Ethanol stimulates glucose uptake and translocation of GLUT-4 in H9c2 myotubes via a Ca2+-dependent mechanism

Bo Yu, Adrienne Schroeder, and Laura E. Nagy

Department of Nutrition, Case Western Reserve University, Cleveland, Ohio 44106


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Short-term exposure to ethanol impairs glucose homeostasis, but the effects of ethanol on individual components of the glucose disposal pathway are not known. To understand the mechanisms by which ethanol disrupts glucose homeostasis, we have investigated the direct effects of ethanol on glucose uptake and translocation of GLUT-4 in H9c2 myotubes. Short-term treatment with 12.5-50 mM ethanol increased uptake of 2-deoxyglucose by 1.8-fold in differentiated myotubes. Pretreatment of H9c2 myotubes with 100 nM wortmannin, an inhibitor of phosphatidylinositol 3-kinase, had no effect on ethanol-induced increases in 2-deoxyglucose uptake. In contrast, preincubation with 25 µM dantrolene, an inhibitor of Ca2+ release from the sarcoplasmic reticulum, blocked the stimulation of 2-deoxyglucose uptake by ethanol. Increased 2-deoxyglucose uptake after ethanol treatment was associated with a decrease in small intracellular GLUT-4 vesicles and an increase in GLUT-4 localized at the cell surface. In contrast, ethanol had no effect on the quantity of GLUT-1 and GLUT-3 at the plasma membrane. These data demonstrate that physiologically relevant concentrations of ethanol disrupt the trafficking of GLUT-4 in H9c2 myotubes resulting in translocation of GLUT-4 to the plasma membrane and increased glucose uptake.

skeletal muscle; glucose tolerance; insulin; phosphatidylinositol 3-kinase; vesicle trafficking


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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GLUCOSE UPTAKE IS MEDIATED by a family of facilitative transporters (GLUT-1-6). GLUT-4, the insulin responsive glucose transporter, is expressed in adipose tissue and muscle. In a resting cell, GLUT-4 proteins are primarily localized to a specialized intracellular vesicular compartment (32). Upon insulin stimulation, GLUT-4 rapidly translocates to the plasma membrane. In muscle, contraction also activates the translocation of GLUT-4 to the cell surface (12). Translocation of GLUT-4 to the cell surface is the primary mechanism for increasing glucose transport into adipose tissue and muscle (32). Current models suggest that GLUT-4 is continuously recycled between its intracellular storage compartment(s) and the plasma membrane, even in the absence of stimulation (32). Insulin treatment rapidly increases the surface-accessible transporter by enhancing the rate of exocytosis and/or decreasing the rate of endocytosis (32). Impaired translocation of GLUT-4 is associated with glucose intolerance in diabetes, insulin resistance, and obesity (32).

Short-term exposure to ethanol disrupts glucose homeostasis. In fasted individuals, ethanol consumption may result in profound hypoglycemia (2). In contrast, in fed individuals, even relatively low amounts of ethanol consumption can impair glucose tolerance. Consumption of moderate amounts of alcohol (equivalent to 2-3 drinks) increases serum glucose levels (9) and decreases glucose oxidation despite increased insulin release (27). This ethanol-induced hyperinsulinemia is associated with enhanced glucose-stimulated release of insulin (1, 21, 22), as well as impaired hepatic extraction of circulating insulin (11). Using hyperinsulinemic-euglycemic clamps, several groups have found that ethanol decreases peripheral glucose utilization (27, 40); however, the site of ethanol action in humans is not clear. Systemic exposure to glucose decreases forearm glucose balance in men, but intrabrachial infusion of ethanol (localized exposure) has no effect on forearm glucose balance (3).

Treatment of rats and mice with ethanol during a glucose load also results in glucose intolerance (10, 26). Erwin and Towell (10) have suggested that ethanol-induced hyperglycemia may result from increased sympathetic output. Brief exposure of rats to ethanol results in insulin resistance (37). During hyperinsulinemic-euglycemic clamping, ethanol decreases insulin-stimulated glycogen synthesis and glucose oxidation in heart and skeletal muscle of overnight-fasted rats (36). Ethanol treatment also decreases glucose-stimulated glycogen synthesis in oxidative muscles (38). Xu et al. (37) proposed that, because ethanol decreases both glycogen synthesis and glucose oxidation, ethanol is likely acting at a metabolic step common to both pathways, such as insulin signaling or glucose transport.

