Abstract <p>Calorie restriction (CR: ∼20–40% below AL: ad libitum intake) can increase insulin sensitivity in various species. We reported that CR did not induce an increase in insulin-stimulated glucose uptake (ISGU) by skeletal muscle from female Akt substrate of 160 kDa knockout (AS160-KO) rats. It seemed possible that the marked decline in muscle GLUT4 abundance, a characteristic of AS160-KO animals, played a role in our observation. We used an adeno-associated virus (AAV) approach to increase muscle GLUT4 abundance in female AS160-KO rats. We injected an AAV-GLUT4 vector into one epitrochlearis muscle. The contralateral epitrochlearis underwent sham injection and served as the control. Rats were randomly assigned to AL or CR (consuming 65% of AL intake) groups for 8-weeks. ISGU was determined in isolated muscles incubated ± insulin. Akt phosphorylation on regulatory sites (Threonine-308, pAkt^Thr308 and Serine-473, pAkt^Ser473) was determined to evaluate a key insulin signaling protein. The abundance of proteins that regulate ISGU (GLUT4 glucose transporter and hexokinase II) was determined by immunoblotting. We found: (1) AAV-delivery of GLUT4 to muscles successfully increased GLUT4 expression compared to sham-treated contralateral muscles in both diet groups; (2) this elevation in muscle GLUT4 did not result in a CR-induced increase in ISGU; (3) no significant diet-related differences were detected for pAkt^Thr308, pAkt^Ser473 or abundance of GLUT4 and hexokinase II in the muscles with either sham treatment or AAV-GLUT4. These observations revealed that the absence of a CR-induced increase in muscle ISGU of female AS160-KO rats was not attributable to low muscle GLUT4 abundance.</p>