Fig 2: Evolutionary analysis of EREG binding to LepR. (A–E) EREG docking to LepR. Evolutionary analysis of 175 open The dependence of EREG-mediated glucose uptake on the ERK phosphorylation cascade was examined using (1) a specific inhibitor of ERK1/2 SCH772984 [56], (2) an inhibitor of ERK dimerization DEL-22379 [57], and (3) a selective inhibitor of MEK1 and MEK2 U0126 [58]. All inhibitors increased basal glucose uptake, which was further increased by leptin (Figure 4F). The inhibition of ERK1/2 and MEK1/2 as well as ERK dimerization prevented stimulatory effect of EREG on FD-glucose uptake but did not decrease it beyond the levels seen in the control cells. Although transient ERK phosphorylation occurred in response to EREG stimulation, this pathway was dispensable for glucose uptake and dependent on PI3K and may be other pathways (Figure 4B and Figure S7B). (F) Hypothetic mechanism suggesting EREG as an alternative ligand for both EGFR and LepR. The canonic leptin/LepR response can induce JAK/STAT3 signaling and required the long form of LepR. The alternative binding of EREG to LepR can induce ERK and PI3K activation increasing GLUT4 translocation and glucose uptake, but not the other canonic effects of leptin, including the regulation of appetite and energy expenditure.

Fig 3: Kinetics of the changes in LepR film thickness in the presence of leptin (A) or EREG (B). Film thickness was measured using QCM and quantified based on the binding kinetics to a gold sensor.

Fig 4: EREG improved glucose tolerance in the absence of leptin in Lepob mice and exhibited no effect in LepR-deficient Leprdb mice. (A) Body weight of Lepob male mice in groups before and after treatment with Veh (PBS, white bar) or EREG (50 ng/g body weight (BW), black bar) for 26 days. Mice were on regular chow diet. Unpaired t-test, n = 7/group. ns, not significant. (B,C) Fat (B) and lean body (C) composition in same groups of mice at the end of the study was measured by Echo-MRI. Fat and lean mass are shown as % of the total weight (100%). (D,E) Glucose tolerance test (GTT) was performed in fasted Lepob mice treated with PBS (Veh, open circles) or EREG (closed circles) (n = 7 per group). GTT kinetics (D) and area under the curve (AUC) (E) are shown. Statistical significance was examined by ANOVA (D) and Student’s t-test (E). (F) Insulin levels in plasma in both mouse groups were measured by ELISA. Unpaired student’s t-test. (G) Weight before and after treatment of Leprdb male mice with Veh (PBS, white bar) or EREG (50 ng/g body weight (BW), black bar) for 4 weeks (n = 6 per treatment). Mice were on regular chow. Unpaired Student’s t-test, n = 6/group. (H,I) Fat (H) and lean body (I) composition (% of total weight) in the same groups of mice at the end of the study were measured by Echo-MRI. (J,K) GTT kinetics (J) and AUC (K) were obtained from Leprdb mice treated with PBS (Veh, open circles) or EREG (closed circles). ANOVA (J) and Student’s t-test (K). (L) Insulin levels in plasma in both mouse groups were measured by ELISA. Unpaired student’s t-test.

Fig 5: EREG mediates glucose uptake via PI3K with transient activation of ERK. (A) FD-glucose uptake in 3T3-L3 preadipocytes treated with or without EREG (50 ng/mL) and in the presence of inhibitors for EGFR-I (AG1478, 10 µM), EGFR and ErbB2 (AST-1306 or CI-1033 10 µM), dual IR/IGF-1R inhibitor (BMS 536924, 1 µM), and SRC-I, AZM475271, 1 µM) for 30 min. Cells were starved for 90 min before stimulation. Dashed line shows glucose uptake in the presence of insulin (Ins, 10 µg/mL). (B) FD-glucose uptake was measured in mouse 3T3-L1 preadipocytes with or without EREG (50 ng/mL) and inhibitors of MEK1/2 and PI3K (MEK1/2-I, U0126 10 µM, and PI3K-I, wortmannin 200 nM). Data (mean ± SD, n = 6) are shown as a percentage of control (Veh 100%). Unpaired Student’s t-test. (C) 3T3-L1 preadipocytes were stimulated with EREG at different concentrations (0–100 ng/mL) for 5 or 15 min. The total and phosphorylated levels of AKT, STAT3, STAT5, and ERK were measured by Western blot in duplicates. Data are shown in a representative Western blot. (D) The kinetics of pERK expression was quantified based on the Western blots. pAKT, p-STAT3, and p-STAT5 analysis are described in Figure S8. Pearson correlation analysis. (E) 3T3-L1 preadipocytes were stimulated with or without EREG or EGF (50 ng/mL, each) for 30 min in the presence and absence of EGFR inhibitor AST1306 (100 nM), and antibody against mouse LepR (Invitrogen, PA1-053, 10 µg/mL). For inhibition, cells were pre-treated 30 min before EREG and EGF stimulation. (F) FD-glucose uptake was measured in mouse 3T3-L3 preadipocytes pre-treated with either Veh (DMSO) or ERK inhibitors (U0126, SCH772984, or DEL 22379, each 10 µM in DMSO) for 40 min. Then, cells were treated with either Veh (PBS), mouse EREG (50 ng/mL), or mouse leptin (Lep, 200 ng/mL) for 80 min. Data are shown as a percentage of Veh-treated control (100%, n = 7 per group). Unpaired Student’s t-test. ns, not significant (p > 0.05).