Using CHO cells expressing individual GLUT isoforms, Krauss, et al. (18) reported that short-term ethanol exposure decreases GLUT-1-dependent glucose transport activity but does not alter the catalytic activity/transport capacity of GLUT-4. Because glucose transport in adipose tissue and muscle is primarily regulated by translocation of GLUT-4 to the plasma membrane (32), we have investigated whether short-term treatment of muscle cells with ethanol affects the intracellular trafficking of GLUT-4. Here, we report that, at physiologically relevant concentrations, ethanol increases glucose uptake in H9c2 myotubes as a result of translocation of GLUT-4 from an intracellular storage vesicle to the plasma membrane.


    MATERIALS AND METHODS
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ABSTRACT
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MATERIALS AND METHODS
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Materials. H9c2 muscle cells were from the American Tissue Culture Collection (Rockville, MD). Cell culture reagents were obtained from GIBCO (Gaithersberg, MD). Antibodies were obtained from the following sources: rabbit polyclonal anti-GLUT-4 (East Acres Biologicals, Cambridge, MA or Biogenesis, Sandown, NH), anti-GLUT-1 and anti-GLUT-3 (Chemicon International, Temecula, CA), monoclonal antibodies to c-myc (Developmental Hybridoma Studies Bank, Iowa City, IA) and alpha -adaptin (AP2; Affinity Bioreagents, Golden, CO). Goat anti-rabbit IgG coupled to horseradish peroxidase was from Boehringer Mannheim (Indianapolis, IN), and those coupled to Texas red and fluorescein isothiocyanate (FITC) were from Molecular Probes (Eugene, OR). Protease inhibitor cocktail was from GIBCO. 2-[3H]deoxyglucose was from Amersham (Arlington Heights, IL), rats were from Harlan Sprague Dawley (Indianapolis, IN), and insulin (regular Iletin) was from Eli Lily (Indianapolis, IN). All other reagent-grade chemicals were from Sigma (St. Louis, MO), Fisher Biochemicals (Santa Clara, CA), or Research Biochemicals International (Natick, MA).

Cell culture. H9c2 cells [wild-type and c-myc GLUT-4 transfected (41)] were propagated as myoblasts in DMEM containing 10% fetal bovine serum (FBS) and 100 U/ml penicillin-streptomycin in a 10% CO2 incubator at 37°C. In some experiments, 0.25 µg/ml of amphotericin B was added to the cell culture media. H9c2 cells were stably transfected with c-myc GLUT-4 (H9c2 c-myc GLUT-4) (41). Cells were seeded at 4,100 cells/cm2 in 150-mm plates (for isolation of membrane fractions), 24-well plates (for 2-deoxyglucose uptake studies) or LabTek slide chambers (for immunofluorescence) in this medium. After 48 h, medium was changed to DMEM containing 2% FBS to promote differentiation (17). Cells were fed DMEM with 2% FBS every 3-4 days until day 14 in culture. At this time, cells were cultured overnight in DMEM containing 0.5% FBS for 16 h before experimental treatments.

2-Deoxyglucose uptake in rat soleus and isolated adipocytes. Adipocytes were isolated from the epididymal fat pads of male Wistar rats (250-350 g), and uptake of 2-[3H]deoxyglucose was measured after treatment with and without 25 mM ethanol or 4 nM insulin, exactly as previously described (35). Soleus muscle was also dissected from the rats, and uptake of 2-[3H]deoxyglucose was measured as previously described (28). Briefly, soleus pieces weighing between 15 and 25 mg were incubated for 30 min in 2 ml of oxygenated Krebs-Henseleit buffer containing 2 mM pyruvic acid, 38 mM mannitol, and 0.1% BSA with or without 10 nM insulin or 25 mM ethanol at 37°C. The gas phase was 95% O2-5% CO2. Transport of 1 mM 2-[3H]deoxyglucose was measured over 20 min in the continued presence or absence of insulin or ethanol and 39 mM [U-14C]mannitol. Muscle samples were then processed, and calculation of intracellular 2-deoxyglucose and extracellular space was carried out as previously described (28). Procedures involving animals were approved by the Institutional Animal Care and Use Committee at Case Western Reserve University.

