Grace M. Meers

Simvastatin impairs exercise training adaptations.

OBJECTIVES This study sought to determine if simvastatin impairs exercise training adaptations. BACKGROUND Statins are commonly prescribed in combination with therapeutic lifestyle changes, including exercise, to reduce cardiovascular disease risk in patients with metabolic syndrome. Statin use has been linked to skeletal muscle myopathy and impaired mitochondrial function, but it is unclear whether statin use alters adaptations to exercise training. METHODS This study examined the effects of simvastatin on changes in cardiorespiratory fitness and skeletal muscle mitochondrial content in response to aerobic exercise training. Sedentary overweight or obese adults with at least 2 metabolic syndrome risk factors (defined according to National Cholesterol Education Panel Adult Treatment Panel III criteria) were randomized to 12 weeks of aerobic exercise training or to exercise in combination with simvastatin (40 mg/day). The primary outcomes were cardiorespiratory fitness and skeletal muscle (vastus lateralis) mitochondrial content (citrate synthase enzyme activity). RESULTS Thirty-seven participants (exercise plus statins: n = 18; exercise only: n = 19) completed the study. Cardiorespiratory fitness increased by 10% (p < 0.05) in response to exercise training alone, but was blunted by the addition of simvastatin resulting in only a 1.5% increase (p < 0.005 for group by time interaction). Similarly, skeletal muscle citrate synthase activity increased by 13% in the exercise-only group (p < 0.05), but decreased by 4.5% in the simvastatin-plus-exercise group (p < 0.05 for group-by-time interaction). CONCLUSIONS Simvastatin attenuates increases in cardiorespiratory fitness and skeletal muscle mitochondrial content when combined with exercise training in overweight or obese patients at risk of the metabolic syndrome. (Exercise, Statins, and the Metabolic Syndrome; NCT01700530).

Altered Hepatic Lipid Metabolism Contributes to Nonalcoholic Fatty Liver Disease in Leptin-Deficient Ob/Ob Mice

Nonalcoholic fatty liver disease (NAFLD) is strongly linked to obesity, insulin resistance, and abnormal hepatic lipid metabolism; however, the precise regulation of these processes remains poorly understood. Here we examined genes and proteins involved in hepatic oxidation and lipogenesis in 14-week-old leptin-deficient Ob/Ob mice, a commonly studied model of obesity and hepatic steatosis. Obese Ob/Ob mice had increased fasting glucose, insulin, and calculated HOMA-IR as compared with lean wild-type (WT) mice. Ob/Ob mice also had greater liver weights, hepatic triglyceride (TG) content, and markers of de novo lipogenesis, including increased hepatic gene expression and protein content of acetyl-CoA carboxylase (ACC), fatty acid synthase (FAS), and stearoyl-CoA desaturase-1 (SCD-1), as well as elevated gene expression of PPARγ and SREBP-1c compared with WT mice. While hepatic mRNA levels for PGC-1α, PPARα, and TFAM were elevated in Ob/Ob mice, measures of mitochondrial function (β-HAD activity and complete (to CO2) and total mitochondrial palmitate oxidation) and mitochondrial OXPHOS protein subunits I, III, and V content were significantly reduced compared with WT animals. In summary, reduced hepatic mitochondrial content and function and an upregulation in de novo lipogenesis contribute to obesity-associated NAFLD in the leptin-deficient Ob/Ob mouse.

PGC-1α overexpression results in increased hepatic fatty acid oxidation with reduced triacylglycerol accumulation and secretion

Studies have shown that decreased mitochondrial content and function are associated with hepatic steatosis. We examined whether peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) overexpression and a subsequent increase in mitochondrial content and function in rat primary hepatocytes (in vitro) and Sprague-Dawley rats (in vivo) would comprehensively alter mitochondrial lipid metabolism, including complete (CO(2)) and incomplete (acid-soluble metabolites) fatty acid oxidation (FAO), tricarboxylic acid cycle flux, and triacylglycerol (TAG) storage and export. PGC-1α overexpression in primary hepatocytes produced an increase in markers of mitochondrial content and function (citrate synthase, mitochondrial DNA, and electron transport system complex proteins) and an increase in FAO, which was accompanied by reduced TAG storage and TAG secretion compared with control. Also, the PGC-1α-overexpressing hepatocytes were protected from excess TAG accumulation following overnight lipid treatment. PGC-1α overexpression in hepatocytes lowered expression of genes critical to VLDL assembly and secretion (apolipoprotein B and microsomal triglyceride transfer protein). Adenoviral transduction of rats with PGC-1α resulted in a liver-specific increase in PGC-1α expression and produced an in vivo liver phenotype of increased FAO via increased mitochondrial function that also resulted in reduced hepatic TAG storage and decreased plasma TAG levels. In conclusion, overexpression of hepatic PGC-1α and subsequent increases in FAO through elevated mitochondrial content and/or function result in reduced TAG storage and secretion in the in vitro and in vivo milieu.

