Abstract
Neuregulin, a growth factor involved in myogenesis, has rapid effects on muscle metabolism. In a manner analogous to insulin and exercise, neuregulins stimulate glucose transport through recruitment of glucose transporters to surface membranes in skeletal muscle. Like muscle contraction, neuregulins have additive effects with insulin on glucose uptake. Therefore, we examined whether neuregulins are involved in the mechanism by which muscle contraction regulates glucose transport. We show that caffeine-induced increases in cytosolic Ca2+ mediate a metalloproteinase-dependent release of neuregulins, which stimulates tyrosine phosphorylation of ErbB4 receptors. Activation of ErbB4 is necessary for Ca2+-derived effects on glucose transport. Furthermore, blockage of ErbB4 abruptly impairs contraction-induced glucose uptake in slow twitch muscle fibers, and to a lesser extent, in fast twitch muscle fibers. In conclusion, we provide evidence that contraction-induced activation of neuregulin receptors is necessary for the stimulation of glucose transport and a key element of energetic metabolism during muscle contraction.
Highlights
Since neuregulin receptors are activated during contraction [8], 3 The abbreviations used are: AMPK, AMP-activated protein kinase; HRG/Hrg, recombinant heregulin-1-(177–244); calmodulindependent kinase II (CAMKII), Ca2ϩ/calmodulin-dependent kinase II; EGF, epidermal growth factor; AICAR, 5-aminoimidazole-4carboxamide-1--D-ribofuranoside; ACC, acetyl coenzyme A carboxylase; PI3K, phosphatidylinositol-3 kinase; Extensor digitorum longus (EDL), extensor digitorum longus; PBS, phosphate-buffered saline
Contraction stimulated phosphorylation of ErbB4 and ErbB2 but not ErbB3 receptors (Fig. 1A). This is consistent with the finding that an in vitro exposure of skeletal muscle to saturating exogenous neuregulins induced tyrosine phosphorylation of ErbB4 and ErbB2 but only induced weak phosphorylation of ErbB3 (Fig. 1, A and B) [15]. These results indicated that ErbB2 and ErbB4 are the main neuregulin receptors in adult skeletal muscle
We tested whether induction of ErbB receptor phosphorylation during contraction was caused by AMPK activation or by increases in cytosolic Ca2ϩ
Summary
Reagents and Materials—Purified porcine insulin was a kind gift from Eli Lilly Co. A recombinant heuregulin 1 isoform containing the bioactive EGF domain, heregulin-1-(177–244) (HRG), was donated by Genentech, Inc. (South San Francisco, CA). Muscles were subjected to contraction as described previously [13] and either frozen immediately to obtain total lysates or further incubated to assess glucose transport activity or cell surface GLUT4 content. Samples were immunoprecipitated to identify ErbB2, ErbB3, ErbB4, and insulin receptor phosphorylation levels by conjugating 30 l of protein G-Sepharose beads with 2–5 g of the corresponding polyclonal antibody (except for ErbB4, which was monoclonal) for 1 h at 4 °C and washing twice in lysis buffer and incubating with 0.5–1 mg of protein lysate overnight with constant shaking at 4 °C. The pellet was washed several times in the lysis buffer and boiled with 50 l of Laemmli sample buffer (LBS) for Western blot analysis using a monoclonal anti-phosphotyrosine antibody or polyclonal ErbB4 antibody to determine phosphorylated or total ErbB4 content. Analysis of variance, assessed by Bonferonni’s multiple comparison test, was used to compare more than two groups
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