Cast immobilization induces BAT thermogenesis via sympathetic activation.

(A) Time course of rectal core body temperature of cast-immobilized (gypsum, n = 6) and control (n = 5) mice subjected to a cold (4°C) challenge test. The test was performed 7 days after bilateral immobilization of hind limbs. (B) Rectal core body temperature of cast-immobilized (n = 5) and control (n = 5) mice during cold (4°C) exposure at 24 h after bilateral cast immobilization. (C) Decline in rectal temperature of cast-immobilized and control (n = 9) mice after cold (4°C) exposure for 4 h performed 2 h after unilateral (UL, n =8) or bilateral (BL, n = 9) immobilization. (D) Reverse transcription (RT) and real-time polymerase chain reaction (PCR) analysis of the Ucp1/Gapdh mRNA abundance ratio in iBAT of control mice and mice subjected to bilateral cast immobilization for the indicated times (n = 7 to 13 per group). (E–G) Immunoblot analysis of PGC-1α (n = 3 or 4 per group) and UCP1 (n = 6 per group) in iBAT of control mice and mice subjected to bilateral cast immobilization for the indicated times. GAPDH was examined as a loading control. Representative blots (E) and densitometric quantitation of the PGC- 1α/GAPDH (F) and UCP1/GAPDH (G) band intensity ratios are shown. (H) Noradrenaline concentrations in iBAT of cast-immobilized (n = 4) and control (n = 4) mice after subsequent biteral cast immobilization. (I) RT and real-time PCR analysis of the Ucp1/Gapdh mRNA abundance ratio in iBAT subjected to surgical denervation or sham surgery, either in control mice or in mice at 24 h after subsequent bilateral cast immobilization (n = 6–9 per group). (J) Rectal core body temperature of mice as in (I) subjected to cold (4°C) exposure for 1 h (n = 3 or 4 per group). All quantitative data are means ± SEM. *p < 0.05, **p < 0.01 as determined by two-way ANOVA followed by Tukey’s post hoc test or the post hoc paired/unpaired t test with Bonferroni’s correction (A, B, I and J), by one-way ANOVA followed by Tukey’s post hoc test (C), by Dunnett’s test (D, F, G and H), or by the unpaired t test (I). See also Figure 1_figure supplement 1 and 2, and Table S1.

Cast immobilization alters systemic metabolic dynamics associated with BAT thermogenesis.

(A and B) Concentrations of carbohydrate metabolites (A) and amino acids (B) in sham-operated or denervated iBAT of mice without (control) or at the indicated times after bilateral cast immobilization. The metabolomics data are presented as heat maps corresponding to the log10 value of fold change in cast- immobilized mice relative to the corresponding control mice and are means of four or five mice in each group. TCA, tricarboxylic acid cycle. (C and D) RT and real-time PCR analysis of mRNA abundance for the fatty acid transporter CD36 and the succinate transporter SLC25A10 in sham-operated or denervated iBAT of control mice or mice subjected to bilateral cast immobilization for 24 h (n = 6 per group). (E) Succinate content of sham-operated or denervated iBAT of control mice or mice subjected to bilateral cast immobilization for 24 h (n = 3–5 per group). (F and G) Oxygen consumption rate (VO2) and respiratory exchange ratio (RER), respectively, for control mice and mice subjected to bilateral cast immobilization for 7 days (n = 6 per group). (H) Weight of eWAT for control mice and mice subjected to bilateral cast immobilization for the indicated times (n = 5–7 per group). (I) Hepatic glycogen content for control mice and mice subjected to bilateral cast immobilization for the indicated times (n = 6–10 per group). Date in (C) through (I) are means ± SEM. *p < 0.05, **p < 0.01 as determined by one-way ANOVA followed by Tukey’s post hoc test (C–E), by two-way ANOVA followed by Tukey’s post hoc test or the unpaired post hoc t test with Bonferroni’s correction (F and G), or by Dunnett’s test (H and I). See also Figure 2_figure supplement 1 and 2.

Free amino acids are transferred from skeletal muscle to BAT and the liver for maintenance of energy homeostasis.