Combining metformin and aerobic exercise training in the treatment of type 2 diabetes and NAFLD in OLETF rats

Here, we sought to compare the efficacy of combining exercise and metformin for the treatment of type 2 diabetes and nonalcoholic fatty liver disease (NAFLD) in hyperphagic, obese, type 2 diabetic Otsuka Long-Evans Tokushima Fatty (OLETF) rats. OLETF rats (age: 20 wk, hyperglycemic and hyperinsulinemic; n = 10/group) were randomly assigned to sedentary (O-SED), SED plus metformin (O-SED + M; 300 mg·kg(-1)·day(-1)), moderate-intensity exercise training (O-EndEx; 20 m/min, 60 min/day, 5 days/wk treadmill running), or O-EndEx + M groups for 12 wk. Long-Evans Tokushima Otsuka (L-SED) rats served as nonhyperphagic controls. O-SED + M, O-EndEx, and O-EndEx + M were effective in the management of type 2 diabetes, and all three treatments lowered hepatic steatosis and serum markers of liver injury; however, O-EndEx lowered liver triglyceride content and fasting hyperglycemia more than O-SED + M. In addition, exercise elicited greater improvements compared with metformin alone on postchallenge glycemic control, liver diacylglycerol content, hepatic mitochondrial palmitate oxidation, citrate synthase, and β-HAD activities and in the attenuation of markers of hepatic fatty acid uptake and de novo fatty acid synthesis. Surprisingly, combining metformin and aerobic exercise training offered little added benefit to these outcomes, and in fact, metformin actually blunted exercise-induced increases in complete mitochondrial palmitate oxidation and β-HAD activity. In conclusion, aerobic exercise training was more effective than metformin administration in the management of type 2 diabetes and NAFLD outcomes in obese hyperphagic OLETF rats. Combining therapies offered little additional benefit beyond exercise alone, and findings suggest that metformin potentially impairs exercise-induced hepatic mitochondrial adaptations.

Female rats selectively bred for high intrinsic aerobic fitness are protected from ovariectomy-associated metabolic dysfunction

Ovariectomized rodents model human menopause in that they rapidly gain weight, reduce spontaneous physical activity (SPA), and develop metabolic dysfunction, including insulin resistance. How contrasting aerobic fitness levels impacts ovariectomy (OVX)-associated metabolic dysfunction is not known. Female rats selectively bred for high and low intrinsic aerobic fitness [high-capacity runners (HCR) and low-capacity runners (LCR), respectively] were maintained under sedentary conditions for 39 wk. Midway through the observation period, OVX or sham (SHM) operations were performed providing HCR-SHM, HCR-OVX, LCR-SHM, and LCR-OVX groups. Glucose tolerance, energy expenditure, and SPA were measured before and 4 wk after surgery, while body composition via dual-energy X-ray absorptiometry and adipose tissue distribution, brown adipose tissue (BAT), and skeletal muscle phenotype, hepatic lipid content, insulin resistance via homeostatic assessment model of insulin resistance and AdipoIR, and blood lipids were assessed at death. Remarkably, HCR were protected from OVX-associated increases in adiposity and insulin resistance, observed only in LCR. HCR rats were ∼30% smaller, had ∼70% greater spontaneous physical activity (SPA), consumed ∼10% more relative energy, had greater skeletal muscle proliferator-activated receptor coactivator 1-alpha, and ∼40% more BAT. OVX did not increase energy intake and reduced SPA to the same extent in both HCR and LCR. LCR were particularly affected by an OVX-associated reduction in resting energy expenditure and experienced a reduction in relative BAT; resting energy expenditure correlated positively with BAT across all animals (r = 0.6; P < 0.001). In conclusion, despite reduced SPA following OVX, high intrinsic aerobic fitness protects against OVX-associated increases in adiposity and insulin resistance. The mechanism may involve preservation of resting energy expenditure.

