Adrenomedullin 2 Enhances Beiging in White Adipose Tissue Directly in an Adipocyte-autonomous Manner and Indirectly through Activation of M2 Macrophages

Abstract

Adrenomedullin 2 (ADM2) is an endogenous bioactive peptide belonging to the calcitonin gene-related peptide family. Our previous studies showed that overexpression of ADM2 in mice reduced obesity and insulin resistance by increasing thermogenesis in brown adipose tissue. However, the effects of ADM2 in another type of thermogenic adipocyte, beige adipocytes, remain to be understood. The plasma ADM2 levels were inversely correlated with obesity in humans, and adipo-ADM2-transgenic (tg) mice displayed resistance to high-fat diet-induced obesity with increased energy expenditure. Beiging of subcutaneous white adipose tissues (WAT) was more noticeably induced in high-fat diet-fed transgenic mice with adipocyte-ADM2 overexpression (adipo-ADM2-tg mice) than in WT animals. ADM2 treatment in primary rat subcutaneous adipocytes induced beiging with up-regulation of UCP1 and beiging-related marker genes and increased mitochondrial uncoupling respiration, which was mainly mediated by activation of the calcitonin receptor-like receptor (CRLR)·receptor activity-modifying protein 1 (RAMP1) complex and PKA and p38 MAPK signaling pathways. Importantly, this adipocyte-autonomous beiging effect by ADM2 was translatable to human primary adipocytes. In addition, M2 macrophage activation also contributed to the beiging effects of ADM2 through catecholamine secretion. Therefore, our study reveals that ADM2 enhances subcutaneous WAT beiging via a direct effect by activating the CRLR·RAMP1-cAMP/PKA and p38 MAPK pathways in white adipocytes and via an indirect effect by stimulating alternative M2 polarization in macrophages. Through both mechanisms, beiging of WAT by ADM2 results in increased energy expenditure and reduced obesity, suggesting ADM2 as a novel anti-obesity target.

Keywords: adipocyte; energy metabolism; macrophage; obesity; uncoupling protein.

FIGURE 1.

Plasma ADM2 levels are inversely correlated with obesity in humans. Plasma samples were collected from a total of 74 Chinese adults, and the ADM2 levels were measured by radioimmunoassay. A, negative correlation of body weight (BW) versus plasma ADM2 levels. B, inverse correlation of BMI and plasma ADM2 levels. C, comparison of the plasma ADM2 levels in normal (BMI ≤ 24), overweight (24 < BMI ≤ 28), and obese (BMI > 28) human subjects. D, no significant correlation of age versus plasma ADM2 levels. E, similar plasma ADM2 levels in the male (n = 38) and female (n = 36) human subjects. For group comparison (C and E), the data are represented as mean ± S.E. One-way ANOVA with Newman-Keuls test (C) or two-tailed Student's t test (E): *, p < 0.05 versus control. For linear regression (A, B, and D), the slope was considered significantly non-zero when p < 0.05.

FIGURE 2.

Adipocyte-ADM2 overexpression improves mitochondrial respiration and thermogenesis in scWAT. A, inverse correlation of body weight (BW) and relative Adm2 mRNA levels in WAT of C57BL/6J male mice (n = 56). Linear regression: the slope was considered significantly non-zero when p < 0.05. B–G, Adipo-ADM2-tg mice and WT controls were fed for 12 weeks on an HFD. B–D, mitochondrial energetics analysis of scWAT. B, OCR measurement curves. C, basal respiration, ATP production, and maximal respiration OCR calculated from A. D, coupling efficiency (ATP production OCR/basal respiration OCR). E–G, relative mRNA levels of mitochondrial respiration and thermogenesis genes (E), beige-selective markers (F), and white adipocyte-selective markers (G) in scWAT. H, flow cytometry analysis of CD137 expression in mature scWAT adipocytes. The data are represented as mean ± S.E. n = 3 independent experiments, 4–6 mice/group. Two-tailed Student's t test: *, p < 0.05 versus WT; **, p < 0.01 versus WT.

FIGURE 3.

Cold-induced beiging was markedly activated in scWAT of adipo-ADM2-tg mice. Adipo-ADM2-tg mice and WT controls were fed an HFD for 12 weeks, followed by a 48-h cold (4 °C) exposure. A and B, H&E staining of scWAT (A) with adipocyte size on sections measured by ImageJ software (B). C, immunohistochemical staining of UCP1 in scWAT. D, immunoblotting analysis of UCP1 and TMEM26 in scWAT, with eIF5 as a loading control. E, body temperature. The data are represented as mean ± S.E. n = 3 independent experiments, 3–6 mice/group. Two-tailed Student's t test: *, p < 0.05 versus WT.

FIGURE 4.

