Previous studies have shown that many metabolic actions of adiponectin are mediated via the activation of AMP kinase and that adiponectin stimulates GLUT4 translocation and glucose uptake in the muscle. In this study, we demonstrate that adiponectin stimulates actin cytoskeleton remodeling, with increased phosphorylation of cofilin, and that blocking of cytoskeletal remodeling with cytochalasin D prevents adiponectin-stimulated AMPK phosphorylation in L6 myoblasts. LKB1 is an upstream kinase of AMPK, and we observed the colocalization of LKB1 with filamentous actin in response to adiponectin. Adiponectin-stimulated translocation of LKB1 from a nuclear to a cytoplasmic location to activate AMPK was also dependent on actin cytoskeleton remodeling. Cytoskeletal remodeling visualized by rhodamine–phalloidin immunofluorescence indicated that adiponectin-stimulated reorganization resulted in the formation membrane ruffles, which were also clearly visible by scanning electron microscopy in L6-GLUT4myc myoblasts. The stimulation of glucose uptake, but not of GLUT4-myc translocation to the cell surface, by adiponectin was also dependent on actin cytoskeleton remodeling. These results suggest that actin remodeling induced by adiponectin is essential for mediating LKB1/AMPK signaling and glucose uptake in skeletal muscle cells.
Vivian Vu, Phuong Bui, Megumi Eguchi, Aimin Xu and Gary Sweeney
Penny Ahlstrom, Esther Rai, Suharto Chakma, Hee Ho Cho, Palanivel Rengasamy and Gary Sweeney
Skeletal muscle insulin resistance is known to play an important role in the pathogenesis of diabetes, and one potential causative cellular mechanism is endoplasmic reticulum (ER) stress. Adiponectin mediates anti-diabetic effects via direct metabolic actions and by improving insulin sensitivity, and we recently demonstrated an important role in stimulation of autophagy by adiponectin. However, there is limited knowledge on crosstalk between autophagy and ER stress in skeletal muscle and in particular how they are regulated by adiponectin. Here, we utilized the model of high insulin/glucose (HIHG)-induced insulin resistance, determined by measuring Akt phosphorylation (T308 and S473) and glucose uptake in L6 skeletal muscle cells. HIHG reduced autophagic flux measured by LC3 and p62 Western blotting and tandem fluorescent RFP/GFP-LC3 immunofluorescence (IF). HIHG also induced ER stress assessed by thioflavin T/KDEL IF, pIRE1, pPERK, peIF2α and ATF6 Western blotting and induction of a GRP78-mCherry reporter. Induction of autophagy by adiponectin or rapamycin attenuated HIHG-induced ER stress and improved insulin sensitivity. The functional significance of enhanced autophagy was validated by demonstrating a lack of improved insulin sensitivity in response to adiponectin in autophagy-deficient cells generated by overexpression of dominant negative mutant of Atg5. In summary, adiponectin-induced autophagy in skeletal muscle cells alleviated HIHG-induced ER stress and insulin resistance.