INTRODUCTION

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Glucose is a potent stimulator of insulin secretion from -cells of the pancreatic islets of Langerhans. Regulation of insulin secretion by glucose is thought to be mediated, at least in part, by increases in the cellular ATP:ADP ratio, resulting in closure of ATP-sensitive K⁺ channels, membrane depolarization, and consequent activation of voltage-dependent Ca²⁺ channels (reviewed in Refs. 1 and 2). In recent years the concept that glucose may also signal via altering lipid metabolism has received significant experimental support (3-5). Thus, glucose administration to HIT insulinoma cells results in an increase in malonyl-CoA levels that precede the rise in insulin secretion. The increase in malonyl-CoA, acting via its capacity to inhibit the mitochondrial enzyme carnitine palmitoyltransferase I (6), results in inhibition of fatty acid oxidation, increased de novo lipid synthesis, and a rise in diacylglycerol content. These findings led Prentki and colleagues (3, 4) to propose that increases in the levels of cytosolic long-chain acyl-CoA (LC-CoA)¹ esters are a signal transduction intermediate in glucose-stimulated insulin secretion, and this idea has come to be known as the "long-chain acyl-CoA hypothesis." Feasible sites at which increased LC-CoA could influence insulin secretion include conversion to bioactive metabolites such as diacylglycerol or inositol trisphosphate, contribution to plasma membrane or secretory granule membrane lipid turnover, or direct acylation of proteins involved in secretory granule trafficking. While the proposed mechanisms are reasonable, direct evidence for any of these actions of lipids on insulin secretion has not been provided to date.

Other recent studies have demonstrated that the effects of lipids on -cell function are complex. Lowering of circulating fatty acids via administration of nicotinic acid (7, 8) or adenovirus-induced hyperleptinemia (9) completely abrogates glucose-stimulated insulin secretion, but full secretory function can be restored by provision of fatty acids to the pancreas of such animals. It has also been established for some time that fatty acids are acute potentiators of the glucose-stimulated insulin secretion response (10, 11). In contrast to these positive acute effects of lipids on islet function, long-term exposure to hyperlipidemic conditions results in a condition of "lipotoxicity," wherein lipid overstorage leads to the syndrome of -cell dysfunction associated with insulin-resistant and diabetic states, including -cell hyperplasia, basal hyperinsulinemia, and loss of glucose responsiveness (12, 13).

In light of the growing emphasis on the role of lipids in -cell function, the current study was undertaken to re-investigate the LC-CoA hypothesis of -cell signal transduction using novel molecular and pharmacologic approaches. The goal of the study was to manipulate the levels of malonyl-CoA and LC-CoA esters during glucose stimulation and to assess the effect of such maneuvers on insulin secretion. The molecular approach chosen was to use recombinant adenovirus to express the enzyme malonyl-CoA decarboxylase, which decarboxylates malonyl-CoA to acetyl-CoA, thus allowing us to specifically block malonyl-CoA accumulation during glucose stimulation. The pharmacologic approach was to use triacsin C, an inhibitor of long-chain acyl-CoA synthetase (14) to block the conversion of fatty acids to LC-CoA. We demonstrate that while both approaches have a large impact on lipid metabolism in the -cell, neither maneuver influences glycolytic flux or glucose-stimulated insulin secretion.

	MATERIALS AND METHODS

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INS-1 Cell Culture and Preparation of Rat Pancreatic Islets-- INS-1 cells were a gift from Drs. Philippe Halban and Claes Wollheim (15), and were cultured as described previously (16). Briefly, INS-1 cells (passages 180-260) were grown on 10-cm tissue culture plates (Corning) in RPMI 1640 (Life Technologies, Inc.) media that contains 11.1 mM glucose, supplemented with 10 mM HEPES, 10% fetal calf serum (Atlanta Biologicals), 2 mM L-glutamine (Life Technologies, Inc.), 100 units/ml penicillin, 10 µg/ml streptomycin (Bio-Whittaker), and 50 µM -mercaptoethanol. Cultures were incubated at 37 °C in a humidified 95% air, 5%CO₂ atmosphere. INS-1 cells were trypsinized (Life Technologies, Inc.) and split (1:2) every 3-4 days for passaging or replated on 12-well tissue culture plates (Corning) for secretion experiments. Floating cell clusters were discarded. Pancreatic islets were prepared from 150-200-g male Sprague-Dawley rats by collagenase digestion and Histopaque density gradient centrifugation as described (17), and used for insulin secretion and metabolic measurements within hours of isolation.

