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Abstract：
BACKGROUND: Diabetic cardiomyopathy (DCM) causes the myocardium to rely on fatty acid β-oxidation for energy. The accumulation of intracellular lipids and fatty acids in the myocardium usually results in lipotoxicity, which impairs myocardial function. Adipsin may play an important protective role in the pathogenesis of DCM. The aim of this study is to investigate the regulatory effect of Adipsin on DCM lipotoxicity and its molecular mechanism.
METHODS: A high-fat diet (HFD)-induced type 2 diabetes mellitus model was constructed in mice with adipose tissue-specific overexpression of Adipsin (Adipsin-Tg). Liquid chromatography-tandem mass spectrometry (LC-MS/MS), glutathione-S-transferase (GST) pull-down technique, Co-immunoprecipitation (Co-IP) and immunofluorescence colocalization analyses were used to investigate the molecules which can directly interact with Adipsin. The immunocolloidal gold method was also used to detect the interaction between Adipsin and its downstream modulator.
RESULTS: The expression of Adipsin was significantly downregulated in the HFD-induced DCM model (P < 0.05). Adipose tissue-specific overexpression of Adipsin significantly improved cardiac function and alleviated cardiac remodeling in DCM (P < 0.05). Adipsin overexpression also alleviated mitochondrial oxidative phosphorylation function in diabetic stress (P < 0.05). LC-MS/MS analysis, GST pull-down technique and Co-IP studies revealed that interleukin-1 receptor-associated kinase-like 2 (Irak2) was a downstream regulator of Adipsin. Immunofluorescence analysis also revealed that Adipsin was co-localized with Irak2 in cardiomyocytes. Immunocolloidal gold electron microscopy and Western blotting analysis indicated that Adipsin inhibited the mitochondrial translocation of Irak2 in DCM, thus dampening the interaction between Irak2 and prohibitin (Phb)-optic atrophy protein 1 (Opa1) on mitochondria and improving the structural integrity and function of mitochondria (P < 0.05). Interestingly, in the presence of Irak2 knockdown, Adipsin overexpression did not further alleviate myocardial mitochondrial destruction and cardiac dysfunction, suggesting a downstream role of Irak2 in Adipsin-induced responses (P < 0.05). Consistent with these findings, overexpression of Adipsin after Irak2 knockdown did not further reduce the accumulation of lipids and their metabolites in the cardiac myocardium, nor did it enhance the oxidation capacity of cardiomyocytes expose to palmitate (PA) (P < 0.05). These results indicated that Irak2 may be a downstream regulator of Adipsin.
CONCLUSIONS: Adipsin improves fatty acid β-oxidation and alleviates mitochondrial injury in DCM. The mechanism is related to Irak2 interaction and inhibition of Irak2 mitochondrial translocation.
METHODS: A high-fat diet (HFD)-induced type 2 diabetes mellitus model was constructed in mice with adipose tissue-specific overexpression of Adipsin (Adipsin-Tg). Liquid chromatography-tandem mass spectrometry (LC-MS/MS), glutathione-S-transferase (GST) pull-down technique, Co-immunoprecipitation (Co-IP) and immunofluorescence colocalization analyses were used to investigate the molecules which can directly interact with Adipsin. The immunocolloidal gold method was also used to detect the interaction between Adipsin and its downstream modulator.
RESULTS: The expression of Adipsin was significantly downregulated in the HFD-induced DCM model (P < 0.05). Adipose tissue-specific overexpression of Adipsin significantly improved cardiac function and alleviated cardiac remodeling in DCM (P < 0.05). Adipsin overexpression also alleviated mitochondrial oxidative phosphorylation function in diabetic stress (P < 0.05). LC-MS/MS analysis, GST pull-down technique and Co-IP studies revealed that interleukin-1 receptor-associated kinase-like 2 (Irak2) was a downstream regulator of Adipsin. Immunofluorescence analysis also revealed that Adipsin was co-localized with Irak2 in cardiomyocytes. Immunocolloidal gold electron microscopy and Western blotting analysis indicated that Adipsin inhibited the mitochondrial translocation of Irak2 in DCM, thus dampening the interaction between Irak2 and prohibitin (Phb)-optic atrophy protein 1 (Opa1) on mitochondria and improving the structural integrity and function of mitochondria (P < 0.05). Interestingly, in the presence of Irak2 knockdown, Adipsin overexpression did not further alleviate myocardial mitochondrial destruction and cardiac dysfunction, suggesting a downstream role of Irak2 in Adipsin-induced responses (P < 0.05). Consistent with these findings, overexpression of Adipsin after Irak2 knockdown did not further reduce the accumulation of lipids and their metabolites in the cardiac myocardium, nor did it enhance the oxidation capacity of cardiomyocytes expose to palmitate (PA) (P < 0.05). These results indicated that Irak2 may be a downstream regulator of Adipsin.