Intrinsic aerobic capacity impacts susceptibility to acute high-fat diet-induced hepatic steatosis

Aerobic capacity/fitness significantly impacts susceptibility for fatty liver and diabetes, but the mechanisms remain unknown. Herein, we utilized rats selectively bred for high (HCR) and low (LCR) intrinsic aerobic capacity to examine the mechanisms by which aerobic capacity impacts metabolic vulnerability for fatty liver following a 3-day high-fat diet (HFD). Indirect calorimetry assessment of energy metabolism combined with radiolabeled dietary food was employed to examine systemic metabolism in combination with ex vivo measurements of hepatic lipid oxidation. The LCR, but not HCR, displayed increased hepatic lipid accumulation in response to the HFD despite both groups increasing energy intake. However, LCR rats had a greater increase in energy intake and demonstrated greater daily weight gain and percent body fat due to HFD compared with HCR. Additionally, total energy expenditure was higher in the larger LCR. However, controlling for the difference in body weight, the LCR has lower resting energy expenditure compared with HCR. Importantly, respiratory quotient was significantly higher during the HFD in the LCR compared with HCR, suggesting reduced whole body lipid utilization in the LCR. This was confirmed by the observed lower whole body dietary fatty acid oxidation in LCR compared with HCR. Furthermore, LCR liver homogenate and isolated mitochondria showed lower complete fatty acid oxidation compared with HCR. We conclude that rats bred for low intrinsic aerobic capacity show greater susceptibility for dietary-induced hepatic steatosis, which is associated with a lower energy expenditure and reduced whole body and hepatic mitochondrial lipid oxidation.

Selective hepatic insulin resistance in a murine model heterozygous for a mitochondrial trifunctional protein defect

Earlier reports suggest a link between mitochondrial dysfunction and development of hepatic insulin resistance. Here we used a murine model heterozygous (HET) for a mitochondrial trifunctional protein (MTP) gene defect to determine if a primary defect in mitochondrial long‐chain fatty acid oxidation disrupts hepatic insulin action. Hyperinsulinemic‐euglycemic clamps and signaling studies were performed for assessment of whole‐body and hepatic insulin resistance/signaling. In addition, hepatic fatty acid oxidation and hepatic insulin action were assessed in vitro using primary hepatocytes isolated from HET and wildtype (WT) mice. In both hepatic mitochondria and isolated primary hepatocytes, heterozygosity of MTP caused an ∼50% reduction in mitochondrial fatty acid oxidation, a significantly impaired glucose disposal during the insulin clamp, and a markedly lower insulin‐stimulated suppression of hepatic glucose production. HET mice also exhibited impaired insulin signaling, with increased hepatic phosphorylation of IRS2 (ser731) and reduced Akt phosphorylation (ser473) in both hepatic tissue and isolated primary hepatocytes. Assessment of insulin‐stimulated FOXO1/phospho‐FOXO1 protein content and PEPCK/G6Pase messenger RNA (mRNA) expression did not reveal differences between HET and WT mice. However, insulin‐induced phosphorylation of GSK3β was significantly blunted in HET mice. Hepatic insulin resistance was associated with an increased methylation status of the catalytic subunit of protein phosphatase 2A (PP2A‐C), but was not associated with differences in hepatic diacylglycerol content, activated protein kinase C‐ϵ (PKC‐ϵ), inhibitor κB kinase β (IKK‐β), c‐Jun N‐terminal kinase (JNK), or phospho‐JNK protein contents. Surprisingly, hepatic ceramides were significantly lower in the HET mice compared with WT. Conclusion: A primary defect in mitochondrial fatty acid β‐oxidation causes hepatic insulin resistance selective to hepatic glycogen metabolism that is associated with elevated methylated PP2A‐C, but independent of other mechanisms commonly considered responsible for insulin resistance. (HEPATOLOGY 2013;)

