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INSULIN-GLUCAGON-GI PEPTIDES-DIABETES MELLITUS |
Department of Medicine (L.-X.L., I.H.J., V.G.), Endocrine Section, Institute of Cancer Research and Molecular Biology (F.S., K.E.), Medical Faculty, Norwegian University of Science and Technology, N-7489 Trondheim, Norway
Address all correspondence and requests for reprints to: Valdemar Grill, M.D., Ph.D., Endocrine Section, Medical Faculty, Norwegian University of Science and Technology, N-7489 Trondheim, Norway. E-mail: . valdemar.grill{at}medisin.ntnu.no
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Abstract |
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ligand, affected neither UCP-2 mRNA nor [1-14C] oleate oxidation. Antioxidants (vitamin E and sodium selenite) did not affect oleate-induced UCP-2 mRNA. We conclude that: 1) UCP-2 mRNA is induced by fatty acid oxidation in ß-cells; and 2) glucose exerts a modulating effect that is coupled to inhibition of fatty acid oxidation |
Introduction |
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A plausible theory on the functional importance of UCP-2 holds that regulation of this protein provides a way to dissipate energy in situations of oversupply to a specific tissue or cell (1, 2). Restraining the build-up of excessive amounts of energy, stored (for example) as glycogen or triglycerides, could be beneficial in many tissues. However, in ß-cells, a decrease in the metabolic efficiency of glucose could negatively affect a glucose signal for secretion, because the latter includes the generation of ATP. This notion is strengthened by studies in ß-cells overexpressing UCP-2 after transfection (4, 5). In these cells, ATP levels are lowered and glucose-induced insulin secretion deficient. Further, evidence exists, in rat pancreatic islets, that fatty acids can induce mitochondrial uncoupling of glucose oxidation (6); and it seems possible that such uncoupling could be important for the ß-cell insensitivity to glucose that follows long-term exposure to fatty acids (7, 8, 9). The regulation of UCP-2 in ß-cells, in relation to metabolism of glucose and fatty acids, however, has not been extensively studied.
Against this background, we have investigated the effects of fatty acids on the expression of UCP-2 gene under varying conditions of fatty acid oxidation. These results were supplemented with data on UCP-2 protein, mitochondrial membrane potential, and insulin secretion.
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Materials and Methods |
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-tocopherol), sodium selenite, rhodamine123, propidium iodide, and oleate (sodium salt) were obtained from Sigma (St. Louis, MO). Sodium 2-(ß-(4-chlorophenoxy)hexyl)oxirane-2-carboxylate (etomoxir) was obtained from ASAT AG (Zug, Switzerland). Rosiglitazone was from GlaxoSmithKline (West Sussex, UK). [1-14C]-oleate, [32P ]-deoxycycidine triphosphate, Hybond N+ membrane, Rediprime DNA labeling system kit, and ECL Western blotting detection reagents were from Amersham Pharmacia Biotech (Buckinghamshire, UK). Collagenase was obtained from Roche Molecular Biochemicals (Mannheim, Germany). Trizol was from Life Technologies, Inc. (Oslo, Norway). ExpressHyb hybridization solution was obtained from CLONTECH Laboratories, Inc. (Palo Alto, CA). Dynabeads mRNA direct kit was obtained from Dynal A.S. (Oslo, Norway). Sprague Dawley rats were from B&K (Stockholm, Sweden). INS-1 cells were a generous gift from Dr. Claes Wollheim (Geneva, Switzerland).
Cell culture and incubations
INS-1 cells (passage between 30–60) were grown in monolayer cultures as described previously (10). The medium used was RPMI-1640 supplemented with 10 mM HEPES, 10% heat-inactivated FCS, 2 mM L-glutamine, 1 mM sodium pyruvate, 50 µM mercaptoethanol, 100 IU/ml penicillin and 100 µg/ml streptomycin, and (when not otherwise mentioned) 11 mM glucose. Cells were cultured at 37 C in a humidified (5% CO2-95% air) atmosphere. Cells were seeded 7 d before use, in 25-cm2 flasks, at a density of 1.2 x 106 cells per flask.
After 6–7 d (60–80% confluence), cells were further cultured for 48 h with or without 0.2 mM oleate or 0.1 mM palmitate together with different glucose concentrations (5.5, 11, or 27 mM). Palmitate and oleate were prepared as described previously (8). The culture medium was changed daily to maintain a constant concentration of fatty acid.
