Epiregulin induces leptin secretion and energy expenditure in high-fat diet-fed mice

in Journal of Endocrinology
Correspondence should be addressed to O Ziouzenkova: ziouzenkova.1@osu.edu
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Adipokine leptin regulates neuroendocrine circuits that control energy expenditure, thermogenesis and weight loss. However, canonic regulators of leptin secretion, such as insulin and malonyl CoA, do not support these processes. We hypothesize that epiregulin (EREG), a growth factor that is secreted from fibroblasts under thermogenic and cachexia conditions, induces leptin secretion associated with energy dissipation. The effects of EREG on leptin secretion were studied ex vivo, in the intra-abdominal white adipose tissue (iAb WAT) explants, as well as in vivo, in WT mice with diet-induced obesity (DIO) and in ob/ob mice. These mice were pair fed a high-fat diet and treated with intraperitoneal injections of EREG. EREG increased leptin production and secretion in a dose-dependent manner in iAb fat explants via the EGFR/MAPK pathway. After 2 weeks, the plasma leptin concentration was increased by 215% in the EREG-treated group compared to the control DIO group. EREG-treated DIO mice had an increased metabolic rate and core temperature during the active dark cycle and displayed cold-induced thermogenesis. EREG treatment reduced iAb fat mass, the major site of leptin protein production and secretion, but did not reduce the mass of the other fat depots. In the iAb fat, expression of genes supporting mitochondrial oxidation and thermogenesis was increased in EREG-treated mice vs control DIO mice. All metabolic and gene regulation effects of EREG treatment were abolished in leptin-deficient ob/ob mice. Our data revealed a new role of EREG in induction of leptin secretion leading to the energy expenditure state. EREG could be a potential target protein to regulate hypo- and hyperleptinemia, underlying metabolic and immune diseases.


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    EREG/EGFR/MAPK axes induced leptin (LEP) secretion from adipose tissues ex vivo. Five iAb epididymal fat explants were excised from WT mice and stimulated. After 2 h of stimulation LEP levels were measured in the media by ELISA. This experiment was repeated in three different WT mice. (A) Concentration-dependent secretion of LEP in the media from iAb fat explants stimulated with different concentrations of EREG. Data (mean ± s.d., n = 5) are shown as percent to control (Veh, 100%). Kruskal–Wallis test. (B and C) LEP concentrations in the iAb fat lysates (B) and media (C) released from iAb fat before (triangles) and after (circles) stimulation with EREG (black bar, 50 ng/mL). Dashed and solid lines show mean value (n = 3 mice) before and after stimulation. (D) Expression levels of Egfr and Erbb4 in epididymal iAb fat of lean WT mice (mean ± s.d., n = 5) were measured by RT-PCR and normalized by Tbp. (E) LEP concentrations in the iAb fat stimulated with EREG (black bar, 50 ng/mL) in the presence and absence of inhibitors of EGFR (10 µM) and MAPK (10 µM). Data (mean ± s.d., n = 5) are shown as percent to control (Veh, white bar, 100%, dashed line). Between subject ANOVA with post-hoc Fisher LSD group comparison (α = 0.05). (F) LEP in the media released following stimulation of mouse explants with and without EREG (50 ng/mL) in the presence and absence of inhibitors of HSL (10 µM), PPARα (10 µM), and PI3K (100 nM). Data (mean ± s.d., n = 5) are shown as percent of control (Veh, 100%, dashed line). Between subject ANOVA. ns, not significant.

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    EREG stimulates leptin secretion and lipolysis in mice with diet-induced obesity (DIO). (A) DIO WT male mice (n = 7/group) were injected with 100 µL PBS (Veh) with and without EREG (1.5 ng/g body weight or 20 ng/per iAb depot) into both epididymal iAb fat pads every other day for 2 weeks. Total mRNA was isolated from one whole iAb fad pad. Expression of Lep was measured using NanoString assay. Normalized data represent mean ± s.d., n = 5; Mann–Whitney U test. (B) Expression of LEP protein levels were measured in plasma in the same mice by ELISA. Data (mean ± s.d., n = 5/group). Independent Student’s t-test. (C) Non-esterified fatty acids (NEFA) and (D) TG concentrations in plasma are shown as mean ± s.d., n = 5. Independent Student’s t-test.

