E3 Ubiquitin Ligase, WWP1, Interacts with AMPK alpha 2 and Down-regulates Its Expression in Skeletal Muscle C2C12 Cells

Abstract

It is known that the activity of AMP-activated protein kinase (AMPK alpha 2) was depressed under high glucose conditions. However, whether protein expression of AMPK alpha 2 is also down-regulated or not remains unclear. In this study, we showed that the expression of AMPK alpha 2 was down-regulated in cells cultured under high glucose conditions. Treatment of proteasome inhibitor, MG132, blocked high glucose-induced AMPK alpha 2 downregulation. Endogenous AMPK alpha 2 ubiquitination was detected by immunoprecipitation of AMPK alpha 2 followed by immunoblotting detection of ubiquitin. The yeast-two hybrid (YTH) approach identified WWP1, an E3 ubiquitin ligase, as the AMPK alpha 2-interacting protein in skeletal muscle cells. Interaction between AMPK alpha 2 and WWP1 was validated by co-immunoprecipitation. Knockdown of WWP1 blocked high glucose-induced AMPK alpha 2 down-regulation. The overexpression of WWP1 down-regulated AMPK alpha 2. In addition, the expression of WWP1 is increased under high glucose culture conditions in both mRNA and protein levels. The level of AMPK alpha 2 was downregulated in the quadriceps muscle of diabetic animal model db/db mice. Expression of WWP1 blocked metformin-induced glucose uptake. Taken together, our results demonstrated that WWP1 down-regulated AMPK alpha 2 under high glucose culture conditions via the ubiquitin-proteasome pathway.