Increase in adipose thermogenesis can augment whole-body energy expenditure, independent of physical activity. We therefore analyzed the adipose tissues from Myo-PGC-1α4 mice for expression of genes related to thermogenesis and/or genes involved in imparting a brown-fat-like program (browning). As shown in Figure 1 E, quantitative PCR (qPCR) analyses of messenger RNA (mRNA) revealed a robust increase in thermogenic, β-oxidation and mitochondrial gene programs, including UCP-1, Acsl1, PGC-1α, and ERR-α in the subQ WAT. In addition, we also observed significant increases in expression of these same genes in the epi WAT ( Figure 1 F), a tissue that has a much lower capacity to induce activity of brown or beige fat thermogenic gene programs. However, no significant changes in these gene expression events were noted in the interscapular “classical” brown adipose tissue (BAT) ( Figure S1 A available online). Notably, PGC-1α4 transgene expression is localized to the skeletal muscle in the Myo-PGC-1α4 mice and is not expressed in the adipose tissues ( Figure S1 B). Taken together, these results indicate that muscle-specific expression of PGC-1α4 promotes browning of the WAT (both subQ and epi), which might contribute to the lean phenotype of the Myo-PGC-1α4 mice. These observations also point toward the existence of a PGC1α4-dependent myokine that mediates muscle-fat crosstalk to promote expression of a broad beige thermogenic gene program.

(B) qPCR analysis of PGC-1α4 transgene expression in the adipose tissue of WT and Myo-PGC1α4 mice. Transgene expression was measured with primers targeting Flag epitope (forward) and Exon 2 of PGC1α4 (reverse).

We recently reported that the muscle-specific PGC-1α4 transgenic mice (Myo-PGC-1α4) demonstrate muscle hypertrophy and increased basal energy expenditure without any changes in food intake or movement (). The molecular basis for the increased energy expenditure was not explored. As shown in Figure 1 A, the Myo-PGC-1α4 mice have an obvious reduction in the size of epididymal (epi) fat depot and demonstrate ∼30%–40% reduction in the weights of the epi and subcutaneous adipose tissue (inguinal) (SubQ WAT), compared to those of controls ( Figure 1 B). In addition, the Myo-PGC-1α4 mice demonstrated an overall lean phenotype, as evidenced by a 43% reduction in whole-body fat content when assessed by MRI ( Figure 1 C). Interestingly, the Myo-PGC-1α4 mice gain significantly less weight after 12 weeks on a high-fat diet (HFD) compared to their wild-type (WT) littermate controls. No significant weight differences were observed prior to the HFD challenge at 4 weeks of age ( Figure 1 D).

(E and 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, and ∗∗∗ p < 0.0001.

Because Metrnl is regulated by exercise, we also explored other physiological stimuli that might regulate its expression. Given the abundance of Metrnl mRNA in the adipose tissues ( Figure 2 C), we investigated its regulation by thermogenic stimuli, specifically acute and chronic exposure to cold. Metrnl expression was measured in BAT, subQ, and epi fat depots of mice chronically housed at 30°C (thermoneutrality) or after an acute (6 hr, 24 hr) and chronic challenge (2 weeks) at 4°C. Acute cold exposure significantly elevated Metrnl gene expression in all three adipose tissues although with different kinetics of expression ( Figure 2 F). Notably, Metrnl mRNA was increased in a transient manner, with maximal induction observed at 6 hr postchallenge. In addition, chronic cold exposure failed to maintain elevated Metrnl expression in all three adipose tissues examined ( Figure S2 E). Importantly, cold exposure for 24 hr increased circulating levels of Metrnl, as assessed by western blotting and ELISA assay ( Figures 2 G and 2H). It is interesting to note that acute cold exposure does not induce Metrnl expression in the skeletal muscle ( Figure S2 F), and downhill running exercise specifically induces Metrnl expression in the skeletal muscle, but not in the adipose tissue ( Figure S2 G). Overall, these results identify Metrnl as a hormone that can be selectively induced in different tissues, depending upon the physiologic stimulus. These data further suggest a potential role for this protein in the physiological adaptations to exercise and cold.