ADM2 treatment promotes beiging in vitro in an adipocyte-autonomous manner. Rat scWAT SVCs were differentiated into adipocytes and treated with 20 nm ADM2 for 8 h. A, heat map of the expression profiles of genes involved in mitochondrial respiration, thermogenesis, and adipocyte phenotype identity by RNAseq analysis. B and C, relative mRNA levels of mitochondrial respiration and thermogenesis genes (B) and beige and white adipocyte markers (C) by qPCR analysis. D, immunofluorescence staining of UCP1 (green) with nuclei stained by Hoechst33258 (blue) and lipid droplets stained by LipidTOX (red). E, immunoblotting analysis of UCP1 with eIF5 used as a loading control (Ctr). F–H, mitochondrial energetics analysis. F, OCR measurement curves. G, basal respiration, ATP production, uncoupling, and maximal respiration OCR calculated from F. H, coupling efficiency (ATP production OCR/basal respiration OCR). I, TMRM staining (red) of adipocytes, with nuclei stained by Hoechst 33258 (blue) and lipid droplets stained by LipidTOX (green). TMRM fluorescence intensity was quantified by ImageJ software and normalized by nucleus number. The data are represented as mean ± S.E. n = 3 independent experiments (3–6 samples/experiment). Two-tailed Student's t test: *, p < 0.05 versus control; **, p < 0.01 versus control.

FIGURE 5.

ADM2 up-regulates UCP1 expression in adipocyte mainly through the CRLR·RAMP1-cAMP/PKA and p38 MAPK pathways. Rat scWAT SVCs were differentiated into adipocytes and treated with ADM2. A and E, Relative mRNA levels of Ucp1 in adipocytes treated with ADM2 for 8 h with or without 1-h pretreatment of CRLR-RAMP receptor antagonists (A) or signaling pathway inhibitors (E). B, cellular cAMP levels. C and D, immunoblotting analysis of phosphorylation levels of PKA-mediated substrates (C) and p38 MAP kinase (D) in adipocytes. The data are represented as the mean ± S.E. n = 3 independent experiments (3–6 samples/experiment). One-way ANOVA with Newman-Keuls test (A and E) or two-tailed Student's t test (B): *, p < 0.05 versus control; **, p < 0.01 versus control; #, p < 0.05 versus ADM2 treatment; ##, p < 0.01 versus ADM2 treatment.

FIGURE 6.

ADM2 treatment activates beiging of primary human white adipocytes. Primary human scWAT SVCs were differentiated into adipocytes and treated with 20 nm ADM2 for 8 h. A and B, relative mRNA levels of UCP1 and adipocyte markers. C, immunofluorescence staining of UCP1 (green) with nuclei stained by Hoechst 33258 (blue) and lipid droplets stained by LipidTOX (red). D, immunoblotting analysis of UCP1 with eIF5 used as a loading control (Ctr). E and F, mitochondrial energetics analysis. E, OCR measurement curves. F, basal respiration, ATP production, and maximal respiration OCR calculated from E. The data are represented as mean ± S.E. n = 3 independent experiments (3–6 samples/experiment). Two-tailed Student's t test: *, p < 0.05 versus control; **, p < 0.01 versus control.

FIGURE 7.

M2 macrophage activation also contributes to the beiging effects of ADM2. A and B, adipo-ADM2-tg mice and WT controls were fed for 12 weeks on an HFD. A, relative mRNA levels of M1/M2 macrophage markers in scWAT. B, flow cytometry analysis of M2 macrophages in the scWAT stromal vascular fraction. C–F, primary mouse peritoneal macrophages were treated with 20 nm ADM2 for 12 h. C, relative mRNA levels of M1 and M2 macrophage markers with 18S rRNA used as a normalization gene. D, immunoblotting analysis of Arg1, and eIF5 was used as a loading control (Ctr). IL4 is an M2 polarization stimulator and was used as a positive control. E, immunoblotting analysis of Th, Ddc, and Dbh, with β-actin as a loading control. F, NE accumulation in the medium of macrophages for the indicated time course. Data are represented as mean ± S.E. n = 3 independent experiments (3–4 mice/group, 3–6 cell samples/experiment). Two-tailed Student's t test: *, p < 0.05 versus control; **, p < 0.01 versus control.

FIGURE 8.

ADM2 promotes beiging in scWAT through cross-talk between adipocytes and macrophages. A and B, conditioned medium test. A, the conditional medium system for adipocytes and macrophages. After treatment with 20 nm ADM2 for 12 h, fresh medium was given to macrophages for catecholamine accumulation. Then the conditioned medium of macrophages was collected and given to adipocytes. After incubation with the medium for 8 h, adipocytes were collected for Ucp1 mRNA analysis. B, relative mRNA levels of Ucp1 in adipocytes treated with conditioned medium from ADM2-treated or control macrophages for 8 h with or without 1-h pretreatment of the β-adrenergic receptor antagonist propranolol (10 μm), and adipocytes treated with normal control medium were used as a negative control. β-Actin was used as a normalization gene. Data are represented as mean ± S.E. n = 3 independent experiments (3–6 samples/experiment). One-way ANOVA with Newman-Keuls test: **, p < 0.01 versus normal control medium; ##, p < 0.01 versus control conditioned medium; &&, p < 0.01 versus ADM2-treated conditioned medium. C, proposed model of the effects of ADM2 on WAT beiging and energy homeostasis. As an endogenous beiging activator, ADM2 interacts with the CRLR-RAMP1 receptor on adipocytes and activates beiging of white adipocytes directly. Additionally, ADM2 acts on resident macrophages in scWAT and promotes M2 polarization and catecholamine production. The catecholamine secreted locally by M2 macrophages in turn activates the β3 adrenergic receptor on adipocytes and enhances white adipocyte beiging. The induced beige adipocytes utilize UCP1 to uncouple oxidative respiration and dissipate excess energy by thermogenesis, thus improving systematic energy homeostasis and attenuating HFD-induced obesity and related metabolic disorders.