Human GLUT4 protein expression in muscle and adipose tissues of transgenic mice decreases plasma insulin and glucose levels and improves glucose tolerance compared with nontransgenic controls (Liu, M.-L., Gibbs, E. M., McCoid, S. C., Milici, A. J., Stukenbrok, H. A., McPherson, R. K., Treadway, J. L., and Pessin, J. E. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 11346-11350). We examined the basis of improved glycemic control in hGLUT4 transgenic mice by determining glucose homeostasis and metabolic profiles in vivo. Glucose turnover experiments indicated a 1.4-fold greater systemic glucose clearance in hGLUT4 mice relative to controls (p < 0.05), whereas hepatic glucose production was similar despite 26% lower (p < 0.05) glucose levels. Glucose infusion rate during an euglycemic-hyperinsulinemic clamp was 2-fold greater (p < 0.05) in hGLUT4 mice versus controls, and skeletal muscle and heart glycogen content were increased 3-5-fold (p < 0.05). The increased peripheral glucose clearance in hGLUT4 mice was associated with increased (25-32%) basal and insulin-stimulated glucose transport rate in soleus muscle (p < 0.01), and increased muscle plasma membrane-associated GLUT4 protein. Fed hGLUT4 mice displayed 20-30% lower plasma glucose and insulin levels (p < 0.05) and 43% elevated glucagon levels (p < 0.001) compared with controls. Triglycerides, free fatty acids, and beta-hydroxy-butyrate were elevated 43-63% (p < 0.05) in hGLUT4 mice due to hypoinsulinemia-induced lipolysis. Free fatty acids and beta-hydroxybutyrate levels in hGLUT4 mice increased further upon fasting, and skeletal muscle glycogen levels decreased markedly compared with controls. The data demonstrate that high level expression of hGLUT4 increases systemic glucose clearance and muscle glucose utilization in vivo and also results in marked compensatory lipolysis and muscle glycogenolysis during a fast.