Erin J. Stephenson

Exercise training reverses impaired skeletal muscle metabolism induced by artificial selection for low aerobic capacity

We have used a novel model of genetically imparted endurance exercise capacity and metabolic health to study the genetic and environmental contributions to skeletal muscle glucose and lipid metabolism. We hypothesized that metabolic abnormalities associated with low intrinsic running capacity would be ameliorated by exercise training. Selective breeding for 22 generations resulted in rat models with a fivefold difference in intrinsic aerobic capacity. Low (LCR)- and high (HCR)-capacity runners remained sedentary (SED) or underwent 6 wk of exercise training (EXT). Insulin-stimulated glucose transport, insulin signal transduction, and rates of palmitate oxidation were lower in LCR SED vs. HCR SED (P < 0.05). Decreases in glucose and lipid metabolism were associated with decreased β₂-adrenergic receptor (β₂-AR), and reduced expression of Nur77 target proteins that are critical regulators of muscle glucose and lipid metabolism [uncoupling protein-3 (UCP3), fatty acid transporter (FAT)/CD36; P < 0.01 and P < 0.05, respectively]. EXT reversed the impairments to glucose and lipid metabolism observed in the skeletal muscle of LCR, while increasing the expression of β₂-AR, Nur77, GLUT4, UCP3, and FAT/CD36 (P < 0.05) in this tissue. However, no metabolic improvements were observed following exercise training in HCR. Our results demonstrate that metabolic impairments resulting from genetic factors (low intrinsic aerobic capacity) can be overcome by an environmental intervention (exercise training). Furthermore, we identify Nur77 as a potential mechanism for improved skeletal muscle metabolism in response to EXT.

Divergent skeletal muscle respiratory capacities in rats artificially selected for high and low running ability: a role for Nor1?

Inactivity-related diseases are becoming a huge burden on Western society. While there is a major environmental contribution to metabolic health, the intrinsic properties that predispose or protect against particular health traits are harder to define. We used rat models of inborn high running capacity (HCR) and low running capacity (LCR) to determine inherent differences in mitochondrial volume and function, hypothesizing that HCR rats would have greater skeletal muscle respiratory capacity due to an increase in mitochondrial number. Additionally, we sought to determine if there was a link between the expression of the orphan nuclear receptor neuron-derived orphan receptor (Nor)1, a regulator of oxidative metabolism, and inherent skeletal muscle respiratory capacity. LCR rats were 28% heavier (P < 0.0001), and fasting serum insulin concentrations were 62% greater than in HCR rats (P = 0.02). In contrast, HCR rats had better glucose tolerance and reduced adiposity. In the primarily oxidative soleus muscle, maximal respiratory capacity was 21% greater in HCR rats (P = 0.001), for which the relative contribution of fat oxidation was 20% higher than in LCR rats (P = 0.02). This was associated with increased citrate synthase (CS; 33%, P = 0.009) and β-hydroxyacyl-CoA (β-HAD; 33%, P = 0.0003) activities. In the primarily glycolytic extensor digitum longus muscle, CS activity was 29% greater (P = 0.01) and β-HAD activity was 41% (P = 0.0004) greater in HCR rats compared with LCR rats. Mitochondrial DNA copy numbers were also elevated in the extensor digitum longus muscles of HCR rats (35%, P = 0.049) and in soleus muscles (44%, P = 0.16). Additionally, HCR rats had increased protein expression of individual mitochondrial respiratory complexes, CS, and uncoupling protein 3 in both muscle types (all P < 0.05). In both muscles, Nor1 protein was greater in HCR rats compared with LCR rats (P < 0.05). We propose that the differential expression of Nor1 may contribute to the differences in metabolic regulation between LCR and HCR phenotypes.

