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, 28 (4), 631-643.e3

Brown Adipose Tissue Controls Skeletal Muscle Function via the Secretion of Myostatin

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Brown Adipose Tissue Controls Skeletal Muscle Function via the Secretion of Myostatin

Xingxing Kong et al. Cell Metab.

Abstract

Skeletal muscle and brown adipose tissue (BAT) are functionally linked, as exercise increases browning via secretion of myokines. It is unknown whether BAT affects muscle function. Here, we find that loss of the transcription factor IRF4 in BAT (BATI4KO) reduces exercise capacity, mitochondrial function, ribosomal protein synthesis, and mTOR signaling in muscle and causes tubular aggregate formation. Loss of IRF4 induces myogenic gene expression in BAT, including the secreted factor myostatin, a known inhibitor of muscle function. Reducing myostatin via neutralizing antibodies or soluble receptor rescues the exercise capacity of BATI4KO mice. In addition, overexpression of IRF4 in brown adipocytes reduces serum myostatin and increases exercise capacity in muscle. Finally, mice housed at thermoneutrality have reduced IRF4 in BAT, lower exercise capacity, and elevated serum myostatin; these abnormalities are corrected by excising BAT. Collectively, our data point to an unsuspected level of BAT-muscle crosstalk driven by IRF4 and myostatin.

Keywords: IRF4; brown adipose tissue; exercise; myopathy; myostatin; thermoneutrality; tubular aggregates.