Given that PGC-1α4 mRNA is expressed in the skeletal muscle of mice and humans upon resistance training (), we investigated the regulation of Metrnl in human skeletal muscle following an acute bout of concurrent exercise (resistance followed by endurance exercise; concurrent training, see Experimental Procedures ). Skeletal muscle biopsies from the vastus lateralis were obtained at rest, 1 hr, and 4 hr following the completion of the exercise session. Figure 2 D shows an increase in Metrnl mRNA expression at both the time-points, with maximal induction at 1 hr postexercise. In addition, we also noted increases in PGC-1α4 (both time points) and PGC-1α1 (1 hr only) mRNA expression ( Figure S2 B). Next, we investigated the regulation of Metrnl in a mouse model of eccentric exercise that promotes muscle strength and hypertrophy. A single bout of downhill treadmill running exercise increases Metrnl mRNA expression in triceps, but not in the quadriceps muscle ( Figure 2 E). Concomitantly, we also observe an ∼2-fold increase in circulating Metrnl at day 1 postexercise, as assessed by ELISA ( Figure 2 E). The specificity of the antibody against Metrnl was demonstrated by antigen-antibody neutralization experiments using ELISA ( Figure S2 C). In addition, late in the manuscript review process, we obtained access to total body knockout (KO) mice for Metrnl; the specificity of the antibody used here was shown definitively with these animals ( Figure S2 D). We did not observe any changes in Metrnl expression upon a program of endurance exercise training (free wheel running) (data not shown).

Based on the above criteria, we focused on a protein called Metrnl. Metrnl mRNA was upregulated about 4-fold in the mRNA isolated from quadriceps muscle from Myo-PGC-1α4 mice ( Figure 2 A), and the protein was increased ∼8-fold in the mass spectrometric analysis from supernatants of cultured myotubes with forced PGC-1α4 expression ( Figure 2 B). To further confirm the identity of Metrnl as a bona fide secreted factor, we expressed Metrnl in Cos7 cell line, using adenoviral vectors expressing this protein fused to a C-terminal V5-His tag (Metrnl-Ad) or a Lac Z (Lac Z Ad) control. Ectopic Metrnl expression produced robust increases in V5-tagged Metrnl protein in the culture supernatants ( Figure S2 A). Next, we analyzed abundance of Metrnl expression across various tissues. In addition to its abundance in the skeletal muscle, it is also expressed at comparable levels in the subQ and epi WAT depots and heart, followed by BAT and kidney ( Figure 2 C).

(G) Metrnl mRNA expression in subQ and epi WAT after an acute bout of downhill running exercise. C57/BL6 mice were divided into groups: sedentary (N = 9) and run (N = 10). The subQ and Epi WAT were harvested 6 hr after run and processed for gene expression by qPCR ∗ p < 0.05, ∗∗ p < 0.001, ∗∗∗ p < 0.0001.

(F) Analysis of Metrnl mRNA expression in quadriceps and back muscle of mice chronically housed at 30°C or acutely subjected to a 4°C cold challenge for 24 hr (N = 5 per group).

(E) 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 2 weeks (N = 5 per group).

(C) Plasma obtained from C57/BL6 mice was subjected to ELISA to measure Metrnl levels. For the panel labeled “antibody + excess antigen” the antibody was incubated with 50ug of recombinant Metrnl for 1 hr before using it in ELISA (N = 4).

(B) Analysis of PGC1α isoform gene expression in skeletal muscle biopsies from human volunteers. Vastus lateralis biopsies were obtained prior to commencement, 1hr and 4hr post-exercise and gene expression was analyzed by qPCR using human PGC-1α isoform specific primers.

(A) Cos 7 were transduced with Lac Z or Metrnl-Ad and 8h post-transduction, medium was changed to serum-free expression medium for overnight incubation. Medium was collected, concentrated using 10KD MW cut-off and 20ul was used for Western blotting against a V5 antibody.

(G and H) Under the same experimental setting as in (F), plasma from mice housed at 30°C or exposed to 4°C for 24 hr 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, and ∗∗∗ p < 0.0001.

(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).

(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 hr after run and processed for gene expression by qPCR. Plasma was collected 24 hr after the run, and Metrnl levels were measured by ELISA.

(D) Analysis of Metrnl gene expression in skeletal muscle biopsies from human volunteers. Vastus lateralis biopsies were obtained prior to commencement and 1 hr and 4 hr postexercise. Gene expression was analyzed by qPCR.

