Meteorin-like is a hormone that regulates immune-adipose interactions to increase beige fat thermogenesis

Abstract

Exercise training benefits many organ systems and offers protection against metabolic disorders such as obesity and diabetes. Using the recently identified isoform of PGC1-α (PGC1-α4) as a discovery tool, we report the identification of meteorin-like (Metrnl), a circulating factor that is induced in muscle after exercise and in adipose tissue upon cold exposure. Increasing circulating levels of Metrnl stimulates energy expenditure and improves glucose tolerance and the expression of genes associated with beige fat thermogenesis and anti-inflammatory cytokines. Metrnl stimulates an eosinophil-dependent increase in IL-4 expression and promotes alternative activation of adipose tissue macrophages, which are required for the increased expression of the thermogenic and anti-inflammatory gene programs in fat. Importantly, blocking Metrnl actions in vivo significantly attenuates chronic cold-exposure-induced alternative macrophage activation and thermogenic gene responses. Thus, Metrnl links host-adaptive responses to the regulation of energy homeostasis and tissue inflammation and has therapeutic potential for metabolic and inflammatory diseases.

Figure 1. Muscle-specific PGC1α4 transgenic mice are lean and display increased thermogenesis in the adipose tissue

See also Figure S1 (A) Representative images of epi fat from wild-type (WT) or Myo-PGC1α4 mice. (B) Determination of SubQ, Epi and BAT weights from WT (N=5) and Myo-PGC1α4 mice (N=7). (C) Determination of percent fat mass between WT and Myo-PGC1α4 mice using MRI (N=6). (D) Body weights of WT and Myo-PGC1α4 mice upon challenging with HFD starting at 4 weeks (N=10). (E, F) Real-time PCR (qPCR) analysis of markers associated with thermogenic, mitochondrial and β-oxidation genes in (E) subQ, and (F) epi adipose tissue of WT and Myo-PGC1α4 (N=6). *p<0.05, **p<0.001, ***p<0.0001. All data are presented as mean +/− s.e.m.

Figure 2. Identification of Metrnl as a PGC1α4 target gene

See also Figure S2 and Supplementary Tables S1–S3 (A) qPCR analysis against Metrnl in quadriceps from Myo-PGC1α4 transgenic mice or WT littermate controls (N=4 each group). (B) Expression of Metrnl in culture supernatants from primary myotubes transduced with Lac Z or PGC1α4 expressing adenovirus using mass spectrometry (N=3 each group). (C) Tissue specific Metrnl expression patterns by qPCR. Bar graphs represent RNA samples from three independent mice pooled together. (D) Analysis of Metrnl gene expression in skeletal muscle biopsies from human volunteers. Vastus lateralis biopsies were obtained prior to commencement, 1hr and 4hr post-exercise. Gene expression was analyzed by qPCR. (E) Metrnl mRNA expression in skeletal muscle and plasma levels after an acute bout of downhill running exercise. C57/BL6 mice were divided into groups: sedentary (N=9) and run (N=10). The quadriceps and triceps muscles were harvested 6 hours after run and processed for gene expression by qPCR. Plasma was collected 24h after the run and Metrnl levels were measured by ELISA (F) Analysis of Metrnl mRNA expression in subQ, epi and brown adipose tissue of mice chronically housed at 30°C or acutely subjected to a 4°C cold challenge for the indicated time points (N=5 per group). (G–H) Under the same experimental setting as in (F), plasma from mice housed at 30°C or exposed to 4°C for 24 hrs were subjected to (G) western blot against Metrnl and Metrnl band is normalized to an invariant nonspecific band, and (H) ELISA against Metrnl. *p<0.05, **p<0.001, ***p<0.0001. All data are presented as mean +/− s.e.m.

Figure 3. Increase in circulating Metrnl promotes increase in thermogenic and anti-inflammatory gene programs in the adipose tissue

See also Figure S3 (A–H) C57/BL6 mice were injected with adenoviral vectors (Ad) expressing Lac Z or Metrnl intravenously, via tail vein injections and (A) Plasma from Lac Z or Metrnl injected mice was subjected to western blotting against Metrnl at day 5 post-injection, (B–C) qPCR analysis of markers associated with thermogenesis and mitochondrial gene programs in (B) subQ, (C) Epi WAT at day 7 (N=6) (D) Western blotting against UCP-1 (N=3), and (E) immunohistochemistry against UCP-1 in subQ WAT at day 7 (N=2). (F) Determination of percent fat mass between Lac Z and Metrnl-injected mice using MRI at day 7 (N=6) (G–H) qPCR analysis of markers associated with β-oxidation and pro/anti-inflammatory gene programs in the subQ WAT at day 7 (N=6). (I–J) C57/BL6 mice fed a HFD for 20 weeks (N=8) were injected daily with saline or Metrnl-Fc protein (10mg/kg) i.p. for 7 days, and (I) 6 hours after the last injection, animals were sacrificed and subQ WAT was analyzed for changes in thermogenic, β-oxidation and pro/anti-inflammatory gene programs, (J) Body weights of mice. *p<0.05, **p<0.001, ***p<0.0001. All data are presented as mean +/− s.e.m.

