Fig 2: Berberine reduced hepatic MPC1 induction with regulation of PDH and PC. (a–b): MPC1 protein expression in the liver of HFD-fed mice and primary mouse hepatocytes incubated with PA for 24 h. (c–d): Lactate production in primary mouse hepatocytes or HepG2 cells transfected with pCDNA3.1-MPC1 in the presence of 10 mM pyruvate for 8 h. (e): Oxygen consumption rate in HepG2 cells pretreated with BBR, Met and UK-5099 for 2 h. (f–h): PDH phosphorylation, PC protein expression and oxaloacetate contents in the liver of HFD-fed mice. (i): The mechanic pathway for berberine to restrain pyruvate-driven hepatic glucose production in the setting of lipid disorder. (BBR, berberine; Met, metformin; TMZ, trimetazidine; THP, mildronate; PA, palmitate). Data were expressed as the mean ± SD (n = 5–6). ⁎p < 0.05 vs. HFD mice, PA or pyruvate-treated cells; #p < 0.05 vs. chow-fed diet mice or untreated cells.

Fig 3: Berberine reduced SIRT3 induction and preserved MPC1 acetylation in the liver. (a): Acetylated-Lysine protein expression in the liver mitochondria. (b): Acetylation in precipitated MPC1 in primary mouse hepatocytes incubated with PA for 24 h. (c–d): SIRT3 gene expression in the liver of HFD-fed mice and PA-treated hepatocytes. (e): SIRT3 protein expression in primary hepatocytes in the presence of 10 mM pyruvate for 8 h. (f): Western blot examination of SIRT3 in precipitated MPC1 protein in hepatocytes incubated with pyruvate for 8 h. (g): Confocal image of mitochondrial MPC1 and SIRT3 colocalization in hepatocytes (Blue: MPC1; Green: SIRT3; Red: MitoTracker Red cMXRos. Scale bars: 5 μm), The view is one of five independent experiments. (h): Knockdown efficiency of three SIRT3 siRNAs transfected in HepG2 cells. (i): MPC1 protein expression in HepG2 with SIRT3 knockdown when cultured with pyruvate (10 mM) for 8 h. (j): Acetylation of MPC1 expression in HepG2 cells overexpressed with SIRT3 when cultured with pyruvate (10 mM) for 8 h. (BBR, berberine; Met, metformin; NAM, nicotinamide; PA, palmitate). Data were expressed as the mean ± SD (n = 5–6). ⁎p < 0.05 vs. HFD mice, PA or pyruvate-treated cells; #p < 0.05 vs. chow-fed diet mice or untreated cells.

Fig 4: Conditional deletion of Mpc1 in brown fat leads to increased ketogenesis.(a) Volcano plot showing changes in serum metabolites between Mpc1F/F and Mpc1F/F::Ucp1Cre mice housed at 4°C for 6 hr. N = 6. (b) Heat map of top 25 metabolites in BAT from Mpc1F/F and Mpc1F/F::Ucp1Cre mice housed at 4°C for 6 hr. Dendograms illustrate hierarchical clustering across metabolites (left) and genotypes (top). N = 6. Data was sum normalized, log transformed, and autoscaled. (c) Serum 3-hydroxybutyrate levels from Mpc1F/F and Mpc1F/F::Ucp1Cre mice housed at 23°C or 4°C for 6 hr. N = 6. (d) Gene expression analysis of livers from Mpc1F/F and Mpc1F/F::Ucp1Cre mice housed at 4°C for 6 hr. N = 6.

Fig 5: Histopathological changes and mitochondrial markers of eWATs in the MPC1± mice.(A) Representative HE-stained images of eWATs from 24-week-old WT and MPC1± mice, scale bars: 50 µm, n = 6; (B) Average adipocyte volume of eWATs in WT and MPC1± mice, n = 6; (C) The percentage of adipocyte volume in picture A (n = 100 adipocyte). The volume (diameters) was tested by the software installed in the pathological microscope (RM2500, Leica), 100 adipocytes in each mouse were tested. (D) Serum TG levels of TG in mice that underwent 5 h and 48 h fasting, n = 6; (E) Serum FFA levels of mice that underwent 5 h and 48 h fasting, n = 6; (F) Fold-changes in mtDNA levels in eWATs, n = 6; (G) The mRNA expression levels of Nrf1, Nrf2, and Tfam in the eWATs, n = 6. * P < 0.05, ** P < 0.01 for MPC1± mice vs. WT mice.