Recombinant Adenovirus Preparation and Use-- A 1.7-kilobase EcoRI fragment of the malonyl-CoA decarboxylase (MCD) cDNA lacking its putative N-terminal mitochondrial localization sequence was excised from plasmid pLDC8 (Ref. 18; a generous gift of Dr. P. E. Kolattukudy, Ohio State University). This fragment was ligated into EcoRI-digested pAC.CMVpLpA (19) to generate pAC.CMV-MCD. A recombinant adenovirus containing the MCD cDNA (AdCMV-MCD) was then prepared by co-transfection of 293 cells with pAC.CMV-MCD and JM17 (20) plasmids as described (21). Viral stocks of AdCMV-MCD and a control virus containing the -galactosidase gene, AdCMV-GAL (22) were purified on a CsCl gradient and titered as described (21). Twenty-four hours after replating on a 12-well plate, INS-1 cells were transduced with either virus at a multiplicity of infection of 1000 plaque forming units/cell. AdCMV-MCD or AdCMV-GAL viruses were added directly to the RPMI tissue culture media and cells were exposed to virus for 24 h prior to initiating metabolic studies or insulin measurements of insulin secretion (see below). Cell viability as assessed by trypan blue exclusion was identical in AdCMV-MCD, AdCMV-GAL, and untreated cells after this 24-h treatment (data not shown).

Insulin Secretion Experiments-- INS-1 cells were grown either in 12-well dishes or in 10-cm tissue culture plates in the culture medium described above. Larger plates (10 cm) were required for those experiments in which malonyl-CoA levels were measured in parallel with insulin secretion. To study insulin secretion from isolated rat islets, approximately 50 islets were placed in a 1.5-ml Eppendorf-style tube. To initiate insulin secretion experiments, the medium was removed, cells were rinsed in PBS, and then each well was preincubated for 1 h in 4 ml (1 ml for islets) secretion assay buffer (SAB), containing 3 mM glucose. SAB contains 114 mM NaCl, 4.7 mM KCl, 1.2 mM KH₂PO₄, 1.16 mM MgSO₄, 2.5 mM CaCl₂, 25 mM NaHCO₃, 20 mM HEPES, and 1% fatty acid-free bovine serum albumin, pH 7.4. SAB was utilized in all subsequent procedures unless otherwise noted. Following this preincubation step, the medium was aspirated and replaced with 0.5 ml (0.18 ml for islets) of SAB containing appropriate secretagogues. In some experiments, insulin secretion from islets or INS-1 cells was studied in the presence or absence of 10 µM (INS-1 cells) or 50 µM (isolated islets) triacsin C (BioMol), an inhibitor of long-chain acyl-CoA synthetase (14). Medium samples were collected between 30 and 180 min after the initiation of an experiment, centrifuged at 700 × g to remove loose cells, and subjected to insulin radioimmunoassay (DPC Coat-A-Count), using a rat insulin standard. The cells remaining in the wells were then washed once with PBS (Life Technologies, Inc.) and used for the various measurements described below.

MCD Activity Assay-- MCD activity was assayed by a carnitine acetyltransferase-linked assay that measures the rate of malonyl-CoA decarboxylation to acetyl-CoA. The rate of acetyl-CoA production was monitored by cleavage of its thioester bond with carnitine acetyltransferase. The thiol group of free CoA was detected with the colorimetric compound 5,5'-dithiobis(nitrobenzoic acid) (Ellman's reagent). The reaction mixture (0.25 ml) contained 10 mM Tris, pH 7.1, 0.2 mM 5,5'-dithiobis(nitrobenzoic acid), 1 mM L-carnitine, 10 mM malonyl-CoA, and 3 units of carnitine acetyltransferase (Sigma). INS-1 cells were lysed in 50 mM HEPES, 1% Triton X-100. To initiate the assay, 500 µg of protein from INS-1 cell homogenates diluted 1:10 in 50 µl of PBS was added to the reaction mixture, and absorbance was continuously measured at 412 nm for 10 min, over which time the rate of product accumulation was linear.

Malonyl-CoA Assay-- INS-1 cells were grown to near confluence in 10-cm plates. The RPMI growth medium was removed and replaced with SAB containing 3 mM glucose for 1 h. Following this preincubation, cells were washed once with SAB containing 3 mM glucose and then incubated with SAB containing 3 or 20 mM glucose for 5 or 30 min. Cells were then collected in 1.2 ml of ice-cold 10% trichloroacetic acid and centrifuged at 7,000 × g. The supernatant containing acid soluble metabolites was collected and washed six times with 0.8 ml of diethyl ether. Samples were dried in a Speed-Vac and stored at 80 °C prior to assay of malonyl-CoA levels. For assay, each sample was resuspended in 0.4 ml of 1 M K₂PO₄, pH 7.1, and divided into two 0.2-ml aliquots. 100 pmol of malonyl-CoA was added to one of the two samples, and malonyl-CoA levels were assayed in both samples according to the method of McGarry and Foster (23), using purified fatty acid synthetase.

