RNA sequencing of pancreatic islets from WT, Mfn1-/-, Mfn2-/- and Mfn1/2-/- mice

The dynamin-like GTPases Mitofusin 1 and 2 (Mfn1 and Mfn2) are essential for mitochondrial function, which has been principally attributed to their regulation of fission/fusion dynamics. Here, we report that Mfn1 and 2 are critical for glucose-stimulated insulin secretion (GSIS) primarily through control of mtDNA content. Whereas Mfn1 and Mfn2 individually were dispensable for glucose homeostasis, combined Mfn1/2 deletion in beta-cells reduced mtDNA content, induced impaired mitochondrial morphology and networking fragmentation, and impaired decreased respiratory function, ultimately resulting in severe glucose intolerance. Importantly, gene dosage studies unexpectedly revealed that Mfn1/2 control of glucose homeostasis was dependent on maintenance of mtDNA content, rather than mitochondrial structure. Indeed, pharmacologic mitofusin agonists rescued islet mtDNA depletion due to mitofusin deficiency independent of changes on mitochondrial structure. Mfn1/2 maintain mtDNA content by regulating the expression of the crucial mitochondrial transcription factor Tfam, as Tfam overexpression ameliorated the reduction in mtDNA content and GSIS in Mfn1/2-deficient beta-cells. Thus, the primary physiologic role of Mfn1 and 2 in beta-cells is coupled to preservation of mtDNA content rather than mitochondrial architecture, and Mfn1 and 2 may be promising targets to overcome mitochondrial dysfunction and restore glucose control in diabetes. RNA libraries from WT, Mfn1-/-, Mfn2-/- and Mfn1/2-/- mouse islets were prepared by standard Illumina protocols and sequenced using the NovaSeq-6000 platform (Illumina).动力蛋白样GTP酶（dynamin-like GTPases）Mitofusin 1与Mitofusin 2（简称Mfn1、Mfn2）是维持线粒体功能的关键因子，既往研究多将其核心功能归结为调控线粒体分裂与融合的动态平衡。本研究发现，Mfn1与Mfn2对葡萄糖刺激胰岛素分泌（glucose-stimulated insulin secretion, GSIS）的调控作用主要通过维持线粒体DNA（mitochondrial DNA, mtDNA）含量实现。尽管单独敲除Mfn1或Mfn2对葡萄糖稳态无显著影响，但在β细胞中联合敲除Mfn1/2会降低mtDNA含量，引发线粒体形态异常与网络碎片化，并削弱呼吸功能，最终导致严重的糖耐量受损。值得注意的是，基因剂量相关研究意外揭示，Mfn1/2对葡萄糖稳态的调控依赖于mtDNA含量的维持，而非线粒体结构完整性。进一步研究证实，药物性Mitofusin激动剂可挽救Mitofusin缺陷导致的胰岛mtDNA耗竭，且该效应与线粒体结构变化无关。Mfn1与Mfn2通过调控关键线粒体转录因子A（mitochondrial transcription factor A, Tfam）的表达维持mtDNA含量；实验显示，过表达Tfam可改善Mfn1/2缺陷β细胞中mtDNA含量与GSIS的降低情况。综上，β细胞中Mfn1与Mfn2的核心生理功能在于维持mtDNA含量，而非线粒体结构形态；Mfn1与Mfn2或可作为改善线粒体功能障碍、恢复糖尿病患者血糖调控的潜在靶点。本研究采用标准Illumina建库流程，制备了野生型（wild type, WT）、Mfn1基因敲除（Mfn1-/-）、Mfn2基因敲除（Mfn2-/-）以及Mfn1/2联合敲除（Mfn1/2-/-）小鼠胰岛的RNA文库，并通过NovaSeq-6000测序平台（Illumina）完成测序。