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Abstract：
OBJECTIVE: A number of studies have highlighted muscle-specific mechanisms of thermogenesis involving futile cycling of Ca2+ driven by sarco (endo)plasmic reticulum Ca2+-ATPase (SERCA) and generating heat from ATP hydrolysis to be a promising strategy to counteract obesity and metabolic dysfunction. However, to the best of our knowledge, no experimental studies concerning the metabolic effects of pharmacologically targeting SERCA in human skeletal muscle cells have been reported. Thus, in the present study, we aimed to explore the effects of SERCA-activating compound, CDN1163, on energy metabolism in differentiated human skeletal muscle cells (myotubes).
METHODS: In this study, we used primary myotube cultures derived from muscle biopsies of the musculus vastus lateralis and musculi interspinales from lean, healthy male donors. Energy metabolism in myotubes was studied using radioactive substrates. Oxygen consumption rate was assessed with the Seahorse XF24 bioanalyzer, whereas metabolic genes and protein expressions were determined by qPCR and immunoblotting, respectively.
RESULTS: Both acute (4 h) and chronic (5 days) treatment of myotubes with CDN1163 showed increased uptake and oxidation of glucose, as well as complete fatty acid oxidation in the presence of carbonyl cyanide 4-(trifluromethoxy)phenylhydrazone (FCCP). These effects were supported by measurement of oxygen consumption rate, in which the oxidative spare capacity and maximal respiration were enhanced after CDN1163-treatment. In addition, chronic treatment with CDN1163 improved cellular uptake of oleic acid (OA) and fatty acid β-oxidation. The increased OA metabolism was accompanied by enhanced mRNA-expression of carnitine palmitoyl transferase (CPT) 1B, pyruvate dehydrogenase kinase (PDK) 4, as well as increased AMP-activated protein kinase (AMPK)Thr172 phosphorylation. Moreover, following chronic CDN1163 treatment, the expression levels of stearoyl-CoA desaturase (SCD) 1 was decreased together with de novo lipogenesis from acetic acid and formation of diacylglycerol (DAG) from OA.
CONCLUSIONS: Altogether, these results suggest that SERCA activation by CDN1163 enhances energy metabolism in human myotubes, which might be favourable in relation to disorders that are related to metabolic dysfunction such as obesity and type 2 diabetes mellitus.
METHODS: In this study, we used primary myotube cultures derived from muscle biopsies of the musculus vastus lateralis and musculi interspinales from lean, healthy male donors. Energy metabolism in myotubes was studied using radioactive substrates. Oxygen consumption rate was assessed with the Seahorse XF24 bioanalyzer, whereas metabolic genes and protein expressions were determined by qPCR and immunoblotting, respectively.
RESULTS: Both acute (4 h) and chronic (5 days) treatment of myotubes with CDN1163 showed increased uptake and oxidation of glucose, as well as complete fatty acid oxidation in the presence of carbonyl cyanide 4-(trifluromethoxy)phenylhydrazone (FCCP). These effects were supported by measurement of oxygen consumption rate, in which the oxidative spare capacity and maximal respiration were enhanced after CDN1163-treatment. In addition, chronic treatment with CDN1163 improved cellular uptake of oleic acid (OA) and fatty acid β-oxidation. The increased OA metabolism was accompanied by enhanced mRNA-expression of carnitine palmitoyl transferase (CPT) 1B, pyruvate dehydrogenase kinase (PDK) 4, as well as increased AMP-activated protein kinase (AMPK)Thr172 phosphorylation. Moreover, following chronic CDN1163 treatment, the expression levels of stearoyl-CoA desaturase (SCD) 1 was decreased together with de novo lipogenesis from acetic acid and formation of diacylglycerol (DAG) from OA.
CONCLUSIONS: Altogether, these results suggest that SERCA activation by CDN1163 enhances energy metabolism in human myotubes, which might be favourable in relation to disorders that are related to metabolic dysfunction such as obesity and type 2 diabetes mellitus.
摘要：
目的：许多研究强调了肌肉特异性的产热机制，涉及由sarco（endo）质网Ca2+-ATPase（SERCA）驱动的Ca2+的无效循环，并通过ATP水解产生热量，这是一种有希望的抵抗肥胖和代谢功能障碍的策略。然而,据我们所知,目前还没有关于药理学靶向SERCA在人骨骼肌细胞中的代谢作用的实验研究报道.因此，在本研究中，我们旨在探索SERCA激活化合物的作用，CDN1163，对分化的人骨骼肌细胞（肌管）能量代谢的影响。
方法：在本研究中，我们使用了来自股外侧肌的肌肉活检和来自瘦肌间肌的原发性肌管培养物，健康的男性捐赠者。使用放射性底物研究了肌管中的能量代谢。用海马XF24生物分析仪评估耗氧率，而代谢基因和蛋白质表达是通过qPCR和免疫印迹确定的，分别。
结果：用CDN1163治疗肌管的急性（4小时）和慢性（5天）均显示葡萄糖的摄取和氧化增加，以及在羰基氰4-（三氟甲氧基）苯基腙（FCCP）存在下的完全脂肪酸氧化。这些影响得到了氧气消耗率测量的支持，其中CDN1163治疗后氧化备用容量和最大呼吸增强。此外,CDN1163的慢性治疗可改善细胞对油酸（OA）的摄取和脂肪酸β-氧化。OA代谢增加伴随着肉碱棕榈酰转移酶（CPT）1B的mRNA表达增强，丙酮酸脱氢酶激酶（PDK）4，以及增加AMP激活的蛋白激酶（AMPK）Thr172磷酸化。此外，慢性CDN1163治疗后，硬脂酰辅酶A去饱和酶（SCD）1的表达水平降低，同时乙酸从头生成脂肪和OA形成二酰甘油（DAG）。
结论：总而言之，这些结果表明，CDN1163激活SERCA可以增强人体肌管的能量代谢，这可能有利于与代谢功能障碍相关的疾病，如肥胖和2型糖尿病。
方法：在本研究中，我们使用了来自股外侧肌的肌肉活检和来自瘦肌间肌的原发性肌管培养物，健康的男性捐赠者。使用放射性底物研究了肌管中的能量代谢。用海马XF24生物分析仪评估耗氧率，而代谢基因和蛋白质表达是通过qPCR和免疫印迹确定的，分别。
结果：用CDN1163治疗肌管的急性（4小时）和慢性（5天）均显示葡萄糖的摄取和氧化增加，以及在羰基氰4-（三氟甲氧基）苯基腙（FCCP）存在下的完全脂肪酸氧化。这些影响得到了氧气消耗率测量的支持，其中CDN1163治疗后氧化备用容量和最大呼吸增强。此外,CDN1163的慢性治疗可改善细胞对油酸（OA）的摄取和脂肪酸β-氧化。OA代谢增加伴随着肉碱棕榈酰转移酶（CPT）1B的mRNA表达增强，丙酮酸脱氢酶激酶（PDK）4，以及增加AMP激活的蛋白激酶（AMPK）Thr172磷酸化。此外，慢性CDN1163治疗后，硬脂酰辅酶A去饱和酶（SCD）1的表达水平降低，同时乙酸从头生成脂肪和OA形成二酰甘油（DAG）。
结论：总而言之，这些结果表明，CDN1163激活SERCA可以增强人体肌管的能量代谢，这可能有利于与代谢功能障碍相关的疾病，如肥胖和2型糖尿病。
