PPARγ-induced upregulation of subcutaneous fat adiponectin secretion, glyceroneogenesis and BCAA oxidation requires mTORC1 activity

Abstract

The nutrient sensors peroxisome proliferator-activated receptor γ (PPARγ) and mechanistic target of rapamycin complex 1 (mTORC1) closely interact in the regulation of adipocyte lipid storage. The precise mechanisms underlying this interaction and whether this extends to other metabolic processes and the endocrine function of adipocytes are still unknown. We investigated herein the involvement of mTORC1 as a mediator of the actions of the PPARγ ligand rosiglitazone in subcutaneous inguinal white adipose tissue (iWAT) mass, endocrine function, lipidome, transcriptome and branched-chain amino acid (BCAA) metabolism. Mice bearing regulatory associated protein of mTOR (Raptor) deletion and therefore mTORC1 deficiency exclusively in adipocytes and littermate controls were fed a high-fat diet supplemented or not with the PPARγ agonist rosiglitazone (30 mg/kg/day) for 8 weeks and evaluated for iWAT mass, lipidome, transcriptome (Rnaseq), respiration and BCAA metabolism. Adipocyte mTORC1 deficiency not only impaired iWAT adiponectin transcription, synthesis and secretion, PEPCK mRNA levels, triacylglycerol synthesis and BCAA oxidation and mRNA levels of related proteins but also completely blocked the upregulation in these processes induced by pharmacological PPARγ activation with rosiglitazone. Mechanistically, adipocyte mTORC1 deficiency impairs PPARγ transcriptional activity by reducing PPARγ protein content, as well as by downregulating C/EBPα, a co-partner and facilitator of PPARγ. In conclusion, mTORC1 and PPARγ are essential partners involved in the regulation of subcutaneous adipose tissue adiponectin production and secretion and BCAA oxidative metabolism.

Keywords: Adiponectin secretion; BCAA oxidation; C/EBPα; Glyceroneogenesis; PPARγ; Subcutaneous adipose tissue; mTORC1.

Figure 1.

Adipocyte mTORC1 deficiency impairs iWAT adiponectin and blocks the activation of its synthesis and secretion by rosiglitazone. Inguinal white adipose tissue (iWAT) Raptor content (A), body weight (B), body weight gain (C), brown adipose tissue (BAT, D), visceral fat (E), and iWAT (F) masses, food intake (G), whole-body oxygen consumption (H), spontaneous motor activity (SMA, I), respiratory exchange ratio (RER, J), serum leptin (K) and adiponectin (L) and iWAT adiponectin mRNA (M) and protein (N) contents, and DsbA-L (O) and Ero1-La (P) mRNA levels in RapWT and RapKO mice fed with high-fat diet (HFD) containing or not rosiglitazone (RSG, 30 mg/kg/day) for 8 weeks. Values are expressed as mean ± SEM. n= 4–12 mice per group. Means not sharing a common superscript are significantly different from each other, p ≤ 0.05.

Figure 2.

Adipocyte mTORC1 deficiency increases iWAT content of free fatty acids and acylcarnitines and oxidative metabolism. Inguinal white adipose tissue (iWAT) contents of the free fatty acids palmitate and oleate (A) and the acylcarnitines palmitoylcarnitine and oleylcarnitine (B), pyruvate incorporation into triacylglycerol-glycerol (C), PEPCK mRNA levels (D), cardiolipin content (E), oxygen consumption (F), citrate synthase (CS) activity (G), PGC1α (H), CPT1a (I), UCP-1 mRNA levels (J) and protein content of UCP-1 (K and O), catalase (L and O), SOD-1 (M and O) and SOD-2 (N and O) in RapWT and RapKO mice fed with high-fat diet (HFD) containing or not rosiglitazone (RSG, 30 mg/kg/day) for 8 weeks. Values are expressed as mean ± SEM. n= 4–10 mice per group. Means not sharing a common superscript are significantly different from each other, p ≤ 0.05.

Figure 3.

Adipocyte mTORC1 deficiency blocks rosiglitazone modulation of a subset of genes in iWAT. Heap map (A) and gene set enrichment analysis (B, ShinyGo) of genes modulated by rosiglitazone in inguinal white adipose tissue (iWAT) of RapWT, but not RapKO mice (A) fed with high-fat diet (HFD) containing or not rosiglitazone (RSG, 30 mg/kg/day) for 8 weeks. n= 2–3 mice per group.

Figure 4.

Adipocyte mTORC1 deficiency reduces iWAT BCAA oxidation and blocks the upregulation in this process induced by rosiglitazone. Serum branched-chain amino acids (BCAA) (A) and inguinal white adipose tissue (iWAT) valine oxidation (B), and mRNA levels of SLC25A44 (C), BCAT2 (D), BCKDHA (E), BCKDHB (F), DBT (G), DLD (H) and BCKDK (I) in RapWT and RapKO mice fed with high-fat diet (HFD) containing or not rosiglitazone (RSG, 30 mg/kg/day) for 8 weeks. Values are expressed as mean ± SEM. n= 5–10 mice per group. Means not sharing a common superscript are significantly different from each other, p ≤ 0.05.

Figure 5.

Adipocyte mTORC1 deficiency reduces iWAT PPARγ and C/EBPα protein contents. Inguinal white adipose tissue (iWAT) mRNA levels of PPARγ1 (A), PPARγ2 (B) and C/EBPα (C), and protein content of PPARγ1 (D and H), PPARγ2 (E and H), C/EBPα p42 (F and H) and C/EBPα p30 (G and H) in RapWT and RapKO mice fed with high-fat diet (HFD) containing or not rosiglitazone (RSG, 30 mg/kg/day) for 8 weeks. Values are expressed as mean ± SEM. n= 6–10 mice per group. Means not sharing a common superscript are significantly different from each other, p ≤ 0.05.