The Baf60c/Deptor pathway links skeletal muscle inflammation to glucose homeostasis in obesity

Abstract

Skeletal muscle insulin resistance in type 2 diabetes is associated with a shift from oxidative to glycolytic metabolism in myofibers. However, whether this metabolic switch is detrimental or adaptive for metabolic homeostasis has not been resolved. We recently demonstrated that the Baf60c/Deptor pathway promotes glycolytic metabolism in the muscle and protects mice from diet-induced insulin resistance. However, the nature of the signals that impinge on this pathway and the role of Baf60c in glucose homeostasis in the severe insulin-resistant state remain unknown. Here we show that expression of Baf60c and Deptor was downregulated in skeletal muscle in obesity, accompanied by extracellular signal-related kinase (ERK) activation. In cultured myotubes, inhibition of ERK, but not Jun NH2-terminal kinase and IκB kinase, blocked the downregulation of Baf60c and Deptor by the proinflammatory cytokine tumor necrosis factor-α. Treatment of obese mice with the ERK inhibitor U0126 rescued Baf60c and Deptor expression in skeletal muscle and lowered blood glucose. Transgenic rescue of Baf60c in skeletal muscle restored Deptor expression and Akt phosphorylation and ameliorated insulin resistance in ob/ob mice. This study identifies the Baf60c/Deptor pathway as a target of proinflammatory signaling in skeletal muscle that may link meta-inflammation to skeletal myofiber metabolism and insulin resistance.

Figure 1

The Baf60c/Deptor pathway is downregulated in skeletal muscle from ob/ob mice. A: qPCR analysis of gene expression in quadriceps muscles from control (CTR) and ob/ob mice. B: Immunoblots of total protein lysates of quadriceps muscles. C: Quantification of Baf60c and Deptor protein levels in B after normalization to α-tubulin. D and E: Immunoblots of total protein lysates from quadriceps muscles using indicated antibodies. LE, long exposure; p, phosphorylated; SE, short exposure; T, total. F: Quantification of phosphorylated JNK1/2 and ERK1/2 in E after normalization to respective total protein levels. Values in C and F indicate mean ± SEM. All of the data shown are representative of at least three independent experiments. *P < 0.05 by two-tailed Student t test.

Figure 2

The Baf60c/Deptor pathway is downregulated in skeletal muscle from db/db and diet-induced obese mice. A: Immunoblots of total protein lysates of quadriceps muscles from control (CTR) and db/db mice. B: Quantification of Baf60c, Deptor, and phosphorylated ERK1/2 protein levels in A. C: Immunoblots of total protein lysates of quadriceps muscles from mice fed with chow or an HFD for 3 months. D: Quantification of Baf60c, Deptor, and phosphorylated ERK1/2 protein levels in C. LE, long exposure; p, phosphorylated; SE, short exposure; T, total. Values in B and D indicate mean ± SEM. All of the data shown are representative of at least three independent experiments. *P < 0.05 by two-tailed Student t test.

Figure 3

TNF-α represses Baf60c and Deptor expression in muscle cells through epigenetic changes. A: qPCR analysis of mRNA expression in C2C12 myotubes treated with IL6 (100 ng/mL), Mstn (200 ng/mL), and TNF-α (50 ng/mL) for 8 h. qPCR analysis of gene expression in C2C12 myotubes (B) and primary human myotubes (C) after TNF-α (30 and 100 ng/mL) treatment for 8 h. D: Immunoblots of total protein lysates from C2C12 and primary human myotubes treated with vehicle (Veh) or TNF-α (50 ng/mL) for 8 h. E: qPCR analysis of TA muscles after intramuscular injection of Veh or TNF-α (5 or 50 µg/mL in 25 µL, n = four). F: Immunofluorescence staining of C2C12 myoblasts expressing vector (Vec) or Flag-HA–tagged Baf60c (FH-Baf60c) with anti-HA antibody and DAPI (nuclei). Scale bar = 10 µm. G: Immunoblots of cytosolic (Cyto) and nuclear (NE) fractions from retroviral transduced C2C12 myoblasts expressing control (Scrb) or siBaf60c shRNA, or from C2C12 myotubes treated with Veh or TNF-α (50 ng/mL) for 3 h. ChIP assays in primary human myotubes treated with Veh or TNF-α (50 ng/mL) for 3 h using primers located at the proximal Baf60c (H) and Deptor (I) promoters. Values in A–C, H, and I indicate mean ± SD, and values in E represent mean ± SEM. All of the data shown are representative of at least three independent experiments. *P < 0.05 by two-tailed Student t test.

