Altering the redox state of skeletal muscle by glutathione depletion increases the exercise-activation of PGC-1α

We investigated the relationship between mitochondrial biogenesis, cell signalling and antioxidant enzymes by depleting skeletal muscle glutathione with diethyl maleate (DEM) which resulted in a demonstrable increase in oxidative stress during exercise. Animals were divided into six groups: (1) sedentary control rats; (2) sedentary rats treated with DEM; (3) exercise control rats euthanized immediately after exercise; (4) exercise rats + DEM; (5) exercise control rats euthanized 4 h after exercise, and; (6) exercise rats + DEM euthanized 4 h after exercise. Exercising animals ran on the treadmill at a 10% gradient at 20 m/min for the first 30 min. The speed was then increased every 10 min by 1.6 m/min until exhaustion. There was a reduction in total glutathione in the skeletal muscle of DEM treated animals compared to the control animals (P<0.05). Within the control group, total glutathione was higher in the sedentary group compared to after exercise (P<0.05). DEM treatment also significantly increased oxidative stress, as measured by increased plasma F2-isoprostanes (P<0.05). Exercising animals given DEM showed a significantly greater increase in peroxisome proliferator activated receptor γ coactivator-1α(PGC-1α) mRNA compared to the control animals that were exercised (P<0.05). This study provides novel evidence that by reducing the endogenous antioxidant glutathione in skeletal muscle and inducing oxidative stress through exercise, PGC-1α gene expression was augmented. These findings further highlight the important role of exercise induced oxidative stress in the regulation of mitochondrial biogenesis.

Resistance exercise with low glycogen increases p53 phosphorylation and PGC-1α mRNA in skeletal muscle

Purpose We determined the effect of reduced muscle glycogen availability on cellular pathways regulating mitochondrial biogenesis and substrate utilization after a bout of resistance exercise. Methods Eight young, recreationally trained men undertook a glycogen depletion protocol of one-leg cycling to fatigue (LOW), while the contralateral (control) leg rested (CONT). Following an overnight fast, subjects completed 8 sets of 5 unilateral leg press repetitions (REX) at 80 % 1 Repetition Maximum (1RM) on each leg. Subjects consumed 500 mL protein/CHO beverage (20 g whey + 40 g maltodextrin) upon completion of REX and 2 h later. Muscle biopsies were obtained at rest and 1 and 4 h after REX in both legs. Results Resting muscle glycogen was higher in the CONT than LOW leg (~384 ± 114 vs 184 ± 36 mmol kg−1 dry wt; P < 0.05), and 1 h and 4 h post-exercise (P < 0.05). Phosphorylation of p53Ser15 increased 1 h post-exercise in LOW (~115 %, P < 0.05) and was higher than CONT at this time point (~87 %, P < 0.05). p38MAPKThr180/Tyr182 phosphorylation increased 1 h post-exercise in both CONT and LOW (~800–900 %; P < 0.05) but remained above rest at 4 h only in CONT (~585 %, P < 0.05; different between legs P < 0.05). Peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α) mRNA was elevated 4 h post-exercise in LOW (~200 %, P < 0.05; different between legs P < 0.05). There were no changes in Fibronectin type III domain-containing protein 5 (FNDC5) mRNA for CONT or LOW legs post-exercise. Conclusion Undertaking resistance exercise with low glycogen availability may enhance mitochondrial-related adaptations through p53 and PGC-1α-mediated signalling.

IHG-1 promotes mitochondrial biogenesis by stabilizing PGC-1α

Increased expression of Induced-by-High-Glucose 1 (IHG-1) associates with tubulointerstitial fibrosis in diabetic nephropathy. IHG-1 amplifies TGF-ß1 signaling, but the functions of this highly-conserved protein are not well understood. IHG-1 contains a putative mitochondrial-localization domain, and here we report that IHG-1 is specifically localized to mitochondria. IHG-1 overexpression increased mitochondrial mass and stabilized peroxisome proliferator-activated receptor ? coactivator-1a (PGC-1a). Conversely, inhibition of IHG-1 expression decreased mitochondrial mass, downregulated mitochondrial proteins, and PGC-1a-regulated transcription factors, including nuclear respiratory factor 1 and mitochondrial transcription factor A (TFAM), and reduced activity of the TFAM promoter. In the unilateral ureteral obstruction model, we observed higher PGC-1a protein expression and IHG-1 levels with fibrosis. In a gene-expression database, we noted that renal biopsies of human diabetic nephropathy demonstrated higher expression of genes encoding key mitochondrial proteins, including cytochrome c and manganese superoxide dismutase, compared with control biopsies. In summary, these data suggest that IHG-1 increases mitochondrial biogenesis by promoting PGC-1a-dependent processes, potentially contributing to the pathogenesis of renal fibrosis.