Skeletal muscle respiratory capacity is enhanced in rats consuming an obesogenic Western diet

Obesity-induced lipid oversupply promotes skeletal muscle mitochondrial biogenesis. Previous investigations have utilized extreme high-fat diets (HFD) to induce such mitochondrial perturbations despite their disparity from human obesogenic diets. Here, we evaluate the effects of Western diet (WD)-induced obesity on skeletal muscle mitochondrial function. Long-Evans rats were given ad libitum access to either a WD [40% energy (E) from fat, 17% protein, and 43% carbohydrate (30% sucrose); n = 12] or a control diet (CON; 16% of E from fat, 21% protein, and 63% carbohydrate; n = 12) for 12 wk. Rats fed the WD consumed 23% more E than CON (P = 0.0001), which was associated with greater increases in body mass (23%, P = 0.0002) and adiposity (17%, P = 0.03). There were no differences in fasting blood glucose concentration or glucose tolerance between diets, although fasting insulin was increased by 40% (P = 0.007). Fasting serum triglycerides were also elevated in WD (86%, P = 0.001). The maximal capacity of the electron transfer system was greater following WD (37%, P = 0.02), as were the maximal activities of several mitochondrial enzymes (citrate synthase, β-hydroxyacyl-CoA dehydrogenase, carnitine palmitoyltransferase). Protein expression of citrate synthase, UCP3, and individual respiratory complexes was greater after WD (P < 0.05) despite no differences in the expression of peroxisome proliferator-activated receptor (PPAR)α, PPARδ, or PPARγ coactivator-1 mRNA or protein abundance. We conclude that the respiratory capacity of skeletal muscle is enhanced in response to the excess energy supplied by a WD. This is likely due to an increase in mitochondrial density, which at least in the short term, and in the absence of increased energy demand, may protect the tissue from lipid-induced impairments in glycemic control.

Low intrinsic exercise capacity in rats predisposes to age-dependent cardiac remodeling independent of macrovascular function.

Rats selectively bred for low (LCR) or high (HCR) intrinsic running capacity simultaneously present with contrasting risk factors for cardiovascular and metabolic disease. However, the impact of these phenotypes on left ventricular (LV) morphology and microvascular function, and their progression with aging, remains unresolved. We tested the hypothesis that the LCR phenotype induces progressive age-dependent LV remodeling and impairments in microvascular function, glucose utilization, and β-adrenergic responsiveness, compared with HCR. Hearts and vessels isolated from female LCR (n = 22) or HCR (n = 26) were studied at 12 and 35 wk. Nonselected N:NIH founder rats (11 wk) were also investigated (n = 12). LCR had impaired glucose tolerance and elevated plasma insulin (but not glucose) and body-mass at 12 wk compared with HCR, with early LV remodeling. By 35 wk, LV prohypertrophic and glucose transporter GLUT4 gene expression were up- and downregulated, respectively. No differences in LV β-adrenoceptor expression or cAMP content between phenotypes were observed. Macrovascular endothelial function was predominantly nitric oxide (NO)-mediated in both phenotypes and remained intact in LCR for both age-groups. In contrast, mesenteric arteries microvascular endothelial function, which was impaired in LCR rats regardless of age. At 35 wk, endothelial-derived hyperpolarizing factor-mediated relaxation was impaired whereas the NO contribution to relaxation is intact. Furthermore, there was reduced β2-adrenoceptor responsiveness in both aorta and mesenteric LCR arteries. In conclusion, diminished intrinsic exercise capacity impairs systemic glucose tolerance and is accompanied by progressive development of LV remodeling. Impaired microvascular perfusion is a likely contributing factor to the cardiac phenotype.

Exercise training enhances white adipose tissue metabolism in rats selectively bred for low- or high-endurance running capacity