2-Deoxyglucose uptake in H9c2 myotubes. H9c2 myotubes were washed twice with PBS (137 mM NaCl, 2.7 mM KCl, 15.2 mM Na2HPO4, 1.5 mM KH2PO4, 0.1 mM CaCl2, 0.1 mM MgCl2 and 10 mM HEPES, pH 7.4). Cells were then treated with or without insulin or ethanol in PBS or 80 mM KCl, 0.1 mM CaCl2, 0.1 mM MgCl2, 10 mM HEPES, 5 mM EDTA, pH 7.4 (80 mM K+)(41) for 20 min at 37°C. After agonist treatment, uptake of 10 µM 2-[3H]deoxyglucose was measured over 10 min. Reactions were terminated by the removal of the permeant solution and rapid washing with ice-cold 20 µM phloretin, a specific inhibitor of equilibrative glucose transport. Cells were then dissolved in 0.1 N KOH, and aliquots were counted by liquid scintillation counting or were used to determine protein concentration. In some experiments, cells were preincubated with 100 nM wortmannin, an inhibitor of phosphatidylinositol 3-kinase, for 30 min or 25 µM dantrolene, an inhibitor of Ca2+ release from the sarcoplasmic reticulum, for 5 min at 37°C before treatment with ethanol.

Subcellular fractionation. Cells were washed twice in PBS and incubated with or without 25 mM ethanol for 15 or 30 min at 37°C. After treatment, cells were washed twice in ice-cold PBS and removed from the cell culture dish by gentle scraping with a cell scraper. Cells were centrifuged at 600 g for 5 min and then homogenized in a Wheaton glass homogenizer with the tight fitting pestle (clearance 0.05 µm) in 150 mM NaCl, 1 mM EGTA, 0.1 mM MgCl2, 10 mM HEPES, pH7.4 (buffer A) with protease inhibitors (14). An S1 fraction was prepared by centrifugation at 16,000 g for 15 min. One to two milligrams of S1 protein were layered on a 5-25% glycerol gradient formed over a 50% sucrose pad and centrifuged at 60,000 g for 70 min (14) in an SW55 rotor, and 0.300-ml fractions were collected. For preparation of plasma membrane-enriched fractions, homogenates were centrifuged at 200 g for 5 min. The supernatant was removed, and plasma membrane containing fractions were pelleted by centrifugation at 16,000 g for 15 min. This fraction contained >95% of the alpha 1-subunit of Na+-K+-ATPase, a plasma membrane marker (41).

Western blotting. Equal volumes of fractions from gradient isolations were solubilized in SDS sample buffer without beta -mercaptoethanol for 15 min at 37°C. Proteins were then separated on 6-18% polyacrylamide gels and transferred to polyvinylidene difluoride for Western blot analysis. The membranes were blocked with 5% nonfat dry milk in Tris-buffered saline (TBS) containing 0.02% Tween-20 (TBST) for 2 h, washed twice with TBST, and then incubated with anti-GLUT-4 (1:500), anti-GLUT-1 (1:1,000), or anti-GLUT-3 (1:10,000) antibody overnight in 1% BSA in TBST at 4°C . Membranes were washed again in TBST with 5% nonfat dry milk and probed with horseradish peroxidase coupled to goat anti-rabbit or anti-mouse IgG for 30 min. Unbound antibody was removed by washing, and membranes were incubated with ECL reagent. Immunoreactive protein quantity was assessed by scanning densitometry within the linear range of detectability. Internal standards were included on each blot to normalize between membranes.

Immunohistochemistry. For immunohistochemistry, cells were washed twice in PBS and incubated with or without 100 nM insulin, 80 mM K+, or 25 mM ethanol for 30 min at 37°C, as described above. Cells were then fixed in freshly prepared 4% paraformaldehyde for 10 min on ice and then for 20 min at room temperature. Slides were then quenched in glycine and blocked for 1 h in 2% BSA, 5% fish gelatin, and 0.02% saponin (34). For experiments visualizing the cell surface labeling of c-myc GLUT-4, all incubations were carried out in the absence of saponin. Slides were then incubated with antibody to GLUT-4 (1:200), 9E10 (anti-c-myc; 1:200), or AP2 (1:100) overnight at 4°C. Slides were washed three times for 15 min in fresh blocking buffer and then incubated for 1 h with Texas red-conjugated anti-rabbit IgG (1:200) or FITC-conjugated anti-mouse IgG (1:200) for 1 h in blocking buffer. Slides were finally washed three times for 15 min in blocking buffer and twice for 5 min in PBS. Slides were then mounted in Vectashield mounting medium. Cells were examined with a Bio-Rad confocal microscope under a 60× lens. Multiple cells from at least four separate preparations were examined. Nonspecific binding was assessed in cells incubated in the absence of primary antibody.

