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Pacific Northwest Research Institute (C.L.K., L.M.J., V.P.) and Department of Medicine, University of Washington (V.P.), Seattle, Washington 98122
Address all correspondence and requests for reprints to: Vincent Poitout, D.V.M., Ph.D., Pacific Northwest Research Institute, 720 Broadway, Seattle, Washington 98122. E-mail: vpoitout{at}pnri.org.
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Introduction |
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Materials and Methods |
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Generation of adenoviruses
A 1.6-kb XhoI/Xba fragment of the mouse DGAT cDNA with an N terminal FLAG epitope was excised from plasmid pBSSK (gift from Dr. Robert V. Farese, Jr., Gladstone Institute of Cardiovascular Disease) and subcloned into pAdTrack-CMV (14). PmeI-linearized pAdTrack-CMV-DGAT was then transformed into Escherichia coli BJ5183 cells along with the adenoviral vector pAdEasy for bacterial recombination. Recombinant adenoviruses (Ad-DGAT) were produced in 293 cells and purified as previously described (14). A firefly luciferase-expressing adenovirus (Ad-Luc) was used as a control (15).
Rat islet isolation and culture
Rat islets were isolated by collagenase digestion as previously described (12). After an overnight culture in RPMI 1640 containing 10% fetal bovine serum (FBS), 100 U/ml penicillin, 100 µg/ml streptomycin, and 11.1 mM glucose to ensure optimal recovery (16), batches of 100–200 islets were infected with 108 plaque-forming units (pfu)/islet of Ad-Luc or Ad-DGAT for 1 h unless otherwise indicated. After centrifugation for 10 min at 1200 rpm, islets were resuspended in fresh medium and incubated in various experimental conditions as described in Results. Preparation of culture medium containing palmitate was previously described (12). The final molar ratio of palmitate/BSA was 5:1. All control conditions contained the same amount of BSA and vehicle (ethanol/H2O, 1:1) as those with palmitate.
Immunoblotting
Islets were harvested in 75 µl lysis buffer [140 mM NaCl, 10 mM Tris-HCl (pH 7.4), 1 mM CaCl2, 1 mM MgCl2, 10% glycerol, 1% Nonidet P-40, 1 mM Na3VO4, 0.5 mM phenylmethylsuflonylfluoride, 0.5 mM dithiothreitol, and 1 µg/ml each of leupeptin, aprotinin, antipain, and pepstatin A], and frozen and thawed three times. Cell debris was removed by centrifugation. Twenty micrograms of protein were separated by SDS-PAGE with 8% acrylamide gels. Resolved proteins were transferred to nitrocellulose membranes (Bio-Rad Laboratories, Inc., Hercules, CA) by electroblotting. The membranes were blocked with 1% nonfat dry milk in PBS with 0.05% Tween for 1 h and then incubated for 1 h at room temperature with a mouse monoclonal anti-FLAG antibody conjugated to horseradish peroxidase (Upstate Biotechnology, Inc., Lake Placid, NY) at a 1:2000 dilution. The bands were visualized by enhanced chemiluminescence detection reagents (Amersham, Little Chalfont, UK) and exposed to X-OMAT Blue Kodak films (Eastman Kodak Co., Rochester, NY) for less than 1 min.
Insulin secretion in static incubations
Batches of 10 islets each were washed twice in Krebs-Ringer buffer (KRB) containing 0.1% BSA (Sigma, St. Louis, MO) and 2.8 mM glucose for 20 min at 37 C, then incubated for 60 min in the presence of various secretagogues, as indicated in the figure legends. Each condition was run in duplicate. Insulin levels in samples collected from the static incubations were measured using the Sensitive Rat Insulin RIA kit (Linco Research, Inc., St. Louis, MO). Total proteins were measured in duplicate batches of 20 islets using the bicinchoninic acid assay (Pierce Chemical Co., Rockford, IL).
Fluorescence-based RT-PCR of DGAT and insulin mRNAs
Comparative analysis of DGAT and insulin mRNA levels in isolated islets was performed using the Gold RT-PCR kit (Perkin-Elmer PE Applied Biosystems, Foster City, CA) and an ABI PRISM 7700 sequence detector equipped with a thermocycler (TaqMan technology) as previously described (12).
Glucose oxidation
Batches of 100 isolated islets each were preincubated twice for 30 min at 37 C in KRB containing 0.1% BSA and then incubated in KRB containing 0.1% BSA, 0.1 µCi/µmol [U-14C]glucose (NEN Life Science Products, Boston, MA), and either 2.8 or 16.7 mM unlabeled glucose for 1 h at 37 C. One milliliter of incubation buffer was then transferred to an Erlenmeyer with a rubber cap and acidified with 100 µl 7% perchloric acid. Benzethonium hydroxide (400 µl) was then injected into small wells suspended to the rubber caps, and after 16 h at room temperature, trapped 14CO2 was measured by liquid scintillation counting. Background counts from a control condition treated side by side with the samples but without islets was subtracted from the counts.
