TET2 expression increases in fasting and HFD mouse livers.

(A) qRT-PCR analysis of Tet2 mRNA levels in mouse livers after 16 h fasting treatment. The data are normalized to the GAPDH expression (n =7). (B) qRT-PCR analysis of Tet2 mRNA levels in mouse livers after high-fat diet treatment for 11 days. The data are normalized to the GAPDH expression (n=7). (C) Western blot analysis and quantification of Tet2 protein levels in mouse livers following 16 h fasting treatment (n=5). (D) Western blot analysis and quantification of Tet2 protein levels in mouse livers following the high-fat diet treatment for 11 days (n=5). (E) Western blot analysis and quantification of Tet2 protein levels in mouse livers following the 12-week high-fat diet treatment (n=5).

TET2 boosts gluconeogenesis.

(A) Glucose production assays were performed in HepG2 cells after TET2 overexpression. Data are represented as the mean±SD (n=3). (B) Glucose production assays were performed in mouse primary hepatocytes after Tet2 overexpression. Data are represented as the mean ± SD (n=3). (C) Glucose production assays were performed in HepG2 cells pre-treated with 20nM glucagon in WT and TET2 KO HepG2 cells. Data are represented as the mean ± SD (n=3). (D) Glucose production assays were performed in mouse primary hepatocytes pre-treated with 20nM glucagon in WT and Tet2 KO cells. Data are represented as the mean ± SD (n=3). (E) PTT was performed following a 16 h fasting treatment and intraperitoneal (i.p.) injection of 1 g/kg sodium pyruvate (n = 5). (F) GTT was performed after a 12 h fasting treatment and i.p. injection of 2 g/kg glucose (n = 5). (G) ITT was performed after a 4 h fasting treatment and i.p. injection of 0.75 U/kg insulin (n = 5). (H) Glucose-stimulated insulin secretion was examined. After fasting and i.p. injection of 2 g/kg glucose, plasma insulin levels were measured at the indicated time points (n = 5). (I) Body weight of 8 or 10-week-old male WT mice and Tet2 KO mice on a normal chow diet (n = 5).

TET2 up-regulates FBP1 expression in liver cells

(A) qPCR analysis of TET2 and FBP1 mRNA expression levels after 20nM glucagon treatment for 48 h in HepG2 cells. Data are represented as the mean ± SD (n=3). (B) qPCR analysis of Tet2 and Fbp1 mRNA expression levels after 20nM glucagon treatment for 48 hours in primary mouse hepatocytes. Data are represented as the mean ± SD (n=3). (C) Western blot analysis of Tet2 and Fbp1 protein levels after 20nM glucagon treatment in mouse primary hepatocyte cells. (D) qPCR analysis of G6Pase and PEPCK mRNA expression levels after 20nM glucagon treatment for 48 hours in HepG2 cells. Data are represented as the mean ± SD (n=3). (E) qPCR analysis of TET2 and FBP1 mRNA expression levels after 20nM glucagon treatment at the indicated time points (n = 5). (F) qPCR analysis of Fbp1 mRNA levels in mouse livers following fasting treatment (n=7). (G) qPCR analysis of Fbp1 mRNA levels in mouse livers following HFD treatment (n=5). (H) Correlation analysis between Tet2 and Fbp1 levels using data from Figure 1A in mouse livers with or without fasting treatment. (I) Correlation analysis between TET2 and FBP1 levels in human livers. Data were collected from GEPIA(29). (J) Western blot analysis of TET2 and FBP1 expression after overexpression of Flag-TET2 in mouse primary hepatocytes and HepG2 cells. (K) qPCR analysis of FBP1 expression levels in control and TET2 knockout HepG2 and LO-2 cells. (L) Western blot analysis of TET2 and FBP1 protein levels in control and TET2 knockout HepG2 and LO-2 cells. (M) Western blot analysis of Tet2 and Fbp1 protein levels in control and Tet2 knockout mouse primary hepatocytes and HepG2 cells treated with or without 20nM glucagon. (N) ChIP-qPCR analysis of TET2 binding to FBP1 promoter in response to glucagon stimulation in control and TET2 knockout HepG2 cells. (O) ChIP-qPCR analysis of 5hmC levels in FBP1 promoter in response to glucagon stimulation in control and TET2 knockout HepG2 cells. (P) ChIP-qPCR analysis of 5mC levels in FBP1 promoter in response to glucagon stimulation in control and TET2 knockout HepG2 cells.