Isolation and culture of islets
Male Sprague Dawley rats were killed by gassing with CO2. Pancreatic islets of Langerhans were isolated by collagenase basically as described (11). Digestion and sedimentation of islets were carried out in Hanks’ solution containing 5.5 mM glucose. Islets were then picked under a stereomicroscope and transferred to Petri dishes containing RPMI 1640 medium and 10% inactivated newborn calf serum. The concentration of glucose in the culture media was 11 mM. Islets were cultured for 48 h, free-floating at 37 C, with an atmosphere of 5% CO2- 95% air.
Assay of UCP-2 mRNA
After incubations, cells were washed twice with ice-cold PBS. The mRNA was isolated according to the Dynabeads mRNA direct kit protocol. Islet total RNA was isolated using Trizol reagent. The RNA was electrophoresed on 1% agarose gels containing 2.2 M formaldehyde, blotted onto Hybond N+ membrane by capillary transfer and cross-linked to the membrane by baking (20 min, at 120 C). Hybridization was carried out as previously described (12). Hybridized blots were placed in a PhosphorImager cassette (Molecular Dynamics, Inc., Sunnyvale, CA) overnight. Hybridization signals were quantified using Molecular Dynamics, Inc. ImageQuant software. Before rehybridization, the membranes were stripped of probe by boiling in 0.5% SDS for 10 min, followed by an additional 10 min in the hot solution after removing it from heat. Membranes were rehybridized with a ß-actin cDNA probe to provide a reference to the amount of RNA present in each lane. The intensity of UCP-2 mRNA band was normalized for the ß-actin signal in each lane. Results were expressed relative to corresponding controls.
Western blot analysis
Total cell protein was lysed in lysate buffer (1% Triton, 0.1% SDS) and left on ice for 10 min. The lysates were centrifuged at 16,000 x g for 20 min (4 C), and the supernatants were secured. Protein concentrations were determined using a commercial kit (Bio-Rad Laboratories, Inc., Hercules, CA). Total protein (70 µg per lane) was denatured by boiling in SDS-PAGE sample buffer, followed by resolution with 10% SDS-PAGE. After electrophoresis, the protein was transferred onto nitrocellulose membranes. The membranes were treated with UCP22-A polyclonal antibody (diluted 1:2000) directed toward the C-terminal domain of UCP2 (Alpha Diagnostic International, Inc., San Antonio, TX), followed by incubation with the second antibody (swine antirabbit Ig conjugated with horseradish peroxidase, diluted 1:5,000). After washing of the blots, the bound primary antibody was detected by reaction with antirabbit IgG antibody-peroxidase conjugate. The antibody complexes were detected using an ECL Western blotting detection reagent and were quantified by Enhanced Laser Densitometer (LKB Productor AB, Bromma, Sweden) using the GelScan XL program, version 2.1 (Pharmacia LKB Biotechnology AB, Bromma, Sweden).
Fatty acid oxidation
INS-1 cells were cultured for 48 h in the presence of 0.2 mM oleate. After trypsinization, cells were preincubated for 1 h at 37 C in Krebs-Ringer’s bicarbonate-HEPES buffer (KRBH) containing 3.3 mM glucose. After centrifugation and cell resuspension in the same medium, INS-1 cells (1 x 106) were incubated for 2 h in 100 µl medium at a low (3.3 mM) or high (27 mM) glucose concentration, in the presence of 0.16 µCi [1-14C]-oleate. The incubations were carried out in 1-ml glass tubes inside 20-ml scintillation vials and stopped by injection of 0.1 ml 0.1-M HCl. The sealed scintillation bottles were subsequently left overnight, at room temperature, to collect 14CO2 into Hyamine. Cells were counted in a Coulter counter. Data were expressed as pmol oleate/2 h x 106 cells.
Triglyceride contents
Triglyceride contents were measured as previously described (9). Briefly, INS-1 cells were trypsinized and resuspended (1.0 x 106) in 50 µl buffer (2 M NaCl/2 M MEDTA/50 mM sodium phosphate, pH 7.4). After sonication, 10 µl of the homogenate was mixed with 10 µl tert-butyl alcohol and 5 µl of a Triton X-100/methyl alcohol mixture (1:1 vol/vol). Contents of triglyceride were then measured by the Sigma triglyceride (GPO-Trinder) kit.
