长期饮酒对正常饮食和高脂饮食大鼠胰岛素敏感性的影响及机制探讨
摘要
背景:
胰岛素抵抗(Insulin resistance,IR)是2型糖尿病(Type 2 Diabetes Mellitus,T2DM)的发病机制之一。流行病学资料显示,长期饮酒与胰岛素敏感性之间存在密切的关系。在正常饮食情况下,酒精摄入量与胰岛素敏感性呈反“U”型或反“J”型曲线关系。换言之,不饮酒、少量饮酒或大量饮酒可导致胰岛素抵抗,而适度饮酒则可以增加胰岛素敏感性。在高脂饮食情况下,酒精影响胰岛素敏感性的研究甚少。尽管Wilkes等报道长期饮酒可使摄入高脂饮食的大鼠脂肪细胞发生胰岛素抵抗,但由于类似研究太少,不足以揭示高脂饮食条件下长期饮酒与2型糖尿病发生的关系。随着饮酒的日益普遍及生活方式的逐渐西方化,探讨长期饮酒伴或不伴高脂饮食对胰岛素敏感性的影响及机制就显得愈发重要,因为相关的研究结果必将成为指导人们建立良好生活方式及预防饮食相关疾病的重要依据。
近年来研究人员发现,脂肪组织中GLUT4的表达在胰岛素抵抗发生过程中发挥着重要作用。例如,脂肪细胞GLUT4表达缺失(GLUT4-/-)的小鼠在发育过程中出现胰岛素抵抗,而脂肪细胞过表达GLUT4的小鼠胰岛素敏感性显著增强。另一项研究显示,在胰岛素抵抗发生过程中,脂肪组织中GLUT4表达受损早于骨骼肌和肝脏。上述研究表明,脂肪组织中GLUT4表达降低可能是机体胰岛素抵抗发生的决定因素及初始环节。
研究发现,GLUT4的表达受到P13K依赖途径及P13K非依赖途径的调节。前者通常指经典的胰岛素信号转导通路,后者如G蛋白通路、Cb1/T10通路等。新的研究发现,AMP活化的蛋白激酶(AMPK)也参与调节GLUT4的表达。在骨骼肌中,活化的AMPK增加GLUT4的表达及转位,而在脂肪组织中AMPK对GLUT4表达调控尚存在争议。除调节糖脂代谢外,AMPK最近还被认为是酒精影响胰岛素敏感性的新靶点。两个研究小组证实饱和脂肪酸与酒精联合应用可以增加新生大鼠心肌细胞及小鼠肝脏中AMPK的活化,从而改善胰岛素敏感性。在脂肪组织中,酒精是否通过作用于AMPK发挥对胰岛素敏感性的调控,目前尚缺乏相关报道。
AMPK调节GLUT4表达的机制尚未阐明。Mora &Pessin发现,在骨骼肌中,活化的AMPK通过肌细胞增强子(myocyte enhancer factor 2,MEF2)来刺激GLUT4的表达。MEF2是在骨骼肌分化过程中发挥重要作用的转录因子,自从位于GLUT4启动子上的MEF2结合位点被发现后,MEF2开始被认为是骨骼肌中GLUT4的转录调节因子。在脂肪组织中,AMPK是否也可以通过MEF2调节GLUT4表达,从而影响机体胰岛素敏感性,目前没有相关研究。
目的:
观察正常饮食及高脂饮食条件下,长期饮酒对机体胰岛素敏感性的影响及相关信号分子的变化;从体内、体外实验两个层面,利用大鼠和人两个种属的成熟脂肪细胞,验证大鼠和人脂肪组织中AMPK/MEF2/GLUT4信号通路的存在,阐述了酒精影响胰岛素敏感性的可能机制。
具体探讨了以下问题:
1.体内实验:
1.1观察长期饮酒对正常饮食和高脂饮食大鼠胰岛素敏感性的影响。
1.2观察长期饮酒对脂肪组织中AMPK、MEF2及GLUT4表达的影响。
1.3验证活体大鼠脂肪组织中AMPK/MEF2/GLUT4信号通路的存在。
2.体外实验:
2.1观察体内主要游离脂肪酸-棕榈酸(Palmitic acid,P)存在或不存在时,酒精对AMPK、MEF2及GLUT4表达的影响。
2.2验证大鼠和人类成熟脂肪细胞中AMPK/MEF2/GLUT4信号通路的存在。
方法:
1.体内实验部分:
1.1观察长期饮酒对胰岛素敏感性及相关信号分子的影响。
1.1.1动物分组及喂养:
分组一:雄性Wistar大鼠36只,按体重随机分为3组,每组12只,即对照组(蒸馏水:5g·kg~(-1)·d~(-1),C组);大剂量饮酒组(酒精量5g·kg~(-1)·d~(-1),H组);小剂量饮酒组(酒精量0.5 g·kg~(-1)·d~(-1),L组)。酒精每日一次灌胃给予,喂养时间22周。
