Abstract
Sortilin has been implicated in the formation of insulin-responsive GLUT4 storage vesicles in adipocytes by regulating sorting events between the trans-Golgi-network and endosomes. We herein show that sortilin serves as a potent myogenic differentiation stimulator for C2C12 myocytes by cooperatively functioning with p75NTR, which subsequently further contributes to development of the insulin-responsive glucose transport system in C2C12 myotubes. Sortilin expression was up-regulated upon C2C12 differentiation, and overexpression of sortilin in C2C12 cells significantly stimulated myogenic differentiation, a response that was completely abolished by either anti-p75NTR- or anti-nerve growth factor (NGF)-neutralizing antibodies. Importantly, small interference RNA-mediated suppression of endogenous sortilin significantly inhibited C2C12 differentiation, indicating the physiological significance of sortilin expression in the process of myogenesis. Although sortilin overexpression in C2C12 myotubes improved insulin-induced 2-deoxyglucose uptake, as previously reported, this effect apparently resulted from a decrease in the cellular content of GLUT1 and an increase in GLUT4 via differentiation-dependent alterations at both the gene transcriptional and the post-translational level. In addition, cellular contents of Ubc9 and SUMO-modified proteins appeared to be increased by sortilin overexpression. Taken together, these data demonstrate that sortilin is involved not only in development of the insulin-responsive glucose transport system in myocytes, but is also directly involved in muscle differentiation via modulation of proNGF-p75NTR.
Highlights
One of the major physiological roles of insulin is the control of postprandial blood glucose levels, and skeletal muscle is the primary tissue responsible for the bulk (70 – 80%) of insulin
The effect of insulin on overall glucose disposal is primarily achieved via stimulation of glucose uptake into insulin-target tissues, and defects in this insulin action in skeletal muscle contribute to development of the insulin resistance which is characteristic of type 2 diabetes [3]
Glucose transport is regulated by a facilitative glucose transport system involving at least two members of the glucose transporter family, GLUT1 and GLUT4 [4], and their expression levels are strictly regulated during myocyte differentiation [5, 6]
Summary
Materials—We obtained 2-deoxy-[3H]glucose (37.2 Ci/mmol) from PerkinElmer Life Sciences. Anti-Myc Antibody Uptake Assay—For the measurement of insulin-induced GLUT4 translocation, the C2C12 cells expressing Myc-GLUT4-ECFP were starved in serum-free DMEM for 4 h, washed three times with KRPH buffer, and placed in a CO2 incubator with 2 ml of KRPH buffer. After the protein concentration had been determined, bromphenol blue and 2-mercaptoethanol were added, denatured by incubation for 5 min at 95 °C, and subjected to Western blotting using anti-mouse IgG antibody, anti-Myc antibody, or anti-Sortilin antibody. For detection of GLUT1, the total membrane fraction was lysed in 100 l of 1ϫ Laemmli sample buffer, and subjected to Western blotting using anti-GLUT1 antibody (1:5840). Cell lysates were subjected to SDS-PAGE, followed by Western blotting analysis using anti-GLUT1 or anti-Myc antibody. Differences were considered to be significant at *, p Ͻ 0.05; **, p Ͻ 0.01; and ***, p Ͻ 0.001
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