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Inhibition of Lipase Activity and Lipolysis in Rat Islets Reduces Insulin Secretion

¹ Department of Cell and Molecular Biology, Lund University, Lund, Sweden
² Department of Medicine, Lund University, Lund, Sweden

	ABSTRACT

TOP ABSTRACT RESEARCH DESIGN AND METHODS RESULTS DISCUSSION REFERENCES

 
Lipids may serve as coupling factors in K_ATP-independent glucose sensing in ß-cells. We have previously demonstrated that ß-cells harbor lipase activities, one of which is the hormone-sensitive lipase. Whether ß-cell lipases are critical for glucose-stimulated insulin secretion (GSIS) by providing lipid-derived signals from endogenous lipids is unknown. Therefore, using a lipase inhibitor (orlistat), we examined whether lipase inhibition reduces insulin secretion. Islet lipolysis stimulated by glucose and diglyceride lipase activity was abolished by orlistat. Incubation of rat islets with orlistat dose dependently inhibited GSIS; this inhibition was reversed by 1 mmol/l palmitate, suggesting that orlistat acts via impaired formation of an acylglyceride-derived coupling signal. Orlistat inhibited the potentiating effect of forskolin on GSIS, an effect proposed to be due to activation of a lipase. In perifused islets, orlistat attenuated mainly the second phase of insulin secretion. Because the rise in islet ATP/ADP levels in response to glucose and oxidation of the sugar were unaffected by orlistat whereas the second phase of insulin secretion was reduced, it seems likely that a lipid coupling factor involved in K_ATP-independent glucose sensing has been perturbed. Thus, ß-cell lipase activity is involved in GSIS, emphasizing the important role of ß-cell lipid metabolism for insulin secretion.

The current view holds that lipids serve as coupling factors in glucose-stimulated insulin secretion (GSIS) that does not rely on closure of the ATP-sensitive K⁺ channel (K_ATP independent) (3). These putative mechanisms presumably drive the second phase of GSIS and accordingly have been termed as amplifying (4), and the coupling factors have been proposed to emanate from intermediary metabolism in the ß-cell (5). Against this background, lipids have become attractive candidates for coupling factors in K_ATP-independent glucose sensing. In fact, a model has been proposed to delineate the combined events in metabolism of glucose and lipids that result in a dose-dependent regulation of insulin secretion, the so-called long-chain acyl-CoA hypothesis (3). This model is based on the observations that an increase in glucose metabolism in ß-cells results in decreased oxidation of fats while esterification of lipids increases. As a consequence, the levels of complex lipids rise, perhaps acyl-CoA moieties, which then act as a signaling molecules coupling stimulus to secretion in the ß-cell.

We have previously demonstrated that different preparations of ß-cells exhibit lipase activities (6). Among these is the hormone-sensitive lipase (HSL), which, in white adipocytes, is the critical enzyme that hydrolyzes triglycerides to fatty acids (7). HSL is unique among lipases in that it is controlled by circulating hormones. Thus, hormones, such as glucagon, which is secreted in the fasted state, and adrenaline stimulate formation of cAMP in adipocytes. Consequently, protein kinase A (PKA) is activated and phosphorylates HSL on three serine residues (8), thereby activating the enzyme and controlling the rate of lipolysis. Insulin, however, is a potent inhibitor of lipolysis. This is achieved by activation of phosphodiesterase 3 B (9), which hydrolyzes cAMP, thereby inactivating PKA. In ß-cells, however, the situation seems to be more complex. Although expression of HSL was confirmed using several techniques, activity assays indicated that lipases other than HSL may be in operation in ß-cells (6). With regard to insulin secretion, it has been reported that the antilipolytic agent 3,5-dimethylpyrazole inhibits insulin secretion from isolated islets in response to glucose, cAMP-raising agents, and the mitochondrial fuel -ketoisocaproic acid (10), suggesting a critical role for a lipase in ß-cell stimulus-secretion coupling. However, observations from genetic mouse models, in which HSL has been inactivated, are in both support (11) and opposition (12) of a crucial role for HSL in control of insulin secretion. In sum, the studies available thus far suggest that lipases, other than HSL and that which remain to be identified, account for some lipase activity in islets and may play a role in ß-cell stimulus-secretion coupling, whereas the role of HSL in these processes requires further clarification.