分组二:雄性Wistar大鼠36只,按体重随机分为3组,每组12只,即正常饮食组(蒸馏水:5g·kg~(-1)·d~(-1),N组);高脂饮食组(蒸馏水总量:5g·kg~(-1)·d~(-1),HF组);高脂饮食加酒精组(酒精总量5g·kg~(-1)·d~(-1),HF+E组)。酒精每日两次灌胃给予,喂养时间22周。
1.1.2大鼠葡萄糖耐量及胰岛素敏感性的评价:处死前3天行口服糖耐量试验,分别测定空腹血糖及糖负荷后30、60、120分钟血糖,计算糖耐量曲线下面积[1/4BG(0min)+1/2BG(30min)+3/4BG(60min)+1/2BG(120min)],用以评价葡萄糖耐量。处死前抽取空腹血,检测空腹血糖(fasting plasma glucose,FPG)、空腹胰岛素(fasting insulin,FINS),并计算HOMA-IR指数(FPG×FINS/22.5),用以评价机体水平胰岛素敏感性。
1.1.3血浆酒精浓度、血清脂联素及游离脂肪酸的检测:大鼠处死时留取空腹血,干化学法检测血浆酒精浓度。酶联免疫吸附实验(ELISA法)检测血清脂联素(adiponectin)及游离脂肪酸(free fatty acid,FFA)。
1.1.4酒精对脂肪组织中AMPK/MEF2/GLUT4信号转导通路的影响:分离附睾及肾周脂肪组织并称重,用以观察内脏脂肪变化。RT-PCR检测附睾脂肪组织中AMPK的催化亚基AMPKα1和α2、MEF2两个亚型MEF2A和MEF2D以及GLUT4的mRNA水平。Western blot检测总AMPKα(total-AMPKα,T-AMPKα)、磷酸化AMPKα(phosphorylated-AMPKα,pAMPKα)、MEF2以及GLUT4的蛋白表达。免疫荧光检测GLUT4的蛋白水平。
1.2验证大鼠脂肪组织中AMPK/MEF2/GLUT4信号通路的存在。
正常雄性Wistar大鼠6只,随机分为2组,每组3只,即AICAR组(0.8 mg/g bodyweight)和对照组(注射相应体积的生理盐水)。RT-PCR检测附睾脂肪组织中GLUT4 mRNA水平。Westem blot检测T-AMPKα、pAMPKα、MEF2以及GLUT4的蛋白表达。
2.体外实验部分:
2.1成熟大鼠和人脂肪细胞的分离:取正常雄性Wistar大鼠的附睾脂肪组织及成年男性的大网膜脂肪组织,小心剔除可见血管,用1mg/ml的胶原酶Ⅰ进行消化,先后经500μm、250μm尼龙筛过滤、800g离心得到成熟脂肪细胞。
2.2细胞分组及处理:将细胞重悬后,种到10mm培养皿中,每皿细胞数为1×10~7。实验分组如下:
分组一:N组,正常对照组;A组,1mM AICAR;C组,20μM compound C;A+C组,AICAR与compound C共培养。
分组二:N组,正常对照组;E1组,100mM酒精;A组,1mM AICAR;E1+A组,100mM酒精与AICAR共培养。
分组三:N组,正常对照组;P组,0.4mM棕榈酸;E2组,20mM酒精;P+E2组,棕榈酸与20mM酒精共培养:C组,20μM compound C;E2+C组,20mM酒精与compound C共培养。
处理时间:1小时。
2.3 AMPKα、MEF2以及GLUT4的检测:RT-PCR检测脂肪细胞中GLUT4mRNA水平;Western blot检测T-AMPKα、pAMPKα、MEF2以及GLUT4的蛋白表达。
结果:
1.观察酒精对胰岛素敏感性和脂肪组织中AMPK、MEF2及GLUT4表达的影响。
1.1体内实验部分:
1.1.1血浆酒精浓度
