We studied five healthy subjects with perfused forearm and euglycemic clamp techniques in combination with a three-tracer (D-[12C]mannitol, not transportable; 3-O-[14C]methyl-D-glucose, transportable but not metabolizable; D-[3-3H]glucose, transportable and metabolizable) intra-arterial pulse injection to assess transmembrane transport and intracellular phosphorylation of glucose in vivo in human muscle. The washout curves of the three tracers were analyzed with a multicompartmental model. A priori identifiability analysis of the tracer model shows that the rate constants of glucose transport into and out of the cells and of glucose phosphorylation are uniquely identifiable. Tracer model parameters were estimated by a nonlinear least-squares parameter estimation technique. We then solved for the tracee model and estimated bidirectional transmembrane transport glucose fluxes, glucose intracellular phosphorylation, extracellular and intracellular volumes of glucose distribution, and extracellular and intracellular glucose concentrations. Physiological hyperinsulinemia (473 +/- 22 pM) caused 2.7-fold (63.1 +/- 7.2 vs. 23.4 +/- 6.1 mumol.min-1.kg-1, P < 0.01) and 5.1-fold (42.5 +/- 5.8 vs. 8.4 +/- 2.2 mumol.min-1.kg-1, P < 0.01) increases in transmembrane influx and intracellular phosphorylation of glucose, respectively. Extracellular distribution volume and concentration of glucose were unchanged, whereas intracellular distribution volume of glucose was increased (approximately 2-fold) and intracellular glucose concentration was almost halved by hyperinsulinemia. In summary, 1) a multicompartment model of three-tracer kinetic data can quantify transmembrane glucose fluxes and intracellular glucose phosphorylation in human muscle; and 2) physiological hyperinsulinemia stimulates both transport and phosphorylation of glucose and, in doing so, amplifies the role of glucose transport as a rate-determining step of muscle glucose uptake
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