ABSTRACT

Lipotoxicity is a form of cellular stress caused by the accumulation of lipids resulting in mitochondrial dysfunction and insulin resistance in muscle. Previously, we demonstrated that the mitophagy receptor BNIP3L/Nix is responsive to lipotoxicity and accumulates in response to a high-fat (HF) feeding. To provide a better understanding of this observation, we undertook gene expression array and shot-gun metabolomics studies in soleus muscle from rodents on an HF diet. Interestingly, we observed a modest reduction in several autophagy-related genes. Moreover, we observed alterations in the fatty acyl composition of cardiolipins and phosphatidic acids. Given the reported roles of these phospholipids and BNIP3L in mitochondrial dynamics, we investigated aberrant mitochondrial turnover as a mechanism of impaired myocyte insulin signaling. In a series of gain-of-function and loss-of-function experiments in rodent and human myotubes, we demonstrate that BNIP3L accumulation triggers mitochondrial depolarization, calcium-dependent activation of DNM1L/DRP1, and mitophagy. In addition, BNIP3L can inhibit insulin signaling through activation of MTOR-RPS6KB/p70S6 kinase inhibition of IRS1, which is contingent on phosphatidic acids and RHEB. Finally, we demonstrate that BNIP3L-induced mitophagy and impaired glucose uptake can be reversed by direct phosphorylation of BNIP3L by PRKA/PKA, leading to the translocation of BNIP3L from the mitochondria and sarcoplasmic reticulum to the cytosol. These findings provide insight into the role of BNIP3L, mitochondrial turnover, and impaired myocyte insulin signaling during an overfed state when overall autophagy-related gene expression is reduced. Furthermore, our data suggest a mechanism by which exercise or pharmacological activation of PRKA may overcome myocyte insulin resistance.

Abbreviations: BCL2: B cell leukemia/lymphoma 2; BNIP3L/Nix: BCL2/adenovirus E1B interacting protein 3-like; DNM1L/DRP1: dynamin 1-like; FUNDC1: FUN14 domain containing 1; IRS1: insulin receptor substrate 1; MAP1LC3A/LC3: microtubule-associated protein 1 light chain 3 alpha; MFN1: mitofusin 1; MFN2: mitofusin 2; MTOR: mechanistic target of rapamycin kinase; OPA1: OPA1 mitochondrial dynamin like GTPase; PDE4i: phosphodiesterase 4 inhibitor; PLD1: phospholipase D1; PLD6: phospholipase D family member 6; PRKA/PKA: protein kinase, AMP-activated; PRKCD/PKCδ: protein kinase C, delta; PRKCQ/PKCθ: protein kinase C, theta; RHEB: Ras homolog enriched in brain; RPS6KB/p70S6K: ribosomal protein S6 kinase; SQSTM1/p62: sequestosome 1; YWHAB/14-3-3β: tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein beta

摘要

脂毒性是由脂质积累引起的细胞应激的一种形式，导致线粒体功能障碍和肌肉中的胰岛素抵抗。以前，我们证明了线粒体自噬受体 BNIP3L/Nix 对脂毒性有反应，并在高脂肪 (HF) 喂养时积累。为了更好地理解这一观察结果，我们在 HF 饮食的啮齿动物比目鱼肌中进行了基因表达阵列和鸟枪代谢组学研究。有趣的是，我们观察到几个自噬相关基因的适度减少。此外，我们观察到心磷脂和磷脂酸的脂肪酰基组成发生了变化。鉴于报告的这些磷脂和 BNIP3L 在线粒体动力学中的作用，我们研究了异常线粒体周转作为肌细胞胰岛素信号传导受损的机制。在啮齿动物和人类肌管的一系列功能获得和功能丧失实验中，我们证明 BNIP3L 积累会触发线粒体去极化、DNM1L/DRP1 的钙依赖性激活和线粒体自噬。此外，BNIP3L 可以通过激活 MTOR-RPS6KB/p70S6 激酶抑制 IRS1 来抑制胰岛素信号传导，这取决于磷脂酸和 RHEB。最后，我们证明了 BNIP3L 诱导的线粒体自噬和葡萄糖摄取受损可以通过 PRKA/PKA 直接磷酸化 BNIP3L 来逆转，从而导致 BNIP3L 从线粒体和肌质网易位到细胞质。这些发现提供了对 BNIP3L 的作用、线粒体转换和在过度喂养状态下受损的肌细胞胰岛素信号传导的作用的见解，当整体自噬相关基因表达降低时。

缩写： BCL2：B 细胞白血病/淋巴瘤 2；BNIP3L/Nix：BCL2/腺病毒 E1B 相互作用蛋白 3 样；DNM1L/DRP1：动力蛋白 1 样；FUNDC1：FUN14 域包含 1；IRS1：胰岛素受体底物 1；MAP1LC3A/LC3：微管相关蛋白 1 轻链 3 α；MFN1：线粒体融合素 1；MFN2：线粒体融合素 2；MTOR：雷帕霉素激酶的机制靶点；OPA1：OPA1 线粒体动力蛋白，如 GTPase；PDE4i：磷酸二酯酶 4 抑制剂；PLD1：磷脂酶 D1；PLD6：磷脂酶D家族成员6；PRKA/PKA：蛋白激酶，AMP 激活的；PRKCD/PKCδ：蛋白激酶 C，δ；PRKCQ/PKCθ：蛋白激酶 C，θ；RHEB：大脑中富含 Ras 同源物；RPS6KB/p70S6K：核糖体蛋白 S6 激酶；SQSTM1/p62：隔离体 1；YWHAB/14-3-3β：酪氨酸3-单加氧酶/色氨酸5-单加氧酶活化蛋白β