Hepatic Mitochondrial Oxidative Capacity Research Articles

582-P: Exercise Training Reverses NAFLD in Mice with Obesity Independent of Changes in Hepatic Mitochondrial Oxidative Capacity

Obesity is a primary cause of hepatic lipid accumulation, leading to nonalcoholic fatty liver disease (NAFLD). As there are no FDA-approved therapies for NAFLD treatment, weight loss through lifestyle intervention is the first-line therapy. Exercise improves hepatic function in patients with obesity related NAFLD. However, the mechanisms whereby exercise mediates the enhancement of liver function are largely unknown. This study evaluated the impact of exercise training on NAFLD in mice with obesity. At 8 weeks of age, mice were randomized to low-fat (LFD) or high-fat diet (HFD). After 4 weeks of dietary intervention, mice were randomized to either control (SED) or exercise training (ExT) with experimental diet for 10 weeks. ExT mice completed 45 minutes of moderate-intensity treadmill running 4 days/week. Body weight was measured weekly, and body composition and maximal treadmill running were evaluated at weeks 0, 4, and 14. Blood lactate accumulation was measured during the final treadmill test. Mice were euthanized 48 hours after the last exercise session for liver histopathology, blood chemistry, and ex vivo mitochondrial function analysis. Liver function was assessed via serum liver panel and quantification of hepatic lipids. High-resolution respirometry was used to determine maximal hepatic fatty acid oxidation, NADH- and succinate-linked oxidative phosphorylation (OXPHOS), and electron transfer capacity in tissue homogenates. HFD increased body mass, which was reduced with ExT. ExT increased maximal running time independent of diet. HFD worsened lactate metabolism, which was improved with ExT. HFD increased liver weight, which was decreased with ExT. HFD-induced increases in hepatic lipid and serum ALT and ALP were reversed with ExT. Hepatic mitochondrial OXPHOS and electron transfer were reduced with HFD relative to LFD. These data suggest that exercise-induced reversal of NAFLD is not mediated by hepatic mitochondrial oxidative capacity. Disclosure E.C. Heintz: None. W.S. Dantas: None. J.E. Stampley: None. G.M. Davis: None. B.A. Irving: None. C.L. Axelrod: None. J.P. Kirwan: None. Funding Nutrition Obesity Research Center (P30DK072476); Louisiana Clinical and Translational Science Center (U54GM104940); Centers of Biomedical Research Excellence (P20GM103528)

Maternal obesity in sheep impairs foetal hepatic mitochondrial respiratory chain capacity

Changes in the nutritional environment in utero induced by maternal obesity (MO) lead to foetal metabolic dysfunction predisposing offspring to later-life metabolic diseases. Since mitochondria play a crucial role in hepatic metabolism and function, we hypothesized that MO prior to conception and throughout pregnancy programmes foetal sheep liver mitochondrial phenotype. Ewes ate an obesogenic diet (150% requirements; MO), or 100% requirements (CTR), from 60days prior to conception. Foetal livers were removed at 0.9 gestation. We measured foetal liver mitochondrial DNA copy number, activity of superoxide dismutase, cathepsins B and D and selected protein content, total phospholipids and cardiolipin and activity of mitochondrial respiratory chain complexes. A significant decrease in activities of mitochondrial complexes I, II-III and IV, but not aconitase, was observed in MO. In the antioxidant machinery, there was a significant increase in activity of total superoxide dismutase (SOD) and SOD2 in MO. However, no differences were found regarding autophagy-related protein content (p62, beclin-I, LC3-I, LC3-II and Lamp2A) and cathepsin B and D activities. A 21.5% decrease in total mitochondrial phospholipid was observed in MO. The data indicate that MO impairs foetal hepatic mitochondrial oxidative capacity and affects total mitochondrial phospholipid content. In addition, MO affects the regulation of foetal liver redox pathways, indicating metabolic adaptations to the higher foetal lipid environment. Consequences of in utero programming of foetal hepatic metabolism may persist and compromise mitochondrial bioenergetics in later life, and increase susceptibility to metabolic diseases.