每日一次小剂量灌酒(0.5g·kg~(-1)·d~(-1)),血浆酒精浓度为4.4±0.6mg/dl,每同一次大剂量灌酒(5g·kg~(-1)·d~(-1)),血浆酒精浓度为87±24.9mg/dl,而每日两次大剂量灌酒(5g·kg~(-1)·d~(-1)),血浆酒精浓度则仅为10.8±4.4mg/dl,表明血浆酒精浓度除与摄入酒精的总剂量有关外,还与摄入酒精的频率有关。结合血浆酒精浓度、以往的研究及本研究的结果,我们推测每日一次小剂量灌酒可能模拟了不饮酒或少量饮酒的效应,而在日饮酒总量相同的情况下,每日一次灌酒可能模拟了大量饮酒的效应,而每日两次则模拟了适量饮酒的效应。
1.1.2胰岛素敏感性评价:
1.1.2.1在正常饮食情况下,酒精喂养22周后,与C组相比,H组和L组大鼠空腹血胰岛素水平分别增加27.6-(P＜0.05)和13.1%(P＞0.05),HOMA-IR指数分别增加32.3-(P＜0.05)和13.3%(P＞0.05),表明长期大剂量和小剂量饮酒均可降低机体胰岛素敏感性,大剂量饮酒对胰岛素敏感性的影响尤为明显。
1.1.2.2在高脂饮食情况下,与N组相比,HF组大鼠空腹血糖、空腹胰岛素和HOMA-IR指数分别增加28.1-(P＞0.05)、28.3-(P＜0.05)和69.8%(P＜0.01)。而与HF组比较,增加适量酒精摄入使空腹血糖、空腹胰岛素和HOMA-IR指数分别下降了7.8-,19.7-和28.2%。上述结果表明,高脂饮食可以诱发机体胰岛素抵抗,而长期适量饮酒能够改善高脂诱导的胰岛素抵抗。
1.1.3长期饮酒对大鼠脂肪组织中AMPK、MEF2、GLUT4表达的影响
1.1.3.1在正常饮食情况下,长期大量和小量饮酒不影响AMPKα1和AMPKα2亚单位的mRNA水平,同时脂肪组织T-AMPKα的蛋白水平也没有明显改变,但H、L组大鼠脂肪组织中pAMPK的蛋白水平显著降低(P<0.05),提示长期摄入乙醇可抑制AMPK活化。与pAMPK变化一致,MEF2及GLUT4的转录和翻译也明显受抑,由于AMPK是MEF2的上游分子,而MEF2又是GLUT4的转录调节因子,因此,长期饮酒降低脂肪组织中GLUT4表达的机制很可能是抑制了AMPK的活化,进而使MEF2的表达降低所致。
1.1.3.2长期高脂饮食降低大鼠脂肪组织中AMPK的活化,进而引起MEF2及GLUT4 mRNA及蛋白表达减少,而长期适量酒精摄入则可以减轻高脂饮食对AMPK活化及MEF2表达的损害,从而增加GLUT4表达。
1.2体外实验部分
1.2.1为选择酒精的最适体外作用浓度,我们观察了酒精的量效关系,如下图所示,20mM酒精使成熟大鼠脂肪细胞中AMPK活化增加,而50、100、150mM酒精抑制AMPK活化,并且这种抑制作用随剂量增加而增强。因此在第一部分实验中我们选择100mM(E1)来模拟大量饮酒的效应,第二部分中选择了20mM(E2)来模拟适量饮酒的效应。
1.2.2大剂量酒精处理抑制AMPK活化,减少MEF及GLUT4表达:在离体大鼠和人类成熟脂肪细胞中,100mM酒精显著降低AMPK活化及MEF2和GLUT4的表达。体外实验结果证实,酒精可以直接抑制上述分子的表达,而不是继发于其他系统的损害。
1.2.3适量酒精处理减轻游离脂肪酸对AMPK、MEF2及GLUT4表达的抑制作用:用棕榈酸处理离体大鼠成熟脂肪细胞后,细胞内AMPK的活化水平、MEF2和GLUT4的表达均显著降低,当与20mM的酒精共培养时,上述分子的表达得到改善,提示适当浓度的酒精可以部分抵消游离脂肪酸对AMPK、MEF2、GLUT4表达的损害。
2.验证脂肪组织中AMPK/MEF2/GLUT4信号通路的存在
2.1体内实验
将AICAR注入大鼠体内可显著增加脂肪组织AMPK的活化,进而使MEF2及GLUT4表达增加,说明活化的AMPK可以正向调节MEF2的表达,从而使GLUT4转录增加,最终导致GLUT4蛋白表达的增加。
2.2体外实验
在大鼠及人类成熟脂肪细胞中,AICAR激活AMPK后,使MEF2和GLUT4的表达显著增加。然而,如果用AMPK阻断剂——Compound C预处理上述细胞,则观察不到AICAR对MEF2及GLUT4的激活作用,提示在大鼠和人类的脂肪细胞中,MEF2和GLUT4确实受到AMPKα正向调节,即存在AMPKα/MEF2/GLUT4这一信号通路。结论:
