Mitochondrial metabolism mediates oxidative stress and inflammation in fatty liver

Abstract

Mitochondria are critical for respiration in all tissues; however, in liver, these organelles also accommodate high-capacity anaplerotic/cataplerotic pathways that are essential to gluconeogenesis and other biosynthetic activities. During nonalcoholic fatty liver disease (NAFLD), mitochondria also produce ROS that damage hepatocytes, trigger inflammation, and contribute to insulin resistance. Here, we provide several lines of evidence indicating that induction of biosynthesis through hepatic anaplerotic/cataplerotic pathways is energetically backed by elevated oxidative metabolism and hence contributes to oxidative stress and inflammation during NAFLD. First, in murine livers, elevation of fatty acid delivery not only induced oxidative metabolism, but also amplified anaplerosis/cataplerosis and caused a proportional rise in oxidative stress and inflammation. Second, loss of anaplerosis/cataplerosis via genetic knockdown of phosphoenolpyruvate carboxykinase 1 (Pck1) prevented fatty acid-induced rise in oxidative flux, oxidative stress, and inflammation. Flux appeared to be regulated by redox state, energy charge, and metabolite concentration, which may also amplify antioxidant pathways. Third, preventing elevated oxidative metabolism with metformin also normalized hepatic anaplerosis/cataplerosis and reduced markers of inflammation. Finally, independent histological grades in human NAFLD biopsies were proportional to oxidative flux. Thus, hepatic oxidative stress and inflammation are associated with elevated oxidative metabolism during an obesogenic diet, and this link may be provoked by increased work through anabolic pathways.

Figure 8. Histological data support a role for oxidative metabolism in NAFLD in humans.

(A) Liver biopsies obtained from 8 individuals with suspected NAFLD were given NAS and Ishak scores. These scores correlated with oxygen consumption calculated from previously reported fluxes (7). (B) The data support a role for oxidative metabolism in facilitating increased anaplerotic work, collateral oxidative stress, and inflammation during NAFLD and hepatic insulin resistance. Correlations were detected by 1-tailed Pearson tests.

Figure 7. Metformin suppressed oxidative metabolism and anaplerosis and lowered inflammation in livers of mice on a HFD.

Mice were treated with metformin during the last 4 weeks of a 16-week HFD. Metformin treatment reduced (A) GNG by suppressing (B) anaplerosis. Oxidative metabolism was suppressed as indicated by reduced (C) TCA cycle flux and (D) calculated oxygen consumption. Markers of inflammation (E) Il6 and (F) Tnfa were reduced in proportion to oxygen consumption. Data are shown as mean ± SEM (n = 4–5). Statistical differences were detected by 2-tailed t test. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 6. Preventing the induction of anaplerosis/cataplerosis protected against hepatic oxidative stress and inflammation during a HFD.

Oxidative stress, indicated by (A) a qPCR gene array, (B) histological DHE ROS staining, and (C) lipid peroxidation (n = 6), was increased by a HFD in WT, but not knockdown mice. TBARS, thiobarbituric acid reactive substances. (D) The Q/QH₂ ratio, estimated from the fumarate/succinate ratio, was oxidized in knockdown mice (n = 6–8). (E) The calculated free energy of complexes I and II was more negative in knockdown mice (n = 6–8). (F) The NADP⁺/NADPH ratio, estimated from the pyruvate/malate ratio, was reduced in knockdown mice (n = 6–8). (G) TCA cycle intermediates with antioxidant properties were increased in knockdown mice (n = 3–4) as were (H) antioxidant genes (n = 6). Knockdown mice were protected from the inflammatory response of a HFD as indicated by (I) fewer inflammatory infiltrates in H&E-stained tissue (n = 6–8) and lower expression of (J) Tnfa and (K) Il6 mRNA (n = 6). (L) NF-κB S536 phosphorylation was reduced in knockdown liver (n = 5–6). Original magnification, ×10 (I); ×20 (B). Data are shown as mean ± SEM. Statistical differences were detected by 2-way ANOVA (J and K), 2-tailed t test (C–H), or 1-tailed t test (I and L). *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 5. Preventing the induction of anaplerosis/cataplerosis during a HFD prevented the rise in oxidative flux through metabolic mechanisms.

(A) TCA cycle flux measured by isotopomer analysis of plasma glucose remained normal in knockdown mice during a HFD. (B) Ketogenesis measured by apparent ketone turnover. (C) Calculated oxygen consumption increased in WT mice, but not knockdown mice, on a HFD (n = 4–9 for A–C). (D) ATP, ADP, and AMP measured by LC-MS. (E) Expression of genes related to oxidative metabolism was normal or elevated in knockdown mice. (F) Mitochondrial NAD⁺/NADH measured by plasma acetoacetate/β-hydroxybutyrate ratio was reduced in liver of knockdown mice. (G) Cytosolic NAD⁺/NADH measured by pyruvate/lactate ratio was reduced in liver of knockdown mice. (H) Hepatic citrate, succinate, and OAA were elevated in knockdown liver (n = 6–9 for D–H). Data are shown as mean ± SEM. Statistical differences were detected by 2-way ANOVA (A–C and E) and 2-tailed t test (D and F–H). *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 4. Preventing the induction of anaplerosis/cataplerosis during a HFD protected against elevated GNG and hepatic insulin resistance.

