Effects And Mechanisms Of Long-Term Ethanol Consumption On Insulin Sensitivity In Rats Fed With Normal Or High-Fat Diet

Background:Insulin resistance (IR) is one of the mechanisms of Type 2 Diabetes Mellitus (T2DM). Epidemiological data showed that long-term ethanol consumption was intimate related to insulin resistance. Under normal diet condition, the association between ethanol consumption and insulin resistance is complex. Studies have demonstrated "U" or "J"-shaped relationships where moderate drinkers have a decreased risk for insulin resistance, whereas nondrinkers have increased risk, and heavy drinkers have the greatest risk. Under high-fat diet condition, few studies were carried out to observe the influence of ethanol on insulin resistance. Wilkes found that long-term ethanol feeding (35% calories from ethanol) in a high-fat diet decreased GLUT4 translocation to the plasma membrane, thus resulting in insulin resistance in rat adipocytes. However, similar studies on consuming high-fat diet plus ethanol are too few to clearly understand their effects on insulin sensitivity although this issue is of most importance to help people with a healthy lifestyle and dietary habits.Expression of GLUT4 in adipose tissue is now recognized to play an important role in determining an individual's insulin sensitivity. For example, adipocyte-specific GLUT4-/- mice developed insulin resistance and glucose intolerance, while mice with adipose-specific over expression of GLUT4 had enhanced insulin sensitivity. These studies manifested that impaired GLUT4 expression in adipose tissue might be the primary step of an individual's insulin resistance, namely, earlier than that in both skeletal muscle and liver.Besides the insulin-dependent pathway, GLUT4 expression was also regulated by AMP-activated protein kinase (AMPK), a fuel gauge for glucose and lipid metabolism. But the connection from AMPK activation to GLUT4 expression was not well understood. Mora & Pessin reported that activated AMPK stimulates GLUT4 expression through up-regulating myocyte enhancer factor 2 (MEF2), a transcription factor that plays a key role in skeletal muscle differentiation. Since a functional MEF2 binding site which locates between -522 and -420 of rat GLUT4 promoter was found, it is believed that MEF2 was a transcription regulator of GLUT4. Moreover, such a regulation was independent of insulin presence, but required activation of AMPK in skeletal muscle. Will AMPK also regulate MEF2, then GLUT4 expression in adipose tissue? No data is available now.Objective:1. In vivo experiments:(1) To observe the influence of long-term ethanol consumption on insulin sensitivity in both normal diet- and high-fat diet-fed rats.(2) To observe the effect of long-term ethanol consumption on AMPK, MEF2, and GLUT4 expression in rat adipose tissue.(3) To test the existence of AMPK/MEF2/GLUT4 pathway in rat adipose tissue.2. In vitro experiments:(1) To observe the effects of ethanol treatment on AMPK, MEF2, and GLUT4 expression in the presence and absence of palmitate.(2) To test the existence of AMPK/MEF2/GLUT4 pathway in both rat and human isolated mature adipocytes.Methods:1. In vivo experiments:(1) Animal feedingâ‘ Grouping 1: Thirty-six male wistar rats, divided into three groups, received either distilled water (controls, group C) or ethanol which was administered by gastric tube once with a total daily dose: 5 g·kg^-1 (high dose ethanol group, group H) and 0.5 g·kg^-1 (low dose group, group L). The total feeding time is 22 weeks.(2) Grouping 2: Thirty-six male wistar rats were randomly allocated into three experimental groups (n = 12 in each group): normal diet group (N), high-fat diet plus ethanol group (HF+E), and the pair-fed high-fat diet group (HF). Rats in group HF+E received edible ethanol twice at a total daily dosage of 5 g/kg and rats in other groups received distilled water by gastric tubes. The total treatment lasted for 22 weeks.(2) Evaluation of glucose tolerance and insulin sensitivity Oral glucose tolerance test (OGTT) was carried out after a 22-week-feeding period. Blood glucose levels were measured at 0, 30, 60, 120 minutes after the glucose load. Area under the curve (AUC) was calculated to assess glucose tolerance. Fasting plasma glucose and insulin levels were measured before rats were sacrificed. HOMA-IR was then calculated by using the following formula: FPG (mmol/ L)×FINS (microunits/ml)/22.5.(3) Effects of ethanol on AMPK activation, MEF2 and GLUT4 expression in rat adipose tissuemRNA levels of AMPKα1, AMPKα2, MEF2A, MEF2D, and GLUT4 were measured by using RT-PCR. Protein levels of total-AMPKα(T-AMPKα), pAMPKα, MEF2 and GLUT4 were measured by using western blotting. GLUT4 protein level was also determined by immunofluorescence method.