Glucose metabolism in isolated fat cells: enhanced response of larger adipocytes from older rats to epinephrine and adrenocorticotropin.

The effects of insulin and of two lipolytic hormones (epinephrine and ACTH1) on the rate and pattern of glucose metabolism were compared during incubation of isolated fat cells, obtained from epididymal fat pads of rats of varying age and degrees of adiposity. Glucose metabolism and the intracellular free fatty acid levels were expressed on a per cell basis and in relation to adipocyte size. The data for total glucose metabolism show that, in contrast to the declining insulin effect observed with adipocyte enlargement, the stimulation of glucose uptake and metabolism by these lipolytic hormones was significantly greater in the larger fat cells from the older fatter rats than in the smaller ones from the younger leaner rats. Lipolytic hormones suppressed, whereas insulin enhanced, fatty acid synthesis; moreover the lipolytic hormones stiumlated glucose ce effect of epinephrine on the intracellular free fatty acid levels was greater in the small fat cells than in the large ones; this effect of epinephrine was markedly curtained by the presence of glucose in the incubation medium, making it unlikely that acceleration of glucose metabolism by the lipolytic stimulus was mediated by an elevation of the intracellular free fatty acid level. The present results show a markedly enhanced capacity of the large adipocytes to accelerate glucose metabolism in response to these liplytic hormones. Thus, in contrast to prevailing notions of declining hormonal responsiveness with expanding fat cell size in older and more obese animals, this study documents an instance of increased hormonal response in enlarged adipocytes and points to the need for a more comprehensive reevaluation of the various hormonal effects in adipocytes of different size.     

Technical Note: Bioluminescence Measurements of the Antilisterial Activity of Nisin: Comparison with Ampicillin and Streptomycin

The influence of nisin on intracellular ATP and cell numbers of Listeria monocytogenes strain Scott A was determined and compared with the effect of ampicillin and streptomycin under similar conditions. In the presence of nisin (3–12 μg/ml), intracellular ATP and cell numbers decreased rapidly during the first hour at 35°C and extracellular ATP increased. Cell numbers and intracellular ATP increased after 3 h of incubation. No effect was observed in cells treated with ampicillin (3–12 μg/ml) and streptomycin (15–60 μg/ml) during the first hour. However, concentrations of ≥3 μg/ml ampicillin and ≥30 μg/ml streptomycin were listeriostatic after 3 h of incubation. Progressive loss of viability and reduction of intracellular ATP were observed in resting cells in PBS (pH 7.2) containing increasing concentrations of the antimicrobials. Rapid accumulation of extracellular ATP, observed immediately after treatment with nisin but not with the antibiotics, supports the reported collapse of proton motive force in L. monocytogenes by nisin.     

Vanadium uptake by yeast cells

During incubation with vanadyl, Saccharomyces cerevisiae yeast cells were able to accumulate millimolar concentrations of this divalent cation within an intracellular compartment. The intracellular vanadyl ions were bound to low molecular weight substances. This was indicated by the isotropic nature of the electron paramagnetic resonance (EPR) spectra of the respective samples. Accumulation of intracellular vanadyl was dependent on presence of glucose during incubation. It could be inhibited by various di- and trivalent metal cations. Of these cations lanthanum displayed the strongest inhibitory action. If yeast cells were exposed to more than 50 microM vanadyl sulfate at a pH higher than 4.0, a potassium loss into the medium was detected. The magnitude of this potassium loss suggests a damage of the plasma membrane caused by vanadyl. Upon addition of vanadate to yeast cells surface-bound vanadyl was detectable after several minutes by EPR. This could be the consequence of extracellular reduction of vanadate to vanadyl. The reduction was followed by a slow accumulation of intracellular vanadium, which could be inhibited by lanthanum or phosphate. Therefore, permeation of vanadyl into the cells can be assumed as one mechanism of vanadium accumulation by yeast during incubation with vanadate.     

Regulation of lipolysis and cyclic AMP synthesis through energy supply in isolated human fat cells.