(A and B) Metabolomics analysis of amino acid concentrations in soleus and EDL muscles (A) as well as in serum and the liver (B) of control mice and mice subjected to bilateral cast immobilization for the indicated times. Results are presented as heat maps of the log10 value of fold change for cast-immobilized mice relative to control mice and are means of three mice per group. (C) Organ-specific [3H]leucine uptake in control mice and mice subjected to bilateral cast immobilization for 24 h (n = 4–6 per group). sWAT, subcutaneous WAT. (D) RT and real-time PCR analysis of SLC25A44 gene expression in sham- operated or denervated iBAT of control mice and mice subjected to subsequent bilateral cast immobilization for 24 h (n = 6 per group). Data in (D) and (E) are means ± SEM. *p < 0.05, **p < 0.01 as determined by the unpaired t test (C) or by one-way ANOVA followed by Tukey’s post hoc test (D). See also Figure 3_figure supplement 1 and 2.

Cast immobilization upregulates Il6 expression in BAT and skeletal muscle.

(A and B) RT and real-time PCR analysis of IL-1β, IL-6, IL-10, IL-15, TNF-α, MCP-1, BMP8B, FGF21, and irisin gene expression in iBAT (A) or soleus (B) of control mice or mice at 10 h (iBAT) or 24 h (soleus) after bilateral cast immobilization (n = 5 or 6 per group). (C and D) RT and real-time PCR analysis of Il6 expression in iBAT (C) and soleus (D) of control mice and mice at the indicated times after bilateral cast immobilization (n = 3–7 per group). (E–L) RT and real-time PCR analysis of Saa3 (E, G, I, and K) and Socs3 (F, H, J, and L) mRNA abundance in iBAT (E and F), soleus (G and H), liver (I and J), and eWAT (K and L) of control mice and mice at the indicated times after bilateral cast immobilization (n = 3–7 per group) (M) RT and real-time PCR analysis of Il6 expression, respectively, in denervated iBAT of control mice or mice at the indicated times after bilateral cast immobilization (n = 4-7 per group). (N) Serum IL-6 concentration in control mice or mice at the indicated times after bilateral cast immobilization (n = 6 per group). All data are means ± SEM. *p < 0.05, **p < 0.01, NS (not significant) as determined by the unpaired t test (A and B) or by Dunnett’s test (C–N). See also Figure 4_figure supplement 1.

IL-6 affects energy metabolism in BAT and skeletal muscle of cast-immobilized mice.

(A) Change in rectal body temperature during cold exposure at 4°C for 4 h for WT (n = 4 or 5) and IL-6 KO (n = 4 or 5) mice that had been subjected (or not) to bilateral cast immobilization for 24 h. (B and C) Concentrations of carbohydrate metabolites (B) and amino acids (C) in iBAT of IL-6 KO (–/–) and WT (+/+) mice without (control) or with bilateral cast immobilization for the indicated times. Results are presented as heat maps of the log10 value of the fold change relative to control WT mice and are means of four mice in each group. (D–F) RT and real-time PCR analysis of the expression of SLC25A44 (D), BCKDHA (E), and CD36 (F) genes in iBAT of IL-6 KO and WT mice without (control) or with bilateral cast immobilization for 24 h (n = 5 or 6). (G) Incorporation of [3H]leucine in iBAT of IL-6 KO and WT mice without (control) or with bilateral cast immobilization for 24 h (n = 4 or 5 per group). (H–J) BCAA (Val, Ile, and Leu, respectively) concentrations in soleus of IL-6 KO and WT mice without (control) or with bilateral cast immobilization for 1 or 7 days (n = 4 per group). Data in (A) and (D) through (J) are means ± SEM. *p < 0.05, **p < 0.01 as determined by one-way ANOVA followed by Tukey’s post hoc test (D–G), by Dunnett’s test (H–J), or by two-way ANOVA followed by Tukey’s post hoc test (A). See also Figure 5_figure supplement 1.

Skeletal-muscle-derived IL-6 increases BCAA concentrations in skeletal muscle for support of BAT thermogenesis.

