Fig 1.
LRPPRC promotes mitochondrially encoded gene expression in liver.
(A) Protein expression and (B) quantification in livers of wild-type littermate controls (WT), hemizygous (Tg/0), and double hemizygous (Tg/Tg) LRPPRC transgenic mice. (C) Protein expression in heart, spleen, brown adipose tissue (BAT) and liver in WT and Tg/Tg mice. (D) Protein expression in WT and Tg/Tg cultured primary hepatocytes. (E) Nuclear Lrpprc, mitochondrial polymerase (Polrmt), mitochondrial transcription factor A (Tfam), and mitochondrial transcription factor B2 (Tfb2m) and (F) mitochondrially encoded respiratory complex subunit gene expression in Tg/Tg and control livers. (G) Genetic determination of mitochondrially encoded DNA content in the samples. For all experiments, n = 3 or 4. Data are mean ± SEM * p<0.05, ** p<0.01, *** p<0.001 vs. WT by two-tailed unpaired Student’s t-test (B, E, G) or 2-way ANOVA with Bonferroni’s post-test (F).
Fig 2.
Liver-specific LRPPRC augments hepatic OXPHOS.
(A) Representative plot, (B) absolute and (C) relative changes in oxygen consumption rate (OCR) in isolated mitochondria from WT and LRPPRC Tg/Tg livers measured on a Clarke-type electrode. (D) FCCP induced maximal OCR vs. LRPPRC protein levels in isolated mitochondria from WT and Tg/Tg livers. (E) P:O ratio calculated from Clarke-electrode data. For all experiments, n = 3 or 4. Data are mean ± SEM (B—E), * p<0.05, ** p<0.01, *** p<0.001 by two-tailed unpaired Student’s t-test (B, C) or linear regression (D).
Fig 3.
Hepatic OXPHOS enhances whole body insulin sensitivity.
(A) Glucose and (B) insulin tolerance tests and area-under-the-curve (insets) and (C) weights in wild-type littermate control mice (WT), LRPPRC single hemizygous (Tg/0), and double hemizygous (Tg/Tg) mice fed a high-fat diet for 12 weeks, n = 10. (D) Hourly activity, (E) daily food intake, and (F) diurnal energy expenditure (7AM—7PM) in WT and Tg/Tg mice fed a high-fat diet > 20 weeks n = 3–6. (G) ER stress and (H) antioxidant gene expression in high-fat fed WT and Tg/Tg mouse liver, n = 9 or 10. (I) Mitochondrial superoxide production and (J) mitochondrial membrane potential (ΔΨm) in HepG2 cells ectopically expressing LacZ (control) or LRPPRC n = 24. (K) Immunoblot and quantification of phospho-AKT in cultured primary hepatocytes from chow-fed WT and Tg/Tg mice treated with 25nM insulin, n = 3. (L) Immunoblot and (M) quantification of membrane or (N) membrane: cytosolic PKCε in high-fat fed WT and Tg/Tg mouse livers, n = 5 or 6. Data are mean ± SEM, *p<0.05, **p<0.01, ***p<0.001 vs. WT by two-way ANOVA (A, B), two-tailed unpaired Student’s t-test (insets, F, H, J, K), mixed model with Bonferroni correction (C) or one-tailed unpaired Student’s t-test (M, N).
Fig 4.
Augmented hepatic OXPHOS interdicts NAFLD.
(A) Representative histological sections of livers from wild-type littermate control mice (WT) and LRPPRC double hemizygous (Tg/Tg) mice. (B) Pathologic classification of inflammation in livers of the same mice. (C) Expression of inflammatory genes in livers of WT and Tg/Tg mice fed a high-fat diet for 12 weeks. (D) Pathologic classification of steatosis in high-fat fed WT and Tg/Tg mouse livers. (E) Biochemical determination of triglyceride content in the same samples. (F) Determination of Fsp27 expression in high-fat fed WT and Tg/Tg livers. (G) Serum triglyceride content in the same mice. Expression of genes involved in (H) lipid export, (I) lipid uptake, (J) Lipogenesis, and (K) fatty acid β-oxidation in high-fat fed WT and Tg/Tg mouse livers. For all experiments, n = 9 or 10. Data are mean ± SEM *p<0.05, **p<0.01, ***p<0.001 vs. WT by chi-squared analysis (B, D), 2-way ANOVA with Bonferroni’s post-test (C), or by two-tailed, unpaired Student’s t-test (D-K).
Fig 5.
OXPHOS dictates NAD+, promoting complete oxidation of fatty acids (A) Fatty acid metabolic pathway in liver: NAD+ dependent β-oxidation catalyzes lipids to acetyl-CoA and ketone bodies (incomplete oxidation).
Acetyl-CoA is further metabolized via the NAD+ dependent TCA cycle, yielding CO2 (complete oxidation). NAD+ allosterically activates TCA cycling, while NADH inhibits this process. (B) NAD+ and (C) [NAD+]/[NADH] levels in chow fed wild-type littermate control mice (WT), LRPPRC single hemizygous (Tg/0), and double hemizygous (Tg/Tg) mice.(D) NAD+ synthetic gene expression in WT and Tg/Tg mice. (E) [NAD+]/[NADH] levels in cultured primary hepatocytes treated with 250 μM palmitic acid, 1 μM rotenone, or 4 μM FCCP. (F) NAD+ levels in nicotinamide (NAM) treated cultured primary hepatocytes. (G) Palmitate oxidation to CO2 in cultured primary hepatocytes treated with NAM. (H) Immunoblot of citrate synthase (CS) expression in cultured primary hepatocytes treated with NAM. (I) Palmitate oxidation in Sirt3 -/- (Sirt3 KO) primary hepatocytes treated with NAM. Palmitate oxidation in primary hepatocytes treated with (J) rotenone or (K) FCCP and NAM. For all experiments, n = 3 or 4 Data are mean ± SEM *p<0.05, **p<0.01, ***p<0.001 vs. vehicle unless otherwise indicated and ###p<0.001 by two-tailed unpaired Student’s t-test (B, C, E, G, I, J, K) or linear regression (F).
Fig 6.
Activation of hepatic OXPHOS increases cellular NAD+ and complete fatty acid oxidation, mitigating NAFLD.
In obesity and nutrient excess, increased ratio of reduced NADH to oxidized NAD+ inhibits complete oxidation of fatty acids in the liver, leading to lipid accumulation, PKCε activation, and insulin resistance. Augmented hepatic OXPHOS, through enhanced redox cycling and/or increased ATP dependent NAD+ synthesis, increases the ratio of [NAD+]/[NADH], thereby driving complete oxidation of fatty acids, reducing steatosis, and promoting insulin sensitivity. Additionally, enhanced OXPHOS reduces reactive oxygen species, which may contribute to increased insulin sensitivity (not shown).