This study further explored whether ß-cell lipase activity is required for GSIS. To circumvent the issue of which particular lipase accounts for hydrolysis of acylglycerides in pancreatic ß-cells, we used an inhibitor of a wide spectrum of lipases. Our choice was tetrahydrolipstatin, or orlistat, which is a lipophilic molecule that irreversibly binds to the catalytic site of a great number of lipases (13) and which has previously been shown to inhibit cAMP-induced insulin secretion in the clonal ß-cell line HIT-T15 (14) and glucagon-like peptide 1 (GLP-1)-stimulated secretion in rat islets (15). The drug is widely used for treatment of obesity (16); it inhibits intestinal lipases, preventing uptake of lipids and thereby restricting caloric input. Orlistat, at therapeutic doses, is not taken up into the circulation, thereby precluding undesired systemic effects. By use of this tool in vitro, we were able to inhibit lipase activity in primary ß-cells, thereby reducing lipolysis. These maneuvers resulted in an inhibition of insulin secretion, suggesting that intracellular mobilization of lipids via activation of lipases in ß-cells is required for GSIS.

	RESEARCH DESIGN AND METHODS

TOP ABSTRACT RESEARCH DESIGN AND METHODS RESULTS DISCUSSION REFERENCES

 
Reagents and animals.
All reagents were from Sigma (St. Louis, MO) unless otherwise stated. Orlistat (tetrahydrolipstatin) was supplied by Dr. Marcel K. Meier (Roche Pharmaceuticals, Basel, Switzerland). The drug was dissolved in DMSO/ethanol (60/40%) as a stock solution of 10 mmol/l. In all experiments, an identical concentration of the vehicle was added. Female Sprague-Dawley rats aged 8–10 weeks were used for the experiments. The studies were approved by the Animal Ethics Committee at Lund University.

Islet isolations, batch incubations, and islet perifusions.
Rat islets from fed rats were isolated by standard collagenase digestion and subsequently handpicked under a stereo microscope. For culture overnight, the isolated islets were kept in RPMI-1640 medium, containing 11.1 mmol/l glucose, 10% FCS, 100 units/ml penicillin, 100 µg/ml streptomycin, and 2.5 µg/ml Fungizone, at 37°C in 95% air and 5% CO₂. For secretion studies, batches (n = 8) of three islets for each condition were kept in HEPES balanced salt solution (HBSS; 125 mmol/l NaCl, 5.9 mmol/l KCl, 1.2 mmol/l MgCl₂, 20 mmol/l HEPES, 1.3 mmol/l CaCl₂, 0.1% BSA [pH 7.35]) containing 2.8 mmol/l glucose for 60 min in an incubator at 37°C. Then, three islets at a time were transferred to a multiwell plate kept on ice and containing 200 µl per well of the same buffer but with the addition of the respective secretagogue and inhibitor. When all islets had been transferred, the plate was again placed in an incubator at 37°C; after 60 min, a sample from the buffer was removed for measurement of insulin by enzyme-linked immunosorbent assay (Mercodia, Uppsala, Sweden). In experiments with orlistat, the lipase inhibitor was also present during the 1-h preincubation at 2.8 mmol/l glucose.

Perifusion of islets was performed as previously described in detail (17). In brief, islets were preincubated in HBSS at 2.8 mmol/l glucose for 60 min. Then, batches of 20 islets were sandwiched between two layers of gel (Bio-Gel P4; Bio-Rad, Richmond, CA). The HBSS was warmed to 37°C. Islets were perifused at a flow rate of 0.5 ml/min, fractions were collected at 1-min intervals, and insulin was measured by enzyme-linked immunosorbent assay.

For the secretion experiments, palmitate was dissolved in ethanol, after which a stoichiometric amount of NaOH was added. The ethanol was evaporated under N₂, and the sodium palmitate was redissolved in H₂O under heat and diluted as a 10-mmol/l stock solution in ice-cold 10% BSA (free fatty acid free). This stock solution was diluted 10 times in the HBSS at the time of the experiment. The free palmitate concentration, using this preparation, was estimated to be 5.6 µmol/l (18).

Lipolysis in islets and adipocytes.
Batches of 100 islets were isolated and subsequently incubated at 37°C in 500 µl of HBSS, containing 2.8 or 16.7 mmol/l glucose with or without 200 µmol/l orlistat; in some experiments, 1 mmol/l 8-bromoadenosine-3',5'-cyclic monophosphate (8-Br-cAMP), a cAMP analog with enhanced stability, was included throughout. Incubations lasted for 3 h. Adipocytes were isolated from gonadal fat pads by collagenase digestion. Samples of the cell suspension were transferred to siliconized tubes containing prewarmed medium; final lipocrit was 1–2%. After a 1-h incubation at 37°C with gentle shaking, cells were centrifuged through silicone oil to separate cells from the incubation medium. Glycerol released into the medium was used as an index of lipolysis and was measured as described (19).

