Fig 2: Phosphorylation of PIMT hampers liver glucose homeostasis(A) Oral Glucose tolerance test (OGTT) in C57BL/6J mice expressing GFP or PIMT (W or mutants) in the liver (n = 5).(B) The area under the curve for Figure 5A. Numerical data are expressed as mean ± SD. Statistical analysis was performed using one-way ANOVA followed by Bonferroni’s post hoc test. dp<0.001 compared to GFP-infected mice, Dp<0.001 compared to PIMT-W-infected mice.(C) Immunoblot analysis of the liver lysates using the indicated antibodies.(D) Densitometric quantification of Figure 5C. Values were normalized with the corresponding loading control, actin. Numerical data are expressed as mean ± SD. Statistical analysis was performed using one-way ANOVA followed by Bonferroni’s post hoc test. ap<0.05, bp<0.01, cp<0.005, dp<0.001 compared to GFP-infected mice, Ap<0.05, Bp<0.01, Cp<0.005 compared to PIMT-W-infected mice.(E) Pyruvate tolerance test (PTT) in C57BL/6J mice expressing GFP or PIMT (W or mutants) in the liver.(F) The area under the curve for Figure 5E. Numerical data are expressed as mean ± SD. Statistical analysis was performed using one-way ANOVA followed by Bonferroni’s post hoc test. dp<0.001 compared to GFP-infected mice, Dp<0.001 compared to PIMT-W-infected mice.(G) Immunoblot analysis of the liver lysates using the indicated antibodies.(H) Densitometric quantification of Figure 5G. Values were normalized with the corresponding loading control, Vinculin. Numerical data are expressed as mean ± SD. Statistical analysis was performed using one-way ANOVA followed by Bonferroni’s post hoc test. bp<0.01, cp<0.005, dp<0.001 compared to GFP infected mice, Cp<0.005, Dp<0.001 compared to PIMT-W-infected mice. PIMT-W: PIMT wild type, PIMT-A: PIMT S656A mutant, PIMT-D: PIMT S656D mutant.

Fig 3: Hepatic PIMT depletion hampers de novo glucose synthesis in diabetic mice(A and E) Oral glucose tolerance test in HFD (A) or OB (E) mice expressing shSCR (NT) or two independent shRNA against Tgs1 in the liver.(B and F) The area under the curve for Figures 7A and B or Figures 7E and F. Numerical data are expressed as mean ± SD. Statistical analysis was performed using one-way ANOVA followed by Dunnett’s post hoc test. ∗∗∗p < 0.005 compared to NT-infected mice.(C and G) Immunoblot analysis of the liver lysates using the indicated antibodies.(D and H) Densitometric quantification of Figures 7C and 7G. Values were normalized with the corresponding loading control, actin. Numerical data are expressed as mean ± SD. Statistical analysis was performed using one-way ANOVA followed by Dunnett’s post hoc test. ∗p<0.05, ∗∗p<0.01, ∗∗∗p<0.005, ∗∗∗∗p<0.001 compared to NT-infected mice.

