Stierwalt Harrison D, Ehrlicher Sarah E, Bergman Bryan C, Robinson Matthew M, Newsom Sean A
School of Biological and Population Health Sciences, College of Public Health and Human Sciences, Oregon State University, Corvallis, Oregon.
Division of Endocrinology, Metabolism and Diabetes, School of Medicine, University of Colorado Denver, Denver, Colorado.
Physiol Rep. 2018 Dec;6(24):e13956. doi: 10.14814/phy2.13956.
Ras-related C3 botulinum toxin substrate 1 (Rac1) is required for normal insulin-stimulated glucose transport in skeletal muscle and evidence indicates Rac1 may be negatively regulated by lipids. We investigated if insulin-stimulated activation of Rac1 (i.e., Rac1-GTP binding) is impaired by accumulation of diacylglycerols (DAG) and ceramides in cultured muscle cells. Treating L6 myotubes with 100 nmol/L insulin resulted in increased Rac1-GTP binding that was rapid (occurring within 2 min), relatively modest (+38 ± 19% vs. basal, P < 0.001), and short-lived, returning to near-basal levels within 15 min of continuous treatment. Incubating L6 myotubes overnight in 500 μmol/L palmitate increased the accumulation of DAG and ceramides (P < 0.05 vs. no fatty acid control). Despite significant accumulation of lipids, insulin-stimulated Rac1-GTP binding was not impaired during palmitate treatment (P = 0.39 vs. no fatty acid control). Nevertheless, phosphorylation of Rac1 effector protein p21-activated kinase (PAK) was attenuated in response to palmitate treatment (P = 0.02 vs. no fatty acid control). Palmitate treatment also increased inhibitory phosphorylation of insulin receptor substrate-1 and attenuated insulin-stimulated phosphorylation of Akt at both Thr308 and Ser473 (all P < 0.05 vs. no fatty acid control). Such signaling impairments resulted in near complete inhibition of insulin-stimulated translocation of glucose transporter protein 4 (GLUT4; P = 0.10 vs. basal during palmitate treatment). In summary, our finding suggests that Rac1 may not undergo negative regulation by DAG or ceramides. We instead provide evidence that attenuated PAK phosphorylation and impaired GLUT4 translocation during palmitate-induced insulin resistance can occur independent of defects in insulin-stimulated Rac1-GTP binding.
Ras相关的C3肉毒杆菌毒素底物1（Rac1）是骨骼肌中正常胰岛素刺激的葡萄糖转运所必需的，并且有证据表明Rac1可能受到脂质的负调控。我们研究了在培养的肌肉细胞中，二酰基甘油（DAG）和神经酰胺的积累是否会损害胰岛素刺激的Rac1激活（即Rac1-GTP结合）。用100 nmol/L胰岛素处理L6肌管会导致Rac1-GTP结合增加，这种增加迅速（在2分钟内发生），相对适度（与基础水平相比增加38±19%，P<0.001），且持续时间短，在连续处理15分钟内恢复到接近基础水平。将L6肌管用500 μmol/L棕榈酸孵育过夜会增加DAG和神经酰胺的积累（与无脂肪酸对照相比，P<0.05）。尽管脂质大量积累，但在棕榈酸处理期间，胰岛素刺激的Rac1-GTP结合并未受损（与无脂肪酸对照相比，P=0.39）。然而，Rac1效应蛋白p21激活激酶（PAK）磷酸化在棕榈酸处理后减弱（与无脂肪酸对照相比，P=0.02）。棕榈酸处理还增加了胰岛素受体底物-1的抑制性磷酸化，并减弱了胰岛素刺激的Akt在Thr308和Ser473位点的磷酸化（与无脂肪酸对照相比，所有P<0.05）。这些信号转导损伤导致胰岛素刺激的葡萄糖转运蛋白4（GLUT4）转位几乎完全受到抑制（在棕榈酸处理期间与基础水平相比，P=0.10）。总之，我们的发现表明Rac1可能不会受到DAG或神经酰胺的负调控。相反，我们提供的证据表明，在棕榈酸诱导的胰岛素抵抗期间，PAK磷酸化减弱和GLUT4转位受损可能独立于胰岛素刺激的Rac1-GTP结合缺陷而发生。