We utilized two independent and unbiased approaches to identify secreted factors controlled by PGC1α4 that might contribute to the browning of white fat: gene expression analyses combined with bioinformatics algorithms and quantitative mass spectrometry of secreted proteins. First, we screened affymetrix data obtained upon PGC1α4 overexpression in primary myotubes () to identify potential candidates that satisfied all of the following criteria (1) >2-fold change in mRNA expression, (2) presence of an N-terminal signal peptide, and (3) absence of a transmembrane domain. The fold change of short-listed genes was independently confirmed in the quadriceps muscle from Myo-PGC-1α4 mice by qPCR (list of the short-listed genes, Table S1 ). Second, we performed quantitative protein mass spectrometric analysis to identify secreted factors that were upregulated (>2-fold) in serum-free culture supernatants of primary myotubes after forced expression of PGC-1α4 ( Table S2 ). The list of short-listed candidates from both these approaches were then cross-referenced, and only those gene candidates that were increased with both of these approaches were selected ( Table S3 ). Potential candidates were further selected based on their mRNA abundance in skeletal muscle from WT mice as quantified by absolute levels of expression by qPCR ( Table S3 ).

To further study the ability of Metrnl to positively regulate browning and thermogenesis, we generated and purified a recombinant protein fused to the Fc portion of IgG to the C terminus of Metrnl (Metrnl-Fc); this was then injected into mice rendered obese by feeding a HFD. This Metrnl protein had a relatively short half-life ( Figure S3 G), so we injected mice daily with a dose of 10 mg/kg for 7 days. Consistent with the adenoviral experiments, recombinant Metrnl protein increased adipose expression of thermogenic, β-oxidation, and anti-inflammatory genes, including UCP-1, DIO2, Acox1, and IL-10 ( Figure 3 I). The magnitude of these changes was weaker than those observed with the viral-mediated expression, presumably reflecting the suboptimal pharmacokinetics seen with this Metrnl fusion protein. Nevertheless, these increases in thermogenic gene expression were accompanied by a small but significant reduction in body weight, compared to controls ( Figure 3 J). Overall, these results identify Metrnl as a hormone that can promote an increase in a broad beige/brown fat thermogenic gene program in vivo.

Increases in brown and beige fat thermogenesis are typically inversely correlated with changes in the expression of inflammatory genes (); we therefore analyzed whether Metrnl also regulated expression of inflammation-linked genes. As shown in Figure 3 H, Metrnl expression promotes increases in expression of anti-inflammatory genes such as IL-10 and TGF-β. We did not detect any changes in expression of proinflammatory genes such as TNF-α and IL-1β. Because mice rendered obese by HFD display higher basal expression of proinflammatory gene markers in adipose tissues (), we next analyzed whether Metrnl can act to suppress expression of proinflammatory genes in these mice. Indeed, expression of Metrnl promotes modest decreases in expression of proinflammatory cytokines, such as TNF-α, IFN-γ, and IL-1β ( Figure S3 F).

To determine whether Metrnl can promote browning of adipose tissues, we performed intravenous injections of adenoviral vectors to deliver full-length Metrnl constructs to the liver. This method generally results in robust expression of proteins in the liver and potential secretion to the plasma (). Notably, serum aspartate aminotransferase (AST) levels were well within the normal range and showed no differences between the control and Metrnl-injected mice ( Figure S3 A). At day 3 postinjection, we observed an ∼20-fold increase in liver Metrnl mRNA ( Figure S3 B) and a 5- to 6-fold increase in plasma Metrnl levels, as detected by western blotting with an anti-Metrnl antibody ( Figure 3 A). Strikingly, the increase in circulating Metrnl produced remarkable increases in broad brown/beige fat thermogenic and mitochondrial gene program in the subQ and epi WAT, including UCP-1, DIO2, PGC-1α, and ERR-α ( Figures 3 B and 3C). The increase in UCP-1 mRNA (∼3.5-fold) was also accompanied by a robust increase in the UCP-1 protein expression in the subQ WAT ( Figures 3 D and 3E). In addition, we also observe moderate increases in thermogenic gene programs in the BAT ( Figure S3 C). Notably, changes in thermogenic gene expression were observed only between days 5 and 7 postinjection, and the expression of UCP-1 mRNA returns to baseline expression by day 10 ( Figure S3 D), even though we detect increases in plasma Metrnl levels as early as day 3. Interestingly, we observe an overall lean phenotype in Metrnl injected mice, as evidenced by a 25% reduction in whole-body fat content when assessed by MRI ( Figure 3 F). Metrnl expression also stimulated elevated mRNA levels for genes associated with β-oxidation such as Acsl1, Acox1, and Cpt1 ( Figure 3 G). Finally, the increases in expression of thermogenic and β-oxidation gene programs due to Metrnl expression were also observed in a different strain of mice, the Balb/c strain ( Figure S3 E).