Palmitate oxidation and esterification
Batches of 100 isolated islets each were prelabeled overnight in RPMI 1640 containing 11.1 mM glucose, 10% FBS, 100 U/ml penicillin, 100 µg/ml streptomycin, 0.1% BSA, 0.5 µCi/µmol [1-14C]palmitate (NEN Life Science Products), 0.1 mM unlabeled palmitate, and 1 mM carnitine at 37 C. Islets were then washed and incubated in KRB containing 0.1% BSA, 2.8 or 16.7 mM glucose, 0.5 µCi/µmol [1-14C]palmitate, 0.1 mM unlabeled palmitate, and 1 mM carnitine for 1 h at 37 C. The production of 14CO2 in the buffer was determined as described above. Islets were harvested at the end of the final incubation for lipid extraction and thin layer chromatographic analysis as previously described (12).
Expression of data and statistics
Data are expressed as the mean ± SE. Intergroup comparisons were performed by paired t test or ANOVA with post hoc Bonferroni adjustment where appropriate. P < 0.05 was considered significant.
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Results |
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, the amount of DGAT mRNA increased with the virus titer and was readily detectable at concentrations of 106 and 108 pfu/islet. Next, isolated islets were infected under the same conditions at concentrations of 107, 108, and 109 pfu/islet, and the expression of DGAT protein was detected by immunoblotting using the anti-FLAG antibody (Fig. 1B, lanes 1–4). A band of the appropriate apparent molecular weight was detected after infection at 108 and 109 pfu/islet. To examine the time course of expression of the protein, islets were infected for 1 h with 108 pfu/islet of Ad-DGAT and harvested at various time points during the expression period (Fig. 1B, lanes 5–8). FLAG immunoreactivity was readily detected after 24 and 48 h of expression. Thus, all subsequent experiments were performed using a titer of 108 pfu/islet and an expression time of at least 24 h. Control islets were infected with the same titer of Ad-Luc.
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). Islets overexpressing DGAT incorporated less palmitate into phospholipids (Fig. 2A; P < 0.01; n = 5) and twice as much palmitate into TG (Fig. 2B; P < 0.01; n = 5) than islets infected with Ad-Luc. Incorporation of palmitate into DG was unchanged (Fig. 2B; P = NS; n = 5). The rates of glucose and palmitate oxidation in islets infected with Ad-Luc or Ad-DGAT are shown in Table 1. As expected, the rate of glucose oxidation was higher at 16.7 mM glucose than at 2.8 mM glucose in both Ad-DGAT-infected (P < 0.05; n = 5) and Ad-Luc-infected (P < 0.05; n = 5) islets. However, DGAT overexpression had no effect on glucose oxidation at either 2.8 mM (P = NS; n = 5) or 16.7 mM (P = NS; n = 5) glucose. Also as expected, glucose inhibited palmitate oxidation in both Ad-DGAT-infected (P < 0.05; n = 4) and Ad-Luc- infected (P < 0.05; n = 4) islets. However, the rate of palmitate oxidation was similar in Ad-DGAT- and Ad-Luc-infected islets at both 2.8 mM (P = NS; n = 4) and 16.7 mM (P = NS; n = 4) glucose. Overall, these results indicate that DGAT overexpression selectively increased the flux through the FA esterification pathway and TG synthesis without affecting the mitochondrial oxidation of either glucose or palmitate.
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). Insulin secretion after culture in 2.8 mM glucose was similar between Ad-DGAT-infected and Ad-Luc-infected islets (Fig. 3A). In contrast, insulin secretion in response to 8.3 mM (P < 0.05; n = 5) and 16.7 mM (P < 0.001; n = 8) glucose was significantly decreased in islets overexpressing DGAT after culture in 16.7 mM glucose (Fig. 3B). Insulin secretion in response to glyburide or potassium chloride, however, was not affected by DGAT overexpression (both P = NS; n=5). DGAT overexpression did not affect insulin secretion after 24 h of expression of the enzyme (data not shown).