(A–C) Concentrations of BCAAs (Val, Ile, and Leu, respectively) in the soleus of control IL-6 KO mice and at 3 h after intraperitoneal administration of recombinant IL-6 (rIL-6, 400 ng) or vehicle in IL-6 KO mice that had been subjected to bilateral cast immobilization for 24 h (n = 4 or 5 per group). (D) BCAA concentrations in C2C12 myotubes incubated in the absence or presence of rIL-6 (50 ng/ml) for 1 h (n = 6 independent experiments) . (E) Rectal core body temperature during cold exposure at 4°C for 1 h for control IL-6 KO mice and for IL-6 KO mice subjected to bilateral cast immobilization for 24 h and injected intraperitoneally with rIL-6 (400 ng) or vehicle 30 min before cold challenge (n = 5 per group). The white circles represent control mice, the red circles indicate cast-immobilized mice, and the red triangles represent cast- immobilized mice that were treated with recombinant IL-6 (rIL-6). (F) Rectal core body temperature during cold exposure at 4°C for 1 h for C57BL/6J mice with denervated iBAT subjected or not to bilateral cast immobilization and treatment with rIL-6 or vehicle as in (E) (n = 4 per group). (G) Serum IL-6 concentration of mice subjected to surgical removal of iBAT and then subjected (or not, control) to bilateral cast immobilization for the indicated times (n = 4-6 per group). The white circles represent control mice, the blue circles indicate C57BL/6J mice with denervated iBAT, and the blue triangles denote C57BL/6J mice with denervated iBAT that were treated with recombinant IL-6 (rIL-6). (H) Weight of eWAT in mice subjected to surgical removal of iBAT (or to sham surgery) followed (or not, control) by bilateral cast immobilization for 7 days (n = 5-7 per group). (I–K) Concentrations of BCAAs (Val, Ile, and Leu, respectively) in soleus of mice subjected to surgical removal of iBAT and then subjected (or not, control) to bilateral cast immobilization for the indicated times (n = 4 per group). All data are means ± SEM. *p < 0.05, **p < 0.01, NS (not significant) as determined by Dunnett’s test (A–C and G and I-K), or by the unpaired t test (D) or by one-way ANOVA followed by Tukey’s post hoc test (H), or by two-way ANOVA followed by the post hoc paired/unpaired t test with Bonferroni’s correction (E and F). See also Figure 6_figure supplement 1 and 2.

Acute cold exposure or restraint stress also induced IL-6 from skeletal muscle to supply BCAA for BAT thermogenesis.

(A) RT and real-time PCR analysis of Il6 expression in iBAT, soleus, liver, eWAT, and spleen of mice subjected (or not) to cold (4°C) exposure for 4 h (control, n = 6–9; cold exposure, n = 5 or 6). (B–D) Concentrations of BCAAs (Val, Ile, and Leu, respectively) in soleus of WT and IL-6 KO mice subjected (or not) to cold exposure at 4°C for 4 h (n = 4 per group). (E and F) RT and real-time PCR analysis of Il6 expression in iBAT (E) and soleus (F) of control mice and mice subjected to restraint stress for 6 h (n = 4 per group). (G–I) Concentrations of BCAAs (Val, Ile, and Leu, respectively) in soleus of WT and IL-6 KO mice subjected (or not) to restraint stress for 6 h (n = 4 per group). All data are means ± SEM. *p < 0.05, **p < 0.01, NS (not significant) as determined by the unpaired t test (A–I). See also Figure 7_figure supplement 1 and 2.

(A–C) Weight of individual soleus (A), gastrocnemius (B), and extensor digitorum longus (EDL) muscles of control mice and mice subjected to bilateral cast immobilization of hind limbs for the indicated times (n = 5-7 per group). (D and E) Locomotor activity during light (D) and dark (E) phases for control mice and mice subjected to bilateral cast immobilization for the indicated times (n = 6 per group). (F) Rectal core body temperature of cast-immobilized (n = 6) and control (n = 4) mice at room temperature after bilateral cast immobilization. (G) RT and real-time PCR analysis of the expression of UCP1 genes in iBAT of control and cast-immobilized mice subjected (or not) to cold exposure at 4°C for 4 h (n = 4-9 per group). (H-K) RT and real-time PCR analysis of Ucp2, Ucp3, Sln and Ppagc1a expression in iBAT of control and cast-immobilized mice subjected (or not) to cold exposure at 4°C for 4 h (n = 4-9 per group). (L) Locomotor activity during cold (4°C) exposure for 4 h determined for control mice and mice subjected to unilateral (UL) or bilateral (BL) cast immobilization (n = 5 per group). These data were obtained in conjunction with those in Figure1C. All quantitative data are means ± SEM. *p < 0.05, **p < 0.01, NS (not significant) as determined by Dunnett’s test (A-C) or by two-way ANOVA followed by Tukey’s post hoc test or the post hoc paired/unpaired t test with Bonferroni’s correction (D-F and L), or by the unpaired t test (G-K)