The effects of glucose and of various inhibitors of glycolysis or of oxidative phosphorylation on stimulated lipolysis and on intracellular cyclic AMP and ATP levels were investigated in isolated human fat cells. The glycolysis inhibitors, NaF and monoiodoacetate, inhibited epinephrine or theophylline-stimulated lipolysis and parallely reduced the intracellular cyclic AMP and ATP levels; however, neither NaF nor monoidoacetate significantly affected dibutyryl cyclic AMP-induced lipolysis. Removal of glucose from the medium also reduced the rate of epinephrine-stimulated lipolysis and the intracellular cyclic AMP and ATP levels but failed to modify the lipolytic activity of dibutyryl cyclic AMP. The oxidative phosphorylation inhibitors, antimycin A and, under fixed conditions, 2,4-dinitrophenol also strongly decreased the adipocyte cyclic AMP and ATP levels but inhibited as well the rate of epinephrine- and of dibutyryl cyclic AMP-induced lipolysis. N-Ethylmaleimide, a mixed glycolysis and oxidative phosphorylation inhibitor, not only reduced the intracellular cyclic AMP and ATP levels and epinephrine- or theophylline-induced lipolysis, but also that stimulated by dibutyryl cyclic AMP. When glycolysis was almost fully inhibited, human fat cells were insensitive to epinephrine but remained fully responsive to dibutyryl cyclic AMP. These results, showing a relationship between ATP availability, cyclic AMP synthesis and lipolysis, suggest a different ATP requirement for cyclic AMP synthesis and triacylglycerol lipase activation, a difference which could explain why ATP issued from glucose breakdown appears to be a determinant factor for cyclic AMP synthesis, but not for triacylglycerol lipase activation in human fat cells.     

Intracellular accumulation of free fatty acids in isolated white adipose cells

A simple, rapid, and accurate method was developed for measuring intracellular FFA levels in isolated white adipose cells using sucrose-(14)C or inulin carboxyl-(14)C as nontransportable, nonutilizable markers of the extracellular space. Following incubation, medium and cells were separated by centrifugation and the infranatant medium was removed by aspiration. The volume of medium trapped between cells was determined by measuring the amount of sucrose-(14)C or inulin carboxyl-(14)C retained in the floating packed adipose cells. In this way the FFA content of the adipose cells could be corrected for contamination by FFA bound to extracellular albumin. With this technique the initial events in hormone-activated lipolysis were studied under conditions of maximal and constant rates of triglyceride hydrolysis. The FFA content of isolated adipocytes of fed rats was 0.5 micro mole/g cell lipid. On addition of norepinephrine in the presence of medium albumin, the concentration of intracellular FFA rapidly increased and reached a plateau at a concentration of 2-2.5 micro moles/g cell lipid. In the presence of medium albumin an initial lag in glycerol release occurred and this was attributed to partial hydrolysis of triglyceride with retention of lower glycerides. After 5 min of incubation FFA and glycerol output was constant. In the absence of medium albumin norepinephrine-stimulated lipolysis was reduced more than 90% and extracellular FFA release was not detected. Nevertheless, intracellular FFA accumulation was identical to that seen in the presence of albumin. The data suggest that most of this intracellular pool of FFA is bound to cytoplasmic constituents.     

Fatty acid transport in adipocytes monitored by imaging intracellular free fatty acid levels

Transport of free fatty acids (FFA) across the adipocyte plasma membrane is critical for maintaining homeostasis. To determine the membrane's role in regulating transport we describe here the first measurements of the intracellular (unbound) FFA concentration ([FFA(i)]) and their use in monitoring transport of FFA across 3T3F442A adipocytes. [FFA(i)] was measured by microinjecting cells with ADIFAB, a fluorescently labeled fatty acid-binding protein that is used to measure unbound FFA levels. We used ratio fluorescence microscopy of intracellular ADIFAB to image unbound FFA levels and determined the time course of [FFA(i)] in response to changing the extracellular unbound FFA concentration ([FFA(o)]). [FFA(o)] was clamped at defined levels using complexes of FFA and bovine serum albumin. We show that FFA influx is slow, requiring about 300 s to reach steady state (rate constant approximately 0.02 s(-1)) and saturable (K(o) approximately 200 nm). Efflux is twice as fast as influx, for zero [FFA(o)], but decreases with increasing [FFA(o)]. Surprisingly, at steady state [FFA(i)] is 2-5-fold (average 2-fold) greater than [FFA(o)] and this [FFA(i)]/[FFA(o)] gradient is abolished by depleting cellular ATP. Our results indicate that FFA transport across adipocyte membranes is highly regulated, involving an ATP-driven pump and a mechanism for gating efflux that is sensitive to [FFA(o)]. These characteristics are well described by a membrane carrier model but are not consistent with FFA transport across the membrane's lipid phase. We suggest that these characteristics are important in regulating circulating FFA levels by the adipocyte.     