(I and J) C57/BL6 mice fed a HFD for 20 weeks (n = 8) were injected daily with saline or Metrnl-Fc protein (10 mg/kg) intraperitoneally (i.p.) for 7 days, and (I) 6 hr 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, and ∗∗∗ p < 0.0001.

(B and C) At day 7 postinjection, qPCR analysis of markers associated with thermogenesis and mitochondrial gene programs in subQ (B) and epi WAT (C). Western blotting against UCP-1 (D) (n = 3) and immunohistochemistry against UCP-1 in subQ WAT (E) at day 7 (n = 2).

(A–H) C57/BL6 mice were injected (i.v.) with adenoviral vectors (Ad) expressing Lac Z or Metrnl (n = 6), and (A) plasma from these mice were subjected to western blotting against Metrnl at day 5 postinjection.

(G) C57/BL6 were injected i.p. with 9.5 mg/mouse of Metrnl-Fc and blood was collected at the indicated time points (N = 3). 2ul of serum were run on a non-reducing SDS-PAGE gel and immunoblotted against Metrnl Ab. The first two lanes represent 2.5 and 5ug/ml of Metrnl-Fc for reference standards. ∗ p < 0.05, ∗∗ p < 0.001, ∗∗∗ p < 0.0001.

(F) C57/BL6 mice fed a HFD for 20 weeks were injected with Lac Z or Metrnl adenovirus (i.v.) and the subQ WAT was analyzed for changes in pro/anti-inflammatory gene programs at day 6 (N = 6).

(D and E) (D) Under the same experimental conditions as in (A), kinetics of UCP-1 mRNA expression in the subQ WAT (N = 5 per time-point) by qPCR (E) BALB/c mice were injected with Lac Z or Metrnl-Ad (i.v.) (N = 6) and the subQ WAT depot was subjected to qPCR analysis of markers associated with thermogenesis and mitochondrial gene programs at day 7.

(A–C) (A-B) C57/BL6 mice were injected with adenoviral vectors (Ad) expressing Lac Z or Metrnl intravenously (N = 6) and (A) measurement of serum AST levels by calorimetric analysis at day 5 post-injection (B) Metrnl mRNA expression in liver at day 3, and (C) markers associated with thermogenesis and mitochondrial gene programs in BAT by qPCR analysis.

Next, intraperitoneal glucose tolerance tests (GTT) were performed in obese mice; Metrnl expression significantly improved glucose tolerance when compared to control mice injected with Lac Z ( Figure 4 E). Collectively, these data illustrate that increases in circulating Metrnl cause an increase in energy expenditure and an improvement in glucose homeostasis in obese/diabetic mice.

Increase in adipose tissue thermogenesis or browning of white fat can be accompanied by increases in whole-body energy expenditure and improved glucose homeostasis in vivo (). We therefore studied these metabolic parameters after delivering Metrnl-expressing adenoviral vectors to mice. Viral vectors were used for these experiments because they require less frequent handling of the mice than protein injections. We first measured energy expenditure using a comprehensive laboratory animal monitoring system (CLAMS) and observed a highly significant increase in energy expenditure in Metrnl-injected mice compared to Lac Z controls ( Figures 4 A–4C). Notably, the increase in oxygen consumption and carbon dioxide production was observed 5 days postinjection, which is consistent with the time course of thermogenic gene expression. This suggests that the action of Metrnl may not directly regulate thermogenesis but might regulate various biological processes that promote remodeling of the adipose tissue in a way conducive for increased browning of the white fat. Importantly, there was no change in respiratory exchange ratio (RER), indicating that Metrnl did not stimulate any substantial shift from carbohydrate to fat-based fuels ( Figure 4 D). Importantly, these changes in energy expenditure were independent of food intake or locomotor activity ( Figures S4 A and S4B).

