Secreted EMC10 is upregulated in human obesity and its neutralizing antibody prevents diet-induced obesity in mice

Abstract

Secreted isoform of endoplasmic reticulum membrane complex subunit 10 (scEMC10) is a poorly characterized secreted protein of largely unknown physiological function. Here we demonstrate that scEMC10 is upregulated in people with obesity and is positively associated with insulin resistance. Consistent with a causal role for scEMC10 in obesity, Emc10^-/- mice are resistant to diet-induced obesity due to an increase in energy expenditure, while scEMC10 overexpression decreases energy expenditure, thus promoting obesity in mouse. Furthermore, neutralization of circulating scEMC10 using a monoclonal antibody reduces body weight and enhances insulin sensitivity in obese mice. Mechanistically, we provide evidence that scEMC10 can be transported into cells where it binds to the catalytic subunit of PKA and inhibits its stimulatory action on CREB while ablation of EMC10 promotes thermogenesis in adipocytes via activation of the PKA signalling pathway and its downstream targets. Taken together, our data identify scEMC10 as a circulating inhibitor of thermogenesis and a potential therapeutic target for obesity and its cardiometabolic complications.

Fig. 1. Serum EMC10 levels in white and Chinese Han cohorts.

A Serum EMC10 levels in white human study participants with leanness (n = 27), overweight (n = 20) and obesity (n = 160). B Serum EMC10 levels in Chinese Han human study participants with leanness (n = 32), overweight (n = 115) and obesity (n = 39). Serum EMC10 levels are presented as box (median with interquartile range) and whisker (1.5x interquartile range) plots. Comparisons among 3 groups of participants were performed by one-way ANOVA with Fisher’s post hoc test, and then multiple comparisons were performed using LSD-t test. A two-sided P-value of <0.05 was considered as significant. C Correlation of serum EMC10 levels with BMI in the white cohort shown in panel A (n = 207). D Correlation of serum EMC10 levels with BMI in the Chinese Han cohort shown in panel B (n = 186). E Correlation of serum EMC10 levels with BMI in a white weight-loss cohort (n = 100). The correlation analyses were performed using Pearson’s bivariate correlation. F–N Correlation of serum EMC10 levels with GIR (glucose infusion rate), FINS (fasting plasma insulin), FPG (fasting plasma glucose), HbA1c, FFA (serum free fatty acid), serum leptin and adiponectin, and subcutaneous (SC) fat area and visceral (VIS) fat area, respectively, in white human study participants who underwent euglycemic-hyperinsulinemic clamp (n = 17). The correlation analyses were performed using Spearman’s rank correlation analysis. Source data are provided in the Source Data file.

Fig. 2. Serum EMC10 levels in white weight-loss cohorts.

A–C BMI, serum EMC10 concentration, or HOMA-IR of white human study participants before and 12 months after bariatric surgery, respectively (n = 50), ***p < 0.001. D–F BMI, serum EMC10 concentration, or HOMA-IR of white human study participants before and 12 months after diet/exercise weight-loss intervention, respectively (n = 50). Comparisons between before and after were performed using Student’s paired t test. G–L Correlations between changes of serum EMC10 levels and changes of BMI, HOMA-IR, HbA1c, serum AST, ALT and TG, respectively, after weight-loss intervention by either bariatric surgery or diet/exercise in the white cohort (n = 100), performed using Pearson’s bivariate correlation. Source data are provided in the Source Data file.

Fig. 3. Effects of EMC10 ablation and scEMC10 overexpression on obesity and metabolic homeostasis.

