R. Scott Rector

Mitochondrial dysfunction precedes insulin resistance and hepatic steatosis and contributes to the natural history of non-alcoholic fatty liver disease in an obese rodent model

BACKGROUND & AIMS In this study, we sought to determine the temporal relationship between hepatic mitochondrial dysfunction, hepatic steatosis and insulin resistance, and to examine their potential role in the natural progression of non-alcoholic fatty liver disease (NAFLD) utilising a sedentary, hyperphagic, obese, Otsuka Long-Evans Tokushima Fatty (OLETF) rat model. METHODS OLETF rats and their non-hyperphagic control Long-Evans Tokushima Otsuka (LETO) rats were sacrificed at 5, 8, 13, 20, and 40 weeks of age (n=6-8 per group). RESULTS At 5 weeks of age, serum insulin and glucose and hepatic triglyceride (TG) concentrations did not differ between animal groups; however, OLETF animals displayed significant (p<0.01) hepatic mitochondrial dysfunction as measured by reduced hepatic carnitine palmitoyl-CoA transferase-1 activity, fatty acid oxidation, and cytochrome c protein content compared with LETO rats. Hepatic TG levels were significantly elevated by 8 weeks of age, and insulin resistance developed by 13 weeks in the OLETF rats. NAFLD progressively worsened to include hepatocyte ballooning, perivenular fibrosis, 2.5-fold increase in serum ALT, hepatic mitochondrial ultrastructural abnormalities, and increased hepatic oxidative stress in the OLETF animals at later ages. Measures of hepatic mitochondrial content and function including beta-hydroxyacyl-CoA dehydrogenase activity, citrate synthase activity, and immunofluorescence staining for mitochondrial carbamoyl phosphate synthetase-1, progressively worsened and were significantly reduced at 40 weeks in OLETF rats compared to LETO animals. CONCLUSIONS Our study documents that hepatic mitochondrial dysfunction precedes the development of NAFLD and insulin resistance in the OLETF rats. This evidence suggests that progressive mitochondrial dysfunction contributes to the natural history of obesity-associated NAFLD.

Daily exercise increases hepatic fatty acid oxidation and prevents steatosis in Otsuka Long-Evans Tokushima Fatty rats

Exercise training is commonly prescribed for treatment of nonalcoholic fatty liver disease (NAFLD). We sought to determine whether exercise training prevents the development of NAFLD in Otsuka Long-Evans Tokushima Fatty (OLETF) rats and to elucidate the molecular mechanisms underlying the effects of exercise on hepatic steatosis. Four-week-old OLETF rats were randomly assigned to either a sedentary control group (Sed) or a group given access to voluntary running wheels for 16 wk (Ex). Wheels were locked 2 days before euthanasia in the Ex animals, and both groups were euthanized at 20 wk old. Voluntary wheel running attenuated weight gain and reduced serum glucose, insulin, free fatty acids, and triglycerides in Ex animals compared with Sed (P < 0.001). Ex animals exhibited significantly reduced hepatic triglyceride levels and displayed fewer lipid droplets (Oil Red O staining) and reduced lipid droplet size compared with Sed. Wheel running increased by threefold the percent of palmitate oxidized completely to CO(2) in the Ex animals but did not alter AMP-activated protein kinase-alpha (AMPKalpha) or AMPK phosphorylation status. However, fatty acid synthase and acetyl-coenzyme A carboxylase (ACC) content were significantly reduced (approximately 70 and approximately 35%, respectively), and ACC phosphorylation and cytochrome c content were significantly elevated (approximately 35 and approximately 30%, respectively) in the Ex animals. These results unequivocally demonstrate that daily physical activity attenuates hepatic steatosis and NAFLD in an obese rodent model and suggest that this effect is likely mediated, in part, through enhancement of hepatic fatty acid oxidation and reductions in key protein intermediates of fatty acid synthesis.

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.

Participation in road cycling vs running is associated with lower bone mineral density in men.

