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 shotgun 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.

Mechanisms of muscle insulin resistance and the cross‐talk with liver and adipose tissue

“…Furthermore, Nix has also been previously demonstrated to become elevated in insulin‐resistant rodents (Mughal et al., 2015). In subsequent studies by our group, we have observed that mitophagy is linked to Nix accumulation in lipotoxic environments leading to impaired insulin signaling via activation of mTOR‐p70S6 kinase and inhibition of IRS1 (da Silva Rosa et al., 2020).…”

“…In a cellular model, we demonstrated that knockdown of the mitochondrial phospholipase‐D, an enzyme responsible for converting cardiolipin to phosphatidic acid, prevented Nix‐induced mTOR‐S6K activation. Furthermore, Nix‐induced mitophagy and impaired insulin signaling could be reversed by direct phosphorylation of Nix at Serine 212 by PKA activating agents, such as clenbuterol and phosphodiesterase‐4 inhibitors (da Silva Rosa et al., 2020). This novel target may represent a future therapeutic strategy to circumvent the mitochondrial defects observed in muscle insulin resistance.…”

“…In turn, it protects the myocyte against nutrient storage stress via activation of mTOR‐dependent desensitization of insulin signaling, via phosphorylation of serine 1,101 of IRS1. Nix induced ER calcium release concurrent DRP1 activation, along with increased levels of mitophagy (da Silva Rosa et al., 2020). As previously described, DRP1 is as an important mediator of mitochondrial fission and mitophagy (Gandhi & Perry, 2020).…”

“…Furthermore, we highlight a link between Nix‐induced mTOR activation and recruitment of lysosomal small GTPases such as Rheb to the mitochondria, to initiate mitophagy. This phenomenon was demonstrated to be dependent on phosphatidic acid availability (da Silva Rosa et al., 2020), which is also an important modulator of mitochondrial dynamics (Hornberger et al., 2006). In a cellular model, we demonstrated that knockdown of the mitochondrial phospholipase‐D, an enzyme responsible for converting cardiolipin to phosphatidic acid, prevented Nix‐induced mTOR‐S6K activation.…”

Antioxidants Targeting Mitochondrial Oxidative Stress: Promising Neuroprotectants for Epilepsy

“…The best known regulators implicated in mitophagy are autophagy-related 32 protein (Atg32), NIP3-like protein X (NIX; also known as BNIP3L), parkin, and PTEN-induced putative kinase protein 1 (PINK1) [ 39 ]. NIX overexpression or downregulation of PINK1 results in induction of mitophagy via activation of mitochondrial depolarization [ 40 , 41 ]. It has shown that mitophagy is activated by a variety of stressors including hypoxia, nutrient deficiency, and increased oxidative phosphorylation activity.…”