Long-chain Acyl-CoA Assay-- To measure total cellular long-chain acyl-CoA levels, INS-1 cell extracts were prepared as for the malonyl-CoA assay, except that cells were incubated in 3 or 20 mM glucose in the presence or absence of triacsin C for 2 h prior to collection of cells in trichloroacetic acid. The acid-insoluble pellet fraction was washed twice with 1 ml of diethyl ether and twice with 200 µl of H₂0. After addition of 250 µl of 10 mM dithiothreitol to the pellet, the pH was raised to 11.5 with 1 M KOH. Samples were then incubated for 10 min at 55 °C to hydrolyze the thioester bonds of the long-chain acyl-CoAs, which are quantitatively recovered in the trichloroacetic acid-insoluble pellet. Samples were neutralized with 1 M HCl, buffered to a final concentration of 50 mM KPO₄, and cleared of debris with a 5000 M_r cut-off spin filter (Millipore). NAD⁺ and -ketoglutarate were added to final concentrations of 500 and 100 µM, respectively. A fluorescence reading was taken prior to addition of enzyme, and then again after addition of 20 milliunits of -ketoglutarate dehydrogenase (Sigma) to initiate the reaction. Within 10 min, the reaction reached completion. The change in flourescence was used to calculate long-chain acyl-CoA concentration in the samples.

-Galactosidase Activity Assays-- -Galactosidase activity was measured by a colorimetric assay. Cells were homogenized as for the MCD assays and approximately 500 µg of total protein was added to a reaction mixture containing 92 mM NaPO₄, 7.5 mM KCl, 0.75 mM MgSO₄, 38 mM -mercaptoethanol, and 0.67 mg/ml o-nitrophenyl--D-galactosidase in a total volume of 250 µl, and absorbance was monitored for 20 min at 420 nM. Histochemical staining was also performed on plated cells as described previously (24). At an multiplicity of infection of 1000, approximately 90% of the AdCMV-GAL-treated cells stained blue.

Palmitate Oxidation-- INS-1 cells were collected in a suspension of SAB and equal aliquots were dispensed into center wells (Kontes) suspended from rubber sleeve stoppers (Fisher). After a 30-min preincubation with SAB containing 3 mM glucose, [1-¹⁴C]palmitate (NEN Life Science Products Inc.), glucose, and L-carnitine were added to final concentrations of 3 or 20 and 0.8 mM, respectively. The center well containing the reaction mixture was sealed with the rubber sleeve stopper within a glass scintillation vial. After a 2-h incubation at 37 °C, 100 µl of 7% perchloric acid (Baker) was injected into the center well and 300 µl of benzethonium hydroxide was injected onto the bottom of the scintillation vial to capture ¹⁴CO₂. Following 2 h at 37 °C, the center well and sleeve stopper were removed, scintillation mixture was dispensed into the vial, and the palmitate derived ¹⁴CO₂ was counted.

Glucose and Palmitate Incorporation into Lipids-- INS-1 cells were subjected to procedures identical to those described for the insulin secretion studies, with the exception that either [U-¹⁴C] or [3-³H]glucose (NEN Life Science Products) or [1-¹⁴C]palmitate were included as tracers (0.5 or 5 Ci/mol, respectively). At the conclusion of the 2-h static incubation, media were collected for insulin assay and 1 ml of methanol:PBS (2:3) was added to the plated cells. Cells were collected with gentle pipeting, centrifuged at 700 × g, and washed once with PBS. 200 µl of 0.2 M NaCl was added to the cell pellet and the mixture was immediately frozen in liquid N₂. The lipid and aqueous soluble products were separated by the following procedure. To the thawed cell suspension, 750 µl of CHCl₃:methanol (2:1) and 50 µl of 0.1 N KOH was added and, after vigorous vortexing, the phases were separated by centrifugation at 2000 × g for 20 min. The top aqueous layer was removed and the bottom lipid-soluble layer was washed with 200 µl of methanol:water:CHCl₃ (48:47:3). 200 µl of either the aqueous or lipid soluble phase was added to BioSafe2 scintillation mixture and incorporation of radiolabel into lipids was quantified.