Impaired visceral white adipose tissue (WAT) metabolism has been implicated in the pathogenesis of several lifestyle-related disease states, with diminished expression of several WAT mitochondrial genes reported in both insulin-resistant humans and rodents. We have used rat models selectively bred for low- (LCR) or high-intrinsic running capacity (HCR) that present simultaneously with divergent metabolic phenotypes to test the hypothesis that oxidative enzyme expression is reduced in epididymal WAT from LCR animals. Based on this assumption, we further hypothesized that short-term exercise training (6 wk of treadmill running) would ameliorate this deficit. Approximately 22-wk-old rats (generation 22) were studied. In untrained rats, the abundance of mitochondrial respiratory complexes I-V, citrate synthase (CS), and PGC-1 was similar for both phenotypes, although CS activity was greater than 50% in HCR (P = 0.09). Exercise training increased CS activity in both phenotypes but did not alter mitochondrial protein content. Training increased the expression and phosphorylation of proteins with roles in β-adrenergic signaling, including β3-adrenergic receptor (16% increase in LCR; P < 0.05), NOR1 (24% decrease in LCR, 21% decrease in HCR; P < 0.05), phospho-ATGL (25% increase in HCR; P < 0.05), perilipin (25% increase in HCR; P < 0.05), CGI-58 (15% increase in LCR; P < 0.05), and GLUT4 (16% increase in HCR; P < 0.0001). A training effect was also observed for phospho-p38 MAPK (12% decrease in LCR, 20% decrease in HCR; P < 0.05) and phospho-JNK (29% increase in LCR, 20% increase in HCR; P < 0.05). We conclude that in the LCR-HCR model system, mitochondrial protein expression in WAT is not affected by intrinsic running capacity or exercise training. However, training does induce alterations in the activity and expression of several proteins that are essential to the intracellular regulation of WAT metabolism.

The relationship between exercise, nutrition and type 2 diabetes

Type 2 diabetes mellitus and its precursor, insulin resistance, are metabolic disease states characterized by impaired regulation in the delivery, transport, and/or storage of energy substrates (primarily carbohydrate- and fat-based fuels). A hallmark feature of patients with type 2 diabetes is prolonged periods of hyperglycemia due to a decreased responsiveness of metabolically active peripheral tissues to the actions of insulin (i.e., metabolic inflexibility). Accordingly, efforts to modify skeletal muscle substrate handling in type 2 diabetes patients so that the capacity for fat oxidation and metabolic flexibility is improved should be a primary goal for the treatment of these disorders. Two potent interventions for improving whole-body glucose homeostasis are exercise and diet. A single bout of either resistance or endurance exercise reduces the prevalence and duration of hyperglycemic excursions in patients with type 2 diabetes, an effect lasting well into the next day. With regard to diet, the carbohydrate content of a meal and the glycemic index (GI) of the carbohydrate consumed are both major determinants of the postprandial glycemic response. Diets containing high-GI carbohydrates have been shown to be independent risk factors for type 2 diabetes onset, while in obese insulin-resistant individuals, low-GI diets are effective for inducing both weight loss and improving insulin action and glucose tolerance. The implementation of physical activity and dietary modifications are effective low-cost treatment options for controlling hyperglycemic episodes in patients with type 2 diabetes.

Androgen receptor agonists increase lean mass, improve cardiopulmonary functions and extend survival in preclinical models of Duchenne muscular dystrophy

Abstract Duchenne muscular dystrophy (DMD) is a neuromuscular disease that predominantly affects boys as a result of mutation(s) in the dystrophin gene. DMD is characterized by musculoskeletal and cardiopulmonary complications, resulting in shorter life‐span. Boys afflicted by DMD typically exhibit symptoms within 3‐5 years of age and declining physical functions before attaining puberty. We hypothesized that rapidly deteriorating health of pre‐pubertal boys with DMD could be due to diminished anabolic actions of androgens in muscle, and that intervention with an androgen receptor (AR) agonist will reverse musculoskeletal complications and extend survival. While castration of dystrophin and utrophin double mutant (mdx‐dm) mice to mimic pre‐pubertal nadir androgen condition resulted in premature death, maintenance of androgen levels extended the survival. Non‐steroidal selective‐AR modulator, GTx‐026, which selectively builds muscle and bone was tested in X‐linked muscular dystrophy mice (mdx). GTx‐026 significantly increased body weight, lean mass and grip strength by 60‐80% over vehicle‐treated mdx mice. While vehicle‐treated castrated mdx mice exhibited cardiopulmonary impairment and fibrosis of heart and lungs, GTx‐026 returned cardiopulmonary function and intensity of fibrosis to healthy control levels. GTx‐026 elicits its musculoskeletal effects through pathways that are distinct from dystrophin‐regulated pathways, making AR agonists ideal candidates for combination approaches. While castration of mdx‐dm mice resulted in weaker muscle and shorter survival, GTx‐026 treatment increased the muscle mass, function and survival, indicating that androgens are important for extended survival. These preclinical results support the importance of androgens and the need for intervention with AR agonists to treat DMD‐affected boys.

