Activation of mTORC1 is essential for β-adrenergic stimulation of adipose browning
Dianxin Liu, Marica Bordicchia, Chaoying Zhang, Huafeng Fang, Wan Wei, Jian-Liang Li, Adilson Guilherme, Kalyani Guntur, Michael P. Czech, Sheila Collins
Dianxin Liu, Marica Bordicchia, Chaoying Zhang, Huafeng Fang, Wan Wei, Jian-Liang Li, Adilson Guilherme, Kalyani Guntur, Michael P. Czech, Sheila Collins
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Research Article Endocrinology

Activation of mTORC1 is essential for β-adrenergic stimulation of adipose browning

Abstract

A classic metabolic concept posits that insulin promotes energy storage and adipose expansion, while catecholamines stimulate release of adipose energy stores by hydrolysis of triglycerides through β-adrenergic receptor (βARs) and protein kinase A (PKA) signaling. Here, we have shown that a key hub in the insulin signaling pathway, activation of p70 ribosomal S6 kinase (S6K1) through mTORC1, is also triggered by PKA activation in both mouse and human adipocytes. Mice with mTORC1 impairment, either through adipocyte-specific deletion of Raptor or pharmacologic rapamycin treatment, were refractory to the well-known βAR-dependent increase of uncoupling protein UCP1 expression and expansion of beige/brite adipocytes (so-called browning) in white adipose tissue (WAT). Mechanistically, PKA directly phosphorylated mTOR and RAPTOR on unique serine residues, an effect that was independent of insulin/AKT signaling. Abrogation of the PKA site within RAPTOR disrupted βAR/mTORC1 activation of S6K1 without affecting mTORC1 activation by insulin. Conversely, a phosphomimetic RAPTOR augmented S6K1 activity. Together, these studies reveal a signaling pathway from βARs and PKA through mTORC1 that is required for adipose browning by catecholamines and provides potential therapeutic strategies to enhance energy expenditure and combat metabolic disease.

Authors

Dianxin Liu, Marica Bordicchia, Chaoying Zhang, Huafeng Fang, Wan Wei, Jian-Liang Li, Adilson Guilherme, Kalyani Guntur, Michael P. Czech, Sheila Collins

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Figure 7

Mutations of RAPTOR PKA site Ser791 have functional consequences for βAR signaling but not for Ins.

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Mutations of RAPTOR PKA site Ser791 have functional consequences for βAR...
(A) NIH3T3 cells were infected with lentivirus harboring mouse RAPTOR shRNA or scramble (Scr) control. Four days later, cells were lysed and assayed for endogenous RAPTOR and internal control GAPDH by immunoblotting. (B) NIH3T3 cells infected with lentivirus harboring RAPTOR shRNA were transfected with WT or S791A mutant myc-RAPTOR plasmid followed by either Iso (10 μM) or Ins (100 nM) treatment for 30 minutes. Phospho- and total S6K1 were measured by immunoblotting. (C) HIB-1B cells stably expressing Flag-RAPTOR (WT or S791A) were differentiated and treated with Iso for 30 minutes, followed by anti-Flag IP. They were detected by PKA substrate and reprobed with anti-Flag. (D) Cells as in C were treated with Iso (1 μM) or Ins (10 nM) for 30 minutes. The lysates were detected by Western blotting for p-S6K1 and p-S6 and their respective total antibodies. GAPDH serves as loading control. (E) HIB-1B cells stably expressing myc-RAPTOR (WT or S791D) were pretreated with rapamycin (Rapa; 100 nM), followed by Iso (1 μM), for 1 hour and analyzed as in B. (F and G) Transcripts of Ppara in stably expressing RAPTOR (WT), S791A, and S791D were detected by qPCR (F), while Ucp1 was detected after 6 hours treatment by Iso (G) (100 nM). One-way ANOVA followed by post-hoc group comparison tests; **P < 0.01. (In HIB-1B cells, endogenous RAPTOR still present.)