Enzyme activity and acylglyceride assays.
The white adipose cell suspension was homogenized and islets were sonicated in buffer (0.25 mol/l sucrose, 1 mmol/l EDTA [pH 7.0], 1 mmol/l dithioerythritol, 20 µg/ml leupeptin, 20 µg/ml antipain, and 1 µg/ml pepstatin A) to allow assay of diglyceride lipase activity, as described elsewhere (20); the diacylglycerol analogue mono-oleoyl-2-O-mono-oleylglycerol (MOME) was used as substrate (21).

ATP and ADP determinations.
Isolated islets were cultured overnight followed by a 1-h incubation in HBSS containing 16.7 mmol/l glucose, 2.5 µmol/l forskolin, and 35 mmol/l KCl. These measures were taken to degranulate ß-cells, thereby depleting them of ATP contained in the insulin granules, which obscures changes in ATP generated by metabolism (22). Islets were washed three times in HBSS, followed by preincubation of batches of 20 freshly isolated rat islets for 1 h in HBSS at 2.8 mmol/l glucose; the buffer was then replaced by HBSS containing 2.8 or 16.7 mmol/l glucose. After an additional 5 min, trichloroacetic acid was added at a final concentration of 2% to the islets, which were kept on ice for 20 min. Cellular debris was collected at the bottom of the tube after maximal centrifugation in a benchtop centrifuge; the supernatant was collected and kept at -20°C until assay for ATP content using bioluminescence (Thermo Labsystems Oy, Helsinki, Finland). ADP was measured after ATP had been depleted by ATP sulfurylase followed by conversion of ADP to ATP by pyruvate kinase, as described in detail (23). The ATP:ADP ratio for the different conditions was calculated.

Glucose oxidation.
After isolation, islets were kept in HBSS containing 2.8 mmol/l glucose for 60 min at 37°C. Batches of islets (n = 30) were transferred to a cup (Kimble-Kontes, Vineland, NJ) suspended from a rubber sleeve stopper (Fisher, Pittsburgh, PA), which was inserted into a glass scintillation vial. A reaction mixture containing 2 DPM/pmol [¹⁴C]glucose was added, and the reaction was terminated after 1 h by injection of 100 µl of 7% perchloric acid into the suspended cup. The rate of [¹⁴C]glucose oxidation was measured as released ¹⁴CO₂ trapped by adding 300 µl of benzethonium hydroxide to the bottom of the sealed vials, followed by an additional 2-h incubation at 37°C. After discarding the cups and rubber stoppers, trapped ¹⁴CO₂ was determined by scintillation counting.

Statistical measures.
Mean ± SE is given. With the exceptions given below, data were compared with a two-tailed unpaired t test. Area under the curve (AUC) in the perifusion experiments was compared by a paired Wilcoxon signed-rank test. When more than two groups were compared, ANOVA was performed, followed by Bonferroni test post hoc. ATP:ADP ratios were compared by nonparametric ANOVA (Kruskal-Wallis test), followed by Bonferroni test post hoc. P < 0.05 was considered statistically significant.

	RESULTS

TOP ABSTRACT RESEARCH DESIGN AND METHODS RESULTS DISCUSSION REFERENCES

 
Lipolysis in rat islets and adipocytes.
We have previously demonstrated that HSL expression is induced by glucose in the clonal ß-cell line INS-1 (24) and that it is paralleled by an increase in lipolysis. Here, our first aim was to demonstrate that lipolysis can be acutely stimulated in primary ß-cells. To this end, fresh isolated rat islets were incubated at 2.8 or 16.7 mmol/l glucose for 3 h, and release of glycerol into the medium was measured as an indicator of lipolysis. As shown in Fig. 1, raising the concentration of glucose and adding 8-Br-cAMP dose dependently increased lipolysis; the higher concentration of glucose stimulated glycerol release 2.3-fold (P < 0.05) from rat islets. Given that HSL is expressed in rat islets (6) and that the lipase is activated by PKA in white adipose tissue (8), the cAMP analog 8-Br-cAMP was added to the islets during the incubation to activate PKA. This maneuver further potentiated the release of glycerol from islets at both low and high glucose (Fig. 1), suggesting that a cAMP-regulated lipase, presumably HSL, accounts for lipolysis in rat islets. Addition of 200 µmol/l orlistat, however, completely blocked glycerol release from the isolated rat islets when lipolysis was maximally stimulated by the addition of 8-Br-cAMP.