Data analysis. Values represent means ± SE. Statistical analysis was performed using SAS for personal computers. Differences between means were assessed by one-way analysis of variance (general linear model procedure) followed by a multiple comparison test (Duncan's or least square means test).


    RESULTS
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RESULTS
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2-Deoxyglucose uptake. In differentiated H9c2 myotubes, exposure to 25 mM ethanol for 20 min increased 2-deoxyglucose uptake by 1.8-fold (Fig. 1). Pretreatment with 10 nM insulin also increased the rate of 2-deoxyglucose uptake, but coincubation with ethanol did not further stimulate uptake (Fig. 1). Additivity between ethanol and insulin was not observed even at submaximal concentrations of insulin: 0.1 nM insulin increased uptake by 1.61 ± 0.21-fold over basal in the absence of ethanol and 1.51 ± 0.17-fold in the presence of 25 mM ethanol (n = 5). Depolarization of the H9c2 myotubes by incubation in 20 mM K+ increased 2-deoxyglucose uptake by twofold over basal. In contrast to the interaction with insulin, coincubation with 25 mM ethanol and 20 mM K+ had an additive effect on uptake. The additive effect of ethanol and depolarization-stimulated 2-deoxyglucose uptake was lost (Fig. 1) when myotubes were maximally activated in the presence of 80 mM K+ (41). Incubation of rat soleus strips with 25 mM ethanol increased 2-deoxyglucose uptake by 65% over basal (Fig. 2), comparable with the stimulation of uptake observed after treatment of soleus with 10 nM insulin (Fig. 2). In contrast to the acute stimulation of 2-deoxyglucose uptake in H9c2 myotubes and rat soleus, exposure of isolated rat adipocytes with 25 mM ethanol had no effect on glucose uptake (Fig. 2).


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Fig. 1.   Stimulation of 2-deoxyglucose uptake by 25 mM ethanol (EtOH) in H9c2 myotubes. H9c2 myotubes were cultured in 0.5% fetal bovine serum (FBS) overnight and then washed twice with incubation buffer. Cells were then incubated in buffer alone, 10 nM insulin, or 20 or 80 mM K+ in the presence or absence of 25 mM ethanol for 20 min at 37°C. Uptake of 10 µM 2-[3H]deoxyglucose was measured over 10 min. Nonspecific uptake was measured in the presence of 20 µM phloretin. Values represent means ± SE; n = 5 (20 mM K+), 13 (insulin), or 18 (basal and 80 mM K+). Values with different superscripts are significantly different, P < 0.05.



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Fig. 2.   Ethanol increases 2-deoxyglucose uptake in rat soleus muscle (A) but not in isolated adipocytes (B). A: strips of rat soleus muscle were incubated with or without 10 nM insulin or 25 mM EtOH for 30 min, and uptake of 1 mM 2-[3H]deoxyglucose was measured over 20 min. B: isolated rat adipocytes were incubated with and without 4 nM insulin or 25 mM EtOH for 30 min. Uptake of 2.5 mM 2-[3H]deoxyglucose was measured over 5 min. Nonspecific uptake was measured in the presence of 20 µM phloretin. Values represent means ± SE; n = 13 for soleus, and n = 10 for adipocytes. Values with different superscripts are significantly different, P < 0.05.

The effect of short-term ethanol treatment on 2-deoxyglucose uptake was biphasic. Concentrations of ethanol as low as 12.5 mM significantly increased 2-deoxyglucose uptake; maximal stimulation was produced in the presence of 25-50 mM ethanol (Fig. 3). In contrast, after treatment with 100-200 mM ethanol, uptake of 2-[3H]deoxyglucose was no longer elevated above basal (Fig. 3).


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Fig. 3.   Dose-dependent stimulation of 2-deoxyglucose uptake by EtOH in H9c2 myotubes. H9c2 myotubes were cultured in 0.5% FBS overnight and then washed twice with incubation buffer. Cells were then incubated with 0-200 mM EtOH for 20 min at 37°C. Uptake of 10 µM 2-[3H]deoxyglucose was measured over 10 min. Nonspecific uptake was measured in the presence of 20 µM phloretin. Values represent means ± SE; n = 14-16, except for 2.5 and 200 mM treatments, where n = 4. *P < 0.05 vs. no EtOH treatment.