A Body weights of male (WT, black circle), and KO (red square) on C57BL/6 background on HFD (n = 6 & 7 per group). B Tissues (iWAT, eWAT, BAT, liver) weight from male WT (open), and KO (red) mice fed with 12-wks of HFD (n = 6 per group). C Body composition of male WT (open) and KO (red) mice fed HFD by DEXA (n = 6 per group). D Glucose tolerance (left) and insulin tolerance (right) in male WT (black diamond), and KO (red square) mice fed with 12-wks of HFD (n = 8 & 7 per group). Plasma glucose and insulin (E); leptin and adiponectin (F); triglyceride (TG), cholesterol (CHO), and non-esterified free fatty acid (NEFA) (G), in male WT (open), and KO (red) mice fed with 12-wks of HFD in the fed or overnight fasted states (n = 6 per group). H Representative images of H&E-stained sections of livers from male WT and KO mice fed with 12-wk of HFD. Scale bar, 100 um. I TG content of liver from male WT (open) and KO (red) mice fed with 12-wks of HFD (n = 6 per group). J Body weights of male C57BL/6 mice expressing LacZ control or hscEMC10 via tail-vein AAV transduction after 20-wks of chow diet (CD) (n = 6 per group). K Tissues (iWAT, eWAT, retroperitoneal (retroWAT), BAT, liver) weight of male C57BL/6 mice expressing LacZ control or hscEMC10 via tail-vein AAV transduction after 20-wks of CD (n = 6 per group). L Glucose tolerance (left) and insulin tolerance (right) of male C57BL/6 mice expressing LacZ control or hscEMC10 via tail-vein AAV transduction after 20-wks of CD (n = 6 per group). M Plasma insulin, leptin, and adiponectin of male C57BL/6 mice in the fed state expressing LacZ control or hscEMC10 via tail-vein AAV transduction after 20-wks of CD (n = 6 per group). All data are presented as mean +/− SEM. Statistical significance was assessed by two-sided Student’s t test and significant differences were indicated with p values. Source data are provided in the Source Data file.

Fig. 4. EMC10 ablation promotes adipose tissue oxygen consumption and whole-body energy expenditure.

A Oxygen consumption (VO2) (left) and carbon dioxide production (VCO2) (right), B Heat production analyzed by indirect calorimetry for 48 h in male WT (black circle) or KO (red square) mice after fed with 12-wks of HFD (n = 8 per group). C Rectal temperature measured for male WT (black) and KO (red) mice fed HFD at room temperature (n = 8 per group). D Energy expenditure analyzed with ANCOVA using lean mass as covariate for WT (black circle) or KO (red square) mice (n = 8 per group). E Respiratory exchange ratio (RER) analyzed by indirect calorimetry in male WT (black circle) and KO (red square) mice fed with HFD (n = 6 per group). F Atgl, Hsl, Adrb3, Glut4, Fasn, Srebp1c, Pgc1a, Ucp1, and Tfam mRNA in BAT from male WT (balck) or KO (red) mice after fed with 12-wks of HFD (n = 6 per group). G Atgl, Hsl, Adrb3, Pgc1a, Ucp1, and Tfam mRNA in iWAT from male WT (balck) or KO (red) mice after fed with 12-wks of HFD (n = 6 per group). H Pgc1a, Ucp1, and Tfam mRNA in BAT and iWAT from male WT (black) or KO (red) mice after fed with 12-wks of LFD (n = 6 per group). I Oxygen consumption in BAT (left) and iWAT (right) from male WT (black) or KO (red) mice fed with 12-wks of LFD. (n = 6 per group). J Body weights of male (WT, black circle), and KO (red square) on HFD at 30 °C (n = 5 & 6 per group). K Oxygen consumption of male WT (black circle) and KO (red square) before and after CL316, 243 (0.1 mg/kg) stimulation (n = 5 & 4 per group). All data are presented as mean +/− SEM. Student’s t test was used for statistical analysis. Statistical significance was assessed by two-way ANOVA followed with Bonferroni’s multiple comparisons test (A, B and E), two-sided Student’s t test (C, F–K) and significant differences were indicated with p values. Source data are provided in the Source Data file.

Fig. 5. EMC10 ablation activates adipocyte adaptive thermogenesis via PKA-mediated CREB and p38MAPK activities.