View larger version (46K): [in this window] [in a new window]   FIG. 1. Lipolysis in isolated rat islets. Fresh isolated rat islets were cultured for 3 h at the indicated glucose concentrations, in the presence or absence of 1 mmol/l 8-Br-cAMP and/or 200 µmol/l orlistat. Lipolysis was monitored by glycerol release; seven independent experiments were performed. *P < 0.05; **P < 0.01.

View larger version (25K): [in this window] [in a new window]   FIG. 2. Lipolysis in primary rat adipocytes. Lipolysis in primary adipocytes exposed to 2.5 µmol/l forskolin for 1 h, with or without 200 µmol/l orlistat, was monitored by glycerol release; four independent experiments were performed. ***P < 0.001 vs. forskolin.

View larger version (44K): [in this window] [in a new window]   FIG. 3. Diglyceride lipase activity in rat islets. Islets from the experiment in Fig. 1 were homogenized, and lipase activity toward a diglyceride substrate was determined. *P < 0.05 for any condition with orlistat vs. without the inhibitor.

View larger version (29K): [in this window] [in a new window]   FIG. 4. Diglyceride lipase activity in primary rat adipocytes. Adipocytes from the experiment in Fig. 2 were homogenized, and lipase activity toward a diglyceride substrate was determined. *P < 0.01 vs. control.

View larger version (14K): [in this window] [in a new window]   FIG. 5. Static incubation of rat islets. Rat islets cultured overnight were preincubated for 1 h at 2.8 mmol/l glucose, after which the conditions were changed to HBSS containing 2.8 mmol/l glucose or 16.7 mmol/l glucose with increasing concentrations of orlistat (A) or to HBSS containing 16.7 mmol/l glucose and 2.5 µmol/l forskolin with increasing concentrations of orlistat (B). Five independent experiments were performed for each condition. ***P < 0.001 vs. control.

View larger version (29K): [in this window] [in a new window]   FIG. 6. Static incubation of rat islets in the presence of orlistat and palmitic acid. The experiments in Fig. 5 were repeated with the addition of 1 mmol/l palmitic acid to 200 µmol/l orlistat. Three independent experiments were performed. ***P < 0.001.

View larger version (23K): [in this window] [in a new window]   FIG. 7. Perifusion of rat islets in the presence of orlistat. Islets were perifused in the presence or absence of 200 µmol/l orlistat (A). First, islets were perifused at 2.8 mmol/l glucose; at 10 min, glucose was raised to 16.7 mmol/l (arrow). At 40 min, glucose was again lowered to 2.8 mmol/l; at 50 min, islets were exposed to 35 mmol/l KCl. Insulin secretion was calculated as AUC (B) and compared by a two-tailed paired Wilcoxon’s signed-rank test. First-phase secretion was defined as insulin release in fractions 14–18 and second-phase secretion as insulin release in fractions 19–40. Five independent experiments were performed for each condition. *P < 0.05.

ATP levels in rat islets exposed to orlistat.
If ATP:ADP ratios, the increase of which closes the K_ATP channel, are altered by orlistat, then it is less likely that the drug exerts its effects on insulin secretion via impaired formation of a lipid-derived signal critical for K_ATP-independent glucose sensing. Instead, a general metabolic incapacitation may have occurred. To address this possibility, we measured islet levels of nucleotides and calculated ATP:ADP ratios. As shown in Fig. 8A, after a rise in glucose from 2.8 to 16.7 mmol/l, the ATP:ADP ratio increased significantly at 5 min; this increase was similar in islets incubated in the presence or absence of 200 µmol/l orlistat, demonstrating that the lipase inhibitor does not interfere with metabolically generated ATP.