We have previously reported that inhibition of phosphatidylinositol 3-kinase activity with wortmannin blocks insulin-stimulated but not depolarization-mediated increases in 2-deoxyglucose uptake in H9c2 myotubes (41). Preincubation of the H9c2 myotubes with 100 nM wortmannin had no effect on ethanol-stimulated increases in 2-deoxyglucose uptake (Fig. 4). Because ethanol activation of 2-deoxyglucose uptake was not inhibited by wortmannin, we hypothesized that ethanol was acting via a mechanism similar to the depolarization-mediated increases in 2-deoxyglucose uptake (41). Because the L-type Ca2+ channel blockers verapamil and nifedipene have nonspecific effects on glucose transport (6), we could not test whether ethanol-stimulated 2-deoxyglucose uptake was mediated by influx of Ca2+ via Ca2+ channels at the plasma membrane. However, the localized release of Ca2+ from the sarcoplasmic reticulum is thought to mediate contraction-induced activation of glucose uptake (12). If ethanol action required the release Ca2+ from the sarcoplasmic reticulum, pretreatment of cells with dantrolene, an inhibitor of Ca2+ release from the sarcoplasmic reticulum, should prevent activation of 2-deoxyglucose uptake in response to ethanol. Consistent with this hypothesis, 25 µM dantrolene completely blocked ethanol-stimulated 2-deoxyglucose uptake (Fig. 4).


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Fig. 4.   Effects of wortmannin and dantrolene on EtOH-stimulated 2-deoxyglucose uptake. H9c2 myotubes were cultured in 0.5% FBS overnight and then washed twice in incubation buffer. Cells were then preincubated with and without 100 nM wortmannin for 30 min or 25 µM dantrolene for 5 min at 37°C before stimulation with 25 mM EtOH. Uptake of 10 µM 2-[3H]deoxyglucose was measured over 10 min. Nonspecific uptake was measured in the presence of 20 µM phloretin. Values represent means ± SE; n = 3-7. Values with different superscripts are significantly different, P < 0.05.

Intracellular localization of GLUT-4. In the resting cell, GLUT-4 is sequestered from the plasma membrane in intracellular vesicles (32). In H9c2 cells, GLUT-4 is distributed to two distinct intracellular vesicles, which can be separated by glycerol velocity gradient centrifugation (41). In nontreated cells, the majority of the GLUT-4 is localized to a small vesicular compartment with a smaller proportion of GLUT-4 in a large vesicular fraction. After stimulation with insulin or K+, there is a reduction in immunoreactive GLUT-4 in the small vesicular compartment (41). Similarly, treatment of H9c2 myotubes with 25 mM ethanol for 30 min decreased GLUT-4 in the small vesicular compartment (Fig. 5). Ethanol decreased the total immunoreactive GLUT-4 in the small vesicle compartment to 59 ± 7% (n = 7, P < 0.02) of basal but had no effect on GLUT-4 in large vesicles. After ethanol treatment, GLUT-4 in the large vesicle peak was 102 ± 20% of basal (n = 7).


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Fig. 5.   Effect of insulin and EtOH on the intracellular distribution of immunoreactive GLUT-4 protein. H9c2 myotubes were cultured in 0.5% FBS overnight, washed twice, and then treated with or without 25 mM EtOH for 30 min at 37°C. A: size distribution of GLUT-4 in intracellular vesicles was assessed by homogenizing cells and preparing an S1 fraction by centrifugation at 16,000 g for 15 min. The S1 fraction was separated on a 5-25% glycerol velocity gradient for 70 min at 75,000 g, and fractions were collected and probed for GLUT-4 by Western blot analysis. B: for quantitation of changes in the intracellular distribution of GLUT-4, small vesicle (SV) and large vesicle (LV) fractions were pooled, and the relative quantity of GLUT-4 in each fraction was measured by Western blot analysis. C: immunoreactive GLUT-4, GLUT-1, and GLUT-3 protein in plasma membrane enriched fractions after treatment with (E) and without (B) 25 mM EtOH. Cells were homogenized and centrifuged at 200 g to remove nuclei. A plasma membrane-enriched fraction was prepared by centrifugation at 16,000 g for 15 min, separated by SDS-PAGE, and probed for GLUT isoforms by Western blot analysis.