	RESULTS

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Effect of MCD Expression on Malonyl-CoA Levels and Secretion-- Our first strategy for testing the long-chain acyl-CoA hypothesis was to specifically reduce malonyl-CoA levels during glucose stimulation of INS-1 cells. The enzyme MCD reverses the reaction catalyzed by acetyl-CoA carboxylase by converting malonyl-CoA to acetyl-CoA. Two sets of experiments were performed with the AdCMV-MCD virus. In order to measure malonyl-CoA levels, it was necessary to use large (10 cm) plates of INS-1 cells. Thus, the first set of experiments measured malonyl-CoA levels and insulin secretion from INS-1 cells grown in large plates and treated with AdCMV-MCD or the control virus, AdCMV-GAL. As shown in Table I, INS-1 cells treated with AdCMV-MCD contained high levels of MCD activity in crude cellular extracts. In contrast, cells treated with a control virus (AdCMV-GAL) contained levels of MCD activity close to the lower detection limit of the assay, and high levels of -galactosidase activity. Centrifugation of the crude cellular extracts of AdCMV-MCD-treated cells to yield mitochondria-enriched and cytosolic fractions (24) revealed that 90% of the MCD was in the latter fraction, as expected because the expressed enzyme lacks its N-terminal mitochondrial localization signal.

View this table: [in this window] [in a new window]   Table I MCD and -galactosidase enzymatic activity measurements in crude extracts of untreated INS-1 cells or INS-1 cells treated with a virus encoding malonyl-CoA decarboxylase (AdCMV-MCD) or -galactosidase (AdCMV-GAL) A depicts activity levels for cells studied in 10-cm plates. B depicts activity levels for cells studied in 12-well dishes. For both panels, data represent the mean ± S.E. for three independent experiments.

View larger version (26K): [in this window] [in a new window]   Fig. 1.   Malonyl-CoA levels in AdCMV-MCD and AdCMV-GAL-treated INS-1 cells. INS-1 cells grown in 10-cm plates were treated with a virus containing the cDNA encoding malonyl-CoA decarboxylase (AdCMV-MCD), or a control virus containing the gene for -galactosidase (AdCMV-GAL). Twenty-four hours after viral treatment, cells were preincubated in SAB containing 3 mM glucose for 1 h, and then switched to SAB containing 3 or 20 mM glucose for 5 (left panel) or 30 (right panel) min. Malonyl-CoA levels were measured in INS-1 cell extracts as described under "Materials and Methods." Data represent the mean ± S.E. for six independent experiments. The asterisk (*) indicates that malonyl-CoA levels were lower in AdCMV-MCD-treated than in AdCMV-GAL-treated INS-1 cells, at a level of significance of p < 0.005.

View larger version (30K): [in this window] [in a new window]   Fig. 2.   Insulin secretion from AdCMV-MCD and AdCMV-GAL-treated INS-1 Cells. Insulin secretion was measured from cells treated as described in Fig. 1 by radioimmunoassay of media samples collected after 30 min of incubation at 3 or 20 mM glucose. Data represent the mean ± S.E. for 12 independent experiments. Statistical analysis revealed no difference in insulin secretion between AdCMV-MCD- and AdCMV-GAL-treated INS-1 cells at either glucose concentration.

Metabolic Impact of MCD Expression-- By reducing malonyl-CoA levels, MCD expression is predicted to decrease glucose-induced LC-CoA accumulation by the two following mechanisms. First, malonyl-CoA is a key substrate for fatty acid synthase. Depriving fatty acid synthase of malonyl-CoA availability will decrease de novo lipogenesis and therefore decrease new LC-CoA biosynthesis. Second, malonyl-CoA is a potent inhibitor of carnitine palmitoyltransferase I (6). Prevention of the normal rise in malonyl-CoA levels during glucose stimulation would therefore be predicted to interfere with LC-CoA accumulation by favoring fatty acid oxidation relative to de novo synthesis and esterification. Thus, in the second set of experiments, INS-1 cells were aliquoted in 12-well plates, treated with AdCMV-MCD, AdCMV-GAL, or left untreated, and used for measurement of palmitate oxidation, or glucose or palmitate incorporation into lipid. An independent set of insulin secretion measurements was also included in this second set of experiments. As shown in Table I, treatment of cells in 12-well plates with AdCMV-MCD caused an even more dramatic increase in MCD enzyme activity in crude extracts than observed in the 10-cm plate experiments described in Table I.