CONCLUSIONS: Adipsin improves fatty acid β-oxidation and alleviates mitochondrial injury in DCM. The mechanism is related to Irak2 interaction and inhibition of Irak2 mitochondrial translocation.
摘要：
背景：糖尿病心肌病（DCM）导致心肌依赖脂肪酸β-氧化来获取能量。细胞内脂质和脂肪酸在心肌中的积累通常会导致脂毒性，损害心肌功能。Adipsin可能在DCM的发病机制中起重要的保护作用。本研究旨在探讨Adipsin对DCM脂毒性的调节作用及其分子机制。
方法：在脂肪组织特异性过表达Adipsin(Adipsin-Tg)的小鼠中构建高脂饮食(HFD)诱导的2型糖尿病模型。液相色谱-串联质谱(LC-MS/MS),谷胱甘肽-S-转移酶（GST）下拉技术，使用免疫共沉淀（Co-IP）和免疫荧光共定位分析来研究可以直接与Adipsin相互作用的分子。免疫胶体金方法也用于检测Adipsin与其下游调节剂之间的相互作用。
结果：在HFD诱导的DCM模型中，Adipsin的表达明显下调（P<0.05）。脂肪组织特异性过表达Adipsin可显著改善DCM患者心功能,减轻心肌重构(P<0.05)。Adipsin过表达也减轻了糖尿病应激中线粒体氧化磷酸化功能（P<0.05）。LC-MS/MS分析,GST下拉技术和Co-IP研究表明，白介素1受体相关激酶样2（Irak2）是Adipsin的下游调节剂。免疫荧光分析还显示Adipsin与Irak2共定位在心肌细胞中。免疫胶体金电镜和Western印迹分析表明，Adipsin抑制了DCM中Irak2的线粒体易位，从而抑制线粒体上Irak2与阻抑素（Phb）-视神经萎缩蛋白1（Opa1）的相互作用，改善线粒体的结构完整性和功能（P<0.05）。有趣的是,在Irak2击倒的情况下，Adipsin过表达不能进一步减轻心肌线粒体破坏和心功能不全,提示Irak2在Adipsin诱导的反应中的下游作用（P<0.05）。与这些发现一致，Irak2敲除后Adipsin的过表达并没有进一步减少脂质及其代谢物在心肌中的积累，它也没有增强暴露于棕榈酸（PA）的心肌细胞的氧化能力（P<0.05）。这些结果表明Irak2可能是Adipsin的下游调节剂。
结论：Adipsin能改善DCM脂肪酸β-氧化，减轻线粒体损伤。其机制与Irak2相互作用和抑制Irak2线粒体易位有关。
方法：在脂肪组织特异性过表达Adipsin(Adipsin-Tg)的小鼠中构建高脂饮食(HFD)诱导的2型糖尿病模型。液相色谱-串联质谱(LC-MS/MS),谷胱甘肽-S-转移酶（GST）下拉技术，使用免疫共沉淀（Co-IP）和免疫荧光共定位分析来研究可以直接与Adipsin相互作用的分子。免疫胶体金方法也用于检测Adipsin与其下游调节剂之间的相互作用。
结果：在HFD诱导的DCM模型中，Adipsin的表达明显下调（P<0.05）。脂肪组织特异性过表达Adipsin可显著改善DCM患者心功能,减轻心肌重构(P<0.05)。Adipsin过表达也减轻了糖尿病应激中线粒体氧化磷酸化功能（P<0.05）。LC-MS/MS分析,GST下拉技术和Co-IP研究表明，白介素1受体相关激酶样2（Irak2）是Adipsin的下游调节剂。免疫荧光分析还显示Adipsin与Irak2共定位在心肌细胞中。免疫胶体金电镜和Western印迹分析表明，Adipsin抑制了DCM中Irak2的线粒体易位，从而抑制线粒体上Irak2与阻抑素（Phb）-视神经萎缩蛋白1（Opa1）的相互作用，改善线粒体的结构完整性和功能（P<0.05）。有趣的是,在Irak2击倒的情况下，Adipsin过表达不能进一步减轻心肌线粒体破坏和心功能不全,提示Irak2在Adipsin诱导的反应中的下游作用（P<0.05）。与这些发现一致，Irak2敲除后Adipsin的过表达并没有进一步减少脂质及其代谢物在心肌中的积累，它也没有增强暴露于棕榈酸（PA）的心肌细胞的氧化能力（P<0.05）。这些结果表明Irak2可能是Adipsin的下游调节剂。
结论：Adipsin能改善DCM脂肪酸β-氧化，减轻线粒体损伤。其机制与Irak2相互作用和抑制Irak2线粒体易位有关。
