AMP activated protein kinase (AMPK) is a heterotrimeric complex that works as an energy sensor to integrate nutritional and hormonal signals. The naturally occurring R225Q mutation in the 3 subunit in pigs is associated with abnormally high glycogen content in skeletal muscle. As skeletal muscle accounts for most of the body's glucose uptake, and 3 is specifically expressed in skeletal muscle, it is important to understand the underlying mechanism of this mutation in regulating glucose and glycogen metabolism. Using skeletal muscle-specific transgenic mice over-expressing wild type 3 (WT3) and R225Q3 (MUT3), we show that both WT3 and MUT3 mice have a 1.5 - 2-fold increase in muscle glycogen content. In WT3 mice, increased glycogen content was associated with elevated total glycogen synthase activity and reduced glycogen phosphorylase activity, whereas alterations in activities of these enzymes could not explain elevated glycogen in MUT3 mice. Basal, 5-Aminoimidazole 4-carboxamide riboside (AICAR),- and phenformin-stimulated AMPK2 isoform specific activities were decreased only in MUT3 mice. Basal rates of 2-DG glucose uptake were decreased in both WT3 and MUT3 mice. However, AICAR- and phenformin-stimulated 2-DG glucose uptake were blunted only in MUT3 mice. In conclusion, expression of either wild type or mutant 3 subunit of AMPK results in increased glycogen concentrations in muscle, but the mechanisms underlying this alteration appear to be different. Furthermore, mutation of the 3 subunit is associated with decreases in AMPK2 isoform specific activity and impairment in AICAR- and phenformin-stimulated skeletal muscle glucose uptake.