Response of white adipocyte of mouse and rabbit to catecholamines and ACTH

Summary Hormone stimulated lipolysis of mouse and rabbit adipocytes as measured by both free fatty acid and glycerol release, is proportionally elevated with increase in the adipocyte cAMP level up to 1 nmole/g. The correlation coefficients are 0.94 and 0.97 for FFA/cAMP and glycerol/cAMP respectively. Increments in cAMP greater than 1 nmole/g show no correlation with increase in lipolysis. The release of lipolytic products, glycerol and free fatty acids, from white adipocytes in response to ACTH, epinephrine or morepinephrine was measured using radiochemical assays in short term incubation systems, with cAMP levels measured at the same time and from the same cell sample. Under the conditions studied, epinephrine is a more effective lipolytic hormone than ACTH in mouse adipocyte, and ACTH is more effective than epinephrine in rabbit adipocyte. The effect of catecholamines on the rabbit adipocyte is not modified by phentolamine (10 μM), but it is potentiated by 1-methyl-3-isobutyl xanthine (0.1 mM). The results suggest that cAMP mediates the action of these lipolytic hormones in white adipocytes of mouse and rabbit.     

In vitro glucose reversal of the inhibitory effect of fasting on epinephrine-induced lipolysis

Cyclic AMP, protein kinase activity and glycerol were measured in adipose tissue from fasted rats incubated with epinephrine with or without glucose. A drastic loss in the sensitivity of the adipose tissue to respond to the lipolytic action of the hormone was observed during fasting, when incubated without glucose. The addition of glucose reverses this process, and a greater lipolytic capacity was observed in the tissue of fasted rats than in fed rats. The three parameters measured were well correlated when there was epinephrine in the medium. Lipolysis is observed with glucose alone, but there was no variation in the cAMP levels nor in the protein kinase activity. These results are discussed in relation to the regulator effect of FFA, which is mobilized during starvation, on lipolysis.     

Effect of extra aeration on extracellular enzyme activities and ATP concentration of dairy Pseudomonas fluorescens

The effect of forced aeration on extracellular enzyme synthesis during batch growth of a Pseudomonas fluorescens strain of dairy origin on pyruvate mineral salts medium at 7 degrees C was studied. Measurement of oxygen tension, electron micrographs to estimate cell volume, luciferase determination of ATP and plate counts were performed in the course of incubation. Cells from the stationary phase of growth had lower energy status (in terms of intracellular ATP concentration) in the cultures receiving surplus aeration. Those cells produced three times more extracellular proteinase and lipase than control cells. Onset time for production of both enzymes coincided with a sharp fall of intracellular ATP levels.     

The effect of temperature on the degradation of triglycerides by a pseudomonad isolated from milk: free fatty acid accumulation as a balance between rates of triglyceride hydrolysis and fatty acid consumption

The lipolysis of triglyceride by a lipolytic pseudomonad, LS107d2, and the net levels of free fatty acids (FFA) in milk cultures of LS107d2 have been studied as a function of temperature. The metabolism of triolein results in the accumulation of oleic acid at 4°C but, at higher temperatures, triolein is hydrolysed without detectable oleate accumulation. Temperature also has a profound effect on the levels of FFA in whole milk cultures of LS107d2, and on the temporal pattern of changes in FFA levels. The results are consistent in demonstrating that FFA consumption plays a significant role in determining the net level of FFA in milk, even in the presence of very high rates of lipolysis. Thus the net levels of FFA as a result of the growth of LS107d2 in milk are the result of a dynamic balance between rates of FFA production (by lipolysis) and subsequent consumption. Temperature can dramatically affect the levels of FFA in milk cultures, and these changes are proposed to be due to opposing influences on FFA consumption and production, affecting the balance between them.     

Carbon-13 nuclear magnetic resonance spectroscopy of high-spin iron(III) porphyrin compounds

1. There was no apparent correlation between the rate of respiration and rate of accumulation of proline in Candida albicans cells. 2. In contrast to normal cells, the respiration in the starved cells became completely cyanide insensitive. The starvation of cells in the presence of cycloheximide prevented the cells from becoming cyanide insensitive. The addition of Fe(III), however, accelerated the process. 3. Oxidizable substrates e.g. NADH, acetate and glucose, when added to cyanide-insensitive starved cells, exhibited 40--280% stimulation in respiration rate. However, this enhancement in oxidation by various substrates was not coupled to a simultaneous increase in the proline uptake or in intracellular ATP levels. 4. There was 6-fold stimulation in proline uptake when cyanide-insensitive cells were preincubated with 50 mM glucose. The preincubation of starved cells resulted in a partial restoration of cyanide sensitivity and increased intracellular ATP levels. The preincubation of starved cells with other oxidizable substrates resulted in a partial restoration of cyanide sensitivity but had no stimulatory effect on intracellular ATP levels and proline accumulation. 5. Both the enhanced uptake and ATP levels in glucose preincubated cells were found to be completely abolished by iodoacetate. 6. It is proposed that the increased proline uptake in cells preincubated with glucose was mainly due to the production of glycolytic energy.     