In parallel with loss of GLUT-4 from the small vesicles in response to insulin or ethanol treatment, GLUT-4 in plasma membrane fractions was increased 1.48 ± 0.10- and 1.85 ± 0.21-fold over basal in cells treated with 25 mM ethanol for 15 min (n = 6, P < 0.03) and 30 min (n = 11, P < 0.01; Fig. 5), respectively. Immunoreactive quantities of GLUT-1 and GLUT-3, two additional isoforms in the equilibrative glucose transporter family that are also expressed in H9c2 myotubes, in isolated plasma membrane fractions were not affected by ethanol treatment with GLUT-1 at 1.11 ± 0.20- (15 min, n = 8) and 0.96 ± 0.21-fold over basal (30 min, n = 7) and GLUT-3 at 0.84 ± 0.14- (15 min, n = 8) and 0.93 ± 0.13-fold over basal (n = 8).

Cell surface localization of GLUT-4. As an independent assessment of GLUT-4 translocation to the cell surface in response to stimulation, H9c2 myotubes stably expressing c-myc-tagged GLUT-4 were treated with and without 25 mM ethanol for 30 min. The c-myc epitope is inserted in the first exofacial loop of the GLUT-4 protein; when c-myc GLUT-4 is translocated to the cell surface, the c-myc epitope is revealed and can be detected at the surface of nonpermeabilized cells (16). In nonstimulated H9c2 myotubes, very little c-myc GLUT-4 was present at the cell surface (Fig. 6). After treatment with 25 mM ethanol or 100 nM insulin for 30 min, surface labeling was increased (Fig. 6). In parallel with increased surface labeling in the H9c2 myotubes transfected with c-myc GLUT-4, 2-deoxyglucose uptake was increased from 24 ± 3 to 56 ± 17 in response to insulin and 50 ± 9 pmol · mg protein-1 · 10 min-1 in response to ethanol in the c-myc GLUT-4 cell line (n = 8). The distribution of GLUT-4 was also visualized by immunohistochemistry in wild-type H9c2 cells. In adipocytes, GLUT-4 is associated with clathrin-coated vesicles at the plasma membrane (7, 30). Using confocal laser microscopy, we compared the localization of GLUT-4 with AP2, a protein involved in the formation of clathrin-coated vesicles. In nonstimulated H9c2 myotubes, AP2 and GLUT-4 were distributed throughout the intracellular space with no concentration at the edges of the cell (Fig. 7A). After treatment with 25 mM ethanol or 80 mM K+, both AP2 and GLUT-4 were concentrated and co-localized at the cell periphery (Fig. 7, C and E), indicating an association of GLUT-4 with clathrin-coated vesicles. GLUT-4 could also be visualized at the cell surface after ethanol treatment or K+ depolarization. In nontreated cells, very little immunoreactive GLUT-4 was observed at the upper surface of the myotube (Fig. 7B). In contrast, after ethanol treatment, intense staining of GLUT-4 was observed when the plane of focus was the upper surface of the myotube (Fig. 7D). This pattern of staining was similar to that observed after K+ depolarization (Fig. 7F).


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Fig. 6.   Translocation of c-myc GLUT-4 to the surface of H9c2 myotubes in response to insulin or ethanol. H9c2 cells, stably transfected with c-myc GLUT-4, were differentiated on LabTek coverslips, serum starved overnight, and then stimulated for 30 min in the absence (A) or presence of 100 nM insulin (B) or 25 mM ethanol (C). Cells were fixed in paraformaldehyde and incubated with antibody to c-myc (9E10) without detergent permeabilization. Cells were examined by confocal laser microscopy. Multiple cells from at least 4 separate experiments were examined.