Mitochondria membrane potential
Mitochondrial membrane potential was measured by double staining with the fluorescent dyes rhodamine123, and propidium iodide. Cells were cultured for 48 h with 0.2 mM oleate, together with 5.5 mM or 11 mM glucose. After aspiration of medium and washing, cells were detached by treating with 0.05% EDTA (in PBS) and resuspended (5.0 x 105/ml) in Hanks’ solution, followed by incubation with 10 µg/ml rhodamine 123 at 37 C for 10 min and, subsequently, incubation with 10 µg/ml propidium iodide for 10 min on ice. Mitochondrial membrane potential was analyzed on a Coulter Epics Elike ESP flow cytometer (Beckman Coulter, Inc., Hialeah, FL) using a 15-mW argon laser (488 nm), a 550-nm dichronic long-pass filter, and a 525 ± 15-nm band pass filter. Total cell count for all samples was 10,000. Each sample was measured in duplicate. According to the values of forward scatter, side scatter, and red fluorescence (propidium iodide), debris, aggregates and dead cells could be identified and were gated out of analysis. Results are expressed as the value of intensity of green fluorescence of the potential sensitive probe rhodamine 123. Fluorescence was measured on a logarithmic scale.
Insulin secretion from INS-1 cells
INS-1 cells were plated (2.5–5 x 105) into 24-well plates and cultured for 2 d. After culture for 48 h during various experimental conditions, cells were washed twice with Krebs-Ringer’s bicarbonate buffer, including 10 mM HEPES, followed by a 30-min preincubation period in 1 ml KRBH without glucose. Cells were then washed once with KRBH and subsequently incubated for 30 min in the presence of 3.3 or 27 mM glucose. Incubation media were collected, and insulin secretion was determined by RIA (13) using an antiporcine insulin antibody (raised in the Department of Endocrinology, Karolinska Hospital, Stockholm).
Presentation of results
All results are expressed as the mean ± SE of the number of experiments indicated in table and figure legends. Comparison between two groups was performed with a two-tailed t test. Comparison between multiple groups was performed by ANOVA, followed by a Bonferroni’s multiple-comparison post hoc test. A P value less than 0.05 was considered significant.
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Results |
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). A significant effect, which was similar in magnitude, was seen in isolated rat pancreatic islets (Fig. 2).
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). In contrast, increasing the glucose concentration to 27 mM during culture abolished a stimulatory effect of culture with oleate. The inhibitory effect of glucose was significant (P < 0.05, by ANOVA) when comparing results from culture of oleate at 27 mM with results at 5.5 mM glucose. Glucose per se did not significantly affect UCP-2 mRNA, the induction of UCP-2 mRNA being 1.0 ± 0.09, 1.0 ± 0.27, and 1.09 ± 0.18, (n = 3–6) after culture with 5.5, 11, and 27 mM glucose, respectively. Also culture with palmitate (0.1 mM) stimulated UCP-2 mRNA expression. The induction of UCP-2 mRNA was 1.34 ± 0.10 vs. 1.0 ± 0.11, 1.26 ± 0.11 vs. 1.0 ± 0.08, and 1.1 ± 0.20 vs.1.0 ± 0.15, relative to control conditions when coculture was performed together with 5.5 (n = 5), 11 (n = 10), and 27 (n = 6) mM glucose, respectively. Thus, a stimulatory effect of palmitate was seen with 5.5 and 11 mM glucose (P < 0.05 for both conditions) but not with 27 mM glucose.
Effects of oleate and glucose on fatty acid oxidation
To assess the role of fatty acid metabolism for UCP-2 expression, we measured oleate oxidation under the same conditions as for UCP-2 expression. In confirmation of previous studies (14, 15, 16, 17), culture with oleate increased the oxidation of this fatty acid in INS-1 cells (Fig. 1B). These effects, however, were dependent on the glucose concentration and were more marked after coculture with 5.5 mM glucose. The glucose effect was significant (P < 0.05, using ANOVA) when comparing results from culture of oleate at 27 mM with the results at 5.5 mM glucose.
Effects of etomoxir
To further test the role of fatty acid oxidation for UCP-2 expression, we investigated the effect of etomoxir, which is a carnitine palmitoyltransferase I (CPTI) inhibitor (18). Inclusion of etomoxir during culture with oleate significantly decreased UCP-2 mRNA level (Table 1), whereas etomoxir per se was without effect. Inclusion of etomoxir during culture with oleate nonsignificantly (tested by ANOVA) affected postculture insulin secretion at 3.3 mM glucose (from 148.7 ± 12.2 vs. 123.2 ± 13.3 µU/30 min·106 cells; n = 6). Previous etomoxir per se failed to affect secretion. (94.0 ± 9.58 vs. 98.8 ± 12.2 µU/30 min·106cells for control, i.e. culture at glucose 5.5 mM alone; n = 5).
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), increased postculture oxidation of [1-14C]-oleate (Fig. 3B), and decreased cellular triglyceride content by 40% (Fig. 3C). However, previous AICAR failed to affect postculture insulin release (Fig. 3D).