View larger version (15K): [in this window] [in a new window]   Fig. 3.   Metabolic impact of malonyl-CoA decarboxylase expression in INS-1 cells. Panel A, [1-14C]palmitate oxidation. INS-1 cells were left untreated, or treated with AdCMV-MCD or AdCMV-GAL viruses for 24 h. After preincubation of cells in SAB containing 3 mM glucose for 1 h, oxidation was measured as a function of 14CO2 generation during a subsequent 2-h incubation with SAB containing radiolabeled palmitate and either 3 or 20 mM glucose, as described under "Materials and Methods." Data represent the mean ± S.E. for three independent experiments, each performed in triplicate. The asterisk (*) indicates that more palmitate was oxidized in AdCMV-MCD-treated INS-1 cells than in either control group at 20 mM glucose, at a level of significance of p < 0.001. Panel B, [1-14C]palmitate incorporation into organically extractable cellular lipids. Cells were treated identically as described for panel A. After incubation of cells for 2 h in SAB containing radiolabeled palmitate and either 3 or 20 mM glucose, cells were collected and extracted for measurement of incorporation of the fatty acid into total cellular lipids as described under "Materials and Methods." Data represent the mean ± S.E. for three independent experiments. The asterisk (*) indicates that less palmitate was incorporated into cellular lipids in AdCMV-MCD-treated INS-1 cells than in either control group at 20 mM glucose, at a level of significance of p < 0.001. Panel C, [U-14C]glucose incorporation into organically extractable cellular lipids. Cells were treated identically as described in panel A, except that [U-14C]glucose was substituted for [1-14C]palmitate. After incubation of cells for 2 h in SAB containing 3 or 20 mM [U-14C]glucose, cells were collected and extracted for measurement of incorporation of labeled sugar into total cellular lipids as described under "Materials and Methods." Data represent the mean ± S.E. for three independent experiments. The asterisk (*) indicates that less glucose was incorporated into cellular lipids in AdCMV-MCD-treated INS-1 cells than in either control group at 20 mM glucose, at a level of significance of p < 0.001.

Effect of Triacsin C Treatment on Metabolism and Insulin Secretion in INS-1 Cells-- Triacsin C potently inhibits LC-CoA synthetase, a microsomal and outer mitochondrial enzyme that condenses long-chain fatty acid and CoASH by thioester linkage (14). Therefore, an alternative strategy for testing the LC-CoA hypothesis was to use triacsin C to block LC-CoA formation from endogenous stores or exogenous fatty acids, thus preventing their incorporation into mono-, di-, and triglycerides, as well as phospholipids and acylated proteins. The feasibility of using triacsin C was supported in a study that demonstrated an 80% reduction in [¹⁴C]oleate incorporation into phospholipids and triglycerides in Raji cells (14) and by our own recent work demonstrating a near-complete block of radiolabeled glycerol incorporation into cellular lipids in glycerol kinase expressing INS-1 cells (16).

View larger version (12K): [in this window] [in a new window]   Fig. 4.   Effect of triacsin C on palmitate metabolism in INS-1 cells. Panel A, [1-14C]palmitate oxidation. INS-1 cells were preincubated in SAB containing 3 mM glucose, in the presence or absence of 10 µM triacsin C, for 2 h, and oxidation was measured as a function of 14CO2 generation during a subsequent 2-h incubation with SAB containing radiolabeled palmitate and either 3 or 20 mM glucose, in the presence or absence of 10 µM triacsin C. Data represent the mean ± S.E. for four independent experiments. The asterisk (*) indicates that less palmitate was oxidized in triacsin C-treated INS-1 cells than in untreated control cells at either glucose concentration, at a level of significance of p < 0.001. Panel B, [1-14C]palmitate incorporation into organically extractable cellular lipids. Cells were treated identically as described for panel A. After incubation of cells for 2 h in SAB containing radiolabeled palmitate and either 3 or 20 mM glucose, with or without 10 µM triacsin C, cells were collected and extracted for measurement of incorporation of the fatty acid into total cellular lipids as described under "Materials and Methods." Data represent the mean ± S.E. for four independent experiments. The asterisk (*) indicates that less palmitate was incorporated into cellular lipids in triacsin C-treated INS-1 cells than in untreated control cells at either glucose concentration, at a level of significance of p < 0.001.

View larger version (10K): [in this window] [in a new window]   Fig. 5.   Effect of triacsin C on glucose incorporation into lipids and glucose-stimulated insulin secretion. Panel A, [3-3H]glucose incorporation into organically extractable cellular lipids. INS-1 cells were preincubated in SAB containing 2 mM glucose in the presence or absence of 10 µM triacsin C for 2 h. Cells were then switched to SAB containing various concentrations of [3-3H]glucose with or without 10 µM triacsin C, and after a further 2-h incubation, cells were collected and extracted for measurement of incorporation of labeled sugar into total cellular lipids as described under "Materials and Methods." Data represent the mean ± S.E. for three independent experiments per glucose concentration. The asterisks (*) indicate that less glucose was incorporated into cellular lipids in triacsin C-treated cells than in controls at the indicated glucose concentrations. Panel B, insulin secretion. Media samples were collected from the cells described in panel A and subjected to insulin radioimmunoassay. Data represent the mean ± S.E. for three independent experiments.