(A) Concentrations of carbohydrate metabolites (acetyl CoA divalent, succinic acid, fumaric acid and malic acid) in soleus and EDL of control mice and mice subjected to bilateral cast immobilization for the indicated times. Results are presented as heat maps of the log10 value of fold change for cast-immobilized mice relative to control mice and are are shown individually for three mice of each group. (B and C) RT and real-time PCR analysis of Ucp3 and Ucp2 expression, respectively, in soleus of control mice and mice subjected to bilateral cast immobilization for the indicated times (n = 5-6 per group). (D and E) RT and real-time PCR analysis of Sln and Camk2a expression, respectively, in soleus of control mice and mice subjected to bilateral cast immobilization for the indicated times (n = 6 per group). (F and G) RT and real-time PCR analysis of Ppargc1a, and Tfam expression, respectively, in soleus of control mice and mice subjected to bilateral cast immobilization for the indicated times (n = 6 per group). (H) Hematoxylin-eosin staining of surgically denervated or sham-operated iBAT from control mice or mice subjected to bilateral cast immobilization for 7 days. Scale bar, 50 µm. (I) Food intake of control mice and mice subjected to bilateral cast immobilization for the indicated times (n = 6–9 per group). (J) Hematoxylin-eosin staining of iBAT of control mice and mice subjected to bilateral cast immobilization for 1 or 7 days. Scale bar, 50 µm. All quantitative data other than those in (A, H and J) are means ± SEM. *p < 0.05, **p < 0.01, NS (not significant) as determined by Dunnett’s test (B-G) or by two-way ANOVA followed by Tukey’s post hoc test or the post hoc paired/unpaired t test with Bonferroni’s correction (I).

(A) Pathway analysis of metabolites upregulated in iBAT by cast immobilization for 10 h. (B and C) Oxygen consumption rate (VO2) and RER, respectively, for mice with denervated iBAT subjected (or not) to bilateral cast immobilization for 7 days (n = 3 per group). Quantitative data other than in (A) are means ± SEM. *p < 0.05, **p < 0.01, as determined by two-way ANOVA followed by Tukey’s post hoc test or the post hoc paired/unpaired t test with Bonferroni’s correction (B and C).

(A and B) RT and real-time PCR analysis of G6Pase (G6pc1) and PEPCK (Pck1) gene expression in the liver of control mice and mice subjected to bilateral cast immobilization for the indicated times (n = 5 or 6 per group). (C) Concentrations of carbohydrate metabolites in the liver of control mice and mice subjected to bilateral cast immobilization for the indicated times. The metabolomics data are presented as heat maps corresponding to the log10 value of fold change in cast-immobilized mice relative to control mice. Results are means of three mice per group. (D) Serum concentration of noradrenaline in control mice and mice subjected to bilateral cast immobilization for the indicated times (n = 5–7 per group). (E) Serum concentration of corticosterone for control mice and mice subjected to bilateral cast immobilization for the indicated times (n = 6–8 per group). (F) RT and real-time PCR analysis of Crh expression in the hypothalamus of control mice and mice subjected to bilateral cast immobilization for the indicated times (n = 5–8 per group). Quantitative data other than in (C) are means ± SEM. *p < 0.05, **p < 0.01, NS (not significant) as determined by Dunnett’s test (A, B and D-F).

(A and B) RT and real-time PCR analysis of Fbxo32 (Atrogin-1) and Trim63 (MuRF-1) expression in the soleus muscle of control mice and mice subjected to bilateral cast immobilization for the indicated times (n = 6 per group). (C–H) RT and real-time PCR analysis of SLC1A5, SLC7A5, SLC38A2, SLC43A1, BCAT2, and BCKHDA gene expression, respectively, in iBAT of control mice or mice subjected to bilateral cast immobilization for the indicated times (n = 3–6 per group). (I–M) RT and real-time PCR analysis of gene expression for SLC1A5, SLC7A5, SLC38A2, SLC43A1, and BCKHDA, respectively, in the liver of control mice or mice subjected to bilateral cast immobilization for the indicated times (n = 6 per group). (N–S) RT and real-time PCR analysis of gene expression for SLC1A5, SLC7A5, SLC38A2, SLC43A1, BCAT2, and BCKHDA in soleus of control mice or mice subjected to bilateral cast immobilization for the indicated times (n = 6 per group). All data are means ± SEM. *p < 0.05, **p < 0.01 as determined by Dunnett’s test.