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(PPAR ) (22)] failed to stimulate UCP-2 mRNA in INS-1 cells (Table 1). During the same experimental conditions, 1 µM of this compound also failed to stimulate fatty acid oxidation. Thus, the oleate oxidation of control culture and rosiglitazone were 173.1 ± 24.3 vs. 169.7 ± 29.1 pmol/2 h·106 and 16.2 ± 1.6 vs. 12.7 ± 2.8 pmol/2 h·106 (n = 6) during subsequent incubation at 3.3 mM and 27 mM glucose, respectively. Rosiglitazone also failed to affect postculture insulin secretion at 3.3. mM glucose (81.3 ± 7.1 vs. 90.3 ± 6.1 µU/30 min·106 cells; n = 6).
Effects of antioxidant treatment
UCP-2 may act as an antioxidant (2), and fatty acid oxidation could possibly enhance UCP-2 expression as a response to increased oxidative stress. This notion was tested by culturing INS-1 cells with oleate, with or without the further addition of vitamin E together with sodium selenite. As reported by us (12), this addition of antioxidants to the culture medium lowered UCP-2 mRNA per se (Table 1). However, coculture with the antioxidant and oleate did not diminish the oleate-induced stimulation of UCP-2 mRNA.
Effects of oleate on UCP-2 protein
Culture with oleate increased UCP-2 protein, as tested at 5.5 mM glucose in INS-1 cells (Fig. 4). This was assessed by immunoblotting from a 32-kDa band that corresponded to the molecular mass of the UCP-2 protein (as shown in the figure, this polyclonal antibody also detected other bands).
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Effects of culture with oleate on insulin secretion
Previous exposure to oleate exerted a dual effect on postculture insulin secretion, with stimulation at low, but inhibition at high, glucose concentrations (Fig. 5). Insulin release was increased 1.5- to 2.1-fold during incubations with 3.3 mM glucose, and such stimulation was seen regardless of the glucose concentration during culture (5.5, 11, or 27 mM glucose). Contrary to the stimulatory effects at low glucose concentrations, culture with oleate, together with 5.5 or 11 mM glucose, inhibited postculture stimulation by 27 mM glucose.
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Discussion |
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An inhibitory effect by ambient glucose on oxidation of fatty acids has been observed and studied for many years. In analogy with the situation in other tissues, it has been proposed that buildup of malonyl-CoA, which inhibits the CPTI enzyme, is a key factor by which glucose inhibits fatty acid oxidation (24). In most tissues, malonyl-CoA is increased by activation of acetyl-CoA carboxylase (21). This enzyme is inhibited by AMPK (21). Glucose decreases AMPK activity in INS-1 cells (19), and such an effect could thus play a part in the rise in malonyl-CoA. It follows that regulation of AMPK is potentially important for regulation by glucose of fatty acid oxidation.
Our results with AICAR add information on the role of AMPK for fatty acid metabolism in ß-cells. Evidence indicates that AICAR is a fairly specific activator of AMPK (21), and it has been shown that AICAR can inhibit AMPK in INS-1 cells (19). In liver, fat, and muscle tissue, AICAR inactivates acetyl-CoA carboxylase, decreases malonyl-CoA, increases fatty acid oxidation, and decreases lipogenesis (21). Our findings of increased fatty acid oxidation and decreased cellular triglycerides, after long-term exposure to AICAR, give evidence for similar effects in INS-1 cells and attest to the importance of AMPK regulation in ß-cells. In relation to a glucose effect, we note that previous culture with AICAR did not affect the inhibitory effect of an acute elevation of glucose (Fig. 3B). Further studies are thus needed to document the role of AMPK regulation for glucose-induced inhibition of fatty acid oxidation in ß-cells.
Importantly, in the present context, our results with AICAR strongly indicate that intracellularly derived fatty acids induce UCP-2 expression similarly to fatty acids that have been added exogenously.
Other observations from our study reinforce the notion of a close coupling of fatty acid oxidation with induction of UCP-2 mRNA. Thus, the CPTI inhibitor etomoxir was found to restrain the effect of oleate on UCP-2 mRNA. Furthermore, a PPAR ligand, rosiglitazone, was without effect.
The rationale for testing a PPAR ligand was to investigate an alternative route by which fatty acids could potentially influence UCP-2 before oxidation. In support of such a notion, the UCP-2 promoter contains a PPAR response element (25), and the PPAR activator troglitazone has been found to increase UCP2 expression in rat pancreatic islets (26). However, our results with rosiglitazone showed no effect on UCP-2 mRNA level or fatty acid oxidation. Different experimental systems, as well as molecular differences, may explain the discrepancy between effects of troglitazone and rosiglitazone. In this context, we note that a third thiozoladindione, ciglitazone, was also found to be without effect on UCP-2 mRNA (23).