PGC-1α and exercise: important partners in combating insulin resistance

Diabetes and obesity are characterised by an impairment in mitochondrial function resulting in a decrease in glucose and fatty acid oxidation, respiration and an increase in intramuscular triglycerides (IMTG's) and insulin resistance. Peroxisome proliferator-activated receptor (PPAR)-ggr coactivator 1agr (PGC-1agr) is a nuclear transcriptional coactivator which regulates several important metabolic processes including, mitochondrial biogenesis, adaptive thermogenesis, respiration, insulin secretion and gluconeogenesis. In addition, PGC-1agr has been shown to increase the percentage of oxidative type I muscle fibres, with the latter responsible for the majority of insulin stimulated glucose uptake. PGC-1agr also co-activates PPAR's agr, bgr/dgr and ggr which are important transcription factors of genes regulating lipid and glucose metabolism. Exercise causes mitochondrial biogenesis, improves skeletal muscle fatty acid oxidation capacity and insulin sensitivity, therefore making it an important intervention for the treatment of insulin resistance. The expression of PGC-1agr mRNA is reduced in diabetic subjects, however, it is rapidly induced in response to interventions which signal alterations in metabolic requirements, such as exercise. Because of the important role of PGC-1agr in the control of energy metabolism and insulin sensitivity, it is seen as a candidate factor in the etiology of type 2 diabetes and a drug target for its therapeutic treatment.

Improved skeletal muscle oxidative enzyme activity and restoration of PGC-1α and PPARß/δ gene expression upon rosiglitazone treatment in obese patients with type 2 diabetes mellitus

Objective: To examine whether rosiglitazone alters gene expression of some key genes involved in mitochondrial biogenesis and oxidative capacity in skeletal muscle of type 2 diabetic patients, and whether this is associated with alterations in skeletal muscle oxidative capacity and lipid content.

Design: Skeletal muscle gene expression, mitochondrial protein content, oxidative capacity and lipid accumulation were measured in muscle biopsies obtained from diabetic patients, before and after 8 weeks of rosiglitazone treatment, and matched controls. Furthermore, whole-body insulin sensitivity and substrate utilization were assessed.

Subjects: Ten obese type 2 diabetic patients and 10 obese normoglycemic controls matched for age and BMI.

Methods: Gene expression and mitochondrial protein content of complexes I–V of the respiratory chain were measured by quantitative polymerase chain reaction and Western blotting, respectively. Histochemical staining was used to quantify lipid accumulation and complex II succinate dehydrogenase (SDH) activity. Insulin sensitivity and substrate utilization were measured during a hyperinsulinemic–euglycemic clamp with indirect calorimetry.

Results: Skeletal-muscle mRNA of PGC-1a and PPARb/d – but not of other genes involved in glucose, fat and oxidative metabolism – was significantly lower in diabetic patients (Po0.01). Rosiglitazone significantly increased PGC-1a (B2.2-fold, Po0.01) and PPARb/d (B2.6-fold, Po0.01), in parallel with an increase in insulin sensitivity, SDH activity and metabolic flexibility (Po0.01). Surprisingly, none of the measured mitochondrial proteins was reduced in type 2 diabetic patients, nor affected by rosiglitazone treatment. No alterations were seen in muscular fat accumulation upon treatment.

Conclusion: These results suggest that the insulin-sensitizing effect of rosiglitazone may involve an effect on muscular oxidative capacity, via PGC-1a and PPARb/d, independent of mitochondrial protein content and/or changes in intramyocellular lipid.

Brief intense interval exercise activates AMPK and p38 MAPK signaling and increases the expression of PGC-1α in human skeletal muscle

