Insulin and Contraction Stimulate Exocytosis, but Increased AMP-activated Protein Kinase Activity Resulting from Oxidative Metabolism Stress Slows Endocytosis of GLUT4 in Cardiomyocytes

Abstract

Stimulations of glucose transport produced by insulin action, contraction, or through a change in cell energy status are mediated by separate signaling pathways. These are the wortmannin-sensitive phosphatidylinositol 3-kinase pathway leading to the intermediate Akt and the wortmannin-insensitive AMP-activated protein kinase (AMPK) pathway. Electrical stimulation of cardiomyocytes produced a rapid, insulin-like, wortmannin-sensitive stimulation of glucose transport activity, but this occurred without extensive activation of Akt. Although AMPK phosphorylation was increased by contraction, this response was not wortmannin-inhibitable and consequently did not correlate with the wortmannin sensitivity of the transport stimulation. Oxidative metabolism stress due to hypoxia or treatment with oligomycin led to increased AMPK activity with a corresponding increase in glucose transport activity. We show here that these separate signaling pathways converge on GLUT4 trafficking at separate steps. The rate of exocytosis of GLUT4 was rapidly stimulated by insulin, but insulin treatment did not alter the endocytosis rate. Like insulin stimulation, electrical stimulation of contraction led to a stimulation of GLUT4 exocytosis without any marked change in endocytosis. By contrast, after oxidative metabolism stress, no stimulation of GLUT4 exocytosis occurred; instead, this treatment led to a reduction in GLUT4 endocytosis.Regulation of cellular energy levels and metabolic processes are intricately dependent on the supply of glucose, and there are now known to be multiple mechanisms controlling this supply to specific tissues. Insulin stimulation of glucose transport occurs in response to raised blood glucose or when stimulated storage of glucose, in the form of glycogen and triglyceride, is necessary. A stimulation of glucose uptake is also necessary during metabolic stress such as that which occurs during hypoxia and ischemia. Recent work in this area has identified AMP-activated protein kinase (AMPK) 1 as the key regulatory intermediate of these processes. This protein functions as an energy level sensor in organisms ranging from yeast to mammals (1, 2). Increased AMPK activity is associated with increased glucose transport in cell lines (3, 4) and skeletal muscle that have been challenged by a metabolic stress stimulus or more direct activation of the kinase by the AMP analogue, ZMP, which is produced from the nucleoside AICAR (5, 6). Contraction of skeletal myocytes is also known to lead to increased levels of glucose transport. Evidence from transgenic mice expressing a dominant inhibitory form of AMPK suggest that although AMPK is involved in regulation of hypoxia-induced glucose transport, it may not be involved in the contraction response (7). Similar conclusions have been reached in studies on animal models for obesity-related type 2 diabetes (8).Electrically stimulated contraction of cardiac myocytes also leads to stimulation of glucose transport. This occurs without a marked stimulation of Akt phosphorylation. Howev...

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