Fig 2: Hepatic Mpc1 Deletion Decreases Hepatocellular Carcinoma Development(A) Body weight of WT and MPC LivKO mice over course of CCl4 treatment; n = 24–29 biological replicates.(B) Liver tumor burden at euthanasia, determined by grossly dissecting each liver lobe and examining for and counting visible tumors; please see STAR Methods section for additional detail; n = 24–29 biological replicatesC) Liver mass at euthanasia, determined by removing and weighing whole livers; n = 24–29 biological replicates.(D) Average tumor size, measured by calipers; n = 152 WT and 54 MPC LivKO tumors measured.(E) Calculated liver-to-body weight ratio for WT and MPC LivKO mice; n = 24–29 biological replicates.(F) Mpc1 and Mpc2 gene expression by qPCR in paired normal-adjacent non-tumor tissue (NT) and tumor tissue (T) in WT and MPC LivKO samples; n = 4 biological replicates per condition per genotype analyzed by one-way ANOVA.(G) Representative western blot of Mpc1 and Mpc2 expression in normal-adjacent non-tumor liver tissue of WT and MPC LivKO mice. VDAC loading control.(H) Representative liver images.(I) 8-OHdG ELISA on day 15 saline-injected (CTRL) or DEN-injected (DEN) pups; n = 4–11 biological replicates.(J) AST and ALT measured in plasma taken from WT and MPC LivKO mice after 9 weeks of CCl4 injections (n = 5, biological replicates) and prior to euthanasia; n = 10 biological replicates.(K) Ki67 staining of WT and MPC LivKO tumors; scale bar, 50 μm; n = 5–6 biological replicates.(L) Cleaved caspase-3 staining of WT and MPC LivKO tumors; scale bar, 50 μm; n = 5–6 biological replicates.Data are presented as mean ± SEM, compared by t test unless otherwise noted (*p < 0.05, **p < 0.01, ***p < 0.001). See also Figures S1, S2, and S3.

Fig 3: (A) Mitochondrial pyruvate carrier (MPC) bioluminescence resonance energy transfer (BRET) assay is activated by 5 mM pyruvate or known MPC inhibitors (5 μM UK-5099 or 10 μM MSDC-0602K) compared to dimethyl sulfoxide (DMSO) vehicle controls. Mutation of MPC1-F66, MPC2-K49, or MPC2-N100 to alanine mostly abolishes the ability of pyruvate or inhibitors to increase BRET, suggesting mutation of these residues decreases binding and MPC conformational change. (B) Western blot images of U2OS cells transfected with WT or mutant MPC constructs indicates mutants are well expressed. Data presented as mean ± S.E.M. Analyzed by two-way ANOVA with Tukey’s correction for multiple comparisons (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; with black symbols for treatment comparisons and grey for genotype comparisons).

Fig 4: OPA1 ASOs increase levels of MPC2 and SDH subunits in megamitochondria-associated NASH(A and B) Levels of serum triglyceride (A) and cholesterol (B). Bars are average ± SD (n = seven to eight mice).(C) Heatmap summary of real-time qPCR analysis. Heatmap representing fold change of mRNA levels relative to those in mice that were fed with the standard diet and treated with non-targeting ASOs (Table S1).(D) Western blotting of livers using the indicated antibodies was performed.(E) Band intensity was quantified. Bars are average ± SD (n = 3 mice).(F) Summary of the data. Protein levels of MPC2, SDHA, SDHB, and SDHC were lowered in the MCD model. OPA1 ASOs restored the level of MPC2, SDHA, and SDHB, and surpassed the original level of SDHC. Statistical analysis was performed using one-way ANOVA with post-hoc Dunnett’s test in (A, B, and E; MCD + NT and other groups): ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Fig 5: Generation of mice with muscle-specific deletion of Mpc1 (MPC SkmKO).(A) Scheme illustrating generation of the muscle-specific Mpc1 null allele. (B) Representative western blots of MPC1 and MPC2 protein abundance in mouse tissues. Loading was normalized to total protein. A reference protein is not shown because of lack of an equally expressed protein across different tissues (age 21 weeks; n = 3, littermates; TA, tibialis anterior; BAT, brown adipose tissue; WAT, white adipose tissue). (C) 14C-pyruvate uptake by muscle mitochondria isolated from WT and MPC SkmKO mice (age 13 weeks, n = 6, four littermates + 2 non-littermates, two-tailed t-test). (D, E) Pyruvate- (D) and glutamate-driven (E) respiration by muscle mitochondria isolated from WT and MPC SkmKO mice. Experimental media contained 1 mM malate and 10 mM pyruvate or 10 mM glutamate (age 16 weeks; n = 6, four littermates + 2 non-littermates; two-tailed t-test; FCCP, trifluoromethoxy carbonylcyanide phenylhydrazone; CHC, 4-alpha-hydroxycinnamatic acid; Rot., rotenone). (F) Representative western blots of components of electron transport chain (ETC) complexes I-V, MPC1, VDAC, and HSP90 proteins in TA muscle from WT and MPC SkmKO mice (age 21 weeks, n = 6, littermates). Data presented as mean ± SEM (*p<0.05, **p<0.01).