Ucp1, Pgc1a, Dio2, and Tfam mRNA in differentiated brown (A) or inguinal (B) primary adipocytes from male WT or KO mice treated with saline control or 0.1 μM CL316, 243 for 24 h (n = 6 per group). C Ucp1 and Pgc1a mRNA in differentiated epididymal primary adipocytes from male WT or KO mice treated with saline control or 0.1 μM of CL316, 243 for 24 h (n = 6 per group). D Ucp1 and Pgc1a mRNA in differentiated brown primary adipocytes from male WT or KO mice treated with control or recombinant human scEMC10 at indicated dose for 24 h (n = 6 per group). E Western blotting for UCP1 and β-actin in differentiated brown primary adipocytes from male WT or KO mice treated with control or recombinant scEMC10 at indicated dose for 24 h. F Ucp1 and Pgc1a mRNA in differentiated brown primary adipocytes from male WT or KO mice treated with control or PKA inhibitor, 1 μM of H89 for 24 h (n = 3 per group). G Western blotting for pCREB, total CREB, pP38MAPK and total p38MAPK proteins in differentiated brown primary adipocytes from male WT and KO mice after treated with saline (0 min) or CL316,243 (5, 15 min) (n = 3 per group). H Ucp1 and Pgc1a mRNA in differentiated brown primary adipocytes from male WT or KO mice treated with control or p38MAPK inhibitor, 1 μM of SB203580 for 24 h (n = 3 per group). I Ucp1 and Pgc1a mRNA in differentiated brown primary adipocytes from male WT or KO mice treated with control or CREB inhibitor, 0.5 μM of HY101120 for 24 h (n = 3 per group). All data are presented as mean +/− SEM. Statistical significance was assessed by two-sided Student’s t test (A–D, F, H & I) and significant differences were indicated with p values. Source data are provided in the Source Data file.

Fig. 6. Extracellular scEMC10 binds PKA Cα and inhibits its stimulatory action on CREB.

A Immunoprecipitation and western blotting of scEMC10, PKA Cα, and AKT1 in cell lysate prepared from 293T cells transfected with either FLAG-scEMC10 plasmid alone, or co-transfected with HA-PKA Cα or HA-AKT1 plasmid. B Immunoprecipitation and western blotting of scEMC10 and endogenous PKA Cα in cell lysate prepared from 293T cells transfected with FLAG-scEMC10 plasmid. C Western blotting for pCREB, total CREB and PKA in 293T cells after treatment with H89-2HCl (10 μM), recombinant scEMC10 protein (2 μg) or inactivation scEMC10 protein (2 μg) as indicated. D Immunofluorescence analysis of Hela cells treated with either scEMC10 labeled with FITC or control. Nuclei were stained blue with 4’,6-diamidino-2-phenylindole (DAPI), and cell membranes were stained red with Octadecyl Rhodamine B Chloride (R18). Scale bar, 5μm. Each experiment was repeated 3 times independently with similar results. Source data are provided in the Source Data file.

Fig. 7. Effects of scEMC10 neutralization on diet-induced obesity and metabolic homeostasis.

A Body weight (left) and body weight gain (right) of C57BL/6J male mice fed with HFD before or after IP injected with 3 mg/kg BW antibody as indicated twice a week. (IgG, black circle. mAb-1F12, gray square. mAb-4C2, red triangle) (n = 9 per group). B Body weight gain of HFD male mice IP injected with 3 mg/kg BW 4C2 antibody or control IgG twice a week. 7 days later, the two antibodies swapped with each other, and then 4 days later, swapped back followed by 3 other days of original treatment (IgG, dashed/black. mAb-4C2, solid/red) (n = 5 per group). C Representative images of H&E-stained sections of liver from male mice treated with IgG or 4C2 antibodies. Scale bar, 50 um. D TG content of liver from male mice treated with IgG (black) or 4C2 (red) antibodies for 3 weeks (n = 6 per group). E Glucose tolerance (left) and insulin tolerance (right) of male mice treated with IgG (black circle), 1F12 (gray square) or 4C2 (red triangle) antibodies (n = 9 per group) for 3 weeks. Plasma glucose after 6 h fasting and fed insulin (F); Fed plasma triglyceride (TG), cholesterol (CHO), non-esterified fatty acid (NEFA), and ALT (G); plasma leptin and adiponectin (H) in male mice treated with IgG (black), 1F12 (gray) or 4C2 (red) antibodies for 3 weeks (n = 8 per group). I Ucp1, Pgc1a, Dio2, Cox8b, and Elvol3 mRNA in BAT from male mice treated with IgG (black) or 4C2 (red) antibodies (n = 6 per group). J Western blotting for UCP1, PGC1a and actin in BAT from male mice treated with IgG or 4C2 antibodies. K Oxygen consumption (VO2), carbon dioxide production (VCO2), and heat production analyzed by indirect calorimetry for 48 h in male mice IP injected with IgG (black circle) or 4C2 (red circle) antibodies (n = 8 per group). All data are presented as mean +/− SEM. Statistical significance was assessed by two-sided Student’s t test (A, B, D–I) and significant differences were indicated with p values. Source data are provided in the Source Data file.