Aerobic capacity and hepatic mitochondrial lipid oxidation alters susceptibility for chronic high-fat diet-induced hepatic steatosis.

Rats selectively bred for high capacity running (HCR) or low capacity running (LCR) display divergence for intrinsic aerobic capacity and hepatic mitochondrial oxidative capacity, both factors associated with susceptibility for nonalcoholic fatty liver disease. Here, we tested if HCR and LCR rats display differences in susceptibility for hepatic steatosis after 16 wk of high-fat diets (HFD) with either 45% or 60% of kcals from fat. HCR rats were protected against HFD-induced hepatic steatosis, whereas only the 60% HFD induced steatosis in LCR rats, as marked by a doubling of liver triglycerides. Hepatic complete fatty acid oxidation (FAO) and mitochondrial respiratory capacity were all lower in LCR compared with HCR rats. LCR rats also displayed lower hepatic complete and incomplete FAO in the presence of etomoxir, suggesting a reduced role for noncarnitine palmitoyltransferase-1-mediated lipid catabolism in LCR versus HCR rats. Hepatic complete FAO and mitochondrial respiration were largely unaffected by either chronic HFD; however, 60% HFD feeding markedly reduced 2-pyruvate oxidation, a marker of tricarboxylic acid (TCA) cycle flux, and mitochondrial complete FAO only in LCR rats. LCR rats displayed lower levels of hepatic long-chain acylcarnitines than HCR rats but maintained similar levels of hepatic acetyl-carnitine levels, further supporting lower rates of β-oxidation, and TCA cycle flux in LCR than HCR rats. Finally, only LCR rats displayed early reductions in TCA cycle genes after the acute initiation of a HFD. In conclusion, intrinsically high aerobic capacity confers protection against HFD-induced hepatic steatosis through elevated hepatic mitochondrial oxidative capacity.

Hepatic mTORC1 Opposes Impaired Insulin Action to Control Mitochondrial Metabolism in Obesity

Dysregulated mitochondrial metabolism during hepatic insulin resistance may contribute to pathophysiologies ranging from elevated glucose production to hepatocellular oxidative stress and inflammation. Given that obesity impairs insulin action but paradoxically activates mTORC1, we tested whether insulin action and mammalian target of rapamycin complex 1 (mTORC1) contribute to altered invivo hepatic mitochondrial metabolism. Loss of hepatic insulin action for 2weeks caused increased gluconeogenesis, mitochondrial anaplerosis, tricarboxylic acid (TCA) cycle oxidation, and ketogenesis. However, activation of mTORC1, induced by the loss of hepatic Tsc1, suppressed these fluxes. Only glycogen synthesis was impaired by both loss of insulin receptor and mTORC1 activation. Mice with a double knockout of the insulin receptor and Tsc1 had larger livers, hyperglycemia, severely impaired glycogen storage, and suppressed ketogenesis, as compared to those with loss of the liver insulin receptor alone. Thus, activation of hepatic mTORC1 opposes the catabolic effects of impaired insulin action under some nutritional states.

Mitochondrial energetics in liver and skeletal muscle after energy restriction in young rats

The present study investigated the effect of 2 weeks of energy restriction on whole body, liver and skeletal muscle energy handling. We measured whole-body oxygen consumption, as well as mitochondrial protein mass, respiratory capacity and energetic coupling in liver and skeletal muscle from food-restricted (FR) rats, age- and weight-matched controls. We also assessed markers of oxidative damage and antioxidant defences. The present results show that, in response to energy restriction, an adaptive decrease in whole-body energy expenditure is coupled with structural and functional changes in mitochondrial compartment, both in liver and skeletal muscle. In fact, liver mitochondrial mass per g of liver significantly increased, whereas total hepatic mitochondrial oxidative capacity was lower in FR than in control rats, because of a significant decrease in liver contribution to total body weight. In skeletal muscle, sub-sarcolemmal (SS) mitochondrial respiratory capacity, as well as SS and inter-myofibrillar (IMF) mitochondrial protein mass per g of tissue, was significantly lower in FR rats, compared to controls. Finally, a decrease in oxidative damage was found in liver but not in skeletal muscle mitochondria from FR rats, whereas an increase in antioxidant defence was found in both tissues. From the present results, it appears that skeletal muscle is involved in the decrease in energy expenditure induced by energy restriction. Energy sparing is achieved through changes in the activity (SS), mass (SS and IMF) and efficiency (IMF) of mitochondrial compartment.