Effect of 2-deoxy-D-glucose on lipolytic processes in blood and adipose tissue of rat

The effect of 2-deoxy-D-glucose on lipolytic processes in the blood and adipose tissue was studied. Rats treated with this antimetabolite showed a significant increase in serum glucose, FFA and glycerol level, as well as in the lipid mobilizing activity. On the other hand, the lipolytic activity of rat serum decreased when compared to control group. From these results it may be concluded that during hypothermia induced by administration of 2-deoxy-D-glucose intracellular, but not intravascular, lipolysis is enhanced.     

Effect of Sorbinil on myo-Inositol Metabolism in Cultured Neuroblastoma Cells Exposed to Increased Glucose Levels

Neuroblastoma cells were used to determine the effect of sorbinil on myo-inositol metabolism in cells exposed to elevated levels of glucose in culture. Exposing cells to elevated levels of glucose led to an increase in levels of intracellular sorbitol. The increase in sorbitol levels was dependent on the extracellular glucose concentration. In contrast, the myo-inositol content of cells was decreased in the presence of increasing concentrations of extracellular glucose. Increasing the concentration of glucose in the culture medium caused a decrease in myo-inositol uptake and in the incorporation of extracellular myo-inositol into phospholipid. The effect of elevated glucose levels on myo-inositol metabolism and sorbitol accumulation was blocked by addition of 0.4 mM sorbinil. The ability of sorbinil to block the decrease in myo-inositol metabolism and sorbitol accumulation caused by 30 mM extracellular glucose was dependent on its concentration. Maximal effects were obtained with 0.4 mM sorbinil. However, there was some variation in the degree of effectiveness among batches of sorbinil. These results at the cellular level suggest that the intracellular accumulation of sorbitol is responsible for the alteration of myo-inositol metabolism observed in neuroblastoma cells exposed to elevated glucose concentrations.     

Bimodal effect of insulin on hormone-stimulated lipolysis: relation to intracellular 3',5'-cyclic adenylic acid and free fatty acid levels

The present study was undertaken to determine the relationship between the antilipolytic and lipolytic effects of insulin on hormone-stimulated lipolysis and the mechanisms of these reactions. The dose-response curve of norepinephrine-stimulated lipolysis in rat adipocytes was not sigmoidal but biphasic in nature. Intracellular free fatty acid levels were linearly related to lipolytic rate and also described a biphasic profile in response to increments in norepinephrine concentration. Intracellular 3',5'-cyclic AMP levels measured 10 min after addition of increasing concentrations of norepinephrine showed a rise and a plateau followed by a secondary rise. Insulin was antilipolytic at low concentrations of norepinephrine and distinctly lipolytic at high concentrations. The combined antilipolytic and lipolytic effect of insulin is termed the "bimodal" effect of insulin on hormone-stimulated lipolysis. The bimodal effect of insulin correlated positively with changes in peak intracellular 3',5'-cyclic AMP levels. In the presence of glucose, insulin invariably enhanced lipolysis. It is suggested that the antilipolytic effect of insulin is achieved by both inhibition of adenyl cyclase activity and activation of low-K(m) 3',5'-cyclic AMP phosphodiesterase, the net effect being a low accumulation of 3',5'-cyclic AMP. On the other hand, the lipolytic effect of insulin probably reflects enhancement of adenyl cyclase activity to an extent that overrides any activation of low-K(m) 3',5'-cyclic AMP phosphodiesterase activity, resulting in an increase in peak adipocyte 3',5'-cyclic AMP levels.     