View larger version (20K): [in this window] [in a new window]   Fig. 6.   Effect of triacsin C on total cellular LC-CoA levels. INS-1 cells were preincubated in SAB, 3 mM glucose, either lacking or containing 10 µM triacsin C for 30 min. This media was removed and replaced with SAB containing 3 or 20 mM glucose in the presence or absence of 10 µM triacsin C for 2 h. Cells were harvested and assayed for total LC-CoA as described under "Materials and Methods." Data represent the mean ± S.E. for three independent groups of cells per condition. The asterisk (*) indicates a lower LC-CoA level in triacsin C-treated cells, with significance of p  0.005.

Effect of Triacsin C on Glucose-stimulated Insulin Secretion from Freshly Isolated Rat Islets-- The LC-CoA hypothesis was originally conceived from data collected from studies on insulinoma cell lines (3, 4). Thus, the work described herein on INS-1 cells, a well differentiated -cell line, would appear to represent a valid test of the hypothesis. It remains possible, however, that perturbations in lipid metabolism could have effects on regulation of insulin secretion in normal islets that were not observed in our INS-1 cell experiments. To deal with this concern, we investigated the impact of triacsin C treatment on freshly isolated rat islets of Langerhans. We found that in order to observe potent perturbation of lipid metabolism in intact islets, a higher concentration of triacsin C was required than used in the INS-1 cells experiments (50 versus 10 µM, respectively). As was the case for INS-1 cells, triacsin C treatment or fresh rat islets resulted in a significant reduction of palmitate oxidation at 3 mM glucose (Fig. 7, inset), verifying that the drug has a similar metabolic impact in the two cell preparations. The freshly isolated rat islets exhibited a 4-fold increase in insulin secretion in response to a change from 3 to 20 mM glucose, but once again, triacsin C had no inhibitory effect on this response (Fig. 7). These data verify the lack of impact of large perturbations of lipid metabolism on glucose-stimulated insulin secretion in two independent in vitro models.

View larger version (20K): [in this window] [in a new window]   Fig. 7.   Effect of triacsin C on insulin secretion from freshly isolated rat islets. Freshly isolated rat islets were incubated in SAB supplemented with 3 or 20 mM glucose, with or without 50 µM triacsin C, for 2 h, and media samples were collected for insulin radioimmunoassay. Data represent the mean ± S.E. for four independent experiments. Statistical analysis revealed no difference in insulin secretion between triacsin C and untreated islets at either glucose concentration. The inset shows the effect of 50 µM triacsin C on [1-14C]palmitate oxidation in freshly isolated rat islets incubated at 3 mM glucose (data represent the mean ± S.E. for three independent determinations per condition).

	DISCUSSION

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In this study we have applied molecular (MCD expression) and pharmacologic (triacsin C treatment) strategies for attenuating glucose-induced alterations in lipid metabolism in insulin-secreting cells. Suprisingly, neither approach impaired glycolysis or affected glucose-stimulated insulin secretion in INS-1 cells, and the lack of impact of triacsin C on regulation of insulin secretion was also confirmed in freshly isolated rat islets. These results suggest that reassessment of the long-chain acyl-CoA hypothesis for stimulus secretion coupling in islet -cells is in order. Two important observations that led to development of the hypothesis were: 1) increasing glycolytic flux in -cells by increasing the extracellular glucose concentration led to an increase in malonyl-CoA levels proportional to insulin secretion (3, 4), and 2) glucose-mediated alterations in malonyl-CoA levels were linked to changes in lipid metabolism and the cytosolic LC-CoA pool, which in turn was suggested to trigger insulin secretion by an undefined mechanism (3-5). In what follows, these two critical observations are re-evaluated in the context of the data obtained in the current study.

