#70 Therapeutic targeting of skeletal muscle Nix in early-onset insulin resistance
Simone da Silva Rosa, Lucas Nguyen, Wajihah Mughal, Matthew Martens, Donald Chapman, Yan Hai, Christof Rampitsch, Vernon Dolinsky, William Diehl-Jones and Joseph W Gordon, Children’s Hospital Research Institute of Manitoba, Department of Human Anatomy and Cell Science, Rady Faculty of Health Sciences, Faculty of Nursing, University of Manitoba.
Fetal exposure to diabetes during pregnancy increases the risk for early- onset insulin resistance in offspring; however, the key molecular regulators responsible for fetal metabolic programming have not been characterized. Previously, we demonstrated that the expression of a mitochondrial death gene, named Nix, was elevated in the skeletal muscle of rats exposed to gestational diabetes. Here, we identify a novel phosphorylation residue, activated by clenbuterol treatment that serves to prevent Nix-induced mitochondrial dysfunction in muscle cells.
Using cell and biochemical approaches, the C2C12 skeletal muscle myotubes were exposed to 200 μM palmitate, or vehicle control. To assess mitochondrial membrane potential, cells were stained with TMRM, while macro-autophagy was determined by LC3-GFP aggregation into autophagosomes (n=10). Plasmid-based PKA biosensor was used to identify clenbuterol activation. Cellular localization of Nix was determined by cell fractionation and protein expression by western blot. Phospho-peptide mapping was performed by mass spectrometry. One-way anova determined multiple comparisons between groups and student t-test compared mean differences.
Exposure to palmitate during differentiation resulted in mitochondrial depolarization, compared to the control myotubes (p<0.05). Furthermore, mitochondrial depolarization was prevented by PKA-activating drug clenbuterol (p<0.05). Consistent with these findings, Nix-induced mitochondrial depolarization was inhibited by clenbuterol, or co-expression of PKA, and clenbuterol restored muscle glucose uptake levels. It has also been observed that phosphorylated version of Nix is located at the cytosol and not in mitochondria. Detailed phospho-peptide mapping of Nix, revealed a novel phosphorylation residue within the transmembrane domain of Nix. Mutational analysis of this novel phosphorylation site attenuated Nix-induced mitochondrial depolarization (p<0.05), without impacting Nix-induced autophagy, determined by LC3-GFP fluorescence.
Our data supports the hypothesis that Nix regulates mitochondrial metabolism in differentiating skeletal muscle and suggest a possible therapeutic strategy to circumvent the mitochondrial dysfunction characteristic of insulin resistance without impacting Nix’s regulation of autophagy.