The effects of regular non-weight-bearing (NWB) exercise on bone health are largely unknown. The objective of the study was to determine the effects of participation in NWB sports on bone health in adult male recreational athletes. Male cyclists (NWB; n = 27) and runners (weight-bearing [WB]; n = 16) aged 20 to 59 years were recruited from the community. Whole-body and regional bone mineral content and bone mineral density (BMD), and body composition were assessed using dual x-ray absorptiometry. Bone formation and resorption markers, and hormones were measured in serum. Bone-loading history was estimated from a sports participation history questionnaire. Nutrient intake and current physical activity were estimated from 7-day written logs. The NWB athletes had significantly lower BMD of the whole body and spine than the WB athletes, despite having similar age, weight, body mass index, body composition, hormonal status, current activity level, and nutrient intakes. Sixty-three percent of NWB athletes had osteopenia of the spine or hip, compared with 19% of WB athletes. Cyclists were 7 times more likely to have osteopenia of the spine than runners, controlling for age, body weight, and bone-loading history. There were no group differences in serum markers of bone turnover. Based on the results of this study, current bone loading is an important determinant of whole-body and lumbar spine BMD. Therefore, bone-loading activity should be sustained during adulthood to maintain bone mass.

Daily exercise vs. caloric restriction for prevention of nonalcoholic fatty liver disease in the OLETF rat model

The maintenance of normal body weight either through dietary modification or being habitually more physically active is associated with reduced incidence of nonalcoholic fatty liver disease (NAFLD). However, the means by which weight gain is prevented and potential mechanisms activated remain largely unstudied. Here, we sought to determine the effects of obesity prevention by daily exercise vs. caloric restriction on NAFLD in the hyperphagic, Otsuka Long-Evans Tokushima Fatty (OLETF) rat. At 4 wk of age, male OLETF rats (n = 7-8/group) were randomized to groups of ad libitum fed, sedentary (OLETF-SED), voluntary wheel running exercise (OLETF-EX), or caloric restriction (OLETF-CR; 70% of SED) until 40 wk of age. Nonhyperphagic, control strain Long-Evans Tokushima Otsuka (LETO) rats were kept in sedentary cage conditions for the duration of the study (LETO-SED). Both daily exercise and caloric restriction prevented obesity and the development of type 2 diabetes observed in the OLETF-SED rats, with glucose tolerance during a glucose tolerance test improved to a greater extent in the OLETF-EX animals (30-50% lower glucose and insulin areas under the curve, P < 0.05). Both daily exercise and caloric restriction also prevented excess hepatic triglyceride and diacylglycerol accumulation (P < 0.001), hepatocyte ballooning and nuclear displacement, and the increased perivenular fibrosis and collagen deposition that occurred in the obese OLETF-SED animals. However, despite similar hepatic phenotypes, OLETF-EX rats also exhibited increased hepatic mitochondrial fatty acid oxidation, enhanced oxidative enzyme function and protein content, and further suppression of hepatic de novo lipogenesis proteins compared with OLETF-CR. Prevention of obesity by either daily exercise or caloric restriction attenuates NAFLD development in OLETF rats. However, daily exercise may offer additional health benefits on glucose homeostasis and hepatic mitochondrial function compared with restricted diet alone.

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‐γ 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.

Changes in visceral adipose tissue mitochondrial content with type 2 diabetes and daily voluntary wheel running in OLETF rats

Using the hyperphagic, obese, Otsuka Long–Evans Tokushima Fatty (OLETF) rat, we sought to determine if progression to type 2 diabetes alters visceral white adipose tissue (WAT) mitochondrial content and if these changes are modified through prevention of type 2 diabetes with daily exercise. At 4 weeks of age, OLETF rats began voluntary wheel running (OLETF‐EX) while additional OLETF rats (OLETF‐SED) and Long–Evans Tokushima Otsuka (LETO‐SED) rats served as obese and lean sedentary controls, respectively, for 13, 20 and 40 weeks of age (n= 6–8 for each group at each age). OLETF‐SED animals displayed insulin resistance at 13 and 20 weeks and type 2 diabetes by 40 weeks. OLETF‐SED animals gained significantly (P < 0.001) more weight and omental fat mass compared with OLETF‐EX and LETO‐SED. Markers of WAT mitochondrial protein content (cytochrome c, COXIV‐subunit I, and citrate synthase activity) significantly increased (P < 0.05) from 13 to 40 weeks in the LETO‐SED, but were significantly attenuated in the OLETF‐SED rats. Daily exercise normalized WAT cytochrome c and COXIV‐subunit I protein content in the OLETF‐EX to the healthy LETO‐SED animals. In conclusion, increases in omental WAT mitochondrial content between 20 and 40 weeks of age in LETO control animals are attenuated in the hyperphagic, obese OLETF rat. These alterations occurred in conjunction with the progression from insulin resistance to type 2 diabetes and were prevented with daily exercise. Reduced ability to increase WAT mitochondrial content does not appear to be a primary cause of insulin resistance, but may play a key role in the worsening of the disease condition.