1、在大鼠和人类成熟脂肪细胞中存在AMPK/MEF2/GLUT4信号转导通路,AMPK正向调节MEF2及GLUT4的表达。
2、在正常饮食情况下,长期小量及大量饮酒降低胰岛素敏感性,脂肪组织中AMPK活化受抑,导致MEF2表达减少,引起GLUT4转录及蛋白表达降低是其可能机制之一。
3、在高脂饮食情况下,适量酒精摄入明显改善高脂诱导的胰岛素抵抗,这可能与酒精减轻高脂对组织中AMPK/MEF2/GLUT4信号通路的抑制作用有关。
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 feeding
①Grouping 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 tissue
mRNA 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 tissue
Six 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 adipocytes
Adipocytes 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 typeⅠ. 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 1.0×10~6 cells/ml.
(2) Grouping and treatment
Isolated adipocytes were adjusted to 1×10~7/100 mm culture dish. Then the cells were incubated at 37°C for lh in the absence or presence of①AICAR (1 mM) and compound C (20μM);②ethanol (100 mM) and AICAR (1 mM);③palmitate (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:
①Plasma 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.
②Evaluation of insulin sensitivity
When 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.
③Effect of ethanol on AMPK、MEF2 and GLUT4 expression in rat adipose tissue
In 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:
①Determination of ethanol concentration in vitro
In 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.
②Inhibition of activated-AMPK, MEF2, and GLUT4 expression after 100mM ethanol treatment
When 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.
③20mM ethanol treatment prevented the inhibition of palmitate on AMPK phosphorylation and restored MEF2 and GLUT4 levels in isolated primary adipocytes
We 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.
