Skeletal muscle ERalpha action is critical for the maintenance of mitochondrial dynamics and metabolic homeostasis in male mice. Mus musculus

Impaired estrogen production and action are associated with obesity and insulin resistance in male males however the tissues critical for estrogen action in the maintenance of metabolic homeostasis remain inadequately understood. Moreover, the cell specific target genes and molecular actions of estrogen in males remain unknown. We generated male mice with a muscle-specific ERa knockout (MERKO) to determine the role of this hormone nuclear receptor in controlling metabolic function and insulin action in skeletal muscle. Male MERKO mice became insulin resistant with age and accumulated more adipose tissue than floxed control mice. Skeletal muscle was distinguished by enlarged hyper-fused mitochondria that produced increased amounts of superoxide, and this was associated with enhanced proinflammatory signaling. The striking mitochondrial phenotype was accompanied by reduced protein abundance of electron transport chain proteins and the mitochondrial biogenesis marker Pgc1a. Muscle from MERKO mice showed imbalanced protein abundance of mitochondrial fission-fusion proteins most notably marked reductions in total DRP1, MFN1, and OPA1 were observed compared with f/f control. To recapitulate the reduction in DRP1 protein leading to impairment in mitochondrial fission, we generated mice with a muscle-specific heterozygous deletion in Drp1 (mDrp1+/-). mDrp1+/- mice phenocopied the aberrant muscle mitochondrial morphology and dysfunction of the MERKO mice and developed insulin resistance with age. Skeletal muscle ERa is critical for the maintenance of mitochondrial function and metabolic homeostasis in male mice. Overall design: Quadriceps mRNA profiles of male 20-week old wild type (WT) and muscle-specific Erα knockout (MERKO) mice were generated by deep sequencing, in triplicate, using Illumina HiSeq 4000.

雌激素（estrogen）生成与功能受损与雄性小鼠的肥胖及胰岛素抵抗（insulin resistance）密切相关，然而，介导雌激素维持代谢稳态（metabolic homeostasis）功能的关键组织仍未得到充分阐明。此外，雌激素在雄性体内的细胞特异性靶基因与分子作用机制仍未明确。本研究构建了肌肉组织特异性敲除雌激素受体α（estrogen receptor α, ERα）的雄性小鼠（命名为MERKO），以探究该激素核受体在骨骼肌（skeletal muscle）代谢功能与胰岛素信号调控中的作用。随着年龄增长，雄性MERKO小鼠会出现胰岛素抵抗，且其脂肪组织（adipose tissue）蓄积量较loxP位点对照（floxed control）小鼠更高。其骨骼肌表现出线粒体（mitochondria）体积增大、过度融合的表型，且生成更多超氧化物（superoxide），这与促炎信号通路（proinflammatory signaling）的活化增强密切相关。这种显著的线粒体表型伴随电子传递链（electron transport chain）蛋白及线粒体生物发生（mitochondrial biogenesis）标志物过氧化物酶体增殖物激活受体γ辅激活因子1α（PPARγ coactivator 1α, Pgc1α）的蛋白丰度降低。MERKO小鼠的骨骼肌中，线粒体分裂融合（mitochondrial fission-fusion）相关蛋白的丰度失衡：与f/f对照小鼠相比，总动力相关蛋白1（dynamin-related protein 1, DRP1）、线粒体融合蛋白1（mitofusin 1, MFN1）及视神经萎缩蛋白1（optic atrophy 1, OPA1）的蛋白水平均出现显著降低，这一现象尤为突出。为了模拟DRP1蛋白水平降低所导致的线粒体分裂功能受损，本研究构建了Drp1基因肌肉组织特异性杂合敲除小鼠（命名为mDrp1+/-）。mDrp1+/-小鼠呈现出与MERKO小鼠一致的骨骼肌线粒体形态异常与功能障碍表型，且随着年龄增长同样出现胰岛素抵抗。综上，骨骼肌ERα对于雄性小鼠的线粒体功能维持与代谢稳态至关重要。实验整体设计：本研究采用Illumina HiSeq 4000测序平台，对20周龄雄性野生型（wild type, WT）及肌肉特异性ERα敲除（MERKO）小鼠的股四头肌（Quadriceps）组织进行了三次生物学重复的mRNA深度测序，以获取其转录组表达谱。