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). The presence of palmitate in the culture medium dramatically reduced the subsequent response to 16.7 mM glucose in islets infected with Ad-Luc (P < 0.001; n = 6). Islets infected with Ad-DGAT secreted less insulin than islets infected with Ad-Luc after 72 h of culture in the absence of palmitate (P < 0.01; n = 6), confirming the results shown in Fig. 3
. The presence of palmitate during the culture did not further impair glucose-induced insulin release in DGAT-overexpressing islets (P = NS; n = 6). Total islet protein levels were not affected by either the presence of palmitate or infection with Ad-DGAT or Ad-Luc (data not shown). To further confirm that the deleterious effects of palmitate and those of DGAT overexpression are not additive, islets infected with either Ad-Luc or Ad-DGAT were cultured for 72 h in the presence of 2.8 or 16.7 mM glucose with increasing concentrations of palmitate, from 0–0.5 mM (Fig. 5). Neither the presence of palmitate nor infection with Ad-DGAT affected the subsequent response to 2.8 mM glucose, confirming the results shown in Fig. 4. In islets infected with Ad-Luc, increasing the concentration of palmitate in the culture medium led to a dose-dependent decrease in the subsequent response to 16.7 mM glucose. In contrast, islets infected with Ad-DGAT showed a reduced response to 16.7 mM glucose even in the absence of palmitate (P < 0.05; n = 5), confirming the results shown in Figs. 3 and 4, and the response was not further impaired with increasing amounts of palmitate. These results suggest a common mechanism of action between palmitate exposure and DGAT overexpression, which might lie at the level of TG accumulation.
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). As expected, insulin mRNA levels were higher in islets cultured in high glucose than in islets cultured in low glucose (P < 0.01; n = 3). As we have previously shown (12, 17), 72-h culture in the presence of palmitate and high glucose led to a significant decrease in insulin mRNA levels (P < 0.05; n = 3). However, DGAT overexpression did not affect insulin mRNA levels in any of the culture conditions (P = NS; n = 3).
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Discussion |
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Prolonged exposure of pancreatic ß-cells to FA inhibits glucose-stimulated insulin secretion (18, 19, 20, 21) and inhibits insulin gene expression in the presence of high glucose levels (17, 22, 23). Two mechanisms have been proposed to explain the inhibitory effects of FA on insulin secretion: the existence of a glucose-fatty acid cycle in the mitochondria and the accumulation of long-chain CoA (LC-CoA) derivatives in cytosol. The glucose-fatty acid cycle hypothesis holds that an increase in mitochondrial FA oxidation inhibits glucose oxidation, as originally demonstrated in cardiac muscle (24). Indeed, a decrease in glucose oxidation in islets has been reported after chronic exposure to elevated FA (11, 18, 19, 20, 21) and has been attributed in part to decreased pyruvate dehydrogenase activity (20, 25, 26). Several observations, however, argue against the existence of a glucose-FA cycle in the ß-cell. First, several studies have failed to observe FA- induced changes in glucose oxidation in islets (27) or insulin-secreting cell lines (28, 29). Second, pharmacological inhibition of FA oxidation only partially prevents the decrease in insulin secretion (18, 20, 21). Third, the rate of FA oxidation is much lower than that of glucose, making it unlikely that the former can significantly reduce the latter (30).
The malonyl-CoA/LC-CoA hypothesis postulates that glucose metabolism generates malonyl CoA, which inhibits carnitine-palmitoyl transferase-1 and transport of LC-CoA into the mitochondria, thus leading to their accumulation in the cytosol (31). Cytosolic LC-CoA then act as signaling molecules, thus playing an important role in stimulus-secretion coupling in the ß-cell (32). This model can be applied to circumstances of prolonged exposure to excessive levels of FA; when both glucose and FA are chronically elevated, sustained inhibition of carnitine-palmitoyl transferase-1 by malonyl-CoA induces a switch in the partitioning of LC-CoA toward complex lipid synthesis. Consequently, deleterious effects of prolonged exposure to FA are only observed in the presence of elevated glucose levels, a hypothesis supported by several lines of experimental evidence (reviewed in Ref. 33). In a previous study we have demonstrated that accumulation of TG in isolated rat islets upon prolonged exposure to elevated FA occurs only in the presence of high glucose (12). We observed an inverse correlation between TG accumulation and insulin mRNA levels, suggesting that the functional effects of prolonged FA might be due to TG accumulation, as proposed in the ZDF rat (3, 4). However, to our knowledge a direct cause and effect relationship between TG accumulation and ß-cell dysfunction has never been established, which was the objective of the present study.
