The phosphoinositide 3-kinase/Akt pathway is thought to be essential for normal insulin action and glucose metabolism in skeletal muscle and has been shown to be dysregulated in insulin resistance. However, the specific roles of and signaling pathways triggered by Akt isoforms have not been fully assessed in muscle in vivo. We overexpressed constitutively active (ca-) Akt-1 or Akt-2 constructs in muscle using in vivo electrotransfer and, after 1 wk, assessed the roles of each isoform on glucose metabolism and fiber growth. We achieved greater than 2.5-fold increases in total Ser473 phosphorylation in muscles expressing ca-Akt-1 and ca-Akt-2, respectively. Both isoforms caused hypertrophy of muscle fibers, consistent with increases in p70S6kinase phosphorylation, and a 60% increase in glycogen accumulation, although only Akt-1 increased glycogen synthase kinase-3beta phosphorylation. Akt-2, but not Akt-1, increased basal glucose uptake (by 33%, P = 0.004) and incorporation into glycogen and lipids, suggesting a specific effect on glucose transport. Consistent with this, short hairpin RNA-mediated silencing of Akt-2 caused reductions in glycogen storage and glucose uptake. Consistent with Akt-mediated insulin receptor substrate 1 (IRS-1) degradation, we observed approximately 30% reductions in IRS-1 protein in muscle overexpressing ca-Akt-1 or ca-Akt-2. Despite this, we observed no decrease in insulin-stimulated glucose uptake. Furthermore, a 68% reduction in IRS-1 levels induced using short hairpin RNAs targeting IRS-1 also did not affect glucose disposal after a glucose load. These data indicate distinct roles for Akt-1 and Akt-2 in muscle glucose metabolism and that moderate reductions in IRS-1 expression do not result in the development of insulin resistance in skeletal muscle in vivo.

Analysis of conventional germ-line or tissue-specific gene manipulation in vivo is potentially confounded by developmental adaptation of animal physiology. We aimed to adapt the technique of in vivo electrotransfer (IVE) to alter local gene expression in skeletal muscle of rodents as a means of investigating the role of specific proteins in glucose metabolism in vivo. We utilized a square-wave electroporator to induce intracellular electrotransfer of DNA constructs injected into rat or mouse muscles and investigated the downstream effects. In initial studies, expression of green fluorescent protein reporter was induced in 53 ؎ 10% of muscle fibers peaking at 7 days, and importantly, the electrotransfer procedure itself did not impact upon the expression of stress proteins or our ability to detect a reduction in 2-deoxyglucose tracer uptake by electroporated muscle of high-fat-fed rats during hyperinsulinemic-euglycemic clamp. To demonstrate functional effects of electrotransfer of constructs targeting glucose transporters, we administered vectors encoding GLUT-1 cDNA and GLUT-4 short hairpin RNAs (shRNAs) to rodent muscles. IVE of the GLUT-1 gene resulted in a 57% increase in GLUT-1 protein, accompanied by a proportionate increase in basal 2-deoxyglucose tracer uptake into muscles of starved rats. IVE of vectors expressing two shRNAs for GLUT-4 demonstrated to reduce specific protein expression and 2-deoxyglucose tracer uptake in 3T3-L1 adipocytes into mouse muscle caused a 51% reduction in GLUT-4 protein, associated with attenuated clearance of tracer to muscle after a glucose load. These results confirm that glucose transporter expression is largely rate limiting for glucose uptake in vivo and highlight the utility of IVE for the acute manipulation of muscle gene expression in the study of the role of specific proteins in glucose metabolism. Diabetes 54:2702-2711, 2005