First, increasing glycolytic flux in -cells by increasing the extracellular glucose concentration leads to an increase in malonyl-CoA levels that is proportional to insulin secretion. Our findings are in agreement with this cornerstone observation of the LC-CoA hypothesis, but only in control INS-1 cells. We find that malonyl-CoA levels are increased in response to a change in glucose concentration from 3 to 20 mM at both 5 and 30 min in control INS-1 cells, the same range over which glucose stimulated-insulin secretion. However, expression of MCD in INS-1 cells sharply attenuates the rise in malonyl-CoA levels in response to stimulatory glucose at the 5-min time point. At 30 min, MCD expression lowers basal malonyl-CoA levels substantially, but allows an increase in the intermediate in response to glucose. However, at both 5 and 30 min, the levels of malonyl-CoA achieved in MCD expressing cells exposed to 20 mM glucose are well below the levels in control cells incubated at 20 mM glucose and are in fact not statistically different than the levels in control cells incubated at 3 mM glucose. These data indicate that the high levels of malonyl-CoA achieved in glucose-stimulated control INS-1 cells do not define a critical threshold of the intermediate that must be achieved in order for insulin secretion to be activated. However, we cannot as yet formally exclude the unlikely possibility that the increment in malonyl-CoA that remains in MCD expressing cells at the 30-min time point is involved in stimulus/secretion coupling.

Second, glucose-mediated alterations in malonyl-CoA levels are linked to changes in lipid metabolism and an increase in the cytosolic LC-CoA pool that regulate insulin secretion. Again, we were able to confirm the correlative linkage between lipid metabolism and insulin secretion (3-5), but only in INS-1 cells not engineered for MCD expression or treated with triacsin C. In such control cells, raising glucose from 3 to 20 mM results in a profound inhibition of fatty acid oxidation, with concomitant and potent activation of incorporation of radiolabeled glucose and palmitate into organically extractable cellular lipids. It is assumed that the increased incorporation of glucose or palmitate into lipids reflects accumulation of cytosolic LC-CoA, the necessary intermediate for synthesis of end products such as phospholipids and triglycerides. Interestingly, previous measurements of LC-CoA during glucose stimulation of a hamster insulinoma cell line (HIT) showed a decrease in the total LC-CoA pool (3). It was suggested that the total LC-CoA pool largely represents material sequestered in the mitochondria, and is an inaccurate measure of changes in the more relevant cytoplasmic pool (3). This represents a technical limitation for evaluating the relationship between cytoplasmic LC-CoA and insulin secretion. However, in the current study, we have utilized triacsin C as a direct inhibitor of long-chain acyl-CoA synthetase, the enzyme responsible for LC-CoA synthesis. We find that this reagent has its anticipated effects of sharply suppressing fatty acid oxidation and esterification in both INS-1 cells and freshly isolated rat islets, but does not influence glucose-stimulated insulin secretion. Importantly, triacsin C also causes a 45% decrease in total LC-CoA levels in INS-1 cells incubated at either low or high glucose concentrations. Similarly, expression of MCD in INS-1 cells significantly attenuates glucose-induced suppression of fatty acid oxidation while decreasing glucose and palmitate incorporation into cellular lipids with no effect on regulation of insulin secretion. Because these two completely independent methods produced similar results, we conclude that there is no direct correlation between the extent to which lipids are directed toward oxidative or esterification pathways and insulin secretion. While we have not directly measured the cytosolic LC-CoA pool, to the extent that the fall in total LC-CoA in response to triacsin C is reflective of this fraction, our data also argue that a rise in LC-CoA is not a critical event in glucose-stimulated insulin secretion. This conclusion is also applicable to other carbohydrate secretagogues, since we have recently shown that blockade of glycerol incorporation into cellular lipids with triacsin C in glycerol kinase-expressing INS-1 cells has no effect on glycerol-stimulated insulin secretion (16).

Note that in a recent study in fibroblasts, triacsin C was shown to be almost completely effective in blocking triglyceride and phospholipid synthesis from glycerol, while incorporation of oleate or arachidonate into phospholipids was less impaired (26). Similarly, using thin layer chromatography analysis of INS-1 cell extracts, we have observed that triacsin C inhibits conversion of [1-¹⁴C]palmitate into triglycerides by more than 90%, while incorporation of the same substrate into phospholipids is reduced by 50% (data not shown). Thus, while the effects of MCD expression or triacsin C treatment on lipid fluxes in our studies were large, they were not complete, and it remains possible that a small residual flux of substrate into specific esterification or biosynthetic pathways is required for maintenance of glucose responsiveness. It could be envisaged, for example, that a lesser efficiency of triacsin C for blockade of phospholipid biosynthesis could allow maintenance of a critical pool of this substrate for phospholipase A₂-mediated hydrolysis and release of potential second messengers such as arachadonic acid (27). Furthermore, our results are not simply reconciled with earlier experiments in which a presumed block of malonyl-CoA synthesis with the citrate lyase inhibitor, hydroxycitrate, in the perfused rat pancreas was found to inhibit glucose-stimulated insulin secretion, an effect that was reversible by palmitate addition (5). It should be noted, however, that studies by other investigators failed to demonstrate an effect of hydroxycitrate on isolated rat islets (28), and in confirmation of this finding, no effect of the drug on insulin secretion was observed in fresh rat islets or INS-1 cells by us (data not shown). Also, addition of the -oxidation inhibitor, etomoxir, to the perfused pancreas preparation potentiates insulin secretion, but only at concentrations 10 times higher than required to inhibit oxidation, suggesting that the effect of this reagent on insulin secretion may be via its structural similarity to saturated fatty acids, which are known potentiators of insulin release (5). We can only speculate that the intact pancreas has a unique susceptibility to hydroxycitrate compared with isolated islets or INS-1 cells, or that this reagent has effects at sites other than inhibition of citrate lyase in the perfused pancreas preparation. Perhaps culturing isolated islets or cell lines in rich media containing essential lipids from the fetal calf serum is sufficient to overcome hydroxycitrate-mediated inhibition of lipogenesis. Additionally, isolated islets and -cell lines may have a greater storage capacity for lipids than the intact pancreas, which would make the pancreas more susceptible to lipogenic inhibitors. Thus, while we feel that our major conclusion that large alterations in lipid metabolism are not linked to perturbations in glucose-regulated insulin secretion is correct for freshly isolated rat islets and INS-1 cells, it remains possible that islets in situ are sensitive to regulation by such a mechanism.

