Chemical Hypoxia-induced Glucose Transporter-4 Translocation In Neonatal Rat Cardiomyocytes

Glucose is the major metabolic substrate in the heart during ischemia/hypoxia. Glucose is transported into cardiomyocytes by members of facilitative glucose transporters(GLUTs).The most abundant glucose transporter in heart is glucose transporter 4(GLUT-4),which translocates to the plasma membrane in response to insulin or ischemia.It has been illustrated that insulin increases glucose uptake through GLUT-4 translocation via a pathway mediated by phosphotidal inositol 3-kinase(PI3K). In contrast,myocardial glucose uptake during ischemia is stimulated by GLUT-4 translocation to the cell surface through a PI3K-independent pathway.AMPK is activated by increased intracellular AMP and decreased creatine phosphate and thought as a metabolic stress protein.It has been demonstrated that AMPK is of vital importance for glucose metabolism in heart and it might be involved in the signaling pathway of GLUT-4 translocation.Meanwhile it has been illustrated that low dosage of azide incubation could induce a successful chemical hypoxia model of primary cultured neonatal rat cardiomyocytes.Thus,in this study we will investigated whether AMPK was involved in GLUT-4 translocation induced by azide-induced chemical hypoxia in primary cultured neonatal rat cardiomyocytes.MethodsWith or without adenine 9-β-D-arabinofuranoside(ara A,an AMPK inhibitor) preincubation,primary cultured rat cardiomyocytes were randomized to several groups as incubated with Sodium Azide(the respiratory chain inhibitor),insulin,or 5-aminoimidazole-4-carboxyamide-1-β-D-ribofuranoside(AICAR,an AMPK activator). Glucose uptake was measured throughγ- scintillation and GLUT-4 protein was detected by western blot for each group.ResultsCell viability was reduced by azide incubation in a significant dose dependent and time dependent manner.Treated with lmmol/L azide for 3 hours,cells were identified as early apoptosis which could retain full viability in additional wash-off experiments.We thus define this condition(lmmol/L azide for 3 hours)as a successful and reproducible chemical hypoxia model.2-[~3H]deoxyglucose uptake in rat neonatal cardiomyocytes incubated with AICAR or azide was significantly increased.This was similar to cells incubated with insulin at a concentration that maximally stimulates glucose uptake.Moreover,the addition of insulin increased glucose uptake in cells incubated with AICAR and reached significant increase in glucose uptake.Meanwhile,cells incubated with azide and AICAR both showed increased translocation of GLUT-4 from the GLUT-4 storage vesicles to the outer membrane.And AICAR/azide coincubation caused the most abundant GLUT-4 translocation.Ara A preincubation inhibited AMPK activation of cardiomyocytes,and it blunted the effect of azide,AICAR/azide conincubation,or insulin/AICAR coincubation on glucose uptake.Furthermore,it completely diminished the AICAR-induced glucose uptake.However,ara A significantly diminished the above effects of stimulants on GLUT-4 in cardiomyocytes except for insulin.ConclusionsObtained results were consistent with previous studies on chemical hypoxia model and thus provided the pathophysiological relevance of this azide system for our following experiments.Azide-induced chemical hypoxia increased glucose uptake and GLUT-4 translocation in neonatal rat cardiomyocytes through a mechanism that is independent of PI3K activation and at least partially be mediated by AMPK activation.