Treating NAFLD in OLETF Rats with Vigorous-Intensity Interval Exercise Training

BACKGROUND There is increasing use of high-intensity interval-type exercise training in the management of many lifestyle-related diseases. PURPOSE This study aimed to test the hypothesis that vigorous-intensity interval exercise is as effective as traditional moderate-intensity aerobic exercise training for nonalcoholic fatty liver disease (NAFLD) outcomes in obese, Otsuka Long-Evans Tokushima Fatty (OLETF) rats. METHODS OLETF rats (age, 20 wk; n = 8-10 per group) were assigned to sedentary (O-SED), moderate-intensity exercise training (O-MOD EX; 20 m·min(-1), 15% incline, 60 min·d(-1), 5 d·wk(-1) of treadmill running), or vigorous-intensity interval exercise training (O-VIG EX; 40 m·min(-1), 15% incline, 6 × 2.5 min bouts per day, 5 d·wk(-1) of treadmill running) groups for 12 wk. RESULTS Both MOD EX and VIG EX effectively lowered hepatic triglycerides, serum alanine aminotransferase (ALT), perivenular fibrosis, and hepatic collagen 1α1 messenger RNA (mRNA) expression (vs O-SED, P < 0.05). In addition, both interventions increased hepatic mitochondrial markers (citrate synthase activity and fatty acid oxidation) and suppressed markers of de novo lipogenesis (fatty acid synthase, acetyl coenzyme A carboxylase, Elovl fatty acid elongase 6, and steroyl CoA desaturase-1), whereas only MOD EX increased hepatic mitochondrial Beta-hydroxyacyl-CoA dehydrogenase (β-HAD) activity and hepatic triglyceride export marker apoB100 and lowered fatty acid transporter CD36 compared with O-SED. Moreover, whereas total hepatic macrophage population markers (CD68 and F4/80 mRNA) did not differ among groups, MOD EX and VIG EX lowered M1 macrophage polarization markers (CD11c, interleukin-1β, and tumor necrosis factor α mRNA) and MOD EX increased M2 macrophage marker, CD206 mRNA, compared with O-SED. CONCLUSIONS The accumulation of 15 min·d(-1) of VIG EX for 12 wk had similar effectiveness as 60 min·d(-1) of MOD EX in the management of NAFLD in OLETF rats. These findings may have important health outcome implications as we work to design better exercise training programs for patients with NAFLD.

Hepatic steatosis development with four weeks of physical inactivity in previously active, hyperphagic OLETF rats

Physical activity-induced prevention of hepatic steatosis is maintained during short-term (7-day) transitions to an inactive state; however, whether these protective effects are present under a longer duration of physical inactivity is largely unknown. Here, we sought to determine whether previous physical activity had protective effects on hepatic steatosis and metabolic health following 4 wk of physical inactivity. Four-week old, hyperphagic, male Otsuka Long-Evans Tokushima fatty (OLETF) rats were randomly assigned to either a sedentary group for 16 wk (OLETF-SED), given access to running wheels for 16 wk with wheels locked 5 h (OLETF-WL5hr) or given access to running wheels for 12 wk with wheels locked 4 wk (OLETF-WL4wk) prior to death. Four weeks of physical inactivity caused hepatic steatosis development, but liver triglycerides remained 60% lower than OLETF-SED (P < 0.01), and this was associated with only a partial loss in activity-induced improvements in body composition, serum lipids, and glycemic control. Total hepatic mitochondrial palmitate oxidation, citrate synthase, and β-HAD activity returned to SED levels following 4 wk of inactivity, whereas markers of fatty acid uptake and lipogenesis remained relatively suppressed following 4 wk of inactivity. In addition, 4 wk of inactivity caused a complete loss of activity-induced increases in serum IL-6 and reductions in regulated upon activation, normal T-cell expressed, and secreted (RANTES), and a partial loss in reductions in leptin, monocyte chemoattractant protein-1, and TNF-α. In conclusion, 4 wk of physical inactivity does not result in a complete loss in physical activity-induced benefits but does cause deterioration in the liver phenotype and overall metabolic health in hyperphagic OLETF rats.

Reduced Hepatic Mitochondrial Respiration following acute High-fat Diet is Prevented by PGC-1α Overexpression

Changes in substrate utilization and reduced mitochondrial respiratory capacity following exposure to energy-dense, high-fat diets (HFD) are putatively key components in the development of obesity-related metabolic disease. We examined the effect of a 3-day HFD on isolated liver mitochondrial respiration and whole body energy utilization in obesity-prone (OP) rats. We also examined if hepatic overexpression of peroxisomal proliferator-activated receptor-γ coactivator-1α (PGC-1α), a master regulator of mitochondrial respiratory capacity and biogenesis, would modify liver and whole body responses to the HFD. Acute, 3-day HFD (45% kcal) in OP rats resulted in increased daily energy intake, energy balance, weight gain, and adiposity, without an increase in liver triglyceride (triacylglycerol) accumulation. HFD-fed OP rats also displayed decreased whole body substrate switching from the dark to the light cycle, which was paired with reductions in hepatic mitochondrial respiration of multiple substrates in multiple respiratory states. Hepatic PGC-1α overexpression was observed to protect whole body substrate switching, as well as maintain mitochondrial respiration, following the acute HFD. Additionally, liver PGC-1α overexpression did not alter whole body dietary fatty acid oxidation but resulted in greater storage of dietary free fatty acids in liver lipid, primarily as triacylglycerol. Together, these data demonstrate that a short-term HFD can result in a decrease in metabolic flexibility and hepatic mitochondrial respiratory capacity in OP rats that is completely prevented by hepatic overexpression of PGC-1α.