Muscle Involvement in Preservation of Metabolic Flexibility by Treatment using n-3 PUFA or Rosiglitazone in Dietary-Obese Mice

Impaired resistance to insulin, the key defect in type 2 diabetes (T2D), is associated with a low capacity to adapt fuel oxidation to fuel availability, i.e., metabolic inflexibility. The hampered metabolic adaptability triggers a further damage of insulin signaling. Since skeletal muscle is the main site of glucose uptake, effectiveness of T2D treatment depends in large on the improvement of insulin sensitivity and metabolic adaptability of the muscle. We have shown previously in mice fed an obesogenic high-fat diet that a combination treatment using n-3 long-chain polyunsaturated fatty acids (n-3 LC-PUFA) and thiazolidinedione (TZD) anti-diabetic drugs preserved metabolic health and synergistically improved muscle insulin sensitivity. We investigated here whether TZD rosiglitazone could elicit the additive beneficial effects on metabolic flexibility when combined with n-3 LC-PUFA. Adult male C57BL/6N mice were fed an obesogenic corn oil-based high-fat diet (cHF) for 8 weeks, or randomly assigned to various dietary treatments: (i) cHF+F, cHF with n-3 LC-PUFA concentrate replacing 15% of dietary lipids; (ii) cHF+ROSI, cHF with 10 mg rosiglitazone/kg diet; and (iii) cHF+F+ROSI, or chow-fed. Indirect calorimetry demonstrated superior preservation of metabolic flexibility to carbohydrates in response to the combination treatment. Metabolomic and gene expression analyses in the muscle suggested distinct and complementary effects of the single treatments, with rosiglitazone augmenting insulin sensitivity by the modulation of branched-chain amino acid metabolism, and n-3 LC PUFA supporting complete oxidation of fatty acids in mitochondria. These beneficial metabolic effects were associated with the activation of the switch between glycolytic and oxidative muscle fibers, especially in the cHF+F+ROSI mice. Our results further support the idea that the combination treatment using n-3 LC-PUFA and TZDs could improve the efficacy of the treatment of obese and diabetic patients. Male C57BL/6N mice had free access to water and Chow. Three-month-old mice were randomly assigned (8 animals per group) to cHF diet (lipid content ~35% wt/wt) or to the following 'treatments' by isocaloric cHF-based diets, namely (i) cHF+F, cHF diet supplemented with n-3 LC-PUFA concentrate (46% DHA, 14% EPA, wt/wt, as triglycerides; product EPAX 1050 TG), which replaced 15% wt/wt of dietary lipids; (ii) cHF+ROSI, cHF diet supplemented with 10 mg rosiglitazone/kg diet; and (iii) cHF+F+ROSI, cHF diet supplemented with both n-3 LC-PUFA concentrate and rosiglitazone. cHF+ROSI at a higher dose, namely 100 mg rosiglitazone/kg diet was also included in the study, but not in the final microarray analysis. The treatment lasted for 8 weeks, whereafter the animals were first fasted for 10 hours during the light phase of the day (between 8.00hr and 18.00hr), than re-fed Chow (starting at 18.00hr), and killed the following day by cervical dislocation under pentobarbital anesthesia (between 9.00hr and 11.00hr); the so-called 'diet-switch protocol'. Gastrocnemius muscle was isolated and used for total RNA isolation.

胰岛素抵抗作为2型糖尿病（T2D）的核心病理缺陷，与机体适配底物氧化以匹配底物可利用性的能力低下（即代谢灵活性受损）密切相关。受损的代谢适应性会进一步加重胰岛素信号通路的损伤。由于骨骼肌是机体摄取葡萄糖的主要部位，2型糖尿病的治疗有效性在很大程度上取决于肌肉胰岛素敏感性与代谢适应性的改善。本团队此前在饲喂致肥胖高脂饲料的小鼠模型中证实，联合使用n-3长链多不饱和脂肪酸（n-3 LC-PUFA）与噻唑烷二酮类（TZD）降糖药物，可维持小鼠代谢健康，并协同改善肌肉胰岛素敏感性。本研究旨在探究：将TZD类药物罗格列酮与n-3 LC-PUFA联合使用时，能否对代谢灵活性产生叠加有益效应。实验选用成年雄性C57BL/6N小鼠，将其分为两组：一组饲喂玉米油基致肥胖高脂饲料（cHF）8周，另一组随机接受不同饮食干预：(i) cHF+F组：用n-3 LC-PUFA浓缩物替代15%膳食脂质的cHF饲料；(ii) cHF+ROSI组：每千克饲料添加10mg罗格列酮的cHF饲料；(iii) cHF+F+ROSI组：同时添加n-3 LC-PUFA浓缩物与罗格列酮的cHF饲料，另设普通饲料饲喂对照组。间接量热法检测结果显示，联合干预组在应答碳水化合物负荷时，可更好地维持代谢灵活性。肌肉组织的代谢组学与基因表达分析表明，单一干预手段具有独特且互补的效应：罗格列酮通过调控支链氨基酸代谢增强胰岛素敏感性，而n-3 LC-PUFA则可促进线粒体中脂肪酸的完全氧化。上述有益代谢效应与肌纤维表型转换（糖酵解型向氧化型肌纤维转化）的激活密切相关，该效应在cHF+F+ROSI组小鼠中尤为显著。本研究结果进一步证实，联合使用n-3 LC-PUFA与TZD类药物，可提升肥胖与糖尿病患者的临床治疗效果。所有实验小鼠可自由饮水并采食普通饲料。将3月龄小鼠随机分为每组8只的队列，分别饲喂脂含量约35%（质量分数）的cHF饲料，或等热量的cHF基干预饲料：(i) cHF+F组：添加n-3 LC-PUFA浓缩物（46%二十二碳六烯酸DHA、14%二十碳五烯酸EPA，质量分数，以甘油三酯形式存在；产品编号EPAX 1050 TG）的cHF饲料，该浓缩物替代了15%质量分数的膳食脂质；(ii) cHF+ROSI组：每千克饲料添加10mg罗格列酮的cHF饲料；(iii) cHF+F+ROSI组：同时添加n-3 LC-PUFA浓缩物与罗格列酮的cHF饲料。本研究还设置了更高剂量的cHF+ROSI组（每千克饲料添加100mg罗格列酮），但该组未纳入最终的微阵列分析。干预持续8周后，小鼠于当日光照周期（8:00至18:00）禁食10小时，随后于18:00起重新饲喂普通饲料，次日在戊巴比妥麻醉下采用颈椎脱臼法处死小鼠（处死时间为9:00至11:00），该处理流程即所谓的"饮食转换方案"。采集腓肠肌组织，用于总RNA提取。