DGAT catalyzes the last and only committed step in TG synthesis. It is a microsomal enzyme whose acyltransferase activity is highly specific for diacylglycerol (34) and which we have shown to be expressed in isolated islets and insulin-secreting cells (12). Invalidation of the DGAT gene in mice dramatically reduces diacylglycerol acyltransferase activity in several tissues, although the animals are still capable of some degree of TG synthesis. Indeed, an additional diacylglycerol acyltransferase gene family, DGAT2, has recently been identified (35, 36). DGAT overexpression in islets resulted in a 2-fold increase in the incorporation of palmitate into TG, without any detectable change in the rate of oxidation of either palmitate or glucose. Glucose-induced insulin secretion was unchanged after 24 h of expression of DGAT; however, it was significantly decreased after 72 h of expression in the presence of elevated glucose levels. The requirement for long-term culture for DGAT overexpression to affect insulin secretion is not due to delayed expression of the protein, which was already maximal after 24 h. It suggests that either several days in culture are necessary to accumulate enough TG to affect insulin secretion or that the effects of TG accumulation are only observed after 72 h in culture. It is interesting to note that exposure of islets to palmitate also requires 72 h to affect ß-cell function (17). The glucose- dependent, long-term effects of DGAT thus have characteristics similar to the effects of palmitate on ß-cell function (17). The glucose dependency of the DGAT effects are also consistent with the concept that FA-induced ß-cell dysfunction is only observed in the presence of high glucose. Interestingly, insulin secretion in response to the nonnutrient secretagogues glyburide and potassium chloride were not perturbed by DGAT expression.
Thus, the effects of DGAT overexpression on glucose-induced insulin secretion share key characteristics with those of chronic FA: they only occur after prolonged culture (72 h), and they require the presence of elevated glucose levels during the culture. Most importantly, the effects of DGAT are not additive to those of palmitate. These findings therefore suggest that one mechanism by which prolonged exposure to FA diminishes insulin secretion is an increase in the esterification pathway and intracellular accumulation of TG. This does not prove, however, that accumulation of TG by itself is the mechanism underlying the effects of FA. In fact, the concept that TG can cause ß-cell dysfunction is somewhat counterintuitive, because storage of fat in the form of inert neutral lipids is usually seen as a defense mechanism by which the cell protects itself from the deleterious effects of FA (37). An alternative possibility is that the effects of FA are mediated by generation of an intermediate signal arising from the esterification pathway, such as DG.
Interestingly, we did not observe any effect of DGAT overexpression on insulin mRNA levels. This is in contrast to our previous observations that prolonged exposure to palmitate decreases insulin mRNA levels in islets in the presence of high glucose (12, 17). Although the reasons for the discrepancy between the effects of palmitate and those of DGAT expression on insulin gene expression are unknown, we speculate that exposure to palmitate is associated with multiple abnormalities in intracellular metabolism, none of which is mimicked by DGAT overexpression. Indeed, our results suggest that TG accumulation is a mechanism for FA-induced impairment of insulin secretion, but might not play a direct causal role in the FA-induced decrease in insulin gene expression. It is conceivable, for instance, that ceramide formation, which occurs upon prolonged exposure to FA (8) impairs insulin gene expression without affecting insulin secretion. Indeed, culture of insulin-secreting cells in the presence of cell-permeable ceramide analogs for 72 h does not impair insulin release (38). In addition, it is important to note that the present study examined the involvement of TG synthesis in defective insulin secretion, which might be one of the early signs of lipotoxicity, but not its potential role in more profound FA-induced abnormalities, such as ß-cell death.
In conclusion, our results uniquely demonstrate that selectively increasing TG synthesis in the presence of elevated glucose levels impairs glucose-induced insulin secretion, supporting the concept that an increase in FA esterification in the presence of high glucose is a key mechanism of lipotoxicity. Whether these in vitro observations over relatively short periods of culture reflect the mechanisms of FA- induced ß-cell dysfunction over many years of exposure of islets to elevated lipid levels in type 2 diabetic patients remains to be determined.
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Acknowledgments |
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Footnotes |
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Abbreviations: Ad-Luc, Firefly luciferase-expressing adenovirus; CoA, coenzyme A; DG, diglycerides; DGAT, diacylglycerol acyltransferase 1; FA, fatty acids; FBS, fetal bovine serum; KRB, Krebs-Ringer buffer; LC-CoA, long-chain fatty acyl-coenzyme A; pfu, plaque-forming units; TG, triglyceride; ZDF, Zucker diabetic fatty.
Received April 15, 2002.
Accepted for publication May 21, 2002.
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Rn, Changes in fluorescence emission. Results are the mean ± SE of triplicate determinations in one representative experiment. B, Islets were infected with increasing titers of Ad-DGAT and cultured for 24 h (lanes 1–4) or were infected with 108 pfu/islet and cultured for 0–48 h (lanes 5–8). Expression of the DGAT protein was assessed by immunoblotting with an anti-FLAG antibody. A representative blot is shown. Similar results were obtained in two replicate experiments.
