TET2 expression increases in fasting and HFD mouse livers. A, B, qRT-PCR analysis of TET2 mRNA levels in mouse livers after 16 hours overnight fasting (A) or 11 days high-fat diet (B). n=7. C-D, WB analysis of TET2 protein levels in mouse livers following the treatment described in A and B (C and D). n=5. E, WB analysis of TET2 protein levels in mouse livers after 12 weeks high-fat diet. n=5.

TET2 boosts gluconeogenesis. A-D, Glucose production assays were performed after TET2 overexpression in HepG2 cells (A) and mouse primary hepatocytes (B), or pre-treated with glucagon in WT and TET2 KO HepG2 cells (C) and mouse primary hepatocytes (D). E-G, PTT(E), GTT(F) and ITT(G) were performed in WT and Tet2 KO mice as described in methods.

TET2 up-regulates FBP1 expression in liver cells A, B, qRT-PCR were performed to analyze TET2 and FBP1 expression levels after glucagon (20nM) treatment for 48 hours in HepG2 cells (A) and primary mouse hepatocytes (B). C, WB analysis of Tet2 and Fbp1 protein levels after glucagon (20nM) treatment in mouse primary liver cells. D, E, qRT-PCR analysis of Fbp1 mRNA levels in mouse livers following the treatment described in Figure 1A (D) and 1B (E). F, Data from Figure 1A were reused to analyze the correlation between Tet2 and Fbp1 levels in mouse livers with or without the treatment. G, The correlation between TET2 and FBP1 levels in human livers was analyzed. Data were collected from GEPIA(29). H, WB analysis was performed to detect TET2 and FBP1 expression after overexpression TET2 in HepG2 cells and mouse primary hepatocytes. I, J, qRT-PCR (I) and WB (J) analysis of FBP1 expression in CRISPR/Cas9-mediated TET2 knockout liver cells. K, TET2 and FBP1 protein levels were determined by WB in WT and Tet2 KO mouse primary hepatocytes and HepG2 cells treated with or without glucagon. L-N, TET2(L), 5hmC(M) and 5mC(N) ChIP-qPCR were performed to measure the ability of TET2 binding to and demethylating FBP1 promoter in response to glucagon stimulation. Each ChIP Ct value is normalized to IgG Ct value.

HNF4α is necessary for TET2 mediated FBP1 up-regulation A, Immunofluorescence analysis of TET2 and HNF4α co-localization in HepG2 cells after glucagon treatment for 48 h. B, The interaction between TET2 and HNF4α was determined in HepG2 cells treated with or without glucagon using IgG and TET2 antibody followed by WB. C, Two siRNAs targeting HNF4α were transfected into HepG2 and the knockdown efficiency were determined by qRT-PCR and WB analysis. D, ChIP analysis of TET2 binding to FBP1 promoter was performed following the treatment with siRNA targeting HNF4α and glucagon as indicated. E, FBP1 protein levels were examined by WB after the treatment with TET2 overexpression and siRNA targeting HNF4α as indicated. F, Glucose production levels were examined after the treatment with glucagon and HNF4α siRNA. G-I, Hnf4α and Fbp1 protein levels in mouse livers were examined by WB after the treatment with 16 hours overnight fasting (G) or 11 days high-fat diet (H), or 12 weeks high-fat diet (I). n=5.

Metformin impairs the ability of HNF4α binding to TET2 and FBP1 expression A, The interaction between HNF4α and TET2 with or without metformin treatment was determined by immunoprecipitation using IgG and TET2 antibody followed by WB. B, TET2 ChIP-qPCR was used to detect the ability of TET2 binding to FBP1 promoter with or without metformin treatment. C, WB analysis of HNF4α, HNF4α phosphorylation at Ser 313 and FBP1 levels after metformin treatment. +:5mM, ++: 10mM. D, The interaction between TET2 and HNF4α wildtype and mutants was determined by WB. E, TET2 ChIP-qPCR was performed to determine the binding of TET2 to FBP1 promoter after transfection of HNF4α wildtype and mutants as indicated. F, qRT-PCR and WB analysis of FBP1 levels after transfection of HNF4α wildtype and mutants as indicated.

Targeting TET2 improves the efficacy of metformin in glucose metabolism in vivo A, The mRNA levels of Tet2 were examined by qPCR in HFD mice infected with AAV-scr or AAV-siTet2 for 10 days. B, C, Fasting blood glucose (B) and insulin (C) levels were determined in HFD mice infected with AAV-scr or AAV-siTet2 for 10 days, and treated with or without metformin for another 10 days. n = 6. D, PTT was examined in HFD mice infected with AAV-scr or AAV-siTet for 10 days, and treated with or without metformin for another 10 days. n = 6. E, WB analysis of Tet2 and Fbp1 levels in livers of HFD mice infected with AAV-scr or AAV-siTet, and treated with or without metformin for another 10 days. n = 6. F, G, GTT (F) and ITT (G) were examined in HFD mice infected with AAV-scr or AAV-siTet for 10 days, and treated with or without metformin for another 10 days. n = 6.