Figure 4

ERK pathway mediates the inhibition of Baf60c and Deptor expression by TNF-α in muscle cells. A and B: Immunoblots of total protein lysates from C2C12 myotubes pretreated without (CTR) or with PD98059 (PD; 50 μmol/L), SP600125 (SP; 2 μmol/L), Amlexanox (AM; 20 μmol/L), CAY10576 (Cay; 5 μmol/L), or Compound VIII (Co VIII; 5 μmol/L) for 30 min, followed by vehicle (Veh) or TNF-α (50 ng/mL) treatments for 15 min. LE, long exposure; p, phosphorylated; SE, short exposure; T, total. C–E: qPCR analysis of gene expression in C2C12 myotubes pretreated with increasing doses of PD (25 and 50 μmol/L), SP (1 and 2 μmol/L), or with AM (20 μmol/L), Cay (5 μmol/L), Co VIII (5 μmol/L) for 30 min, followed by Veh or TNF-α (50 ng/mL) treatments for additional 8 h. Data shown in C–E indicate mean ± SD. All of the data shown are representative of at least three independent experiments. *P < 0.05 by two-tailed Student t test.

Figure 5

In vivo ERK inhibition lowers blood glucose and restores Baf60c and Deptor expression in obese mice. A–D: HFD-fed mice were treated with vehicle (Veh) or U0126 (10 mg/kg) by intraperitoneal injection for 2 weeks (n = eight mice per group). Shown are blood glucose levels after 8 h of starvation (A); qPCR analysis of Baf60c and Deptor expression in quadriceps muscles (B); immunoblots of total (T) protein lysates of quadriceps muscles (C); and quantification of Baf60c, Deptor, and phosphorylated (p) ERK1/2 protein levels in C (D). E–H: Male db/db mice (8 weeks old) treated with Veh or U0126 (20 mg/kg) by intraperitoneal injection for 2 weeks (n = seven to eight mice per group). Shown are blood glucose levels after 8 h of starvation (E); qPCR analysis of Baf60c and Deptor expression in quadriceps muscles (F); immunoblots of total protein lysates of quadriceps muscles (G); and quantification of Baf60c, Deptor, and phosphorylated ERK1/2 protein levels in G (H). Values in A, B, D–F, and H indicate mean ± SEM. All of the data shown are representative of at least three independent experiments. *P < 0.05 by two-tailed Student t test.

Figure 6

Deptor is required for the maintenance of Akt activation and glycolytic metabolism in adult muscles. A: Deptor mRNA expression in TA muscle transduced with indicated adenoviral vectors (n = six mice per group). B: Immunoblots of total (T) and phosphorylated (p) protein lysates from transduced TA muscles. Shown are representative blots from two mice. C: Histochemical staining (top) and quantification of indicated enzymes (bottom) on transduced TA muscle sections. Scale bar = 100 µm. D: LDH enzymatic activity in transduced TA muscle (n = nine mice per group). E: Glucose and lactate concentrations in culture media from transduced C2C12 myotubes. F: Glycolytic flux in transduced C2C12 myotubes treated with vehicle (Veh) or insulin (10 nmol/L) for 1 h. Values represent mean ± SEM in A and D and represent mean ± SD in C, E, and F. All of the data shown are representative of at least three independent experiments. *P < 0.01 by two-tailed Student t test.

Figure 7

Transgenic expression of Baf60c in ob/ob mice promotes glycolytic metabolism in skeletal muscle. A: qPCR analysis of gene expression in quadriceps muscle. B: Relative mRNA levels of Deptor in quadriceps muscle (left) and quantification of Deptor protein levels in C after normalization to α-tubulin (right). C: Immunoblots of total protein lysates from quadriceps muscle. D: Representative histochemical staining (top) and quantification of indicated enzymes (bottom) on frozen TA and extensor digitorum longus (EDL) muscle sections. Scale bar = 100 µm. Complete palmitate oxidation (n = six muscles per genotype) (E), and glycolytic flux (n = six muscles per genotype) (F) in isolated soleus muscles from ob/ob and ob/ob Tg mice. WT, wild-type. Values represent mean ± SEM in A, B, E, and F and represent mean ± SD in D. All of the data shown are representative of at least three independent experiments. *P < 0.01 by two-tailed Student t test.

Figure 8

Transgenic expression of Baf60c improves glucose metabolism in ob/ob mice. A: Fasting blood glucose and plasma insulin levels in ob/ob and ob/ob Tg mice, (n = five to seven mice per group). B: GTT and ITT in 4-month-old male mice (n = five to seven mice per group). C: Plasma levels of indicated metabolites from mice fasted overnight (n = five to seven mice per group). D: Immunoblots of total protein lysates from quadriceps muscle. E: Relative phosphorylation (p) levels of Akt on Thr308 and Ser473 residues in D after normalization to total (T) Akt levels. F: qPCR analysis of gene expression in quadriceps muscle. NEFA, nonesterified fatty acid; WT, wild-type. Values indicate mean ± SEM. All of the data shown are representative of at least three independent experiments. *P < 0.05 by two-tailed Student t test.