AMP kinase activation and glut4 translocation in isolated cardiomyocytes.
Activation of AMP-activated protein kinase (AMPK) results in glucose transporter 4 (GLUT4) translocation from the cytosol to the cell membrane, and glucose uptake in the skeletal muscles. This increased activation of AMPK can be stimulated by a pharmacological agent, AICAR (5' -aminoimidazole-4-carboxamide ribonucleoside), which is converted intracellularly into ZMP (5' -aminoimidazole-4-carboxamideribonucleosidephosphate), an AMP analogue. We utilised AICAR and ZMP to study GLUT4 translocation and glucose uptake in isolated cardiomyocytes. Adult ventricular cardiomyocytes were treated with AICAR or ZMP, and glucose uptake was measured via [3H] -2-deoxyglucose accumulation. PKB/Akt, AMPK and acetyl-CoA-carboxylase phosphorylation and GLUT4 translocation were detected by Western blotting or flow cytometry. AICAR and ZMP promoted AMPK phosphorylation. Neither drug increased glucose uptake but on the contrary, inhibited basal glucose uptake, although GLUT4 translocation from the cytosol to the membrane occurred. Using flow cytometry to detect the exofacial loop of the GLUT4 protein, we showed ineffective insertion in the membrane under these conditions. Supplementing with nitric oxide improved insertion in the membrane but not glucose uptake. We concluded that activation of AMPK via AICAR or ZMP was not sufficient to induce GLUT4-mediated glucose uptake in isolated cardiomyocytes. Nitric oxide plays a role in proper insertion of the protein in the membrane but not in glucose uptake.
Department of Biomedical Sciences, Division of Medical Physiology, Faculty of Health Sciences, University of Stellenbosch, Tygerberg, South Africa.
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Webster
S O
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Friedrich
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A
Lochner
B
B
Huisamen
D000263:Adenylate Kinase / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D015153:Blotting, Western; D002478:Cells, Cultured; D004789:Enzyme Activation / Q000235:genetics*; D005434:Flow Cytometry; D005947:Glucose / Q000378:metabolism; D051275:Glucose Transporter Type 4 / Q000235:genetics* / Q000378:metabolism; D008297:Male; D032383:Myocytes, Cardiac / Q000166:cytology / Q000201:enzymology*; D051381:Rats; D017208:Rats, Wistar; D014178:Translocation, Genetic / Q000235:genetics*
D051275:Glucose Transporter Type 4; C495694:Slc2a4 protein, rat; D000263:Adenylate Kinase; D005947:Glucose