(A–C) RT and real-time PCR analysis of SLC25A44 gene expression in iBAT (A), liver (B), and soleus (C) of control mice or mice subjected to bilateral cast immobilization for the indicated times (n = 6 per group). (D–J) RT and real-time PCR analysis of gene expression for SLC25A44, SLC1A5, SLC7A5, SLC38A2, SLC43A1, BCAT2, and BCKDHA, respectively, in denervated iBAT of control mice or mice subjected to bilateral cast immobilization for the indicated times (n = 4–6 per group). All data are means ± SEM. *p < 0.05, **p < 0.01 as determined by Dunnett’s test.

(A–G) RT and real-time PCR analysis of TNF-α, MCP-1, IL-1β, IL-10, IL-15, FGF21, and BMP8B gene expression, respectively, in iBAT of control mice and mice at the indicated times after bilateral cast immobilization (n = 3–6 per group). (H–N) RT and real-time PCR analysis of TNF-α, MCP-1, IL-1β, IL-10, IL-15, FGF21, and irisin gene expression, respectively, in soleus of control mice and mice at the indicated times after bilateral cast immobilization (n = 5 or 6 per group). (O–Q) RT and real-time PCR analysis of Il6 expression in liver (O), eWAT (P), and spleen (Q) of control mice and mice at the indicated times after bilateral cast immobilization (n = 5-6 per group). (R) Serum concentration of TNF-α in control mice and mice at the indicated times after bilateral cast immobilization (n = 6 per group). All data are means ± SEM. *p < 0.05, **p < 0.01, NS (not significant) as determined by Dunnett’s test.

(A) RT and real-time PCR analysis of Ucp1 mRNA abundance in iBAT of WT and IL-6 KO mice without (control) or with bilateral cast immobilization for 1 or 7 days (n = 5 or 6 per group). (B and C) Oxygen consumption rate for WT (B) and IL-6 KO (C) mice subjected (or not) to bilateral cast immobilization for 7 days (n = 4 per group). (D) Hematoxylin-eosin staining of iBAT from IL-6 KO and WT mice without (control) or with bilateral cast immobilization for 7 days. Scale bar, 50 µm. (E and F) RER for WT (E) and IL-6 KO (F) mice subjected (or not) to bilateral cast immobilization for 7 days (n = 4 per group). (G) Weight of eWAT in WT (+/+) and IL-6 KO (-/- mice subjected (or not, control) to bilateral cast immobilization for 7 days (n = 5-7 per group). (H–J) Serum concentrations of BCAA (Val, Ile, and Leu, respectively) concentrations of IL-6 KO and WT mice without (control) or with bilateral cast immobilization for 1 or 7 days (n = 4 per group). All data other than those in (F) are means ± SEM. *p < 0.05, **p < 0.01 as determined by one-way ANOVA followed by Tukey’s post hoc test (A and G) or by two-way ANOVA followed by Tukey’s post hoc test or the post hoc paired/unpaired t test with Bonferroni’s correction (B-C and E-F), or by Dunnett’s test (H-I).

(A) Amino acid concentrations in soleus of control IL-6 KO mice and at 3 h after intraperitoneal administration of recombinant IL-6 (rIL-6, 400 ng) or vehicle in IL- 6 KO mice that had been subjected to bilateral cast immobilization for 24 h. Results are presented as heat maps of the log10 value of the fold change relative to control mice and are means of four or five mice in each group. (B and C) RT and real-time PCR analysis of Saa3 (B) and Socs3 (C) mRNA abundance in C2C12 myotubes incubated in the absence or presence of rIL-6 (50 ng/ml) for 1 h (n = 8 or 9 independent experiments). (D) Amounts of carbohydrate metabolites in iBAT of mice as in (A). Results are presented as heat maps of the log10 value of the fold change relative to control mice and are means of four mice in each group. (E) Amino acid concentrations in iBAT of mice as in (A). Results are presented as heat maps of the log10 value of the fold change relative to control mice and are means of four or five mice in each group. (F–H) RT and real-time PCR analysis of UCP1 (F), SLC25A44 (G), and BCKDHA (H) gene expression in iBAT of mice as in (A) (n = 5 per group). (I) Change in rectal core body temperature of mice of the indicated Il6 genotypes during cold exposure at 4°C for 4 h (+/+ and –/–, n = 5; +/–, n = 6). (J) RT and real-time PCR analysis of Ucp1 mRNA abundance in iBAT of mice of the indicated Il6 genotypes (n = 5 or 6 per group). (K–M) RT and real-time PCR analysis of UCP1 (K), SLC25A44 (L), and BCKDHA (M) gene expression in cultured brown adipocytes incubated in the absence or presence of rIL-6 (50 ng/ml) or CL316 243 (1 µM) for 24 h (n = 3 or 4 independent experiments). (N–P) RT and real-time PCR analysis of UCP1 (N), SLC25A44 (O) and BCKDHA (P) gene expression in iBAT of WT (+/+, n = 5 per group) or IL-6 KO (–/–, n = 8 per group) mice after intraperitoneal treatment with CL316 243 (0.1 mg/kg per day) or vehicle for 7 days. All data other than those in (A),(D) and (E) are means ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 as determined by the unpaired t test (B and C), by one-way ANOVA followed by Tukey’s post hoc test (F–H and J-P), or by two-way ANOVA followed by the post hoc paired/unpaired t test with Bonferroni’s correction (I).