The muscle–liver axis: does aerobic fitness induce intrahepatic protection against non‐alcoholic fatty liver disease?

Hepatic steatosis often occurs in the context of insulin resistance, dyslipidaemia, type 2 diabetes mellitus and obesity. It is associated with a risk for development of more severe forms of non-alcoholic fatty liver disease (NAFLD), including steatohepatitis, fibrosis and cirrhosis. The amount of triglycerides in an intrinsically normal liver is not fixed, but can readily be modified by nutritional conditions. For instance, short term fasting increases hepatic triglyceride content (through increased release of fatty acids from the adipose tissue). Conversely, prolonged caloric restriction reduces liver fat content. In addition, many other (patho)physiological factors can modulate hepatic triglyceride storage, including alcohol, drugs and dietary composition. Importantly, the propensity to develop steatosis in a certain (patho)physiological state may be determined by genes and/or physical fitness. In this issue of The Journal of Physiology, Thyfault et al. (2009) report the results of a careful study on the biochemical and molecular differences in the livers of rats, which were selectively bred to robustly differ with respect to their intrinsic aerobic capacity. They show that sedentary rats with genetically determined low aerobic fitness develop steatosis and more severe hepatic fibrosis at natural death compared to sedentary rats with intrinsically high aerobic capacity. They also demonstrate that low aerobic capacity is linked to decreased mitochondrial content and mitochondrial oxidative capacity in liver. Isolated hepatic mitochondria from high capacity runners oxidized palmitate more completely (to CO2) and in greater amounts, whereas markers of hepatic peroxisomal activity, acyl CoA oxidase and catalase, were decreased in these animals. A key transcriptional factor for fatty acid synthesis, SREBP-1c, was lower in livers of high capacity runners. These remarkable observations require careful interpretation. The study design has some limitations. In particular, the two groups of rats differed not only with respect to their aerobic capacity, but also in terms of other factors that clearly affect hepatic triglyceride content. For instance, the rats with low aerobic capacity were 42% heavier and the adipocyte cell volume of their omental fat pad was 50% larger. Moreover, they were hyperinsulinaemic, hypertriglyceridaemic, and considerably less active than the high capacity runners. The authors postulate that reduced mitochondrial oxidation in hepatocytes of rats with low aerobic capacity is directly responsible for their propensity to store triglycerides in liver. However, it is conceivable that reduced muscle mitochondrial content, a putative determinant of insulin resistance, hyperinsulinaemia and obesity, indirectly caused hepatic triglyceride accumulation in these animals. The design of the current study does not allow precise conclusions as to which of these potential pathophysiological factors underlies their findings. It is likely that different mechanistic routes contribute. Indeed, the liver may not be merely an innocent bystander, passively adapting to the changes in fatty acid metabolism associated with muscle fitness. Rather, the results raise the possibility that there are active intrahepatic biochemical and molecular mechanisms associated with aerobic fitness that affect hepatic fatty acid metabolism, and, as a consequence, liver triglyceride content. This concept is also supported by a previous study of the authors, which documented that sudden cessation of daily physical activity in hyperphagic/obese rats resulted in stimulation of biochemical pathways known to initiate hepatic steatosis, including decreased hepatic mitochondrial oxidative capacity, increased hepatic expression of de novo lipogenesis proteins, and increased hepatic malonyl CoA levels (Rector et al. 2008). This perspective contrasts with the concept of the liver acting as an innocent bystander, passively storing fatty acids in the form of triglycerides. Rather, aerobic fitness may not be reflected by adaptations of skeletal muscle only, but also involve the liver (and perhaps other tissues as well). The findings reported by Thyfault and colleagues warrant further investigation of the mechanistic underpinnings of this putative muscle–liver axis, because deeper insight may open new alleys for the treatment of non-alcoholic fatty liver disease.

