Differential contribution of insulin receptor substrates 1 versus 2 to insulin signaling and glucose uptake in l6 myotubes.
Insulin receptor substrates-1 and 2 (IRS-1 and IRS-2) are pivotal in relaying insulin signaling in insulin-responsive tissues such as muscle. However, the precise contribution of IRS-1 vis-a-vis IRS-2 in insulin-mediated metabolic and mitogenic responses has not been compared directly in differentiated muscle cells. This study aimed to determine the relative contribution of IRS-1 versus IRS-2 in these responses, using small interfering RNA (siRNA)-mediated specific gene silencing. In L6 myotubes, transfection of siRNA targeted specifically against IRS-1 (siIRS-1) or IRS-2 (siIRS-2) reduced the cognate protein expression by 70-75%. Insulin-induced ERK phosphorylation was much more sensitive to IRS-2 than IRS-1 ablation, whereas p38MAPK phosphorylation was reduced by 43 or 62% in myotubes treated with siIRS-1 or siIRS-2, respectively. Insulin-induced Akt1 and Akt2 phosphorylation was reduced in myotubes treated with siIRS-1, but only Akt2 phosphorylation was reduced in myotubes treated with siIRS-2. In contrast, siIRS-1 treatment caused a marked reduction in insulin-induced actin remodeling, glucose uptake, and GLUT4 translocation, and siIRS-2 was without effect on these responses. Notably, combined siIRS-1 and siIRS-2, although reducing each IRS by around 75%, caused no further drop in glucose uptake than that achieved with siIRS-1 alone, but abolished p38MAPK phosphorylation. We conclude that insulin-stimulated Akt1 phosphorylation, actin remodeling, GLUT4 translocation, and glucose uptake are regulated mainly by IRS-1, whereas IRS-2 contributes selectively to ERK signaling, and Akt2 and p38MAPK lie downstream of both IRS in muscle cells.
Huang
Carol
C
Program in Cell Biology, The Hospital for Sick Children, Toronto, Ontario M5G 1X8, Canada.
Thirone
Ana C P
AC
Huang
Xudong
X
Klip
Amira
A
D000199:Actins / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D002454:Cell Differentiation; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D020868:Gene Silencing; D005947:Glucose / Q000378:metabolism*; D051275:Glucose Transporter Type 4; D047468:Immunoprecipitation; D007328:Insulin / Q000378:metabolism*; D055504:Insulin Receptor Substrate Proteins; D047908:Intracellular Signaling Peptides and Proteins; D051379:Mice; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D009004:Monosaccharide Transport Proteins / Q000378:metabolism; D009124:Muscle Proteins / Q000378:metabolism; D010750:Phosphoproteins / Q000502:physiology*; D010766:Phosphorylation; D020033:Protein Isoforms; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D021381:Protein Transport; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt; D034741:RNA, Small Interfering / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D014162:Transfection; D014443:Tyrosine / Q000737:chemistry; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
D000199:Actins; D051275:Glucose Transporter Type 4; D007328:Insulin; D055504:Insulin Receptor Substrate Proteins; D047908:Intracellular Signaling Peptides and Proteins; C526295:Irs1 protein, mouse; C526298:Irs2 protein, mouse; D009004:Monosaccharide Transport Proteins; D009124:Muscle Proteins; D010750:Phosphoproteins; D020033:Protein Isoforms; D011518:Proto-Oncogene Proteins; D034741:RNA, Small Interfering; C495693:Slc2a4 protein, mouse; D014443:Tyrosine; C494922:Akt2 protein, mouse; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D048049:Extracellular Signal-Regulated MAP Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D005947:Glucose