胰岛素抵抗(Insulin resistance,IR)是2型糖尿病(Type 2 Diabetes Mellitus,T2DM)的发病机制之一。流行病学资料显示,长期饮酒与胰岛素敏感性之间存在密切的关系。在正常饮食情况下,酒精摄入量与胰岛素敏感性呈反“U”型或反“J”型曲线关系。换言之,不饮酒、少量饮酒或大量饮酒可导致胰岛素抵抗,而适度饮酒则可以增加胰岛素敏感性。在高脂饮食情况下,酒精影响胰岛素敏感性的研究甚少。尽管Wilkes等报道长期饮酒可使摄入高脂饮食的大鼠脂肪细胞发生胰岛素抵抗,但由于类似研究太少,不足以揭示高脂饮食条件下长期饮酒与2型糖尿病发生的关系。随着饮酒的日益普遍及生活方式的逐渐西方化,探讨长期饮酒伴或不伴高脂饮食对胰岛素敏感性的影响及机制就显得愈发重要,因为相关的研究结果必将成为指导人们建立良好生活方式及预防饮食相关疾病的重要依据。
近年来研究人员发现,脂肪组织中GLUT4的表达在胰岛素抵抗发生过程中发挥着重要作用。例如,脂肪细胞GLUT4表达缺失(GLUT4-/-)的小鼠在发育过程中出现胰岛素抵抗,而脂肪细胞过表达GLUT4的小鼠胰岛素敏感性显著增强。另一项研究显示,在胰岛素抵抗发生过程中,脂肪组织中GLUT4表达受损早于骨骼肌和肝脏。上述研究表明,脂肪组织中GLUT4表达降低可能是机体胰岛素抵抗发生的决定因素及初始环节。
研究发现,GLUT4的表达受到P13K依赖途径及P13K非依赖途径的调节。前者通常指经典的胰岛素信号转导通路,后者如G蛋白通路、Cb1/T10通路等。新的研究发现,AMP活化的蛋白激酶(AMPK)也参与调节GLUT4的表达。在骨骼肌中,活化的AMPK增加GLUT4的表达及转位,而在脂肪组织中AMPK对GLUT4表达调控尚存在争议。除调节糖脂代谢外,AMPK最近还被认为是酒精影响胰岛素敏感性的新靶点。两个研究小组证实饱和脂肪酸与酒精联合应用可以增加新生大鼠心肌细胞及小鼠肝脏中AMPK的活化,从而改善胰岛素敏感性。在脂肪组织中,酒精是否通过作用于AMPK发挥对胰岛素敏感性的调控,目前尚缺乏相关报道。
AMPK调节GLUT4表达的机制尚未阐明。Mora &Pessin发现,在骨骼肌中,活化的AMPK通过肌细胞增强子(myocyte enhancer factor 2,MEF2)来刺激GLUT4的表达。MEF2是在骨骼肌分化过程中发挥重要作用的转录因子,自从位于GLUT4启动子上的MEF2结合位点被发现后,MEF2开始被认为是骨骼肌中GLUT4的转录调节因子。在脂肪组织中,AMPK是否也可以通过MEF2调节GLUT4表达,从而影响机体胰岛素敏感性,目前没有相关研究。
目的:
观察正常饮食及高脂饮食条件下,长期饮酒对机体胰岛素敏感性的影响及相关信号分子的变化;从体内、体外实验两个层面,利用大鼠和人两个种属的成熟脂肪细胞,验证大鼠和人脂肪组织中AMPK/MEF2/GLUT4信号通路的存在,阐述了酒精影响胰岛素敏感性的可能机制。
具体探讨了以下问题:
1.体内实验:
1.1观察长期饮酒对正常饮食和高脂饮食大鼠胰岛素敏感性的影响。
1.2观察长期饮酒对脂肪组织中AMPK、MEF2及GLUT4表达的影响。
1.3验证活体大鼠脂肪组织中AMPK/MEF2/GLUT4信号通路的存在。
2.体外实验:
2.1观察体内主要游离脂肪酸-棕榈酸(Palmitic acid,P)存在或不存在时,酒精对AMPK、MEF2及GLUT4表达的影响。
2.2验证大鼠和人类成熟脂肪细胞中AMPK/MEF2/GLUT4信号通路的存在。
方法:
1.体内实验部分:
1.1观察长期饮酒对胰岛素敏感性及相关信号分子的影响。
1.1.1动物分组及喂养:
分组一:雄性Wistar大鼠36只,按体重随机分为3组,每组12只,即对照组(蒸馏水:5g·kg~(-1)·d~(-1),C组);大剂量饮酒组(酒精量5g·kg~(-1)·d~(-1),H组);小剂量饮酒组(酒精量0.5 g·kg~(-1)·d~(-1),L组)。酒精每日一次灌胃给予,喂养时间22周。
分组二:雄性Wistar大鼠36只,按体重随机分为3组,每组12只,即正常饮食组(蒸馏水:5g·kg~(-1)·d~(-1),N组);高脂饮食组(蒸馏水总量:5g·kg~(-1)·d~(-1),HF组);高脂饮食加酒精组(酒精总量5g·kg~(-1)·d~(-1),HF+E组)。酒精每日两次灌胃给予,喂养时间22周。