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    EREG increases metabolic rate and core temperature in DIO mice. (A, B, C and D) DIO mice (n = 4/group) were placed in individual metabolic cages equipped with CLAMS. Number of mice was limited by capacity of facility. Mice were randomly selected from whole group (A) Metabolic rate in PBS-(open circles) and EREG-injected (closed red circles) mice were analyzed at room temperature (RT, left panel) and during cold exposure (4°C, right panel). Data represent mean ± s.e.m. (B) Metabolic rate (MR) kinetics during cold exposure was used to measure time until control (white bar) and EREG-treated mice (black bar) reached MR maximum. Independent Student’s t-test (C) locomotor XYZ activity at room temperature (RT, left panel) and during cold exposure (4°C, right panel) (mean ± s.d.), Independent Student’s t-test. (D) RER during cold exposure (mean ± s.d.). (E) Body temperature scans (thermomap) DIO mice groups measured after cold exposure (mean ± s.d., n = 4/group). Arrow indicates the injected sites after cold exposure. Yellow color indicates increased temperature in DIO mice treated with EREG vs mice injected with PBS (Veh). Temperature was quantified within same size iAb areas (Numbers inside animals were generated by camera’s software to indicate highest and lowest point of measurements that are irrelevant to iAb temperature). Student’s t-test.

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    EREG reduces iAb obesity in DIO mice. (A) Initial and final body weight comparison in Veh- and EREG treated DIO mice. The changes in body weights within groups (n = 7/group, paired Student’s t-test). Triangles and circles show weight of individual mice in the control and EREG-treated groups, respectively. (B) Food intake in the pair fed Veh- and EREG treated DIO mice. Mann–Whitney U test. (C, D, E and F) Weight of liver (C), brown fat (D), subcutaneous fat (E), and iAb fat (F). Organ weight was normalized to body weight. Data are shown as individual values, means are indicated as dashed (Veh) and solid (EREG-treated) lines. Within-Subjects ANOVA, with sphericity a = 0.25 and non-parametric one-sample run test.

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    EREG upregulates expression of thermogenic and PPARα-target genes, and downregulates inflammatory genes. (A, B, C, D, E, F and G) Gene expression was measured in total mRNA isolated from whole iAb fat pads of Veh- (white bars) and EREG-treated (black bars) DIO mice using NanoString assay. Gene expression was normalized to the expression of three housekeeping genes. Expression of markers for adipogenesis: (A) Pparg; mitochondrial fatty acid oxidation: (B) Ppara, Mcad, Cpt2; oxidative phosphorylation: (C) CoxIV; thermogenesis: (D) Pgc1a, Cidea, (E) Rip140, (F) Prdm16, Ucp1, Dio2; and inflammatory gene expression: (G) CD137, Tnfa, Ccl2/Mcp1 were analyzed. Data represent mean ± s.d., n = 5; Mann–Whitney U test (Ucp1) and independent Student’s t-test (except Ucp1). (H) Similar Ereg expression analysis was performed in mRNA isolated from whole iAb fat pads of lean, DIO, and ob/ob mice (n = 8/group) using NanoString assay. Data show mean ± s.d., independent Student’s t-test.

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    Leptin deficiency abolishes EREG-mediated energy expenditure. (A) Body weight in ob/ob mice (n = 5/group) before and after treatment with PBS (Veh) (open circles) or EREG (closed circles, 2.7 ng/g body weight). Insert shows food intake in these ob/ob mouse groups, which were pair-fed a high-fat diet throughout this study. (B) Liver, (C) BAT, (D) subcutaneous and iAb fat pad weight. Organ weight was normalized to body weight. (E, F, G and H) Metabolic parameters were analyzed in the same Veh and EREG-injected ob/ob mice (n = 4/group) at RT and after cold exposure in metabolic cages equipped with CLAMS. (E) RER (mean ± s.d.), (F) locomotor activity (mean ± s.d.), and (G and H) metabolic rate kinetics were measured in Veh-treated (white bars, or open circles) and EREG-treated (black bars, or closed circles) ob/ob mice. (D) and (L) represent ‘dark’ and ‘light’ cycles. (I) Expression of thermogenic (Pgc1a, Cidea) and PPARα, and its target gene Mcad in iAb fat from ob/ob mice was analyzed using NanoString mouse metabolic panel. Data show mean ± s.d., Independent Student’s t-test (Cidea) and Mann–Whitney U test (except Cidea). A full colour version of this figure is available at https://doi.org/10.1530/JOE-18-0289.


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