Abstract
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.
Highlights
Stimulating brown adipose tissue (BAT) activity represents a promising therapy for overcoming metabolic diseases. mammalian target of rapamycin (mTOR) complex 2 (mTORC2) is important for regulating BAT metabolism, but its downstream targets have not been fully characterized
We recently identified some AKT substrates with partial sensitivity to mTORC2 loss in brown preadipocytes, including ATP citrate lyase (ACLY) S455, whose phosphorylation is important for brown adipocyte differentiation, histone acetylation, and expression of de novo lipogenesis genes and GLUT4 in differentiated brown adipocytes [5]
Our results show that RICTOR deletion mildly dampens insulindependent phosphorylation; AKT substrate sites more strongly affected by RICTOR include ACLY S455, eukaryotic translation initiation factor 4B (EIF4B) S422, and VIM S39
Summary
We applied DDA proteomics along with DDA and PRM phosphoproteomics to study brown adipocytes lacking RICTOR, an essential subunit of mTOR complex 2, over a time series of insulin stimulation. We recently identified some AKT substrates with partial sensitivity to mTORC2 loss in brown preadipocytes, including ACLY S455, whose phosphorylation is important for brown adipocyte differentiation, histone acetylation, and expression of de novo lipogenesis genes and GLUT4 in differentiated brown adipocytes [5] These studies suggest that loss of mTORC2-dependent AKT hydrophobic motif phosphorylation may differentially affect AKT substrate phosphorylation yet leave many open questions regarding the role of mTORC2 in regulating the global proteome and the dynamic insulin-AKT signaling response. MTORC2 Phosphoproteomics mass spectrometry (MS)-based proteomics to profile protein and dynamic, insulin-stimulated phosphorylation in wild-type and mTORC2-deficient differentiated, mature brown adipocytes in which Rictor is deleted using an inducible knockout system Applying both discovery-driven and targeted proteomic approaches, we assessed changes to baseline protein abundance, insulin response amplitude, and temporal dynamics. This study expands on our prior work [5] by offering a global view of how the brown adipocyte proteome is remodeled in the absence of functional mTORC2, and provides the most comprehensive proteomic analysis to date of the effect of mTORC2 loss on insulin/AKT signaling in mature brown adipocytes
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