1.1.2大鼠葡萄糖耐量及胰岛素敏感性的评价:处死前3天行口服糖耐量试验,分别测定空腹血糖及糖负荷后30、60、120分钟血糖,计算糖耐量曲线下面积[1/4BG(0min)+1/2BG(30min)+3/4BG(60min)+1/2BG(120min)],用以评价葡萄糖耐量。处死前抽取空腹血,检测空腹血糖(fasting plasma glucose,FPG)、空腹胰岛素(fasting insulin,FINS),并计算HOMA-IR指数(FPG×FINS/22.5),用以评价机体水平胰岛素敏感性。
1.1.3血浆酒精浓度、血清脂联素及游离脂肪酸的检测:大鼠处死时留取空腹血,干化学法检测血浆酒精浓度。酶联免疫吸附实验(ELISA法)检测血清脂联素(adiponectin)及游离脂肪酸(free fatty acid,FFA)。
1.1.4酒精对脂肪组织中AMPK/MEF2/GLUT4信号转导通路的影响:分离附睾及肾周脂肪组织并称重,用以观察内脏脂肪变化。RT-PCR检测附睾脂肪组织中AMPK的催化亚基AMPKα1和α2、MEF2两个亚型MEF2A和MEF2D以及GLUT4的mRNA水平。Western blot检测总AMPKα(total-AMPKα,T-AMPKα)、磷酸化AMPKα(phosphorylated-AMPKα,pAMPKα)、MEF2以及GLUT4的蛋白表达。免疫荧光检测GLUT4的蛋白水平。
1.2验证大鼠脂肪组织中AMPK/MEF2/GLUT4信号通路的存在。
正常雄性Wistar大鼠6只,随机分为2组,每组3只,即AICAR组(0.8 mg/g bodyweight)和对照组(注射相应体积的生理盐水)。RT-PCR检测附睾脂肪组织中GLUT4 mRNA水平。Westem blot检测T-AMPKα、pAMPKα、MEF2以及GLUT4的蛋白表达。
2.体外实验部分:
2.1成熟大鼠和人脂肪细胞的分离:取正常雄性Wistar大鼠的附睾脂肪组织及成年男性的大网膜脂肪组织,小心剔除可见血管,用1mg/ml的胶原酶Ⅰ进行消化,先后经500μm、250μm尼龙筛过滤、800g离心得到成熟脂肪细胞。
2.2细胞分组及处理:将细胞重悬后,种到10mm培养皿中,每皿细胞数为1×10~7。实验分组如下:
分组一:N组,正常对照组;A组,1mM AICAR;C组,20μM compound C;A+C组,AICAR与compound C共培养。
分组二:N组,正常对照组;E1组,100mM酒精;A组,1mM AICAR;E1+A组,100mM酒精与AICAR共培养。
分组三:N组,正常对照组;P组,0.4mM棕榈酸;E2组,20mM酒精;P+E2组,棕榈酸与20mM酒精共培养:C组,20μM compound C;E2+C组,20mM酒精与compound C共培养。
处理时间:1小时。
2.3 AMPKα、MEF2以及GLUT4的检测:RT-PCR检测脂肪细胞中GLUT4mRNA水平;Western blot检测T-AMPKα、pAMPKα、MEF2以及GLUT4的蛋白表达。
结果:
1.观察酒精对胰岛素敏感性和脂肪组织中AMPK、MEF2及GLUT4表达的影响。
1.1体内实验部分:
1.1.1血浆酒精浓度
每日一次小剂量灌酒(0.5g·kg~(-1)·d~(-1)),血浆酒精浓度为4.4±0.6mg/dl,每同一次大剂量灌酒(5g·kg~(-1)·d~(-1)),血浆酒精浓度为87±24.9mg/dl,而每日两次大剂量灌酒(5g·kg~(-1)·d~(-1)),血浆酒精浓度则仅为10.8±4.4mg/dl,表明血浆酒精浓度除与摄入酒精的总剂量有关外,还与摄入酒精的频率有关。结合血浆酒精浓度、以往的研究及本研究的结果,我们推测每日一次小剂量灌酒可能模拟了不饮酒或少量饮酒的效应,而在日饮酒总量相同的情况下,每日一次灌酒可能模拟了大量饮酒的效应,而每日两次则模拟了适量饮酒的效应。
1.1.2胰岛素敏感性评价:
1.1.2.1在正常饮食情况下,酒精喂养22周后,与C组相比,H组和L组大鼠空腹血胰岛素水平分别增加27.6-(P＜0.05)和13.1%(P＞0.05),HOMA-IR指数分别增加32.3-(P＜0.05)和13.3%(P＞0.05),表明长期大剂量和小剂量饮酒均可降低机体胰岛素敏感性,大剂量饮酒对胰岛素敏感性的影响尤为明显。
1.1.2.2在高脂饮食情况下,与N组相比,HF组大鼠空腹血糖、空腹胰岛素和HOMA-IR指数分别增加28.1-(P＞0.05)、28.3-(P＜0.05)和69.8%(P＜0.01)。而与HF组比较,增加适量酒精摄入使空腹血糖、空腹胰岛素和HOMA-IR指数分别下降了7.8-,19.7-和28.2%。上述结果表明,高脂饮食可以诱发机体胰岛素抵抗,而长期适量饮酒能够改善高脂诱导的胰岛素抵抗。