(A–E) HFD-fed C57/BL6 mice were injected with Lac Z or Metrnl adenovirus (i.v.) (n=7), and energy expenditure—(A) oxygen consumption and (B) Carbon dioxide production—was measured. (C) Quantification of oxygen consumption between day 5 and 6 and (D) respiratory exchange ratio (RER). (E) Under the same experimental setting as in (A), IP-glucose tolerance test was performed at day 6 (n = 8). ∗ p < 0.05, ∗∗ p < 0.001, and ∗∗∗ p < 0.0001.

Metrnl Induces IL-4/IL-13 Cytokine Expression and Promotes Alternative Macrophage Activation in Adipose Tissue In Vivo

Fisher et al., 2012 Fisher F.M.

Kleiner S.

Douris N.

Fox E.C.

Mepani R.J.

Verdeguer F.

Wu J.

Kharitonenkov A.

Flier J.S.

Maratos-Flier E.

Spiegelman B.M. FGF21 regulates PGC-1α and browning of white adipose tissues in adaptive thermogenesis. Figure 5 Metrnl Expression Induces an Increase in Alternative Activation of Adipose Tissue Macrophages Show full caption (A) SVF from the inguinal fat depot was differentiated into adipocytes for 6 days and treated with saline, recombinant Metrnl-Fc (5 μg/ml), or Fgf21 (100 ng/ml) during the last 2 days of differentiation. qPCR analysis was performed for indicated genes 48 hr posttreatment (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, uninjected) were harvested (B and C) at day 3 postinjection to analyze for increase in Metrnl expression (B) by qPCR (C) western blotting and (D) at day 5 postinjection to assess for changes in thermogenic and β-oxidation genes by qPCR. (E and 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 (10 mg/kg) (i.p.) for 7 days and analyzed for changes in markers of M2 macrophage activation in the subQ WAT. (H–J) (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 and J) under the same experimental setting as in (E and 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, and ∗∗∗∗p < 0.00001. All data are presented as mean ± SEM. See also Figure S5 Figure S5 Metrnl Promotes Alternative Activation of Adipose Tissue Macrophages, Related to Figure 5 Show full caption (A) Primary cortical neurons were treated with the indicated doses of recombinant Metrnl-Fc protein for 30 min and cell lysates were immuno-blotted with an antibody against phospho STAT3. (B) SVF from the inguinal fat depot was differentiated into adipocytes for 6 days and treated with recombinant Metrnl (R&D systems) during last two days of differentiation or transduced with adenovirus expressing Lac Z, Metrnl or Fgf21 during last four days of differentiation. qPCR analysis was performed for indicated genes at day 6 (N = 4). (C–E) C57/BL6 mice were injected with Lac Z or Metrnl-Ad (i.v.) (N = 6) and (C) analyzed for markers associated with alternative macrophage activation in the epi and brown adipose tissue at day 7, and (D) flow cytometric analysis of adipose tissue macrophages (defined as CD11b+ and F4/80+) in the SVF from subQ WAT at day 5. (E) analyzed for markers of classical (M1) macrophage activation in the subQ WAT at day 7. (F and G) SubQ WAT of C57/BL6 mice was injected with Lac Z and Metrnl-Ad (N = 5) and the injected (left) and contralateral adipose tissue (right, un-injected) was harvested (F) at day 3 to analyze for changes in IL4/IL13 gene expression, and (G) at day 5 post-injection to assess for changes in markers associated with alternative macrophage activation, by qPCR. ∗p < 0.05, ∗∗p < 0.001. All data are presented as mean ± SEM. The mechanisms underpinning these effects of Metrnl were first studied by applying recombinant Metrnl-Fc protein directly to the stromal vascular fraction (SVF) of subcutaneous white adipocytes during differentiation in vitro. Interestingly, there was no detectable effect on the regulation of thermogenic or β-oxidation genes such as UCP-1, DIO2, and Acsl1, etc., at varying doses and duration of treatments tested. ( Figure 5 A and data not shown). We used Fgf21 as a positive control, as it has been previously demonstrated to induce expression of thermogenic genes such as UCP-1 and Cidea, in adipose cultures (). Notably, the inability of Metrnl to induce changes in gene expression was not due to inactivity of the Metrnl-Fc protein, in that the same preparations of protein caused adipose tissue browning in vivo ( Figure 3 I) and were able to induce STAT3 phosphorylation in a dose-dependent manner in cultured primary cortical neuron cells ( Figure S5 A). In addition, we tested a commercially available recombinant Metrnl protein and adenoviral-mediated transduction of these same SVF cultures and got similar negative results ( Figure S5 B). These results suggest that Metrnl may induce adipose tissue thermogenesis in vivo independent of a simple, direct action on adipocytes.