HNF4α is necessary for TET2 mediated FBP1 up-regulation

(A) Immunofluorescence analysis of TET2 and HNF4α co-localization in HepG2 cells after 20nM glucagon treatment for 48 h. Scale bar: 10μm. (B) Immunofluorescence analysis of Tet2 and Hnf4α co-localization in liver sections from standard chow and fasting mice. Scale bar: 30μm. (C) Immunofluorescence analysis of Tet2 and Hnf4α co-localization in liver sections from standard chow and HFD mice. Scale bar: 30μm. (D) Endogenous co-immunoprecipitation followed by western blot analysis of the interaction between HNF4α and TET2 with or without glucagon treatment in HepG2 cells. (E)qRT-PCR and western blot analysis of HNF4α expression levels in HepG2 cells transfected with two specific siRNAs. (F) ChIP-qPCR analysis of TET2 binding to FBP1 promoter in HepG2 cells treated with siRNA targeting HNF4α and glucagon as indicated. (G) ChIP-qPCR analysis of 5hmC levels in FBP1 promoter in HepG2 cells treated with siRNA targeting HNF4α and glucagon as indicated. (H) ChIP-qPCR analysis of 5mC levels in FBP1 promoter in HepG2 cells treated with siRNA targeting HNF4α and glucagon as indicated. (I) Western blot analysis of FBP1 protein levels in HepG2 cells treated with TET2 overexpression and siRNA targeting HNF4α as indicated. (J) Glucose production assays were performed in HepG2 cells treated with glucagon and transfected with HNF4α siRNA as indicated. (K-M), Western blot analysis and quantification of Hnf4α and Fbp1 protein levels in mouse livers from the mice treated with 16 h overnight fasting (K) or 11-day HFD (L), or 12-week HFD treatment (M). n=5.

Metformin impairs the ability of HNF4α binding to TET2 and FBP1 expression

(A) Endogenous co-immunoprecipitation followed by western blot analysis of the interaction between HNF4α and TET2 with or without metformin (10 mM) treatment in HepG2 cells. (B) ChIP-qPCR analysis of TET2 binding to FBP1 promoter in HepG2 cells treated with or without metformin (10 mM). (C) Western blot analysis of TET2, HNF4α, HNF4α phosphorylation at Ser 313 and FBP1 levels in HepG2 cells treated with metformin as indicated (+: 5 mM, ++: 10 mM). (D) Endogenous co-immunoprecipitation followed by western blot analysis of the interaction between TET2 and HNF4α wildtype and S313 mutants as indicated. (E) ChIP-qPCR analysis of TET2 binding to FBP1 promoter in HepG2 cells transfected with HNF4α wildtype and S313 mutants as indicated. (F) qRT-PCR and western blot analysis of FBP1 levels in HepG2 cells transfected with HNF4α wildtype and S313 mutants as indicated.

Targeting TET2 improves the efficacy of metformin in glucose metabolism in vivo

(A) qRT-PCR analysis of Tet2 mRNA levels in mouse livers from the HFD mice infected with AAV8 or AAV8-shTet2 for 10 days. (B, C) Analysis of fasting blood glucose (B) and plasma insulin (C) levels in the HFD mice infected with AAV8 or AAV8-shTet2 for 10 days, and treated with or without metformin (300 mg/kg/day) for another 10 days as indicated. n = 6. (D) PTT was performed in the HFD mice infected with AAV8 or AAV8-shTet2 for 10 days, and treated with or without metformin (300 mg/kg/day) for another 10 days as indicated (n = 6). (E) Western blot analysis of Tet2 and Fbp1 levels in livers from the HFD mice infected with AAV8 or AAV8-shTet2 for 10 days, and treated with or without metformin (300 mg/kg/day) for another 10 days as indicated. (F, G) GTT (F) and ITT (G) were performed in the HFD mice infected with AAV8 or AAV8-shTet2 for 10 days, and treated with or without metformin (300 mg/kg/day) for another 10 days as indicated (n = 6).