associated with inhibition of fatty acid oxidation, increased de novo lipid synthesis and a rise in diacylglycerol content. Succinyl-CoA levels, which may reflect anaplerotic influx into the citric acid cycle, were elevated in the presence of glucose. The concentration of acetyl-CoA and the ratio of free CoASH to acetyl-CoA was unchanged. The data are consistent with a metabolic model in which malonyl-CoA mediates the switch from fatty acid catabolism to lipid synthesis during glucose stimulation of beta-cells. We suggest that these changes in lipid metabolism, by leading to increased diacylglycerol synthesis or protein acylation could play a pivotal role in the regulation of the sustained phase of insulin secretion.