We therefore considered the possibility that this increase in adipose tissue thermogenesis caused by Metrnl in vivo could require actions on nonadipose cell types. To explore this further, we first investigated whether Metrnl can induce a thermogenic phenotype when expressed locally in adipose tissues in vivo. Adenoviral injections were performed directly into the subQ fat pad using Metrnl or control Lac Z adenovirus and analyzed for changes in gene expression 5 days postinjection. This method results in robust and localized Metrnl expression (mRNA and protein) only at the site of injection as compared to the uninjected contralateral side on the same mouse ( Figures 5 B and 5C). In addition, we did not detect any increase in Metrnl expression in the liver upon fat pad injections (data not shown). Importantly, this forced expression of Metrnl produces significant increases in both thermogenic and β-oxidation genes when compared to mice injected with Lac Z control ( Figure 5 D). These results demonstrate that, when expressed at the adipose tissue level, Metrnl can act to induce expression of thermogenic and β-oxidation genes, despite its inability to do so in primary adipocyte cultures in vitro.

Odegaard and Chawla, 2011 Odegaard J.I.

Chawla A. Alternative macrophage activation and metabolism. Boström et al., 2012 Boström P.

Wu J.

Jedrychowski M.P.

Korde A.

Ye L.

Lo J.C.

Rasbach K.A.

Boström E.A.

Choi J.H.

Long J.Z.

et al. A PGC1-α-dependent myokine that drives brown-fat-like development of white fat and thermogenesis. While the characteristic cell type of adipose tissue is the adipocyte, this tissue consists of a heterogeneous population of multiple different cell types such as preadipocytes and many immune cells, including macrophages, eosinophils, T cells, B cells, and mast cells. In addition to changes in the expression of thermogenic and β-oxidation genes, we also observed an increase in anti-inflammatory genes (e.g., IL-10) in the adipose tissue ( Figure 3 H). A major source of IL-10 in the adipose tissue is known to be alternatively activated macrophages (M2) that protect adipocytes from inflammation and improve glucose homeostasis (). To investigate whether Metrnl induces a phenotypic switch in adipose tissue macrophages, we examined mRNA from the subQ WAT of Metrnl-treated animals (adenoviral-mediated expression) and observed significant increases in several genes associated with alternative macrophage activation, including Arg1, Mrc-1, Clec10a, and Retnla ( Figure 5 E). Notably, these changes in gene expression associated with alternative macrophage are robust with ∼5- to 6-fold increase in mRNA for Arg1 and Retnla and ∼3- to 4-fold increase in Mrc-1 and Clec10a. These changes were also observed in epi WAT and BAT, albeit with different magnitude of gene expression changes ( Figure S5 C). In addition to the switch in phenotype, Metrnl caused a significant increase in the number of CD11b+ F4/80+ macrophages (∼2.2-fold) in the subQ WAT, as assessed by flow cytometry ( Figure S5 D) In contrast, gene expression for markers of classical (M1) macrophage activation such as TNF-α, Nos2, and CD274 was unchanged ( Figure S5 E). More importantly, Metrnl expression also increased expression of cytokines IL-4 and IL-13 in the adipose tissue; these are the cytokines that are dominant regulators of the macrophage alternative activation program ( Figure 5 F). The changes in IL4/IL13 gene expression were consistently observed at early time points (∼days 4 to 5) after Metrnl expression and had returned to baseline at day 7 when we observe the increases in expression of thermogenic and alternative macrophage activation genes. We also observed increases in expression of genes associated with alternative macrophage activation and cytokines IL4/IL13 in the adipose tissue upon infusion of the recombinant Metrnl-Fc fusion protein in vivo and with localized Metrnl expression in the fat pad using adenoviral vectors ( Figures 5 G, S5 F, and S5G). Notably, these increases in IL4/IL13 gene expression and alternative macrophage activation in vivo were not observed with Irisin, a secreted form of Fndc5 that has been previously shown to stimulate adipose tissue thermogenesis via a direct action on adipocytes ( Figure 5 H) ().