BackgroundDiabetic sarcopenia is a disease‐related skeletal muscle disorder that causes progressive symptoms. The complete understanding of its pathogenesis is yet to be unravelled, which makes it difficult to develop effective therapeutic strategies. This study investigates how MFG‐E8 affects mitophagy and the protective role of D‐pinitol (DP) in diabetic sarcopenia.MethodsIn vivo, streptozotocin‐induced diabetic SAM‐R1 (STZ‐R1) and SAM‐P8 (STZ‐P8) mice (16‐week‐old) were used, and STZ‐P8 mice were administrated of DP (150 mg/kg per day) for 6 weeks. Gastrocnemius muscles were harvested for histological analysis including transmission electron microscopy. Proteins were evaluated via immunohistochemistry (IHC), immunofluorescence (IF), and western blotting (WB) assay. In vitro, advanced glycation end products (AGEs) induced diabetic and D‐galactose (DG) induced senescent C2C12 models were established and received DP, MFG‐E8 plasmid (Mover)/siRNA (MsiRNA), or 3‐MA/Torin‐1 intervention. Proteins were evaluated by IF and WB assay. Immunoprecipitation (IP) and co‐immunoprecipitation (CO‐IP) were used for hunting the interacted proteins of MFG‐E8.ResultsIn vivo, sarcopenia, mitophagy deficiency, and up‐regulated MFG‐E8 were confirmed in the STZ‐P8 group. DP exerted protective effects on sarcopenia and mitophagy (DP + STZ‐P8 vs. STZ‐P8; all P < 0.01), such as increased lean mass (8.47 ± 0.81 g vs. 7.08 ± 1.64 g), grip strength (208.62 ± 39.45 g vs. 160.87 ± 26.95 g), rotarod tests (109.7 ± 11.81 s vs. 59.3 ± 20.97 s), muscle cross‐sectional area (CSA) (1912.17 ± 535.61 μm2 vs. 1557.19 ± 588.38 μm2), autophagosomes (0.07 ± 0.02 per μm2 vs. 0.02 ± 0.01 per μm2), and cytolysosome (0.07 ± 0.03 per μm2 vs. 0.03 ± 0.01 per μm2). DP down‐regulated MFG‐E8 in both serum (DP + STZ‐P8: 253.19 ± 34.75 pg/mL vs. STZ‐P8: 404.69 ± 78.97 pg/mL; P < 0.001) and gastrocnemius muscle (WB assay. DP + STZ‐P8: 0.39 ± 0.04 vs. STZ‐P8: 0.55 ± 0.08; P < 0.01). DP also up‐regulated PINK1, Parkin and LC3B‐II/I ratio, and down‐regulated P62 in gastrocnemius muscles (all P < 0.01). In vitro, mitophagy deficiency and MFG‐E8 up‐regulation were confirmed in diabetic and senescent models (all P < 0.05). DP and MsiRNA down‐regulated MFG‐E8 and P62, and up‐regulated PINK1, Parkin and LC3B‐II/I ratio to promote mitophagy as Torin‐1 does (all P < 0.05). HSPA1L was confirmed as an interacted protein of MFG‐E8 in IP and CO‐IP assay. Mover down‐regulated the expression of Parkin via the HSPA1L‐Parkin pathway, leading to mitophagy inhibition. MsiRNA up‐regulated the expression of PINK1 via SGK1, FOXO1, and STAT3 phosphorylation pathways, leading to mitophagy stimulation.ConclusionsMFG‐E8 is a crucial target protein of DP and plays a distinct role in mitophagy regulation. DP down‐regulates the expression of MFG‐E8, reduces mitophagy deficiency, and alleviates the symptoms of diabetic sarcopenia, which could be considered a novel therapeutic strategy for diabetic sarcopenia.

中文翻译：

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MFG-E8通过HSPA1L-Parkin通路对糖尿病肌少症线粒体自噬的调节及D-松醇的作用

背景糖尿病肌少症是一种与疾病相关的骨骼肌疾病，会导致进行性症状。对其发病机制的完整了解尚未阐明，这使得制定有效的治疗策略变得困难。本研究探讨了 MFG-E8 如何影响线粒体自噬以及 D-松醇 (DP) 在糖尿病肌少症中的保护作用。方法在体内，链脲佐菌素诱导的糖尿病 SAM-R1 (STZ-R1) 和 SAM-P8 (STZ-P8) 小鼠（使用 16 周龄的 STZ-P8 小鼠，给予 DP（每天 150 mg/kg）6 周。收获腓肠肌用于组织学分析，包括透射电子显微镜。通过免疫组织化学 (IHC)、免疫荧光 (IF) 和蛋白质印迹 (WB) 测定评估蛋白质。在体外，建立了晚期糖基化终末产物（AGEs）诱导的糖尿病和D-半乳糖（DG）诱导的衰老C2C12模型，并接受DP、MFG-E8质粒（Mover）/siRNA（MsiRNA）或3-MA/Torin-1干涉。通过 IF 和 WB 测定评估蛋白质。使用免疫沉淀（IP）和免疫共沉淀（CO-IP）来寻找 MFG-E8 的相互作用蛋白。结果在体内，STZ-P8 组证实了肌少症、线粒体自噬缺陷和 MFG-E8 上调。 DP 对肌肉减少症和线粒体自噬发挥保护作用（DP + STZ-P8 与 STZ-P8；所有磷< 0.01），例如增加瘦体重（8.47 ± 0.81 g vs. 7.08 ± 1.64 g）、握力（208.62 ± 39.45 g vs. 160.87 ± 26.95 g）、转杆测试（109.7 ± 11.81 s vs. 59.3 ± 20.97 s） ），肌肉横截面积（CSA）（1912.17 ± 535.61 μm2对比 1557.19 ± 588.38 μm2）、自噬体（0.07 ± 0.02/μm2与 0.02 ± 0.01 每微米2）和细胞溶酶体（0.07 ± 0.03/μm2与 0.03 ± 0.01 每微米2）。 DP 下调两种血清中的 MFG-E8（DP + STZ-P8：253.19 ± 34.75 pg/mL vs. STZ-P8：404.69 ± 78.97 pg/mL；磷< 0.001）和腓肠肌（WB 测定。DP + STZ-P8：0.39 ± 0.04 对比 STZ-P8：0.55 ± 0.08；磷< 0.01）。 DP 还上调腓肠肌中的 PINK1、Parkin 和 LC3B-II/I 比率，并下调 P62（所有磷< 0.01）。在体外，糖尿病和衰老模型中证实了线粒体自噬缺陷和 MFG-E8 上调（所有磷< 0.05）。 DP 和 MsiRNA 下调 MFG-E8 和 P62，并上调 PINK1、Parkin 和 LC3B-II/I 比率，以促进线粒体自噬，就像 Torin-1 一样（所有磷< 0.05）。在 IP 和 CO-IP 测定中，HSPA1L 被证实是 MFG-E8 的相互作用蛋白。 Mover 通过 HSPA1L-Parkin 通路下调 Parkin 的表达，从而抑制线粒体自噬。 MsiRNA通过SGK1、FOXO1和STAT3磷酸化途径上调PINK1的表达，从而刺激线粒体自噬。结论MFG-E8是DP的重要靶蛋白，在线粒体自噬调节中发挥着独特的作用。 DP下调MFG-E8的表达，减少线粒体自噬缺陷，减轻糖尿病肌少症的症状，这可以被认为是糖尿病肌少症的一种新的治疗策略。