BNIP3L/Nix-induced mitochondrial fission, mitophagy, and impaired myocyte glucose uptake are abrogated by PRKA/PKA phosphorylation.

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/14-3-3ß tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein beta.