Fig 4: PIMT enhanced Ep300 coactivation activity(A) HepG2 cells were transfected with pGL3-PEPCK-Luc promoter along with Ep300, PIMT (W and mutants) in the presence or absence of PKAc. Post 36h of transfection, cells were lysed, and luciferase signals were quantified. Renilla luciferase signals were used as an internal control. The values were normalized with corresponding Renilla luciferase activity and expressed relative to PEPCK-Luc (unphosphorylated) (column 1), set to 1. Data are representative of 5 independent experiments and expressed as mean ± SD. Statistical analysis was performed using one-way ANOVA followed by Bonferroni’s post hoc test. ap<0.05, dp<0.001 vs PECK-Luc (unphosphorylated) αp<0.001 compared to PEPCK-Luc + PIMT + PKAc transfected cells, Dp<0.001 compared to PEPCK-Luc + PIMT (W) without PKAc, #p<0.001 compared to PEPCK-Luc + PIMT (W)+ Ep300 + PKAC.(B) Primary hepatocytes isolated from female mice infected with either PIMT (w) or GFP were treated with Forskolin for 4h and then subjected to IP with anti-V5 antibody followed by immunoblots with the defined antibodies (n = 4).(C) Densitometric quantification of Figure 6B. The signals were normalized with the corresponding input signals and neutralized with the PIMT-V5. DMSO-treated samples were set to 1. Data are representative of 3 independent experiments and expressed as mean ± SD. Statistical analysis was performed using unpaired Student’s t-test (two-tailed) ∗∗∗∗p<0.001, versus the DMSO, treated cells.(D) Primary hepatocytes isolated from female mice infected with either PIMT (W or mutants) (n = 3). Post-infection (48h), cells were lysed and subjected to IP with anti-V5 antibody followed by immunoblots with the defined antibodies.(E) Densitometric quantification of Figure 6D. The signals were normalized with the corresponding input signals and neutralized with the PIMT-V5 (W) set to 1. Numerical data are expressed as mean ± SD. Statistical analysis was performed using one-way ANOVA followed by Bonferroni’s post hoc test. ∗p<0.05, ∗∗∗∗p<0.001 compared to PIMT(W) infected cells.(F) 8h fasted liver lysates were subjected to IP with anti-CBP or anti-Ep300 antibodies followed by immunoblots with the defined antibodies (n = 3).(G) Densitometric quantification of Figure 6F. The interaction signals were normalized with the corresponding enriched protein signals. Statistical analysis was performed using unpaired Student’s t-test (two-tailed) ∗∗p<0.01, versus the fed liver lysates.

Fig 5: Hepatic PKA regulates PIMT protein expression(A) HepG2 cells were transfected with an increasing concentration of active catalytic subunit of PKA (PKAc). Post 48h of transfection, cells were lysed, and immunoblots assessed the protein levels of PIMT.(B) Densitometry quantification of Figure 2A. Values were normalized with the corresponding loading control, Vinculin. Data are representative of 3 independent experiments and are expressed as mean ± SD. Statistical analysis was performed using one-way ANOVA followed by Bonferroni’s post hoc test. ∗∗∗∗p< 0.001 compared to pcDNA3.1 transfected cells, ap<0.05, dp<0.001 vs PKAc (4 μg) transfected cells.(C) qPCR analysis of the indicated genes upon PKAc overexpression in HepG2 cells. Values were normalized using 18S as a reference gene. Data are representative of 3 independent experiments and are expressed as mean ± SD. Statistical analysis was performed using one-way ANOVA followed by Bonferroni’s post hoc test. ∗∗p<0.01, ∗∗∗p<0.005, ∗∗∗∗p<0.001 compared to pcDNA3.1 transfected cells, ap<0.05, bp<0.01, dp<0.001 vs PKAc (4 μg) transfected cells.(D) HepG2 cells were treated with increasing concentrations of Rp-Br-cAMPs (RP). Post 8h of treatment, cells were lysed, and immunoblots assessed the protein levels of PIMT.(E) Densitometry quantification of Figure 2D. Values were normalized with the corresponding loading control, Vinculin. Data are representative of 3 independent experiments and are expressed as mean ± SD. Statistical analysis was performed using one-way ANOVA followed by Bonferroni’s post hoc test. ∗∗∗∗p<0.001 compared to DMSO treated cells, bp<0.01, dp<0.001 vs RP (50 μM) treated cells.(F) qPCR analysis of the indicated genes upon RP treatment in HepG2 cells for 8h. Values were normalized using 18S as a reference gene. Data are representative of 3 independent experiments and are expressed as mean ± SD. Statistical analysis was performed using one-way ANOVA followed by Dunnett’s post hoc test. ∗∗p<0.01, ∗∗∗p<0.005, ∗∗∗∗p<0.001 compared to DMSO treated cells, cp<0.005, dp<0.001 vs RP (50 μM) treated cells.(G) HepG2 cells were treated with increasing concentration of RP for 4h followed by treatment with MG132 for 4h (10 μM). Post-treatment cells were lysed and probed for endogenous PIMT protein levels.(H) Densitometry quantification of Figure 2G. Values were normalized with the corresponding loading control, Vinculin. Data are representative of 3 independent experiments and are expressed as mean ± SD. Statistical analysis was performed using one-way ANOVA followed by Bonferroni’s post hoc test. ∗∗∗∗p<0.001 compared to DMSO treated cells, ns: non-significant.(I) qPCR of the sucrose gradient fractions from fed and fasting liver lysates of mice. The values were normalized with extracellular Luciferase RNA (n = 4).