Human Glycated Albumin Affects Glucose Metabolism in L6 Skeletal Muscle Cells by Impairing Insulin-induced Insulin Receptor Substrate (IRS) Signaling through a Protein Kinase Cα-mediated Mechanism

Abstract Transduction of the insulin signal is mediated by multisite Tyr and Ser/Thr phosphorylation of the insulin receptor substrates (IRSs). Previous studies on the function of single-site phosphorylation, particularly phosphorylation of Ser-302, -307, and -318 of IRS-1, showed attenuating as well as enhancing effects on insulin action. In this study we investigated a possible cross talk of these opposedly acting serine residues in insulin-stimulated skeletal muscle cells by monitoring phosphorylation kinetics, and applying loss of function, gain of function, and combination mutants of IRS-1. The phosphorylation at Ser-302 was rapid and transient, followed first by Ser-318 phosphorylation and later by phosphorylation of Ser-307, which remained elevated for 120 min. Mutation of Ser-302 to alanine clearly reduced the subsequent protein kinase C-ζ-mediated Ser-318 phosphorylation. The Ser-307 phosphorylation was independent of Ser-302 and/or Ser-318 phosphorylation status. The functional consequences of these phosphorylation patterns were studied by the expression of IRS-1 mutants. The E302A307E318 mutant simulating the early phosphorylation pattern resulted in a significant increase in Akt and glycogen synthase kinase 3 phosphorylation. Furthermore, glucose uptake was enhanced. Because the down-regulation of the insulin signal was not affected, this phosphorylation pattern seems to be involved in the enhancement but not in the termination of the insulin signal. This enhancing effect was completely absent when Ser-302 was unphosphorylated and Ser-307 was phosphorylated as simulated by the A302E307E318 mutant. Phospho-Ser-318, sequentially phosphorylated at least by protein kinase C-ζ and a mammalian target of rapamycin/raptor-dependent kinase, was part of the positive as well as of the subsequent negative phosphorylation pattern. Thus we conclude that insulin stimulation temporally generates different phosphorylation statuses of the same residues that exert different functions in insulin signaling.

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Myostatin regulates glucose metabolism via the AMP-activated protein kinase pathway in skeletal muscle cells

The International Journal of Biochemistry & Cell Biology ◽

10.1016/j.biocel.2010.09.017 ◽

2010 ◽

Vol 42 (12) ◽

pp. 2072-2081 ◽

Cited By ~ 53

Author(s):

Yuewen Chen ◽

Jianwei Ye ◽

Lingzhi Cao ◽

Yong Zhang ◽

Weibo Xia ◽

...

Keyword(s):

Skeletal Muscle ◽

Protein Kinase ◽

Glucose Metabolism ◽

Muscle Cells ◽

Skeletal Muscle Cells ◽

Amp Activated Protein Kinase ◽

Kinase Pathway

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Loss of HIF-1α impairs GLUT4 translocation and glucose uptake by the skeletal muscle cells

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.00597.2012 ◽

2014 ◽

Vol 306 (9) ◽

pp. E1065-E1076 ◽

Cited By ~ 31

Author(s):

Hidemitsu Sakagami ◽

Yuichi Makino ◽

Katsutoshi Mizumoto ◽

Tsubasa Isoe ◽

Yasutaka Takeda ◽

...

Keyword(s):

Type 2 Diabetes ◽

Skeletal Muscle ◽

Protein Kinase ◽

Glucose Metabolism ◽

Glucose Uptake ◽

Intracellular Signaling ◽

Muscle Cells ◽

Skeletal Muscle Cells ◽

Glut4 Translocation

Defects in glucose uptake by the skeletal muscle cause diseases linked to metabolic disturbance such as type 2 diabetes. The molecular mechanism determining glucose disposal in the skeletal muscle in response to cellular stimuli including insulin, however, remains largely unknown. The hypoxia-inducible factor-1α (HIF-1α) is a transcription factor operating in the cellular adaptive response to hypoxic conditions. Recent studies have uncovered pleiotropic actions of HIF-1α in the homeostatic response to various cellular stimuli, including insulin under normoxic conditions. Thus we hypothesized HIF-1α is involved in the regulation of glucose metabolism stimulated by insulin in the skeletal muscle. To this end, we generated C2C12myocytes in which HIF-1α is knocked down by short-hairpin RNA and examined the intracellular signaling cascade and glucose uptake subsequent to insulin stimulation. Knockdown of HIF-1α expression in the skeletal muscle cells resulted in abrogation of insulin-stimulated glucose uptake associated with impaired mobilization of glucose transporter 4 (GLUT4) to the plasma membrane. Such defect seemed to be caused by reduced phosphorylation of the protein kinase B substrate of 160 kDa (AS160). AS160 phosphorylation and GLUT4 translocation by AMP-activated protein kinase activation were abrogated as well. In addition, expression of the constitutively active mutant of HIF-1α (CA-HIF-1α) or upregulation of endogenous HIF-1α in C2C12cells shows AS160 phosphorylation comparable to the insulin-stimulated level even in the absence of insulin. Accordingly GLUT4 translocation was increased in the cells expressing CA-HIF1α. Taken together, HIF-1α is a determinant for GLUT4-mediated glucose uptake in the skeletal muscle cells thus as a possible target to alleviate impaired glucose metabolism in, e.g., type 2 diabetes.

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