(A-E) RT and real-time PCR analysis of SLC43A1, SLC1A5, SLC7A5, BCAT2, and BCKDHA gene expression, respectively, in soleus of iBAT-depleted mice subjected (or not, control) to bilateral cast immobilization for the indicated times (n = 4–7 per group). (F) Change in soleus and gastrocnemius muscle weights of iBAT-depleted or sham-operated mice subjected to bilateral cast immobilization for 7 days (n = 6 per group). All data are means ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 as determined by the unpaired t test (F) or by Dunnett’s test (A-E). See also Tables S2.

(A-E) RT and real-time PCR analysis of Il6, Saa3, Socs3, Fbxo32 and Trim63 mRNA abundance in BBM of mice subjected (or not, control) to bilateral cast immobilization for the indicated times (n = 4 per group). (F-H) BCAA (Val, Ile, and Leu, respectively) concentrations in BBM of mice subjected (or not, control) to bilateral cast immobilization for the indicated times (n = 4 per group). (I and J) RT and real-time PCR analysis of Saa3 (I) and Socs3 (J) mRNA abundance in soleus of mice subjected (or not) to cold (4°C) exposure for 4 h (control, n = 6; cold exposure, n = 5). (K and L) RT and real-time PCR analysis of Fbox32 (K) and Trim63 (L) mRNA abundance in soleus of mice subjected (or not) to cold (4°C) exposure for 4 h (control, n = 6; cold exposure, n = 5). (M and N) Weight of individual soleus (M) and gastrocnemius (N) muscles of control mice and mice subjected to. cold exposure at 4°C for 4 h (n = 5 or 6 per group). (O) Amino acid concentrations in soleus of WT (+/+) and IL-6 KO (–/–) mice subjected (or not) to cold exposure at 4°C for 4 h. Results are presented as heat maps of the log10 value of fold change relative to WT control mice and are means of four mice in each group. (P) BCAA concentrations in soleus of iBAT-depleted mice subjected (or not, control) to cold exposure at 4°C for 4 h (n = 3 or 4 per group). All data other than in (O) are means ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 as determined by the unpaired t test (I-N and P) or by Dunnett’s test (A-H).

(A) RT and real-time PCR analysis of Ucp1 mRNA abundance in iBAT of mice subjected (or not) to restraint stress for 6 h (n = 4 per group). (B and C) RT and real-time PCR analysis of Saa3 (B) and Socs3 (C) mRNA abundance in soleus of mice subjected (or not) to restraint stress for 6 h (n = 4 per group). (D-F) Weight of soleus (D), gastrocnemius (E) and EDL (F) in mice subjected to restraint stress for 6 h (n = 4 per group). (G and H) RT and real-time PCR analysis of Fbox32 (G) and Trim63 (H) mRNA abundance in soleus of mice subjected (or not) to restraint stress for 6 h (n = 4 per group). (I) Amino acid concentrations in soleus of WT (+/+) and IL-6 KO (–/–) mice subjected (or not) to restraint stress for 6 h. Results are presented as heat maps of the log10 value of fold change relative to WT control mice and are means of four mice in each group. All data are means ± SEM. *p < 0.05 as determined by the unpaired t test (A-H).