Figures

Figure 1:
Figure 1:. Mice lacking IRF4 in BAT have reduced exercise capacity.
BATI4KO mice showed lower exercise capacity with both a Low Intensity (LI) (A) and High Intensity (HI) (B) regimen. The low intensity exercise regimen was also performed in adipose-specific PRDM16 KO (C) and UCP1KO male mice (D). *p < 0.05.
Figure 2:
Figure 2:. BATI4KO mice exhibit myopathy.
(A) H&E staining of vastus lateralis of BATI4KO and control mice (X40). Black arrows indicate nuclei that have migrated to the center of the myofiber after 1 hour of running. The percentage of myofibers with centralized nuclei was determined by manual counting. (100–150 myofibers from 4 mice per group, *p < 0.05 vs. Flox mice). (B) Tubular aggregates (yellow arrows) in the vastus lateralis of male BATI4KO mice. (C) Atp2a2 expression in the vastus lateralis of male BATI4KO and control mice. Results are expressed as mean ± SEM (n=5–6, *p <0.05). (D) Measurement of oxygen consumption rate (OCR) in isolated mitochondria from vastus of male BATI4KO and control mice in the sedentary state. (n=7 mice per group, ***p<0.0001). (E) Mitochondrial DNA content in the vastus lateralis of male BATI4KO and control mice. Results are expressed as mean ± SEM (n=4–5, *p<0.05). (F) Western blot analysis of isolated muscle mitochondria from BATI4KO and control mice. Protein amount was quantified using Image J. (n=3, *p < 0.05 vs. Flox mice). (G) Q-PCR analysis of mitochondrial gene expression in the vastus of male BATI4KO and control mice (n = 8–10, *p<0.05). (H) Volcano plot of vastus RNA-seq. Blue and red dots are significantly different between BATI4KO and flox mice; red dots represent ribosomal subunit genes. (I) GO analysis (biological process) of significantly different genes from RNA-seq analysis. (J) Western blot analysis of mTOR signaling in vastus of male BATI4KO vs. control mice. Activity was quantified using Image J. (n=4, *p < 0.05 vs. Flox mice).
Figure 3:
Figure 3:. Loss of IRF4 induces a myogenic gene expression signature in BAT, including the TGF𝛃-family member myostatin.
(A) Volcano plot of BAT RNA-seq. Blue and red dots represent significantly different genes between BATI4KO and flox groups. Red dots represent muscle-related transcripts. (B) GO analysis (biological process) of significantly different genes from RNA-seq analysis. (C) Q-PCR analysis of selected muscle-related genes in BAT of male BATI4KO vs. control mice (n=5–6, *p<0.05). Tissue (D) and serum (E) myostatin levels in BATI4KO vs. control mice. Protein amount was quantified using Image J. (n=4, *p < 0.05 vs. Flox mice).
Figure 4:
Figure 4:. Brown adipocytes from BATI4KO mice secrete myostatin.
(A) Schematic illustration of co-culture. (B) Elevated Mstn mRNA in primary brown adipocytes from BATI4KO mice. Results are expressed as mean ± SEM (n=5–6, *p<0.05). (C) Western blot analysis of coculture medium myostatin levels. Protein amount was quantified using Image J (n=3). Q-PCR (D, E) and OCR (H) analysis of C2C12 myotubes, co-cultured with BAT primary adipocyte from BATI4KO or control mice for 6 hours. (*p < 0.05). (F) Western blot analysis of mitochondria protein from co-cultured C2C12 myotubes. Protein amount was quantified using Image J (n=3, *p < 0.05). (G) Western blot analysis of mTOR signaling pathway in co-cultured C2C12 myotubes. Activity was quantified using Image J (n=3, *p < 0.05).
Figure 5:
Figure 5:. Myostatin mediates the effect of BAT IRF4 loss on exercise capacity.
(A) Low intensity exercise regimen was performed in mice following a single injection of myostatin. (n=12–16, *p < 0.05.). (B) Exercise capacity determined in BATI4KO and control littermates before, 36 hours or 10 days following intraperitoneal injection of soluble ActRIIB (sActRIIB) with 10 mg/kg per mouse. (n=6–7, *p<0.05). (C) Exercise capacity was measured in BATI4KO and control littermates before, 60 hours or 10 days following a single intraperitoneal injection of myostatin neutralizing antibodies or isotype control. (n=6–7, *p<0.05). (D) Q-PCR analysis of gene expression in vastus lateralis from sedentary mice treated as in (C), but samples were harvested before and 60 hours after injection (n=5, *p<0.05).
Figure 6:
Figure 6:. Mice overexpressing IRF4 in BAT run better than wild-type mice, and have reduced serum myostatin.
(A) Exercise capacity in male BATI4OE mice (n=7, *p<0.05). (B) Continuous measurement of oxygen consumption rate (OCR) in isolated mitochondria from vastus of BATI4OE and control mice in the sedentary state. (C) Mitochondrial DNA content in the vastus of male BATI4OE and control mice. Results are expressed as mean ± SEM (n=4–5, *p<0.05). (D, E) Serum and tissue myostatin level in BATI4OE vs. control mice. Protein amount was quantified using Image J. (n=4, *p < 0.05 vs. control mice). (F) Western blot analysis of mTOR signaling pathway in vastus lateralis of BATI4OE and control mice. Activity was quantified using Image J (n=4, *p < 0.05 vs. control mice).
Figure 7:
Figure 7:. Thermoneutrality induces expression of myostatin in BAT and reduces exercise capacity.
(A) Exercise capacity was measured in male wild-type C57Bl/6J mice after exposure to 30°C for 7 days, (n=10–12, * p<0.05). (B) Serum, BAT and vastus (C) levels of myostatin in mice housed at thermoneutrality vs. RT. Protein amount was quantified using Image J. (n=4, *p < 0.05 vs. RT). (D) Exercise capacity in male iBATx mice (n=8, *p<0.05). (E) Serum myostatin level in mice before and after iBATx and exposure to 30°C. Protein amount was quantified using Image J. (n=3–7, *p < 0.05 vs. RT; #p < 0.05 vs. iBATx). (F) Western blot analysis of isolated muscle mitochondria from mice before and after iBATx and exposure to 30°C. Protein amount was quantified using Image J (n=4–5, *p < 0.05 vs. RT; #p < 0.05 vs. iBATx).

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