Possible mechanisms of weight loss of Siberian hamsters (Phodopus sungorus sungorus) exposed to short photoperiod

Several weeks of short day photoperiod (SD) exposure promote a dramatic decrease of white adipose tissue (WAT) mass in Siberian hamsters(Phodopus sungorus sungorus). This slimming effect is accompanied by changes in the adipocyte responsiveness to adrenergic stimulation that are still under debate. We investigated whether possible changes in the antilipolytic responses, and/or lipogenic activities could be involved in such lipid deposition/mobilisation imbalance. Male Siberian hamsters were exposed for 11 weeks to SD or long day photoperiod and basal or stimulated lipolytic and lipogenic activities were measured on white adipocytes. As expected, the body mass of SD-animals was decreased. Besides a slight reduction in the basal lipolysis and in the maximal response to dibutyryl-cAMP, the responses to adrenergic and non-adrenergic lipolytic agents (forskolin, adenosine deaminase) were similar in both groups. Fat mass loss was likely not resulting from changes in the lipolytic responses of adipocytes to biogenic amines (e.g. octopamine), which were unaltered, or to a direct lipolytic stimulation by melatonin or histamine, which were inactive. Antilipolytic responses to insulin or tyramine were slightly decreased in SD-adipocytes. Basal or insulin-stimulated lipid accumulation in WAT, measured by glucose incorporation into lipids, did not change after SD-exposure. However, a significant decrease in the lipoprotein lipase activity was observed in the WAT of SDanimals. Despite the observed changes, the weight loss of SD-exposed Siberian hamsters was likely not resulting only from impaired antilipolytic orde novo lipogenic activities in white adipocytes, but either from other dramatic changes occurring during seasonal photoperiod-sensitive body weight regulation.     

The correlation between glycogen–glycolysis metabolite pyruvate and vancomycin antibiotic productions of Amycolatopsis orientalis grown in glucose medium

The effect of glucose concentration in the growth medium on the relationship between glycolysis, glycogen accumulation and vancomycin production of Amycolatopsis orientalis was investigated depending on the incubation time. After a lag phase, bacterial growth of A. orientalis began and biomass concentration increased continuously up to 36th or 48th hours while glucose concentration in the culture medium was consumed rapidly in the same time of incubation. In addition, increase in glucose concentrations of the growth medium lead to increase intracellular glucose as well as glycerol levels. Intracellular pyruvate levels increased significantly up to 15 g/L while extracellular pyruvate levels with respect to increases in glucose concentration. A positive correlation between glucose kinase activities and glucose concentration was determined during the incubation period. Pyruvate kinase activity increased up to 15 g/L glucose and 48th hour of incubation. As a glycopeptide antibiotic, vancomycin production increased with the increases in glucose concentrations up to 15 g/L. These results indicated that glycogen accumulation with respect to glucose concentration of the growth medium was concomitant with the sporulation of A. orientalis. When the initial glucose concentration exceeded 15 g/L, pyruvate excretions as well as intracellular glycogen and glycerol productions were supported in spite of repression in vancomycin production of A. orientalis.     

The metabolic requirements for transcellular movement and secretion of collagen.

Cultures of chick tendon fibroblasts were capable of normal ATP production and protein synthetic activity even though the normally high rate of glycolysis was markedly reduced by substitution of pyruvate for glucose. Iodoacetate and 2-deoxyglucose reduced ATP levels and protein synthesis even in the presence of pyruvate. Under these conditions, both inhibitors were shown to have effects on the energy metabolism of cells which were apparently unrelated to an inhibition of glycolysis. Selective inhibition of either glycolysis, by incubation in glucose-free medium, or of oxidative phosphorylation, by incubation with an uncoupler, was shown to have little effect on cellular ATP levels or intracellular transport and secretion of collagen. However, inhibition of both glycolysis and oxidative phosphorylation resulted in decreased cellular ATP levels and an inhibition of collagen secretion. This effect was not due to a requirement for continued protein synthesis, since inhibition of protein synthesis with cycloheximide or puromycin had little effect on collagen secretion. The ATP requirement for intracellular transport and secretion is discussed in relation to the secretory pathway for collagen.     