Rats selectively bred for low aerobic capacity have reduced hepatic mitochondrial oxidative capacity and susceptibility to hepatic steatosis and injury

Fatty liver has been linked to low aerobic fitness, but the mechanisms are unknown. We previously reported a novel model in which rats were artificially selected to be high capacity runners (HCR) and low capacity runners (LCR) that in a sedentary condition have robustly different intrinsic aerobic capacities. We utilized sedentary HCR/LCR rats (generation 17; max running distance equalled 1514 +/- 91 vs. 200 +/- 12 m for HCR and LCR, respectively) to investigate if low aerobic capacity is associated with reduced hepatic mitochondrial oxidative capacity and increased susceptibility to hepatic steatosis. At 25 weeks of age, LCR livers displayed reduced mitochondrial content (reduced citrate synthase activity and cytochrome c protein) and reduced oxidative capacity (complete palmitate oxidation in hepatic mitochondria (1.15 +/- 0.13 vs. 2.48 +/- 1.1 nm g(-1) h, P < 0.0001) and increased peroxisomal activity (acyl CoA oxidase and catalase activity) compared to the HCR. The LCR livers also displayed a lipogenic phenotype with higher protein content of both sterol regulatory element binding protein-1c and acetyl CoA carboxylase. These differences were associated with hepatic steatosis in the LCR including higher liver triglycerides (6.00 +/- 0.71 vs. 4.20 +/- 0.39 nmol g(-1), P = 0.020 value), >2-fold higher percentage of hepatocytes associated with lipid droplets (54.0 +/- 9.2 vs. 22.0 +/- 3.5%, P = 0.006), and increased hepatic lipid peroxidation compared to the HCR. Additionally, in rats aged to natural death, LCR livers had significantly greater hepatic injury (fibrosis and apoptosis). We provide novel evidence that selection for low intrinsic aerobic capacity causes reduced hepatic mitochondrial oxidative capacity that increases susceptibility to both hepatic steatosis and liver injury.

Cessation of daily exercise dramatically alters precursors of hepatic steatosis in Otsuka Long‐Evans Tokushima Fatty (OLETF) rats

The purpose of this study was to delineate potential mechanisms initiating the onset of hepatic steatosis following the cessation of daily physical activity. Four-week-old, hyperphagic/obese Otsuka Long-Evans Tokushima Fatty rats were given access to voluntary running wheels for 16 weeks to prevent the development of hepatic steatosis. The animals were then suddenly transitioned to a sedentary condition as wheels were locked (wheel lock; WL) for 5 h (WL5), 53 h (WL53) or 173 h (WL173). Importantly after the cessation of daily exercise (5-173 h), no changes occurred in body weight, fat pad mass (omental and retroperitoneal), food intake, serum insulin, hepatic triglycerides or in the exercise-suppressed hepatic stearoyl-CoA desaturase-1 and peroxisome proliferator-activated receptor-gamma protein content. However, complete hepatic fatty acid oxidation and mitochondrial enzyme activities were highest at WL5 and WL53 and dropped significantly to SED levels by WL173. In addition, cessation of daily exercise quickly increased the hepatic protein contents of fatty acid synthase and acetyl-coenzyme A carboxylase (ACC), reduced ACC phosphorylation status, and dramatically increased hepatic malonyl-CoA concentration. This study is the first to show that the sudden cessation of daily exercise in a hyperphagic/obese model activates a subgroup of precursors and processes known to initiate hepatic steatosis, including decreased hepatic mitochondrial oxidative capacity, increased hepatic expression of de novo lipogenesis proteins, and increased hepatic malonyl CoA levels; each probably increasing the susceptibility to non-alcoholic fatty liver disease.

