Home » Key Scientific Articles » Electrical stimuli release ATP to increase GLUT4 translocation and glucose uptake via PI3K{Gamma}-Akt-AS160 in skeletal muscle cells.

Electrical stimuli release ATP to increase GLUT4 translocation and glucose uptake via PI3K{Gamma}-Akt-AS160 in skeletal muscle cells.

Osorio-Fuentealba C, Contreras-Ferrat AE, Altamirano F, Espinosa A, Li Q, Niu W, Lavandero S, Klip A, Jaimovich E.

Diabetes. 2013 May;62(5):1519-26.

Center for Molecular Studies of the Cell, Biomedical Sciences Institute, Universidad de Chile, Santiago, Chile.




Skeletal muscle glucose uptake in response to exercise is preserved in insulin-resistant conditions, but the signals involved are debated. ATP is released from skeletal muscle by contractile activity and can autocrinely signal through purinergic receptors, and we hypothesized it may influence glucose uptake. Electrical stimulation, ATP, and insulin each increased fluorescent 2-NBD-Glucose (2-NBDG) uptake in primary myotubes, but only electrical stimulation and ATP-dependent 2-NBDG uptake were inhibited by adenosine-phosphate phosphatase and by purinergic receptor blockade (suramin). Electrical stimulation transiently elevated extracellular ATP and caused Akt phosphorylation that was additive to insulin and inhibited by suramin. Exogenous ATP transiently activated Akt and, inhibiting phosphatidylinositol 3-kinase (PI3K) or Akt as well as dominant-negative Akt mutant, reduced ATP-dependent 2-NBDG uptake and Akt phosphorylation. ATP-dependent 2-NBDG uptake was also inhibited by the G protein {Beta}{Gamma} subunit-interacting peptide {Beta}ark-ct and by the phosphatidylinositol 3-kinase-{Gamma} (PI3K{Gamma}) inhibitor AS605240. ATP caused translocation of GLUT4myc-eGFP to the cell surface, mechanistically mediated by increased exocytosis involving AS160/Rab8A reduced by dominant-negative Akt or PI3K{Gamma} kinase-dead mutants, and potentiated by myristoylated PI3K{Gamma}. ATP stimulated 2-NBDG uptake in normal and insulin-resistant adult muscle fibers, resembling the reported effect of exercise. Hence, the ATP-induced pathway may be tapped to bypass insulin resistance.


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Additional Information

We have described a novel mechanism for both GLUT4 translocation and glucose uptake that depends on extracellular ATP released by cultured muscle cells after electrical stimulation. This stimulus acts via action potentials, mimicking the physiological activation of skeletal muscle during exercise. Electrical stimulation is able to elicit a cascade of events triggered by ATP release and binding to P2Y purinergic receptors, followed by activation of the serine/threonine kinase Akt that depends on PI3K{Gamma}. Each of the above-mentioned elements is required for ATP-dependent GLUT4 translocation, revealing an unrealized connection between electrical stimulation and Akt activation that can now be studied in the context of exercise.

We show that exogenously-added ATP leads to phosphorylation of the two activator sites on Akt. Though contractile activity does not result in tyrosine phosphorylation of IRS1 or associated class I PI3K activation, it can lead to Akt phosphorylation. Our study shows that ATP engages PI3K{Gamma} to mediate its downstream effect on glucose uptake. PI3K{Gamma} is a well-known target of G-coupled receptors, consistent with ATP signaling via purinergic G-coupled receptors.

A further finding of the present study is that exogenous ATP causes phosphorylation of the Akt substrate AS160. This Rab-GAP is believed to be inactivated upon phosphorylation by upstream kinases, allowing activation of Rab proteins required for GLUT4 translocation to the plasma membrane. This pathway has been mapped for insulin-stimulated GLUT4 translocation. In addition to stimulating glucose uptake, exercise sensitizes skeletal muscle to the action of insulin, improving insulin-mediated glucose uptake by mechanisms independent of enhanced insulin receptor phosphorylation. AS160 has been proposed as potential sites for the convergence of insulin and exercise signaling leading to stimulation of glucose transport in skeletal muscle. It is tantalizing to hypothesize that ATP may be an element conveying the insulin-sensitizing effect of exercise, by increasing AS160 phosphorylation.

We further found that ATP largely promotes GLUT4 exocytosis and partly reduced its endocytosis. The exocytic route likely involves GSV that fuse via TeTx-sensitive SNAREs such as VAMP2, and GSV traffic is promoted by Rab8A but not Rab10.

Exercise enhances insulin action in skeletal muscle from diabetic patients and, considering that the effects of ATP seen in myotubes were also seen in adult muscle fibers and are still present in an insulin resistance model, this study suggest the possibility that extracellular ATP or the signaling pathway it enacts, may be tested to design strategies to treat diabetic insulin resistance.