Ethanol Exposure Decreases The Expression Of AMP-activated Protein Kinase And GLUT4 In Rat Cardiac Myocytes

Background:Insulin resistance means that physiological level of insulin does not play the corresponding biological effect. It can be caused by environmental and genetic factors. In recent years, with raising standard of living and changes of life style, the incidence of type 2 diabetes increases. Of them, ethanol plays an important role in insulin sensitivity.Ethanol is the most abused drugs used at present. Long term's consumption may be one of the causes leading to insulin resistance. It suggests that ethanol exposure is as an independent factor to the impairment of insulin sensitivity.At present, it seems that the effect of alcohol intake on insulin sensitivity remain obscure. Both in vivo and in vitro studies have demonstrated that ethanol induce insulin sensitive drop accompanied with the decline in several molecules expression of insulin signal pathway such as insulin receptor (IR), insulin receptor substrate (IRS) and Glucose transporter 4 (GLUT4), which is proposed as possible contributor to insulin resistance. However, it has been reported that normal molecules expression of insulin signal pathway occurred in those mice with IR induced by ethanol. Thus, insulin signaling pathway only can not well explained the decreased insulin sensitivity induced by ethanol.AMPK, AMP-activated protein kinase, is a key regulator of intracellular fatty acid metabolism. Recently, a few studies have addressed that AMPK is necessary for GLUT4 regulating glucose uptake. Noticeable, the regulation by AMPK is through a common insulin-independent manner. It is unclear whether AMPK also participates in the pathophysiological process of ethanol inducing insulin resistance. Further studies demonstrate that the effect of AMPK on GLUT4 is mediated by myocyte enhancer factor 2 (MEF2), a muscle-specific regulator of GLUT4 gene transcription. The information of published studies suggested that phosphorylated-AMPK upregualtes MEF2 gene transcription leading to an increase in MEF2 protein level, subsequently enhancing its binding activity with GLUT4 promoter to impel GLUT4 gene transcription. The adenosine analog 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR) is a potent activator of AMPK in intact cells. In skeletal muscle, acute administration by in vivo injections of AICAR has proven to activate the AMPK and stimulate glucose uptake. However, there are few reports that whether it has similar function in heart tissue.Although some researches have reported that ethanol can decrease AMPK's expression in skeletal muscle and hepatic cells, there is lacking research in rat cardiomyocytes.Objective:Our aims of the current study were to investigate whether there existed these effects of AMPK on MEF2 and GLUT4 in rat cardiac muscles. If so, whether this event was involved in ethanol effects.Methods:1) Animal experiment:Male Wistar rats weighing 180±5g were randomly divided into two groups: AICAR (an AMPK activator) group and control group. AICAR-treated rats were received a subcutaneously injection with 0.8 mg AICAR/g body weight. Control animals were injected with a corresponding volume of 0.9% NaCl. Treatment for 2 hours, all rats were anesthetized and heart ventricles were dissected out for an analysis of the mRNA levels of MEF2A and 2D subtypes as well as GLUT4 by RT-PCR.2) Isolation and Culture of Rat Ventricular Cardiomyocytes: Neonatal rat ventricular cardiomyocytes were prepared from 1- to 3-day-old Wistar rats. Briefly, neonatal rat ventricles were dispersed in a series of incubations at 37℃in 0.125% tripsin . To reduce fibroblast contamination to <5%, dispersed cells were preplated for 30 minutes, and the unattached cells were replated on culture flask. Cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 20% Fetal Bovine Serum and 100 U/ml penicillin/streptomycin at 37℃in the presence of 5% CO₂ in a humidified incubator. After 24 hours, cells were maintained in supplemented with 0.5ug/ml mytomycin for over 24 hours. Experiments were performed between days 2 and 3 in culture. The final myocyte cultures were assessed to contain >90% cardiac myocytes by immunofluorescence with Anti-α-Actinin (Sarcomeric) antibody (SIGMA A7811), cardiomyocyte specific proteins. Protein levels of phospho-AMPK alpha subunit and The mRNA levels of GLUT4 in cardiac myocytes after ethanol and AICAR treated were measured by western blotting and RT-PCR, respectively.Results:1) Effects of AICAR on the expression of mRNA level of MEF2 and GLUT4 of rats' heart muscle: Compared with the controls, AICAR obviously up-regulated the mRNA levels of MEF2A. As a result, the GLUT4 mRNA contents were correspondingly increased (p < 0.05). We, however, failed to detect the significant change of MEF2D mRNA, which is in agreement with a previous report of strong MEF2A while weak MEF2D expression in heart tissues.2) Effects of ethanol and AICAR on the expression of protein level of AMPK-αof neonatal cardiac myocytes: Compared with the controls, ethanol decreases the protein level of AMPK-α, while AICAR increases it.3) Effects of ethanol and AICAR on the expression of mRNA level of GLUT4 of neonatal cardiac myocytes: Compared with the controls, ethanol decreases the mRNA level of GLUT4, while AICAR increases it.Conclusions:AICAR may enhance AMPK activity and increase the expression of MEF2A and GLUT4 in rat cardiac myocytes. While ethanol is able to decline AMPK activity associated with a decrease in GLUT4 expression in rat cardiac myocytes. This maybe partly indicates that ethanol impairs insulin sensitivity in rat heart muscle via, at a certain extent, decreasing the expression of AMPK activity, MEF2A and GLUT4.