AMP kinase activation and glut4 translocation in isolated cardiomyocytes

Abstract

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.

Fig. 1.

A: Lysates of cardiomyocytes were prepared after stimulation for 30 min with either AI CAR (1 mM) or ZMP (1 mM) as described in Methods; 50 μg of protein were loaded per lane, separated and, after Western blotting, probed with an antibody against the Thr¹⁷²-phosphorylated form of AMPK. Cells made anoxic by bubbling nitrogen through the incubation medium were used as a positive control. After visualisation, blots were analysed with laser scanning densitometry. B: A representative Western blot of phosphorylation of AMPK and ACC as substrate of AMPK to demonstrate activation. C: A Ponceau red-stained membrane to show equal loading of proteins. All values are expressed as mean ± SEM (n = 4); *p < 0.05 vs basal level; **p < 0.01 vs basal level.

Fig. 2.

A: Glucose uptake of cardiomyocytes as measured by the accumulation of 2-deoxy-D-[³H] glucose over a 30-min incubation period after stimulation with 1 mM AI CAR , 1 mM ZMP or 100 nM insulin. Values are given as multi-fold stimulation over a baseline of 1. B: PI staining of the cells was performed after treatment with insulin, AICAR and ZMP to demonstrate cell viability. All values are expressed as mean ± SEM (n = 8 individual preparations, assayed in duplicate). *p < 0.05 vs basal level; ^#p < 0.05 vs insulin.

Fig. 3.

A: Sarcolemmal membrane distribution of GLUT4 in basal, insulin- (100 nM), AI CAR - (1 mM) and ZMP- (1 mM) treated cardiomyocytes. GLUT4 was determined via Western blotting as described in Methods, on membrane fractions obtained by differential centrifugation and analysed with laser scanning densitometry. All values are expressed as mean ± SEM, (n = 5–10 individual preparations); **p < 0.01 vs basal. B: A representative Western blot to show content of GLUT 4 in the membrane fraction of cardiomyocytes treated with AI CAR (1 mM), ZMP (1 mM) or insulin (100 nM).

Fig. 4.

A. Cardiomyocytes were stimulated as described in Methods, with 1 mM AI CAR , 100 nM insulin and 100 μM SNP. GLUT4 protein was visualised with Alexa Fluor 488 coupled to an antibody directed against the exofacial loop of the protein. Positive cells were defined by a fixed gate and expressed as a percentage of the total cell population. All values are expressed as mean ± SEM (n = 4 individual preparations); *p < 0.05 vs basal level.

Fig. 5.

Cardiomyocytes were stimulated as described in Fig. 4 where after they were allowed to accumulate 2-DG for a period of 30 min to determine glucose uptake. All values are expressed as mean ± SEM (n = 4 individual preparations); ***p < 0.0001 vs basal level, SN P, AI CAR and AICAR + SNP.