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Fig. 7.   Co-localization of GLUT-4 with alpha -adaptin (AP2) at the cell surface after treatment with 25 mM ethanol or K+ depolarization. Wild-type H9c2 cells were differentiated on LabTek chamber slides, serum starved overnight, and then stimulated for 30 min at 37°C in the absence (A and B) or presence of 25 mM ethanol (C and D) or 80 mM K+ (E and F). Cells were then fixed in paraformaldehyde and incubated with polyclonal antibody to GLUT-4 (1:200) and monoclonal antibody to AP2 (1:100) in the presence of 0.2% saponin. Cells were then incubated with Texas red-labeled anti-rabbit and fluorescein isothiocyanate-labeled anti-mouse secondary antibody and then examined by confocal laser microscopy. Focus planes are through the center of the myotube (A, C, and E) or at the upper surface of the cell (B, D, and F). Multiple cells from at least 3 separate experiments were examined.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Short- and long-term exposure to ethanol disrupts glucose homeostasis in both animals and humans. However, very little is known about the direct actions of ethanol on specific components of the pathways mediating glucose disposal. Translocation of GLUT-4, the insulin-recruitable glucose transporter, from its intracellular storage site to the plasma membrane is a critical step in increasing glucose uptake after a meal or in response to exercise. Here we have shown that short-term treatment with 25 mM ethanol increases 2-deoxyglucose uptake into rat soleus muscle in vitro but has no effect in isolated adipocytes. Short-term treatment of H9c2 myotubes with physiologically relevant concentrations of ethanol, ranging from 12.5 to 50 mM, also increased uptake of 2-deoxyglucose. At 25 mM ethanol, the 1.8-fold increase in uptake was associated with a 1.9-fold increase in GLUT-4 at the plasma membrane and required the release of Ca2+ from the sarcoplasmic reticulum. Stimulation of 2-deoxyglucose uptake by ethanol was additive with stimulation by 20 mM K+ but not with 80 mM K+, a condition which stimulates maximal glucose uptake in this cell line (41). In contrast, ethanol and insulin did not exhibit additive effects on 2-deoxyglucose uptake. This lack of additivity may be due to a suppression of insulin receptor-mediated signal transduction by ethanol reported in skeletal muscle in vivo as well as in other cell types (5, 25, 39).

In skeletal muscle, GLUT-4 translocation and stimulation of glucose uptake occur in response to insulin stimulation and muscle contraction by independent signaling mechanisms (12). Wortmannin blocks insulin-stimulated GLUT-4 translocation in skeletal muscle and cultured myotubes (12, 41) but has no effect on contraction- or depolarization-mediated GLUT-4 translocation in skeletal muscle (12) or in H9c2 myotubes (41). In most reports, insulin stimulation proceeds independently of Ca2+ concentration in both skeletal muscle and cardiac myocytes (12). Under some conditions, insulin-stimulated glucose uptake in skeletal muscle is inhibited by L-type Ca2+ channel antagonists such as nifedipine and verapamil. However, the potency of these antagonists for inhibition of Ca2+ channels is not consistent with their effects on insulin-stimulated glucose uptake (6), suggesting that these compounds might be acting by a nonspecific mechanism unrelated to their function as L-type Ca2+ channel antagonists.

Conversely, contraction-mediated GLUT-4 translocation is Ca2+ dependent. Elevation of intracellular Ca2+ increases glucose transport in isolated skeletal muscle independent of contraction per se (12). When Ca2+ release from the sarcoplasmic reticulum is blocked with dantrolene, contraction- and K+-evoked increases in glucose uptake are inhibited with no effect on insulin stimulation (12, 41). Activation of glucose uptake by ethanol was inhibited by dantrolene but was resistant to the effects of wortmannin, suggesting that the mechanism for ethanol action in increasing glucose uptake in H9c2 myotubes was similar to the action of K+-mediated depolarization. Consistent with this hypothesis, short-term treatment with 25 mM ethanol had no effect on uptake of 2-deoxyglucose in isolated rat adipocytes (Fig. 2), which are also insensitive to K+ depolarization (data not shown).

In neurons, short-term exposure to ethanol decreases the activity of the voltage-gated L-type Ca2+ channel (20), as well as N- and P/Q-type Ca2+ channels (31). However, inhibition of voltage-gated channel activity by ethanol would not be consistent with the stimulation of 2-deoxyglucose uptake observed after ethanol exposure in H9c2 myotubes. In contrast, dantrolene prevented ethanol-induced increases in glucose uptake, indicating that it is more likely that ethanol increases the release of Ca2+ from the sarcoplasmic reticulum in H9c2 myotubes. In cardiac myocytes, ethanol impairs the ability of the sarcoplasmic reticulum to sequester Ca2+, resulting in derangements in excitation-contraction coupling (8, 33). In isolated sarcoplasmic reticulum from frog skeletal muscle, ethanol (2.2 mM) potentiates the release of Ca2+ in response to caffeine but does not directly stimulate the release of Ca2+ from the sarcoplasmic reticulum (23). Similarly, higher concentrations of ethanol (108-217 mM) enhanced Ca2+-induced Ca2+ release from isolated sarcoplasmic reticulum of rabbit skeletal muscle (24). The data presented here suggest that ethanol increases the release of Ca2+ from the sarcoplasmic reticulum independently of the activation of this channel by other regulators. It is possible that the differences in ethanol action between isolated sarcoplasmic reticulum (e.g., no effect on basal release of Ca2+) (23, 24) and intact myotubes is due to the action of a regulatory factor or modulator that is lost during the preparation of isolated sarcoplasmic vesicles. Such a regulatory mechanism would be analagous to the involvement of phospholipase C and inositol trisphosphate in ethanol-induced release of Ca2+ from endoplasmic reticulum in isolated hepatocytes (15).

