Fig 2: Lipid metabolism in global Epgn knockout (KO) mice(A) Circulating TG level, CHO level (B), LDL and HDL levels (C), and FFA levels (D) after 6 months of HFD feeding. Hepatic TG (E) and CHO (F) levels in liver after 6 months of HFD feeding. Representative H&E staining images (G) and quantification of liver changes (H) after 6 months of HFD feeding. (I) Serum levels of AST and ALT after 6 months of HFD feeding. n = 6 WT, 7 KO. Data are the mean ± SEM. Statistical analysis was performed using unpaired two-tailed Student’s t test or Welch's t test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Fig 3: Glucose homeostasis in Epgn KO mice on an HFD(A) Schematic diagram of the experiment. (B) Schematic diagram of the Epgn knockout and genotyping strategy. (C) Genotyping results using PCR with genomic DNA and the relative positions for the primers used specified in (B). (D) Epgn mRNA levels in the tongue. (E) Circulating levels of epigen. (F) Fasting glucose levels. (G) Body weight measurements. (H) Fat and lean mass measurements. (I) Percentage of fat and lean mass relative to body weight. (J) Percentage of sWAT and eWAT mass relative to body weight. (K) H&E staining of sWAT and eWAT. (L) Quantification of adipocyte size in sWAT and eWAT. (M) Quantification of immune cell infiltration in sWAT. (N) GTT results after 14 weeks of HFD feeding. (O) AUC, (P) percentage of basal glucose levels, and (Q) GSIS from the GTT in (M). (R) ITT results after 15 weeks of HFD feeding. (S) AUC and (T) percentage of basal glucose levels from the ITT in panel Q. (U) PTT results after 16 weeks of HFD feeding. (V) AUC and (W) percentage of basal glucose levels from the PTT in (T). n = 6 WT, 7 KO. Data are the mean ± SEM. Statistical analysis was performed using unpaired two-tailed Student’s t test or Welch's t test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Fig 4: Glucose tolerance in mice following acute recombinant epigen treatment(A) GTT results in 18-week HFD-fed mice. AUC (B) and GSIS (C) from the GTT in (A). (D) GTT results in STZ-treated mice. AUC (E) and GSIS (F) from the GTT in (D). (G and H) Epigen-induced insulin secretion in beta cells under high-glucose stimulation. (G) Insulin secretion in isolated mouse islets with and without EGF receptor inhibitor AG1478. (H) Insulin secretion in isolated mouse islets with and without inhibitors for insulin secretion. (I) IVIS image showing label-epigen protein distribution, and (J) IVIS image showing NBDG distribution in mice after injection of either PBS or label-epigen protein for an hour. (K) Glucose uptake in SVF-differentiated adipocytes with epigen treatment in a dose-dependent manner. (L) Glucose uptake in SVF-differentiated adipocytes with and without EGF receptor inhibitors. (M) Glucose uptake in primary myoblast-differentiated muscle cells with epigen treatment in a dose-dependent manner. (N) Glucose uptake in myoblast-differentiated muscle cells with and without EGFR inhibitors AG1478 and Tucatinib. (O) Glucose uptake in primary hepatocytes with epigen treatment. (P) Glucose production in primary hepatocytes under glucagon and pyruvate induction, with and without overexpression of EPGN via adenovirus. (Q) Gluconeogenesis in primary hepatocytes with epigen protein treatment. (A–F), n = 4. (G) and (H), n = 6. (K–Q), n = 6. Data are the mean ± SEM. Statistical analysis was performed using unpaired two-tailed Student’s t test or Welch's t test. Group differences were determined via two-tailed analysis of variance (ANOVA) with Tukey’s post hoc test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Fig 5: Circulating epigen levels in mice and humans(A) Epgn mRNA levels (n = 3) and (B) epigen protein levels were measured in various mouse tissues. (C) Circulating epigen levels were assessed in mice during feeding, after a 16-h fast, and after 2 h of refeeding, with mice having been fed either STC or HFD for 6 weeks. (D) Corresponding glucose levels for (C) are also provided (n = 8 STC, 9 HFD). (E) A scatter correlation and (F) differential analysis box plot epigen levels and BMI in human serum samples (Table S1). (G) A scatter correlation and (H) differential analysis box plot for epigen levels and HbA1c levels in human serum samples (Table S2). Data are the mean ± SEM. Group differences were determined via two-tailed analysis of variance (ANOVA) with Tukey’s post hoc test. Significance in correlations were determined with Spearman’s rho. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.