OBJECTIVE: Insulin and contraction increase skeletal muscle glucose uptake through distinct and additive mechanisms. However, recent reports have demonstrated that both signals converge on the Akt substrate of 160 kDa (AS160), a protein that regulates GLUT4 translocation. Although AS160 phosphorylation is believed to be the primary factor affecting its activity, AS160 also possesses a calmodulin-binding domain (CBD). This raises the possibility that contraction-stimulated increases in Ca(2+)/calmodulin could also modulate AS160 function. RESEARCH DESIGN AND METHODS: To evaluate the AS160 CBD in skeletal muscle, empty-vector, wild-type, or CBD-mutant AS160 cDNAs were injected into mouse muscles followed by in vivo electroporation. One week later, AS160 was overexpressed by approximately 14-fold over endogenous protein. RESULTS: Immunoprecipitates of wild-type and CBD-mutant AS160 were incubated with biotinylated calmodulin in the presence of Ca(2+). Wild-type AS160, but not the CBD-mutant AS160, associated with calmodulin. Next, we measured insulin- and contraction-stimulated glucose uptake in vivo. Compared with empty-vector and wild-type AS160, insulin-stimulated glucose uptake was not altered in muscles expressing CBD-mutant AS160. In contrast, contraction-stimulated glucose uptake was significantly decreased in CBD-mutant-expressing muscles. This inhibitory effect on glucose uptake was not associated with aberrant contraction-stimulated AS160 phosphorylation. Interestingly, AS160 expressing both calmodulin-binding and Rab-GAP (GTPase-activating protein) domain point mutations (CBD + R/K) fully restored contraction-stimulated glucose uptake. CONCLUSIONS: Our results suggest that the AS160 CBD directly regulates contraction-induced glucose uptake in mouse muscle and that calmodulin provides an additional means of modulating AS160 Rab-GAP function independent of phosphorylation. These findings define a novel AS160 signaling component, unique to contraction and not insulin, leading to glucose uptake in skeletal muscle.
OBJECTIVE: Insulin and contraction increase skeletal muscle glucose uptake through distinct and additive mechanisms. However, recent reports have demonstrated that both signals converge on the Akt substrate of 160 kDa (AS160), a protein that regulates GLUT4 translocation. Although AS160 phosphorylation is believed to be the primary factor affecting its activity, AS160 also possesses a calmodulin-binding domain (CBD). This raises the possibility that contraction-stimulated increases in Ca(2+)/calmodulin could also modulate AS160 function. RESEARCH DESIGN AND METHODS: To evaluate the AS160 CBD in skeletal muscle, empty-vector, wild-type, or CBD-mutant AS160 cDNAs were injected into mouse muscles followed by in vivo electroporation. One week later, AS160 was overexpressed by approximately 14-fold over endogenous protein. RESULTS: Immunoprecipitates of wild-type and CBD-mutant AS160 were incubated with biotinylated calmodulin in the presence of Ca(2+). Wild-type AS160, but not the CBD-mutant AS160, associated with calmodulin. Next, we measured insulin- and contraction-stimulated glucose uptake in vivo. Compared with empty-vector and wild-type AS160, insulin-stimulated glucose uptake was not altered in muscles expressing CBD-mutant AS160. In contrast, contraction-stimulated glucose uptake was significantly decreased in CBD-mutant-expressing muscles. This inhibitory effect on glucose uptake was not associated with aberrant contraction-stimulated AS160 phosphorylation. Interestingly, AS160 expressing both calmodulin-binding and Rab-GAP (GTPase-activating protein) domain point mutations (CBD + R/K) fully restored contraction-stimulated glucose uptake. CONCLUSIONS: Our results suggest that the AS160 CBD directly regulates contraction-induced glucose uptake in mouse muscle and that calmodulin provides an additional means of modulating AS160 Rab-GAP function independent of phosphorylation. These findings define a novel AS160 signaling component, unique to contraction and not insulin, leading to glucose uptake in skeletal muscle.
Authors: Samaneh Mafakheri; Ralf R Flörke; Sibylle Kanngießer; Sonja Hartwig; Lena Espelage; Christian De Wendt; Tina Schönberger; Nele Hamker; Stefan Lehr; Alexandra Chadt; Hadi Al-Hasani Journal: J Biol Chem Date: 2018-10-01 Impact factor: 5.157
Authors: Hong Yang; Jie Tang; Dong Guo; Qingqing Zhao; Jiagen Wen; Yanjuan Zhang; Obinna N Obianom; Shiwei Zhou; Wei Zhang; Yan Shu Journal: Kidney Int Date: 2019-11-26 Impact factor: 10.612