The primary mechanism by which insulin and contraction increase uptake of glucose is via the translocation of GLUT-4 from intracellular storage vesicles to the cell surface (12, 32). Short-term treatment of H9c2 myotubes with ethanol also mobilized GLUT-4 from small intracellular vesicles to the cell surface. In adipocytes, kinetic studies indicate that insulin increases the rate of exocytosis of GLUT-4 vesicles as well as slightly decreasing the rate of endocytosis (32). Similar kinetic studies on the mechanisms of contraction-/depolarization-dependent translocation have not been carried out. Short- and long-term ethanol exposure disrupts protein trafficking in a number of model systems (4). Short-term treatment with ethanol decreases the endocytosis of the epidermal growth factor receptor in hepatocytes (13) and the maltose transporter in yeast (19). In contrast, ethanol does not inhibit the fusion (or exocytosis) of Golgi vesicles with the plasma membrane in a cell-free fusion assay derived from hepatocytes (29). In H9c2 myotubes, ethanol treatment did not interfere with GLUT-4 association with clathrin-coated vesicles; qualitatively similar distributions of GLUT-4 and AP2 were observed in the periphery of the myotube after K+ depolarization and ethanol treatment (Fig. 7). However, kinetic analysis of GLUT-4 translocation in response to ethanol will need to be carried out in the future to determine whether ethanol is acting to enhance the exocytosis and/or inhibit the endocytosis of GLUT-4.

Although ethanol exposure can have profound effects on whole body glucose homeostasis, the mechanisms for ethanol action are not well understood. Short-term ethanol exposure can have an impact on hepatic glucose production (2), insulin release and extraction (1, 11, 21, 22), as well as peripheral utilization of glucose (27, 37). Acute ethanol exposure results in peripheral insulin resistance in both rats and humans (27, 37, 40), but it is not clear whether this is a direct effect of ethanol on skeletal muscle to impair insulin signaling and/or glucose utilization or an indirect, systemic response (3). Using an isolated myotube culture model, we investigated the direct effects of ethanol on glucose uptake and GLUT-4 translocation. Brief exposure to physiological concentrations of ethanol increased glucose uptake independently of any hormonal activation. This increased glucose uptake by ethanol was associated with a translocation of GLUT-4 from its intracellular storage site to the plasma membrane and required the release of Ca2+ from the sarcoplasmic reticulum. These results suggest that impaired glucose disposal by acute ethanol exposure in vivo is most likely mediated by ethanol-induced changes in the hormonal regulation of glucose transport rather than by a direct inhibition of glucose transport by ethanol. Furthermore, because acute and chronic alcohol consumption results in the development of a variety of skeletal muscle abnormalities (37), the inability of H9c2 myotubes to maintain normal GLUT-4 localization after ethanol exposure suggests that disruption of intracellular Ca2+ homeostasis by ethanol may also contribute to the development of skeletal muscle abnormalities.


    ACKNOWLEDGEMENTS

We would like to thank Dr. Regis Kelly and laboratory members (University of California, San Francisco) for their generous help in the development of the stable H9c2 cell lines expressing c-myc GLUT-4. The monoclonal antibody against c-myc (9E10), developed by J. M. Bishop, was obtained from the Developmental Studies Hybridoma Bank maintained by the University of Iowa, Dept. of Biological Sciences, Iowa City, Iowa, under contract NO1-HD-7- 3263.


    FOOTNOTES

This work was supported in part by United States Public Health Service Grant RO1 AA-11876.

Address for reprint requests and other correspondence: L. E. Nagy, Dept. of Nutrition, Case Western Reserve Univ., 2123 Abington Rd., Rm 201, Cleveland, Ohio 44106-4906 (E-mail: len2{at}po.cwru.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 6 April 2000; accepted in final form 11 July 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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