View larger version (20K): [in this window] [in a new window]   FIG. 8. ATP levels (A) and glucose oxidation (B) in rat islets in the presence of orlistat. For ATP determinations, islets were incubated at 2.8 mmol/l glucose for 1 h, after which the buffer was switched to 16.7 mmol/l glucose with or without 200 µmol/l orlistat. After 5 min, ATP and ADP were extracted and measured by bioluminescence. For each condition, ATP and ADP in three batches of islets were measured in triplicate; the data from two independent experiments have been averaged. ***P < 0.001 vs. 2.8 mmol/l glucose; 16.7 mmol/l glucose vs. 16.7 mmol/l glucose plus 200 µmol/l orlistat was not significantly different. Oxidation of [14C]glucose was assessed at 2.8 and 16.7 mmol/l. The experiment was performed in quadruplicate and repeated twice. ***P < 0.001, 2.8 mmol/l glucose vs. 2.8 mmol/l glucose plus 200 µmol/l orlistat or 16.7 mmol/l glucose vs. 16.7 mmol/l glucose plus 200 µmol/l orlistat was not significantly different.

	DISCUSSION

TOP ABSTRACT RESEARCH DESIGN AND METHODS RESULTS DISCUSSION REFERENCES

 
Despite the widely embraced view that lipids play a role in the regulation of insulin secretion (25), little is known about the actual details of this involvement. A number of possibilities exist, one being that a lipid moiety in the ß-cell arises as a result of glucose metabolism and participates in nutrient sensing (3,26). This is the long-chain acyl-CoA model, in which malonyl-CoA levels rise as a result of the cataplerotic exit of citrate from the tricarboxylic acid cycle, ultimately forming malonyl-CoA; this metabolite switches lipid metabolism from oxidation to esterification by virtue of its inhibition of the carnitine palmitoyl transferase I (27), the transporter of long-chain acyl-CoA into the mitochondrion for subsequent oxidation. Although there is a body of circumstantial evidence supporting this model (28,29), studies addressing its molecular mechanisms have both agreed (30,31) and disagreed (32,33) with how the model was initially put forth. Another possibility is that lipids provided from the circulation play an important role in insulin secretion, particularly during fasting, when the cellular lipid stores may be low (2).

An interesting new idea, however, has arisen from the observation that ß-cells exhibit lipase activity (6). This was not a surprising finding, considering that it has long been recognized that ß-cells store triglycerides and utilize lipids as fuel when ambient glucose is low (34). If it is assumed that generation of a lipid is involved in control of insulin secretion, then regulation of its level is a potential regulatory mechanism. Clearly, a lipase that mobilizes lipids from triglyceride stores and that is regulated by intracellular second messengers is an attractive candidate for such a role. These characteristics apply to HSL (7). In adipocytes, this lipase is activated by reversible phosphorylation of serine residues by PKA (8). Thus, triglyceride hydrolysis is stimulated by a rise in intracellular cAMP. Indeed, we were able to demonstrate that HSL is expressed and active in ß-cells (6). From immunoinhibition experiments, in which an antibody to HSL is used to block diglyceride lipase activity (6,24), it was deduced that 25% of such activity in INS-1 cells is accounted for by HSL. Moreover, chronic exposure to high glucose induces expression and activity of HSL in INS-1 cells and rat islets and, in parallel, stimulates lipolysis, as determined in INS-1 cells (24). Recently, it was shown that the incretin hormone and cAMP-raising agent GLP-1 stimulates an intracellular acidification of HIT-T15 cells and an efflux of fatty acids (14); these events were attributed to a stimulation of lipolysis, presumably accounted for by HSL, given that cAMP mediates the effect observed. Furthermore, orlistat was demonstrated to inhibit forskolin- and GLP-1–stimulated insulin secretion, while being less effective in GSIS (14), and yet another antilipolytic agent, 3,5-dimethylpyrazole, also inhibits insulin secretion induced by glucose, cAMP-raising agents, and -ketoisocaproic acid (10).

Although these observations support a role for lipases generating lipid signals in GSIS, studies in HSL null mice have been less clarifying. In one line of HSL null mice, GSIS is impaired in vivo and in vitro (11). However, another line of HSL null mice, created in our laboratory, fails to exhibit an impairment of insulin secretion (12). The reason for this apparent discrepancy is unclear but could be explained by genetic redundancy of other lipid-hydrolyzing enzymes. In fact, the existence of such lipases is highly likely, given our previous observation that lipase activity in vitro in INS-1 cells is only partially blocked by HSL-neutralizing antibodies (6,24). Instead, the outstanding finding concerning metabolism in our line of HSL null mice is reduced insulin sensitivity (12); in vivo, the mice appropriately adapt to insulin resistance by hypersecretion of insulin, in part accounted for by islet hypertrophy.