Figure 4. Metrnl expression increases energy expenditure and improves glucose tolerance

See also Figure S4 (A–E) C57/BL6 mice fed a HFD for 20 weeks were injected with Lac Z or Metrnl adenovirus (i.v.) and (A–D) energy expenditure was measured (N=7), (A) oxygen consumption, (B) Carbon dioxide production, (C) quantification of oxygen consumption between day 5 and 6 and (D) respiratory exchange ratio (RER), (E) IP-glucose tolerance test was performed at day 6 (N=8). *p<0.05, **p<0.001, ***p<0.0001. All data are presented as mean +/− s.e.m.

Figure 5. Metrnl expression induces an increase in alternative activation of adipose tissue macrophages

See also Figure S5 (A) SVF from the inguinal fat depot was differentiated into adipocytes for 6 days and treated with either saline, recombinant Metrnl-Fc (5ug/ml) or Fgf21 (100ng/ml), during last two days of differentiation. qPCR analysis was performed for indicated genes, 48h post-treatment (N=4). (B–D) SubQ WAT (left pad) of C57/BL6 mice was injected with either Lac Z and Metrnl adenovirus (N=5) and the injected and contralateral WAT (right, un-injected) were harvested (B–C) at day 3 post-injection to analyze for increase in Metrnl expression (B) by qPCR (C) western blotting, and (D) at day 5 post-injection to assess for changes in thermogenic and β-oxidation genes by qPCR. (E–F) C57/BL6 mice were injected with adenoviral vectors expressing Lac Z or Metrnl (i.v.) (N=6), and (E) analyzed for markers associated with alternative macrophage activation in the subQ WAT at day 7, and (F) IL4/IL13 cytokine expression at day 5. (G) C57/BL6 mice fed a HFD for 20 weeks (N=8) were injected daily with saline or Metrnl-Fc protein (10mg/kg) (i.p.) for 7 days and analyzed for changes in markers of M2 macrophage activation in the subQ WAT. (H) BALB/c mice were injected with Lac Z or Fndc5-Ad (i.v.). Animals were sacrificed 7 days later and subQ WAT was assessed for indicated genes by qPCR (I–J) Under the same experimental setting as in (E–F), (I) tyrosine hydroxylase mRNA expression, and (J) norepinephrine content of subQ adipose tissue at day 7. *p<0.05, **p<0.001, ***p<0.0001, ****p<0.00001. All data are presented as mean +/− s.e.m.

Figure 6. Metrnl induced browning response requires IL4/IL13 signaling

See also Figure S6 (A–B) Wild-type (WT) and STAT6^−/− mice (N=5) were injected with Lac Z or Metrnl-Ad (i.v.) and analyzed at day 7 post-injection for markers associated with (A) M2 macrophage activation, and (B) thermogenic and β-oxidation genes. (C–D) Under the same experimental setting as in (A–B) (C) tyrosine hydroxylase mRNA expression and (D) norepinephrine content of subQ WAT was assessed at day 7. (E) C57/BL6 mice were injected with Lac Z or Metrnl-Ad (i.v.) (N=4) and flow cytometric analysis of eosinophils (defined as CD11b⁺ and Siglec F⁺; gating strategy in Figure S6F) in the SVF from subQ WAT at day 4. Numbers represent percentage of CD11b⁺/Siglec F⁺ cells in total SVF. (F–G) Wild-type (WT) and ΔdblGATA mice (N=5) were injected with Lac Z or Metrnl-Ad (i.v.) and analyzed for (F) IL-4/IL13 mRNA expression at day 4, and (G) M2 macrophage and thermogenic genes at day 7. *p<0.05, **p<0.001, ***p<0.0001 comparison between WT mice injected with Lac Z and Metrnl-Ad. ^#p<0.05, ^##p<0.001, ^###p<0.0001 comparison between STAT6^−/− or ΔdblGATA mice injected with Lac Z and Metrnl-Ad. ⁺p<0.05, ⁺⁺p<0.001, ⁺⁺⁺p<0.0001 comparison between WT or STAT6^−/− / ΔdblGATA mice injected with Metrnl-Ad. All data are presented as mean +/− s.e.m

Figure 7. Metrnl is required for physiologic adaptation to cold temperatures

See also Figure S7 (A–B) Mice chronically housed at 30°C were subjected to a 4°C cold challenge for 24 h and (A) number of adipose eosinophils (gated as described in Figure S7F) is shown per g adipose tissue (B) mRNA expression of IL4/IL13 in the subQ WAT. (C–D) Mice chronically housed at 30°C were injected with isotype or Metrnl antibody (i.p.) and moved to cold 6 hrs later. Sub Q WAT was harvested and analyzed for (C) mRNA expression of the indicated genes and number of eosinophils per g adipose tissue at 24hrs post cold-challenge, and (D) markers of genes associated with thermogenic and M2 macrophage activation at 72hrs post cold-challenge. **p<0.001, ***p<0.0001 comparison between mice at 30°C and 4°C or isotype Ab treatment of mice at 30°C and 4°C. ^#p<0.05, ^##p<0.001 comparison between Metrnl Ab treatment of mice at 30°C and 4°C. All data are presented as mean +/− s.e.m