Expression of microRNAs and target proteins in skeletal muscle of rats selectively bred for high and low running capacity

Impairments in mitochondrial function and substrate metabolism are implicated in the etiology of obesity and Type 2 diabetes. MicroRNAs (miRNAs) can degrade mRNA or repress protein translation and have been implicated in the development of such disorders. We used a contrasting rat model system of selectively bred high- (HCR) or low- (LCR) intrinsic running capacity with established differences in metabolic health to investigate the molecular mechanisms through which miRNAs regulate target proteins mediating mitochondrial function and substrate oxidation processes. Quantification of select miRNAs using the rat miFinder miRNA PCR array revealed differential expression of 15 skeletal muscles (musculus tibialis anterior) miRNAs between HCR and LCR rats (14 with higher expression in LCR; P < 0.05). Ingenuity Pathway Analysis predicted these altered miRNAs to collectively target multiple proteins implicated in mitochondrial dysfunction and energy substrate metabolism. Total protein abundance of citrate synthase (CS; miR-19 target) and voltage-dependent anion channel 1 (miR-7a target) were higher in HCR compared with LCR cohorts (~57 and ~26%, respectively; P < 0.05). A negative correlation was observed for miR-19a-3p and CS (r = 0.32, P = 0.015) protein expression. To determine whether miR-19a-3p can regulate CS in vitro, we performed luciferase reporter and transfection assays in C2C12 myotubes. MiR-19a-3p binding to the CS untranslated region did not change luciferase reporter activity; however, miR-19a-3p transfection decreased CS protein expression (∼70%; P < 0.05). The differential miRNA expression targeting proteins implicated in mitochondrial dysfunction and energy substrate metabolism may contribute to the molecular basis, mediating the divergent metabolic health profiles of LCR and HCR rats.

Exposure to environmentally persistent free radicals during gestation lowers energy expenditure and impairs skeletal muscle mitochondrial function in adult mice

We have investigated the effects of in utero exposure to environmentally persistent free radicals (EPFRs) on growth, metabolism, energy utilization, and skeletal muscle mitochondria in a mouse model of diet-induced obesity. Pregnant mice were treated with laboratory-generated, combustion-derived particular matter (MCP230). The adult offspring were placed on a high-fat diet for 12 wk, after which we observed a 9.8% increase in their body weight. The increase in body size observed in the MCP230-exposed mice was not associated with increases in food intake but was associated with a reduction in physical activity and lower energy expenditure. The reduced energy expenditure in mice indirectly exposed to MCP230 was associated with reductions in skeletal muscle mitochondrial DNA copy number, lower mRNA levels of electron transport genes, and reduced citrate synthase activity. Upregulation of key genes involved in ameliorating oxidative stress was also observed in the muscle of MCP230-exposed mice. These findings suggest that gestational exposure to MCP230 leads to a reduction in energy expenditure at least in part through alterations to mitochondrial metabolism in the skeletal muscle.

Glucocorticoid-Induced Metabolic Disturbances Are Exacerbated in Obese Male Mice

The purpose of this study was to determine the effects of glucocorticoid-induced metabolic dysfunction in the presence of diet-induced obesity. C57BL/6J adult male lean and diet-induced obese mice were given dexamethasone, and levels of hepatic steatosis, insulin resistance, and lipolysis were determined. Obese mice given dexamethasone had significant, synergistic effects on fasting glucose, insulin resistance, and markers of lipolysis, as well as hepatic steatosis. This was associated with synergistic transactivation of the lipolytic enzyme adipose triglyceride lipase. The combination of chronically elevated glucocorticoids and obesity leads to exacerbations in metabolic dysfunction. Our findings suggest lipolysis may be a key player in glucocorticoid-induced insulin resistance and fatty liver in individuals with obesity.