(A) Pck1 mRNA in overnight-fasted WT, Pck1 knockdown (Pck1^{lox+neo/lox+neo}), and liver-specific knockout mice (Pck1^lox/lox Alb-Cre) (n = 8–12). (B) Anaplerosis, (C) endogenous glucose production, and (D) GNG remained normal in knockdown mice on a HFD (n = 4–9). (E) Gluconeogenic genes were decreased in knockdown mice on a HFD (n = 4–5). (F) The rate of glucose disposal during a hyperinsulinemic-euglycemic clamp (n = 4–8) was reduced by a HFD in WT and knockdown mice. (G) Knockdown mice on a HFD maintained normal suppression of hepatic glucose production during the clamp. (H) Western blot analysis of p-Akt/Akt ratios in liver before (basal) and 3 minutes after portal insulin injection (+insulin) and (I) fold induction of p-Akt/Akt by insulin (n = 4). Ctrl, control. Data are shown as mean ± SEM. Statistical differences were detected by 2-way ANOVA (A–D, F, and G), 2-tailed t test (E), and 1-tailed paired t test (H and I). *P < 0.05; **P < 0.01.

Figure 3. The induction of oxidative metabolism by NEFA requires increased anaplerosis/cataplerosis to cause oxidative stress and inflammation.

Increasing circulating NEFA by intralipid infusion (n = 4–6) caused a rise in fat oxidation indicated by a rise in (A) ketogenesis and TCA cycle flux and (B) GNG and anaplerosis measured by tracer methods. (C) Calculated oxygen consumption was increased by intralipid infusion in proportion to the rise in circulating NEFA. Oxidative stress, as indicated by (D) Sod2 mRNA and (E) lipid peroxidation, increased in proportion to the rise in oxygen consumption. The inflammatory response, as indicated by (F) Tnfa and (G) Il6 mRNA, was elevated by NEFA in proportion to the rise in oxygen consumption. (H) Administration of an inhibitor of PEPCK, 100 μM mercaptopicolinate, blocked the induction of ROS in H4IIE rat hepatoma cells treated with high NEFA (n = 3). Data are shown as mean ± SEM. Statistical differences were detected by 2-tailed t tests, except for H, which used a 2-way ANOVA. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 2. Oxidative metabolism is linked to anaplerosis and GNG in liver.

(A) Oxygen consumption in fed, fasted, or octanoate-perfused mouse liver correlated with oxygen consumption calculated from isotopomer analysis using Equations 1 and 2 (see Methods). (B) Fasting oxygen consumption increased in proportion to anaplerosis (n = 4 × 3 repeated measures). A separate group of livers from fed mice (n = 4) was perfused with either low or high NEFA (0.2 mM or 0.8 mM). High NEFA increased oxidative metabolism measured by (C) ketone output and TCA cycle flux. High NEFA also increased (D) anaplerosis and GNG. (E) Addition of insulin suppressed glycogenolysis, but not anaplerosis or GNG regardless of NEFA concentration (n = 3–4). Data are shown as mean ± SEM. Statistical differences were detected by a 2-tailed t test. *P < 0.05; ^#P < 0.05 versus insulin perfusion.

Figure 1. Propionate tracers do not perturb basal hepatic flux.

Propionate (0.8 μmol•min^–1) did not alter (A) glucose production or (B) O₂ consumption in livers perfused with gluconeogenic substrates and NEFA (n = 4). Propionate infusion (0.5 μmol•min^–1) into conscious and unrestrained mice did not alter (C) endogenous glucose production (n = 3) and (D) resulted in tracer level (<4%) glucose enrichment (n = 4–8). (E) Isotopomers of glucose formed by [U-¹³C]lactate/pyruvate during liver perfusion and reported in the ¹³C NMR spectrum of glucose C2 were not altered by the addition of propionate (n = 3). Glucose isotopomers in carbons 1–3 (black circles are ¹³C) that contribute to the NMR signal are indicated above the corresponding signal. (F) Modeling the effect of incomplete OAA/fumarate equilibration in the TCA cycle predicted that [U-¹³C]lactate/pyruvate (white circles) would underestimate pyruvate cycling (PK+ME) and overestimate GNG more severely than [U-¹³C]propionate (gray circles). The highlighted area around 80% to 85% is the experimentally expected degree of randomization (3, 19). (G) Relative fluxes reported by [U-¹³C]lactate/pyruvate underestimated pyruvate cycling and overestimated GNG relative to [U-¹³C]propionate when simple equations (64) were used (left panel), but gave identical values when randomization was fit using a regression model (right panel) (n = 3–4). Data are shown as mean ± SEM. Statistical differences were detected by 2-tailed t test. *P < 0.05; **P < 0.001.