INTERRELATIONSHIPS AMONG THE LEVELS OF ATP, ADENOSINE AND CYCLIC AMP IN INCUBATED SLICES OF GUINEA-PIG CEREBRAL CORTEX: EFFECTS OF DEPOLARIZING AGENTS, PSYCHOTROPIC DRUGS AND METABOLIC INHIBITORS

—Adenine nucleotides of guinea-pig cerebral cortical slices were labelled during a 40 min incubation with [¹⁴C]adenine. Subsequent incubation of cortical slices with depolarizing agents, such as veratridine, ouabain, batrachotoxin and high concentrations of potassium ions, or with certain psychotropic drugs such as chlorpromazine, chlorimipramine or prenylamine resulted in a reduction in both endogenous and radioactive ATP, accompanied by a marked increase in levels of both endogenous and radioactive cyclic AMP. Reduction of ATP levels during incubation with depolarizing agents, such as veratridine, is probably associated with increased activity of membranal Na⁺-K⁺-activated ATPase, while the reduction elicited by psychotropic drugs is proposed to be due to inhibition of mitochondrial synthesis of ATP. With both classes of compounds reduction of ATP levels results in enhanced formation and efflux of adenosine which stimulates formation of cyclic AMP from intracellular ATP in the compartments of brain slices which contain the cyclic AMP-generating systems. Certain classical metabolic inhibitors such as 2,4-dinitrophenol, azide, 1,2-naphthoquinone-8-sulfonate and cyanide also reduce ATP levels and in the case of 2,4-dinitrophenol, cyanide, and azide elicit small but significant accumulations of cyclic AMP. With certain metabolic inhibitors reduction of ATP within the cyclic AMP generating compartments would appear to prevent or reduce the accumulation of cyclic AMP elicited by amines, adenosine or veratridine.     

Adenosine, Inosine, and Guanosine Protect Glial Cells During Glucose Deprivation and Mitochondrial Inhibition: Correlation Between Protection and ATP Preservation

Abstract: The purpose of this study was to determine the mechanism by which adenosine, inosine, and guanosine delay cell death in glial cells (ROC-1) that are subjected to g lucose d eprivation and m itochondrial respiratory chain inhibition with amobarbital (GDMI). ROC-1 cells are hybrid cells formed by fusion of a rat oligodendrocyte and a rat C6 glioma cell. Under GDMI, ATP was depleted rapidly from ROC-1 cells, followed on a much larger time scale by a loss of cell viability. Restoration of ATP synthesis during this interlude between ATP depletion and cell death prevented further loss of viability. Moreover, the addition of adenosine, inosine, or guanosine immediately before the amobarbital retarded the decline in ATP and preserved cell viability. The protective effects on ATP and viability were dependent on nucleoside concentration between 50 and 1,500 µ M . Furthermore, protection required nucleoside transport into the cell and the continued presence of nucleoside during GDMI. A significant positive correlation between ATP content at 16 min and cell viability at 350 min after the onset of GDMI was established ( r = 0.98). Modest increases in cellular lactate levels were observed during GDMI (1.2 nmol/mg/min lactate produced); however, incubation with 1,500 µ M inosine or guanosine increased lactate accumulation sixfold. The protective effects of inosine and guanosine on cell viability and ATP were >90% blocked after treatment with 50 µ M BCX-34, a nucleoside phosphorylase inhibitor. Accordingly, lactate levels also were lower in BCX-34-treated cells incubated with inosine or guanosine. We conclude that under GDMI, the ribose moiety of inosine and guanosine is converted to phosphorylated glycolytic intermediates via the pentose phosphate pathway, and its subsequent catabolism in glycolysis provides the ATP necessary for maintaining plasmalemmal integrity.     

Acute stimulation with long chain acyl-CoA enhances exocytosis in insulin-secreting cells (HIT T-15 and NMRI beta-cells)

Non-insulin-dependent diabetes mellitus is associated with, in addition to impaired insulin release, elevated levels of free fatty acids (FFA) in the blood. Insulin release is stimulated when beta-cells are acutely exposed to FFA, whereas chronic exposure may inhibit glucose-induced insulin secretion. In the present study we investigated the direct effects of long chain acyl-CoA (LC-CoA), the active intracellular form of FFA, on insulin exocytosis. Palmitoyl-CoA stimulated both insulin release from streptolysin-O-permeabilized HIT cells and fusion of secretory granules to the plasma membrane of mouse pancreatic beta-cells, as measured by cell capacitance. The LC-CoA effect was chain length-dependent, requiring chain lengths of at least 14 carbons. LC-CoA needed to be present to stimulate insulin release, and consequently there was no effect following its removal. The stimulatory effect was observed after inhibition of protein kinase activity and in the absence of ATP, even though both kinases and ATP, themselves, modulate exocytosis. The effect of LC-CoA was inhibited by cerulenin, which has been shown to block protein acylation. The data suggest that altered LC-CoA levels, resulting from FFA or glucose metabolism, may act directly on the exocytotic machinery to stimulate insulin release by a mechanism involving LC-CoA protein binding.