1.1.3长期饮酒对大鼠脂肪组织中AMPK、MEF2、GLUT4表达的影响
1.1.3.1在正常饮食情况下,长期大量和小量饮酒不影响AMPKα1和AMPKα2亚单位的mRNA水平,同时脂肪组织T-AMPKα的蛋白水平也没有明显改变,但H、L组大鼠脂肪组织中pAMPK的蛋白水平显著降低(P<0.05),提示长期摄入乙醇可抑制AMPK活化。与pAMPK变化一致,MEF2及GLUT4的转录和翻译也明显受抑,由于AMPK是MEF2的上游分子,而MEF2又是GLUT4的转录调节因子,因此,长期饮酒降低脂肪组织中GLUT4表达的机制很可能是抑制了AMPK的活化,进而使MEF2的表达降低所致。
1.1.3.2长期高脂饮食降低大鼠脂肪组织中AMPK的活化,进而引起MEF2及GLUT4 mRNA及蛋白表达减少,而长期适量酒精摄入则可以减轻高脂饮食对AMPK活化及MEF2表达的损害,从而增加GLUT4表达。
1.2体外实验部分
1.2.1为选择酒精的最适体外作用浓度,我们观察了酒精的量效关系,如下图所示,20mM酒精使成熟大鼠脂肪细胞中AMPK活化增加,而50、100、150mM酒精抑制AMPK活化,并且这种抑制作用随剂量增加而增强。因此在第一部分实验中我们选择100mM(E1)来模拟大量饮酒的效应,第二部分中选择了20mM(E2)来模拟适量饮酒的效应。
1.2.2大剂量酒精处理抑制AMPK活化,减少MEF及GLUT4表达:在离体大鼠和人类成熟脂肪细胞中,100mM酒精显著降低AMPK活化及MEF2和GLUT4的表达。体外实验结果证实,酒精可以直接抑制上述分子的表达,而不是继发于其他系统的损害。
1.2.3适量酒精处理减轻游离脂肪酸对AMPK、MEF2及GLUT4表达的抑制作用:用棕榈酸处理离体大鼠成熟脂肪细胞后,细胞内AMPK的活化水平、MEF2和GLUT4的表达均显著降低,当与20mM的酒精共培养时,上述分子的表达得到改善,提示适当浓度的酒精可以部分抵消游离脂肪酸对AMPK、MEF2、GLUT4表达的损害。
2.验证脂肪组织中AMPK/MEF2/GLUT4信号通路的存在
2.1体内实验
将AICAR注入大鼠体内可显著增加脂肪组织AMPK的活化,进而使MEF2及GLUT4表达增加,说明活化的AMPK可以正向调节MEF2的表达,从而使GLUT4转录增加,最终导致GLUT4蛋白表达的增加。
2.2体外实验
在大鼠及人类成熟脂肪细胞中,AICAR激活AMPK后,使MEF2和GLUT4的表达显著增加。然而,如果用AMPK阻断剂——Compound C预处理上述细胞,则观察不到AICAR对MEF2及GLUT4的激活作用,提示在大鼠和人类的脂肪细胞中,MEF2和GLUT4确实受到AMPKα正向调节,即存在AMPKα/MEF2/GLUT4这一信号通路。结论:
1、在大鼠和人类成熟脂肪细胞中存在AMPK/MEF2/GLUT4信号转导通路,AMPK正向调节MEF2及GLUT4的表达。
2、在正常饮食情况下,长期小量及大量饮酒降低胰岛素敏感性,脂肪组织中AMPK活化受抑,导致MEF2表达减少,引起GLUT4转录及蛋白表达降低是其可能机制之一。
3、在高脂饮食情况下,适量酒精摄入明显改善高脂诱导的胰岛素抵抗,这可能与酒精减轻高脂对组织中AMPK/MEF2/GLUT4信号通路的抑制作用有关。
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 feeding
①Grouping 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 tissue
mRNA 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 tissue
Six 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 adipocytes
Adipocytes 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 typeⅠ. 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 1.0×10~6 cells/ml.
(2) Grouping and treatment