It's all in the genes, so pick your parents wisely

Aerobic exercise capacity is a complex phenotype influenced by a myriad of environmental and genetic factors. At one end of the continuum, elite endurance athletes represent a rare union of genetic potential that, given appropriate environmental stimulus (e.g., training, nutrition) can culminate in

Effect of cold exposure on energy balance and liver respiratory capacity in post-weaning rats fed a high-fat diet.

Variations in energy balance, body composition, and nutrient partitioning induced by high-fat feeding, cold exposure or by concomitant high-fat feeding and cold exposure were studied in young Wistar rats. Changes in hepatic metabolism as well as in serum free triiodothyronine and leptin levels were also evaluated. Rats were exposed to either 24 or 4 degrees C and fed either a low- or high-fat diet (10 % or 50 % energy respectively) for 2 weeks. Relative to low-fat feeding at 24 degrees C, both energy intake and expenditure were increased by high-fat feeding or by cold exposure, and these changes were accompanied by increased serum triiodothyronine levels. In response to concomitant high-fat feeding and cold exposure, serum triiodothyronine tended to be further elevated, but no further increases in energy intake or energy expenditure were observed. Independently of diet, the increased energy expenditure in cold-exposed rats was not completely balanced by adaptive hyperphagia, with consequential reductions in protein and fat gain, accompanied by marked decreases in serum leptin. Furthermore, unlike high-fat feeding at 24 degrees C, cold exposure enhanced hepatic mitochondrial oxidative capacity both in the low-fat- and high-fat-fed groups. It is concluded that in this strain of young Wistar rats, despite similarly marked stimulation of energy expenditure by high-fat feeding at 24 degrees C, by cold exposure and by concomitant high-fat feeding and cold exposure, an increased hepatic oxidative capacity occurred only in the presence of the cold stimulus.

Hepatic Mitochondrial Gene Expression is Stimulated by Factors Other Than Oxygen at Birth 275

The liver is an important organ in the transition from fetal to neonatal life. A significant component of this transition is an increase in hepatic mitochondrial oxidative capacity. Previous studies by Jacobs et al report an elevation in site I mitochondrial oxygen consumption after birth in rabbit hepatic mitochondria. This increase is blunted by 4 hours of neonatal hypoxia(10% FiO2). Site I oxygen consumption occurs when electrons enter the respiratory chain at complex I. We hypothesized that gene expression of NADH dehydrogenase subunit 2 (ND2) (a component of complex I encoded in the mitochondrial genome) and the 39 kD protein of complex I (encoded in the nuclear genome) would increase after birth and be altered by neonatal hypoxia. Rabbit pups were delivered by Csection at term into a 10% inspired O2 chamber. Pups were sacrificed and liver harvested either at birth (B0) or after maintenence for four hours of either normoxia (N4) (21% FiO2) or hypoxia (H4)(10%FiO2). To measure mRNA levels, we used RTPCR with an internal control. PCR products were quantified and standardized. mRNA levels of ND2 increased by 50% in the N4 animals compared to the B0 animals (B0=1.0, N4ND2=1.54±0.12*, N439 kD=1.35±0.11; *p < 0.05). We conclude that hepatic gene expression of ND2 of complex I increases with age in the normoxic rabbit pup. Interestingly, gene expression of ND2 and 39kD protein also increased in the H4 group when compared to the N4 animals (N4=1.0, H4ND2=1.81±0.22*, H439kD=1.53±0.06*; *p < 0.05). This result is intriguing because oxygen has been traditionally presumed to be the primary positive regulator of mitochondrial gene expression. This study suggests that other factors may also significantly alter mitochondrial gene expression. We speculate that this increased gene expression may be important upon reperfusion, and thereby leading to increased free radical production and enhance the injury associated with hypoxia.