Abstract
mTORC2 phosphorylates AKT in a hydrophobic motif site that is a biomarker of insulin sensitivity. In brown adipocytes, mTORC2 regulates glucose and lipid metabolism, however the mechanism has been unclear because downstream AKT signaling appears unaffected by mTORC2 loss. Here, by applying immunoblotting, targeted phosphoproteomics and metabolite profiling, we identify ATP-citrate lyase (ACLY) as a distinctly mTORC2-sensitive AKT substrate in brown preadipocytes. mTORC2 appears dispensable for most other AKT actions examined, indicating a previously unappreciated selectivity in mTORC2-AKT signaling. Rescue experiments suggest brown preadipocytes require the mTORC2/AKT/ACLY pathway to induce PPAR-gamma and establish the epigenetic landscape during differentiation. Evidence in mature brown adipocytes also suggests mTORC2 acts through ACLY to increase carbohydrate response element binding protein (ChREBP) activity, histone acetylation, and gluco-lipogenic gene expression. Substrate utilization studies additionally implicate mTORC2 in promoting acetyl-CoA synthesis from acetate through acetyl-CoA synthetase 2 (ACSS2). These data suggest that a principal mTORC2 action is controlling nuclear-cytoplasmic acetyl-CoA synthesis.
Highlights
Many models show mTORC2 as an obligatory AKT activator[1,5]; its exact function in downstream AKT signaling in vivo remains elusive because, in many tissue-specific conditional Rictor knockout (KO) models, mTORC2 loss has seemingly minimal-to-no effect on the phosphorylation of several AKT substrates[2,10]
To test the hypothesis that some AKT substrates are more sensitive to mTORC2 loss than for others and to identify the mechanism by which mTORC2 drives brown preadipocyte differentiation, we conducted a targeted phosphoproteomics assay comparing the insulin response between Rictor-iKO precursor brown adipocytes (Rictor-iKOPBAs) and isogenic control brown preadipocytes
Solving this mystery is important because a similar state of adipose tissue “selective insulin resistance” in which insulin-stimulated glucose uptake, but not AKT activity per se, is defective, occurs in several models of adipocyte insulin resistance[36,37]
Summary
Many models show mTORC2 as an obligatory AKT activator[1,5]; its exact function in downstream AKT signaling in vivo remains elusive because, in many tissue-specific conditional Rictor knockout (KO) models, mTORC2 loss has seemingly minimal-to-no effect on the phosphorylation of several AKT substrates[2,10]. Expressing recombinant AKT1 containing a phospho-mimetic S473 residue in Rictordeficient preadipocytes rescues differentiation The latter observation suggests that mTORC2 may be more essential for some AKT actions that are required for brown adipocyte differentiation and de novo lipogenesis, but dispensable for many others. We provide evidence that ACLY functions downstream of a uniquely mTORC2-dependent AKT pathway required for DNL, differentiation, histone acetylation, and ChREBPβ and glucolipogenic gene expression. Substrate utilization studies suggest an additional role for mTORC2 in promoting acetyl-CoA synthesis from acetate through acyl-CoA synthetase short chain family member 2 (ACSS2) These data uncover a previously unappreciated selectivity in mTORC2-dependent AKT signaling in Rictor-deleted brown adipocytes and reveal mTORC2’s central role in controlling nuclear–cytoplasmic acetyl-CoA synthesis
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