RESUMO
Stimulating brown adipose tissue (BAT) activity represents a promising therapy for overcoming metabolic diseases. mTORC2 is important for regulating BAT metabolism, but its downstream targets have not been fully characterized. In this study, we apply proteomics and phosphoproteomics to investigate the downstream effectors of mTORC2 in brown adipocytes. We compare wild-type controls to isogenic cells with an induced knockout of the mTORC2 subunit RICTOR (Rictor-iKO) by stimulating each with insulin for a 30-min time course. In Rictor-iKO cells, we identify decreases to the abundance of glycolytic and de novo lipogenesis enzymes, and increases to mitochondrial proteins as well as a set of proteins known to increase upon interferon stimulation. We also observe significant differences to basal phosphorylation because of chronic RICTOR loss including decreased phosphorylation of the lipid droplet protein perilipin-1 in Rictor-iKO cells, suggesting that RICTOR could be involved with regulating basal lipolysis or droplet dynamics. Finally, we observe mild dampening of acute insulin signaling response in Rictor-iKO cells, and a subset of AKT substrates exhibiting statistically significant dependence on RICTOR.
Assuntos
Adipócitos Marrons/efeitos dos fármacos , Adipócitos Marrons/metabolismo , Insulina/farmacocinética , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Proteoma/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteína Companheira de mTOR Insensível à Rapamicina/metabolismo , Animais , Cromatografia Líquida , Técnicas de Inativação de Genes , Ontologia Genética , Glicólise/efeitos dos fármacos , Insulina/metabolismo , Lipogênese/efeitos dos fármacos , Camundongos , Mitocôndrias/efeitos dos fármacos , Fosforilação , Proteína Companheira de mTOR Insensível à Rapamicina/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Espectrometria de Massas em TandemRESUMO
Leigh syndrome is a fatal neurometabolic disorder caused by defects in mitochondrial function. Mechanistic target of rapamycin (mTOR) inhibition with rapamycin attenuates disease progression in a mouse model of Leigh syndrome (Ndufs4 knock-out (KO) mouse); however, the mechanism of rescue is unknown. Here we identify protein kinase C (PKC) downregulation as a key event mediating the beneficial effects of rapamycin treatment of Ndufs4 KO mice. Assessing the impact of rapamycin on the brain proteome and phosphoproteome of Ndufs4 KO mice, we find that rapamycin restores mitochondrial protein levels, inhibits signalling through both mTOR complexes and reduces the abundance and activity of multiple PKC isoforms. Administration of PKC inhibitors increases survival, delays neurological deficits, prevents hair loss and decreases inflammation in Ndufs4 KO mice. Thus, PKC may be a viable therapeutic target for treating severe mitochondrial disease.