The experiments in the present study were carried out in an attempt to confirm that lipases play a role in insulin secretion. To this end, orlistat, an inhibitor of a wide spectrum of lipases, was used to inhibit all lipases in primary ß-cells. Orlistat is highly lipophilic, is insoluble in water, and binds avidly to proteins. Despite these features, it is known that orlistat at high concentrations traverses cellular membranes (35) and blocks intracellular lipases (14,36). Orlistat binds irreversibly to the catalytic site of lipases and has been shown to inhibit HSL (13). Here, we were able to show that acute exposure to glucose stimulated lipolysis in rat islets. This was further potentiated by 8-Br-cAMP, suggesting that HSL plays a role in these processes, because the nucleotide will activate HSL via PKA (7). Addition of orlistat to the islets blocked lipolysis as well as diglyceride lipase activity, strongly indicating that the efflux of glycerol from the islets emanated from lipolysis. This conclusion assumes that glycerol is not generated from other sources, because an enzyme, such as glycerol-3-phosphatase in yeast, has not been reported in ß-cells. However, that inhibition of diglyceride lipase activity by orlistat was more extensive than the abrogation of glycerol efflux could indicate that glyceroneogenesis in fact occurs in islets. This is difficult to reconcile with the lacking activity of, for example, phosphoenolpyruvate carboxykinase in islets (37), and therefore warrants further study. Alternatively, the residual lipase activity after orlistat treatment may be sufficient to generate glycerol. Nevertheless, experiments in adipocytes yielded similar results, showing that the effect of orlistat is not restricted to one cell type. In static incubations of rat islets, orlistat dose dependently inhibited GSIS; the effect on forskolin-induced insulin secretion was also marked. That this inhibition, in fact, stems from a block of lipid mobilization is supported by the observation that exogenous lipid, in the form of palmitic acid, recovered insulin secretion in the presence of orlistat. To exclude that the effect of orlistat was due to nonspecific toxic action of the lipase, we also determined the ATP:ADP ratio in the islets and oxidation of glucose. We found that the increase in the ATP:ADP ratio provoked by a rise in glucose as well as oxidation of the sugar was unaffected by orlistat, yet insulin secretion was clearly inhibited. This indicates that the insulinostatic effect of orlistat cannot be attributed to inhibition of glucose metabolism with impaired production of ATP or that a more general incapacitation of ß-cell function has occurred.

It is interesting that perifusion of islets revealed that mainly the second phase of insulin secretion was inhibited by orlistat. A more pronounced effect on the second phase of insulin secretion than on the first phase suggests that K_ATP-independent glucose sensing is impaired. This fits well with the presumed action of orlistat in ß-cells, namely impaired mobilization of lipids from stored acylglycerides. The implication of our findings is therefore that a lipid generated by acylglyceride hydrolysis plays a critical role in insulin secretion. At this point, attention must be turned toward the identity of these lipid factors and the lipase(s) by which they are generated. Whether HSL is one of these or perhaps the only one remains to be determined.

	ACKNOWLEDGMENTS

 
The studies were supported by the Swedish Research Council (14196 [H.M.], 11284 [C.H.], and 6834 [B.A.]); a Center of Excellence Grant from the Juvenile Diabetes Foundation, U.S.A., and the Knut and Alice Wallenberg Foundation, Sweden (C.H.); the Juvenile Diabetes Research Foundation International (10-2000-676 [H.M.]); the European Union (QLRT-2001-0288 [B.A.]); the Swedish Diabetes Association; the Novo Nordisk, Crafoord, Thelma Zoega, Ingrid and Fredrik Thuring, Åke Wiberg, and Albert Påhlsson Foundations; and the Medical Faculty at Lund University.

We thank Kristina Göransson for reestablishing the islet perifusion technique in the laboratory. Ms. Göransson performed all experiments with islets and statistically analyzed the data therefrom. We also thank Birgitta Danielsson for technical assistance with lipase activity assays.

Address correspondence and reprint requests to Hindrik Mulder, MD, PhD, Section for Molecular Signaling, Department of Cell and Molecular Biology, Lund University, BMC C11 SE-221 84 Lund, Sweden. E-mail: hindrik.mulder{at}medkem.lu.se

Received for publication April 17, 2003 and accepted in revised form September 25, 2003

Abbreviations: 8-Br-cAMP, 8-bromoadenosine-3',5'-cyclic monophosphate; AUC, area under the curve; GLP-1, glucagon-like peptide 1; GSIS, glucose-stimulated insulin secretion; HBSS, HEPES balanced salt solution; HSL, hormone-sensitive lipase; MOME, mono-oleoyl-2-O-mono-oleylglycerol; PKA, protein kinase A

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