Isolated adipocytes were adjusted to 1×10~7/100 mm culture dish. Then the cells were incubated at 37°C for lh in the absence or presence of①AICAR (1 mM) and compound C (20μM);②ethanol (100 mM) and AICAR (1 mM);③palmitate (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:
①Plasma 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.
②Evaluation of insulin sensitivity
When 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.
③Effect of ethanol on AMPK、MEF2 and GLUT4 expression in rat adipose tissue
In 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:
①Determination of ethanol concentration in vitro
In 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.
②Inhibition of activated-AMPK, MEF2, and GLUT4 expression after 100mM ethanol treatment
When 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.
③20mM ethanol treatment prevented the inhibition of palmitate on AMPK phosphorylation and restored MEF2 and GLUT4 levels in isolated primary adipocytes
We 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.
引文
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[14] Ravnskjaer K, Boergesen M, Rubi B, Larsen JK, Nielsen T, Fridriksson J, et al. 2005 Peroxisome proliferator-activated receptor a (PPARa) potentiates, whereas PPARγ attenuates, glucose-stimulated insulin secretion in pancreatic β-cells. Endocrinology 146: 3266-3276.
[15] Zhou YT, Shimabukuro M, Wang MY, Lee Y, Higa M, Milburn JL, et al. 1998 Role of peroxisome proliferator-activated receptor-α in disease of pancreatic β cells. Proc Natl Acad Sci USA 95: 8898-8903.
[16] You M, Matsumoto M, Pacold CM, Cho WK, Crabb DW 2004 The role of AMP-activated protein kinase in the action of ethanol in the liver. Gastroenterology 127: 1798-1808.
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[44] Holmes BF, Sparling DP, Olson AL, Winder WW, Dohm GL. Regulation of muscle GLUT4 enhancer factor and myocyte enhancer factor 2 by AMP-activated protein kinase. Am J Physiol Endocrinol Metab 2005; 289(6):E1071-6.
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