Matheny Ronald W, Abdalla Mary N, Geddis Alyssa V, Leandry Luis A, Lynch Christine M
Military Performance Division, US Army Research Institute of Environmental Medicine, 10 General Greene Ave, Building 42, Natick, MA, 01760, USA.
Military Performance Division, US Army Research Institute of Environmental Medicine, 10 General Greene Ave, Building 42, Natick, MA, 01760, USA.
Biochem Biophys Res Commun. 2017 Jan 22;482(4):1420-1426. doi: 10.1016/j.bbrc.2016.12.051. Epub 2016 Dec 10.
Skeletal muscle metabolic homeostasis is maintained through numerous biochemical and physiological processes. Two principal molecular regulators of skeletal muscle metabolism include AMP-activated protein kinase (AMPK) and phosphatidylinositol 3-kinase (PI3K); however, PI3K exists as multiple isoforms, and specific metabolic actions of each isoform have not yet been fully elucidated in skeletal muscle. Given this lack of knowledge, we performed a series of experiments to define the extent to which PI3K p110β mediated expression and (or) activation of AMPK in skeletal muscle. To determine the effect of p110β inhibition on AMPK expression and phosphorylation in cultured cells, C2C12 myoblasts were treated with a pharmacological inhibitor of p110β (TGX-221), siRNA against p110β, or overexpression of kinase-dead p110β. Expression and phosphorylation of AMPK were unaffected in myoblasts treated with TGX-221 or expressing kinase-dead p110β. However, expressions of total and phosphorylated AMPK at T172 were reduced in myoblasts treated with p110β siRNA. When normalized to expression of total AMPK, phosphorylation of AMPK S485/491 was elevated in p110β-deficient myoblasts. Similar results were observed in tibialis anterior muscle from mice with conditional deletion of p110β (p110β-mKO mice). Analysis of AMPK transcript expression revealed decreased expression of Prkaa2 in p110β-deficient myoblasts and in p110β-mKO muscle. Loss of p110β had no effect on oligomycin-stimulated phosphorylation of AMPK or phosphorylated Acetyl-CoA carboxylase (ACC), although oligomycin-induced AMPK and ACC phosphorylation were increased in p110β-deficient myoblasts compared to oligomycin-stimulated control myoblasts when normalized to levels of total AMPK or ACC. Overall, these results suggest that p110β positively regulates expression of AMPK in cultured myoblasts and in skeletal muscle in vivo; moreover, despite the reduced abundance of AMPK in p110β-deficient myoblasts, loss of p110β does not appear to impair AMPK activation following stimulus. These findings thus reveal a novel role for p110β in mediating skeletal muscle metabolic signaling.
骨骼肌代谢稳态通过众多生化和生理过程得以维持。骨骼肌代谢的两个主要分子调节因子包括AMP活化蛋白激酶（AMPK）和磷脂酰肌醇3激酶（PI3K）；然而，PI3K以多种异构体形式存在，且每种异构体在骨骼肌中的具体代谢作用尚未完全阐明。鉴于这方面知识的欠缺，我们进行了一系列实验，以确定PI3K p110β在骨骼肌中介导AMPK表达和（或）激活的程度。为了确定p110β抑制对培养细胞中AMPK表达和磷酸化的影响，用p110β的药理学抑制剂（TGX-221）、针对p110β的小干扰RNA（siRNA）或激酶失活的p110β过表达处理C2C12成肌细胞。用TGX-221处理或表达激酶失活p110β的成肌细胞中，AMPK的表达和磷酸化不受影响。然而，用p110β siRNA处理的成肌细胞中，T172位点的总AMPK和磷酸化AMPK的表达均降低。当以总AMPK的表达进行标准化时，p110β缺陷型成肌细胞中AMPK S485/491位点的磷酸化水平升高。在条件性缺失p110β的小鼠（p110β-mKO小鼠）的胫前肌中也观察到了类似结果。对AMPK转录本表达的分析显示，p110β缺陷型成肌细胞和p110β-mKO肌肉中Prkaa2的表达降低。p110β的缺失对寡霉素刺激的AMPK磷酸化或磷酸化乙酰辅酶A羧化酶（ACC）没有影响，尽管当以总AMPK或ACC的水平进行标准化时，与寡霉素刺激的对照成肌细胞相比，寡霉素诱导的p110β缺陷型成肌细胞中AMPK和ACC的磷酸化增加。总体而言，这些结果表明，p110β在培养的成肌细胞和体内骨骼肌中正向调节AMPK的表达；此外，尽管p110β缺陷型成肌细胞中AMPK的丰度降低，但p110β的缺失似乎并不损害刺激后AMPK的激活。因此，这些发现揭示了p110β在介导骨骼肌代谢信号传导中的新作用。