(4) To test the existence of AMPK/MEF2/GLUT4 pathway in rat adipose tissueSix normal male Wistar rats were randomly divided into two groups:subcutaneously injected with AICAR (an AMPK activator, 0.8 mg/ g body weight, AICAR group) or with a corresponding volume of 0.9% NaCl (control group). Two hours later, epididymal adipose tissues were obtained for measuring expression of AMPKα, MEF2 and GLUT4.2. In vitro experiments:(1) Isolation of rat and human mature adipocytesAdipocytes were isolated from epididymal fat pad of normal male wistar rats (weighing 250-300 g) and omental adipose tissue of male patients aged 25-55 who received polyp intestinal surgery. Visible blood vessels were carefully removed. The minced fat pads were carefully digested by 1 mg/ml collagenase typeâ… . After being filtered sequentially through 500- and 250-μm nylon mesh, the adipocyte cell suspension was centrifuged at 800g at room temperature for 2 min. Cell concentration was adjusted to 1.0×10⁶ cells/ml.(2) Grouping and treatmentIsolated adipocytes were adjusted to 1×10⁷/100 mm culture dish. Then the cells were incubated at 37°C for lh in the absence or presence ofâ‘ AICAR (1 mM) and compound C (20μM);â‘¡ethanol (100 mM) and AICAR (1 mM);â‘¢palmitate (0.4 mM), ethanol (20 mM), and compound C (20μM).(3) Measurement of AMPKα,MEF2 and GLUT4 expression mRNA level of GLUT4 was measured by using RT-PCR. Protein expression of T-AMPKα, pAMPKα, MEF2 and GLUT4 was determined by using western blotting.Results:1. Effects of ethanol on insulin sensitivity and the expression of AMPK,MEF2and GLUT4 in adipose tissue.(1) In vivo experiments:â‘ Plasma ethanol concentration:Plasma ethanol concentration in rats received once administration at the total dosage of 0.5g·kg^-1·d^-1 was 4.4±0.6mg/dl, in rats received once administration at the total dosage of 5g·kg^-1·d^-1 was 87±24.9mg/dl, while in rats received twice administrations at the total dosage of 5g·kg^-1·d^-1 was only 10.8±4.4mg/dl. These data indicate that the plasma ethanol concentration was determined by not only total daily ethanol dosage, but also the frequency of ethanol intake, which prompt us to suppose that at the same total daily dosage of ethanol (5g·kg^-1·d^-1), once administration might mimic the effect of heavy ethanol, while twice administrations might mimic the effect of moderate ethanol.â‘¡Evaluation of insulin sensitivityWhen rats were fed with normal diet for 22 weeks, the fasting serum insulin levels were increased by 27.6- (P＜0.05) and 13.1% (P＞0.05) , HOMA-IR index was elevated by 32.3- (P＜0.05) fP13.3% (P＞0.05) in group H and L, respectively. These results indicate that long-term heavy and light ethanol consumption, especially the heavy ethanol administration, decreases insulin sensitivity in rats.HF diet increased fasting glucose level by 28.1% (P＞0.05 vs. N), fasting insulin concentration by 28.3% (P＜0.05 vs. N), and HOMA value by 69.8% (P＜0.01 vs. N) in relative to normal diet, which indicated that insulin resistance was presented in HF diet-fed rats. However, in rats fed with HF diet plus ethanol, fasting glucose, fasting insulin, and HOMA value was decreased by 7.8-, 19.7-, and 28.2% in comparison with that in group HF (table 1-2), respectively, indicating an improved insulin sensitivity appeared after long-term moderate ethanol consumption.â‘¢Effect of ethanol on AMPK,MEF2 and GLUT4 expression in rat adipose tissueIn the setting of normal diet, long-term heavy and light ethanol consumption markedly reduced pAMPK level in rat adipose tissue, but had no significant effect on mRNA levels of AMPKα1,α2 isoforms and protein levels of T-AMPKα, indicating that ethanol impairs AMPK activation, but not its expression. Coincidence with the changes of pAMPKα, mRNA levels of MEF2 and GLUT4 were also decreased, accordingly, their protein expression was reduced as the result. These data indicate that the decrease of GLUT4 expression after ethanol feeding most probably results from the inhibition of AMPK activity, then decreased MEF2 expression.Both HF diet and ethanol had no significant effect on transcription and expression of AMPKα. But the relative expression of pAMPKαto T-AMPKαwas reduced to 39.08% of that in group N (P＜0.01 vs. N) and ethanol addition to HF diet recovered the ratio to 88.87% of that in group N (P＜0.01 vs. HF). In parallel with the changes of pAMPKa expression, mRNA levels of MEF2D isoform were markedly reduced in group HF (40.32% of that in group N, P＜0.01 vs. N) and elevated in group HF+E (85.23% of that in group N, P＜0.05 vs. HF). As the result, the total protein expression of MEF2 was diminished in group HF (31.68% of that in group N, P＜0.01 vs. N) and recovered in group HF+E (75.24% of that in group N, P＜0.01 vs. HF). Due to the transcription regulation of MEF2, GLUT4 mRNA levels were reduced by 65.06% (P＜0.01 vs. N) in group HF in comparison with that in group N and increased by 154.15% (P＜0.01 vs. HF) in group HF+E in relative to that in group HF. In immunofluorescence microscopy observation, weak signals were present for the rats fed with HF diet alone, while the stronger signals were observed for the animals fed with a combination of HF diet and ethanol. Taken together, long-term ethanol consumption ameliorated both GLUT4 gene transcription and mRNA translation in the setting of the HF diet.(2) In vitro experiments:â‘ Determination of ethanol concentration in vitroIn the preliminary experiment, rat adipocytes were incubated in the presence or absence of different ethanol concentration (20, 50, 100, 150 mM) for 1h. Results showed that AMPK activation was enhanced when cells were incubated with 20 mM ethanol, but was significantly inhibited when cells were incubated with 50, 100, or 150 mM ethanol. Thus, the 100 mM ethanol concentration was chosen to mimic the effect of heavy ethanol consumption, while 20 mM was chosen to mimic moderate ethanol effect in vitro.â‘¡Inhibition of activated-AMPK, MEF2, and GLUT4 expression after 100mM ethanol treatmentWhen isolated mature rat and human adipocytes were treated with ethanol (100 mM), the activation of AMPK was markedly inhibited, then expression of MEF2 and GLUT4 was decreased accordingly. These finding demonstrated that 100 mM ethanol had direct negative effect on AMPK activation, then MEF2 and GLUT4 expression, but not secondly occurred after impairment of other systems.â‘¢20mM ethanol treatment prevented the inhibition of palmitate on AMPK phosphorylation and restored MEF2 and GLUT4 levels in isolated primary adipocytesWe incubated adipocytes in the presence or absence of palmitate and moderate ethanol in order to observe the sole and combined effect of free fatty acids and ethanol on AMPK activation, MEF2 and GLUT4 expression. The weak bands representing pAMPK alpha, pACC, MEF2 or GLUT4 were detected in cells treated with palmitate alone. The stronger stainings for these proteins were observed when the cells were treated with a combination of palmitate and ethanol.2. To test the existence of AMPK/MEF2/GLUT4 pathway in adipose tissue(1) In vivo experiments:To test the existence of AMPK/MEF2/GLUT4 pathway in adipose tissue and observe the regulation of AMPK on MEF2 and GLUT4 in vivo, rats were injected with AICAR. Results showed that AICAR injection led to a marked increase in AMPKαphosphorylation (1.9-fold over control, P＜0.01) in rat adipose tissues. Following the activation of AMPK, MEF2 protein was elevated by 60.6% (P＜0.05). As the result, GLUT4 mRNA levels were increased by 62.8% (P＜0.05) and subsequently its protein was enhanced by 58.94% (P＜0.05). Our data showed that the activated-AMPK exerted positive regulation on MEF2, subsequently GLUT4 expression in vivo.(2) In vitro experiments:Rat and human primary adipocytes were treated with and without AICAR or compound C in vitro. In AICAR treated-rat and human adipocytes, pAMPK a expression was increased by 83.37- (P＜0.01) and 90.87% (P＜0.01). Subsequently, MEF2 was enhanced by 73.31- (P＜0.01) and 53.92% (P＜0.05). Accordingly, for GLUT4, the mRNA levels were elevated by 51.89- (P＜0.01) and 54.89% (P＜0.01), the protein expression was increased by 48.81- (P＜0.05) and 39.9% (P＜0.05). However, if cells were supplied with compound C prior to AICAR for 20min, the augmented effects of AICAR on pAMPK, MEF2, and GLUT4 were inhibited to nearly normal levels. These data indicated that, in rat and human adipocytes, AMPK was an upstream positive regulator for MEF2, then GLUT4. Namely, an AMPK/MEF2/GLUT4 pathway was existed in adipocytes.Conclusions:1. An AMPK/MEF2/GLUT4 pathway exists in rat and human mature adipocytes, activated-AMPK positively regulate MEF2 and GLUT4 expression.2. Feeding rats with normal diet, we found that long-term low-dose and high-dose ethanol consumption decreased insulin sensitivity. Inhibition of AMPK activation, then reduction of MEF2 and GLUT4 expression in adipose tissue might be the possible mechanism.3. Long-term moderate ethanol consumption reversed the adverse effect of saturated fatty acid on insulin sensitivity, which was likely to be a result from AMPK activation and subsequent upregulation of MEF2 and GLUT4 expression in adipose tissue.