It has, in fact, recently been shown that complete depletion of lipid stores in islets blocks normal responsiveness to glucose and other secretagogues. Thus, animals treated with nicotinic acid to lower circulating free fatty acid exhibit no increase in insulin secretion in response to a glucose challenge (7, 8). Similarly, islets in the perfused pancreas of animals rendered hyperleptinemic for a period of 1 week are completely devoid of triglyceride and fail to respond to glucose or arginine as a secretagogue, while animals pair-fed to the lower rate of food intake of hyperleptinemic animals retain normal islet function and some residual triglyceride (9). Insulin secretion in response to glucose can be rapidly restored by lipid infusion into nicotinic acid-treated animals (7, 8), or by inclusion of fatty acids during perfusion of pancreata from fasted or hyperleptinemic animals (7-9). These data argue that lipids must be present, at least at some minimal level, in order for islet -cells to function normally.

In light of all of these results, we suggest the following modification of the long-chain acyl-CoA hypothesis. Our work shows that large impairment in the normal link between glucose and lipid metabolism in -cells is tolerated with no impact on glucose-stimulated insulin secretion, as long as glycolytic flux is not perturbed. This allows us to narrow the search for metabolic pathways and coupling factors involved in acute glucose-stimulated insulin secretion. With the apparent elimination of changes in malonyl-CoA levels and attendant alterations in lipid metabolism, the most likely candidates now include a glycolytic signal, oxidative events, or anaplerosis (1, 2, 29). The primary signal may include the well recognized glucose-driven increase in ATP:ADP ratio, leading to inhibition of ATP-sensitive K⁺ channels and influx of extracellular Ca²⁺ (1, 2).

Our experiments were designed to evaluate the effect of acute perturbation of the link between glucose and lipid metabolism. The abrogation of glucose-stimulated insulin secretion by more long term maneuvers such as nicotinic acid administration or adenovirus-induced hyperleptinemia (7-9) may be explained by lipid depletion and the resultant absence of essential modulators of secretory granule trafficking and/or exocytosis. Examples of distal sites at which lipids may be acting include generation of signaling molecules such as diacylglycerol or inositol trisphosphate, or via contribution to secretory granule or plasma membrane turnover (3-5). That lipids are acting at a distal rather than a proximal site is supported by the finding that lipid-depleted islets not only fail to respond to glucose but also show no response to arginine, leucine, or the sulfonylurea, glibenclamide (9, 30). Arginine is thought to exert its secretory effects by directly affecting membrane polarization, while sulfonylureas are believed to bring about the same effect by inhibiting ATP-sensitive K⁺ channel activity. Thus, both agents act distal to any anticipated early metabolic signal. Yet with all secretagogues tested to date, the attenuated insulin response in lipid-depleted islets is restored to normal by inclusion of fatty acids in the perfusate (7-9, 30). The mechanism by which fatty acids exert these important modulatory effects on insulin secretion remain to be established.

Finally, it should be emphasized that the data do not discount the possibility that the malonyl-CoA/carnitine palmitoyltransferase I metabolic signaling network may play important roles in long-term processes related to insulin secretion, i.e. -cell growth, apoptosis, regulation of important metabolic genes, and effects related to chronic exposure of -cells to high concentrations of fatty acids (i.e. changes in glucose sensitivity, "lipotoxicity") (2, 12, 31, 32). The molecular and pharmacologic tools described in this paper should be useful for future investigation of these issues.