Does physical inactivity cause nonalcoholic fatty liver disease

While physical activity represents a key element in the prevention and management of many chronic diseases, we and others believe that physical inactivity is a primary cause of obesity and associated metabolic disorders. Unfortunately, accumulating evidence suggests that we have engineered physical activity out of our normal daily living activity. One such consequence of our sedentary and excessive lifestyle is nonalcoholic fatty liver disease (NAFLD), which is now considered the most common cause of chronic liver disease in Westernized societies. In this review, we will present evidence that physical inactivity, low aerobic fitness, and overnutrition, either separately or in combination, are an underlying cause of NAFLD.

Elevated skeletal muscle irisin precursor FNDC5 mRNA in obese OLETF rats

OBJECTIVE There is debate as to whether fibronectin type III domain containing 5 (FNDC5) and its protein product irisin are therapeutic targets for obesity-associated maladies. Thus, we sought to examine FNDC5 mRNA within skeletal muscle of obese/diabetic-prone Otsuka Long-Evans Tokushima Fatty (OLETF) rats versus lean/healthy Long Evans Tokushima Otsuka (LETO) rats. We hypothesized that FNDC5 expression would be greater in obese (OLETF) versus lean (LETO) animals. MATERIALS/METHODS Triceps muscle of 30-32week old OLETF and LETO rats were assayed for FNDC5 and PGC1α mRNA levels. Body composition and circulating biomarkers of the OLETF and LETO rats were also correlated with skeletal muscle FNDC5 mRNA expression patterns in order to examine potential relationships that may exist. RESULTS OLETF rats exhibited twice the amount of triceps FNDC5 mRNA compared to LETO rats (p<0.01). Significant positive correlations existed between triceps muscle FNDC5 mRNA expression patterns versus fat mass (r=0.70, p=0.008), as well as plasma leptin (r=0.82, p<0.001). PGC1α mRNA levels were also highly correlated with FNDC5 mRNA (r=0.85, p<0.001). In subsequent culture experiments, low and high physiological doses of leptin had no effect on PGC1α mRNA or FNDC5 mRNA levels in C2C12 myotubes. Paradoxically, circulating irisin concentrations tended to be higher in a second cohort of LETO versus OLETF rats (p=0.085). CONCLUSION These results reveal a positive association between total body adiposity and skeletal muscle FNDC5 gene expression. Of interest, circulating irisin levels tended to be lower in OLETF rats. Further research is needed to examine whether other adipose tissue-derived factors up-regulate FNDC5 transcription and/or inhibit irisin biosynthesis from FNDC5.

Daily physical activity enhances reactivity to insulin in skeletal muscle arterioles of hyperphagic Otsuka Long-Evans Tokushima Fatty rats.

Insulin-mediated glucose disposal is dependent on the vasodilator effects of insulin. In type 2 diabetes, insulin-stimulated vasodilation is impaired as a result of an imbalance in NO and ET-1 production. We tested the hypothesis that chronic voluntary wheel running (RUN) prevents impairments in insulin-stimulated vasodilation associated with obesity and type 2 diabetes independent of the effects of RUN on adiposity by randomizing Otsuka Long Evans Tokushima Fatty (OLETF) rats, a model of hyperphagia-induced obesity and type 2 diabetes, to 1) RUN, 2) caloric restriction (CR; diet adjusted to match body weights of RUN group), or 3) sedentary control (SED) groups (n = 8/group) at 4 wk. At 40 wk, NO- and ET-1-mediated vasoreactivity to insulin (1-1,000 μIU/ml) was assessed in the presence of a nonselective ET-1 receptor blocker (tezosentan) or a NO synthase (NOS) inhibitor [N(G)-nitro-L-arginine methyl ester (L-NAME)], respectively, in second-order arterioles isolated from the white portion of the gastrocnemius muscle. Body weight, fasting plasma glucose, and hemoglobin A1c were lower in RUN and CR than SED (P < 0.05); however, the glucose area under the curve (AUC) following the intraperitoneal glucose tolerance test was lower only in the RUN group (P < 0.05). Vasodilator responses to all doses of insulin were greater in RUN than SED or CR in the presence of a tezosentan (P < 0.05), but group differences in vasoreactivity to insulin with coadministration of L-NAME were not observed. We conclude daily wheel running prevents obesity and type 2 diabetes-associated declines in insulin-stimulated vasodilation in skeletal muscle arterioles through mechanisms that appear to be NO mediated and independent of attenuating excess adiposity in hyperphagic rats.