It is interesting to view our data in relation to a recent report on INS-1 cells overexpressing UCP-2 (27). Overexpression was, in that study, associated with increased oxidation of fatty acids. A positive feedback system may operate by which increased availability of fatty acids will lead to increases in UCP-2 mRNA and protein, which, in turn, will further enhance oxidation, thus providing signal amplification. Such mechanisms could facilitate a swift adaptation to an increase in fatty acid availability. This speculative hypothesis is testable in future studies.
It has been suggested that UCP-2 may play a role in antioxidant defense in different cells (2, 3, 28). This notion is supported by our own studies in INS-1 cells (12). The reported toxicity of long-term elevated fatty acids during different conditions (7, 9, 29) makes it plausible that UCP-2 would be induced as a reaction to fatty-acid-induced oxidative stress. If such were the case, we reasoned that antioxidants, such as vitamin E and selenite, would decrease or abolish stimulation by fatty acids. However, this was not found in the present experiments. Our observations thus suggest (but do not rule out) that regulation of UCP-2 by fatty acids occurs independently from regulation by oxidative stress.
The present study focused on regulation of UCP-2 gene expression, and only supplementary data on function are included. By Western immunoblotting, we found an increase in UCP-2 protein after oleate exposure, as assessed from a 32-kDa band, a band that was preferentially present in mitochondrial preparations (data not shown) and corresponds to the size of the UCP-2 protein. However, a recent publication, surveying antibodies against UCP-2, found that no commercially available antibodies are completely specific for the UCP-2 protein (28). Thus, we have indications, but not absolute proof, of a link between UCP-2 expression and protein in our experimental system.
A previous study showed that overexpression of the UCP-2 gene in INS-1 cells is associated with uncoupling (27). However, our data on mitochondrial membrane potential during basal conditions do not reveal any decrease in mitochondrial membrane potential after culture with oleate. Measuring the potential during dynamic testing of mitochondrial function may be needed to detect uncoupling after the moderate increase in gene expression that we observe.
The question arises as to what extent UCP-2 influences insulin secretion. It is clear that moderate reductions both in ATP/ADP ratios and insulin secretion are found in ß-cells that overexpress UCP-2 (4, 5). Whether more moderate effects on UCP-2 will affect insulin secretion is less obvious. Here, we confirm, for the present experimental conditions, the well-known stimulatory effect of fatty acids (here, oleate) on insulin secretion at basal glucose as well as inhibition of glucose-induced insulin (8, 14). The inhibitory effects on glucose-induced insulin secretion were seen after 5.5 and 11 mM glucose, but not after 27 mM glucose, and could hence be compatible with the glucose dependency for UCP-2 induction that we observe. However, we note that culture with AICAR did not significantly affect postculture glucose- induced insulin secretion, despite increasing UCP-2mRNA and fatty acid oxidation. All in all, relationships of insulin secretion to effects of fatty acids on UCP need further elucidation.
In summary, induction of UCP-2 mRNA by fatty acids is paralleled by their oxidation, and this coupling includes an inhibitory effect of elevated glucose. The novel finding of inhibition by glucose of fatty acid-induced stimulation of UCP-2 mRNA contradicts the notion of a role for UCP-2 in dissipating excess energy in ß-cells. Our findings thus indicate that fatty acid oxidation, rather than total oxidative phosphorylation, acts as an inducer of UCP-2 mRNA. Further studies are needed to investigate a putative relationship of fatty-acid-induced effects on UCP-2 and effects on glucose-induced insulin secretion.
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Footnotes |
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Abbreviations: AICAR, Aminoimidazole-4-carboxamide-1-ß-D-ribofuranoside; AMPK, AMP-activated protein kinase; CoA, coenzyme A; CPT I, carnitine palmitoyltransferase I; KRBH, Krebs-Ringer’s bicarbonate medium-HEPES buffer; PPAR, peroxisome proliferator-activated receptor; UCP-2, uncoupling protein 2.
Received May 1, 2001.
Accepted for publication December 6, 2001.
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and mediate in vivo regulation of uncoupling protein (UCP-1, UCP-2, UCP-3) gene expression. Endocrinology 139:4920–4927This article has been cited by other articles:
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32 kDa) from one representative experiment; *, P < 0.05, compared with controls, using paired t test.