From a cell signaling perspective, short-duration intense muscular work is typically associated with resistance training and linked to pathways that stimulate growth. However, brief repeated sessions of sprint or high-intensity interval exercise induce rapid phenotypic changes that resemble traditional endurance training. We tested the hypothesis that an acute session of intense intermittent cycle exercise would activate signaling cascades linked to mitochondrialbiogenesis in human skeletal muscle. Biopsies (vastus lateralis) were obtained from six young men who performed four 30-s "all out" exercise bouts interspersed with 4 min of rest (<80 kJ total work). Phosphorylation of AMP-activated protein kinase (AMPK; subunits 1 and 2) and the p38 mitogen-activated protein kinase (MAPK) was higher (P  0.05) immediately after bout 4 vs. preexercise. Peroxisome proliferator-activated receptor- coactivator-1(PGC-1) mRNA was increased approximately twofold above rest after 3 h of recovery (P  0.05); however, PGC-1protein content was unchanged. In contrast, phosphorylation of protein kinase B/Akt (Thr³⁰⁸ and Ser⁴⁷³) tended to decrease, and downstream targets linked to hypertrophy (p70 ribosomal S6 kinase and 4E binding protein 1) were unchanged after exercise and recovery. We conclude that signaling through AMPK and p38 MAPK to PGC-1 may explain in part the metabolic remodeling induced by low-volume intense interval exercise, including mitochondrial biogenesis and an increased capacity for glucose and fatty acid oxidation.

Striated muscle activator of Rho signalling (STARS) is a PGC-1α/oestrogen-related receptor-α target gene and is upregulated in human skeletal muscle after endurance exercise

Exercise improves the ability of skeletal muscle to metabolise fats and sugars. For these improvements to occur the muscle detects a signal caused by exercise, resulting in changes in genes and proteins that control metabolism. We show that endurance exercise increases the amount of a protein called striated muscle activator of Rho signalling (STARS) as well as several other proteins influenced by STARS.We also show that the amount of STARS can be increased by signals directed from proteins called peroxisome proliferator-activated receptor gamma co-activator 1-α (PGC-1α) and oestrogen-related receptor-α (ERRα). We also observed that when we reduce the amount of STARS in muscle cells, we block the ability of PGC-1α/ERRα to increase a gene called carnitine palmitoyltransferase-1β (CPT-1β), which is important for fat metabolism. Our study has shown that the STARS pathway is regulated by endurance exercise. STARS may also play a role in fat metabolism in muscle.

Exercise early in life in rats born small does not normalize reductions in skeletal muscle PGC-1α in adulthood

We have previously shown that 4 wk of exercise training early in life normalizes the otherwise greatly reduced pancreatic β-cell mass in adult male rats born small. The aim of the current study was to determine whether a similar normalization in adulthood of reduced skeletal muscle mitochondrial biogenesis markers and alterations in skeletal muscle lipids of growth-restricted male rats occurs following early exercise training. Bilateral uterine vessel ligation performed on day 18 of gestation resulted in Restricted offspring born small (P < 0.05) compared with both sham-operated Controls and a sham-operated Reduced litter group. Offspring remained sedentary or underwent treadmill running from 5–9 (early exercise) or 20–24 (later exercise) wk of age. At 24 wk of age, Restricted and Reduced litter offspring had lower (P < 0.05) skeletal muscle peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) protein expression compared with Control offspring. Early exercise training had the expected effect of increasing skeletal muscle markers of mitochondrial biogenesis, but, at this early age (9 wk), there was no deficit in Restricted and Reduced litter skeletal muscle mitochondrial biogenesis. Unlike our previous observations in pancreatic β-cell mass, there was no “reprogramming” effect of early exercise on adult skeletal muscle such that PGC-1α was lower in adult Restricted and Reduced litter offspring irrespective of exercise training. Later exercise training increased mitochondrial biogenesis in all groups. In conclusion, although the response to exercise training remains intact, early exercise training in rats born small does not have a reprogramming effect to prevent deficits in skeletal muscle markers of mitochondrial biogenesis in adulthood.

The role of PGC-1α and PGC-1β in myotube protein synthesis

Skeletal muscle mass is regulated by a balance between protein synthesis and protein degradation. Using molecular techniques, this PhD thesis identified that two proteins, PGC-1α and PGC-1β, can increase protein synthesis in myotubes in vitro. This has implications for the treatment of diseases associated with skeletal muscle atrophy.

PGC-1α and PGC-1β increase CrT expression and creatine uptake in myotubes via ERRα

Intramuscular creatine plays a crucial role in maintaining skeletal muscle energy homeostasis, and its entry into the cell is dependent upon the sodium chloride dependent Creatine Transporter (CrT; Slc6a8). CrT activity is regulated by a number of factors including extra- and intracellular creatine concentrations, hormones, changes in sodium concentration, and kinase activity, however very little is known about the regulation of CrT gene expression. The present study aimed to investigate how Creatine Transporter (CrT) gene expression is regulated in skeletal muscle. Within the first intron of the CrT gene, we identified a conserved sequence that includes the motif recognized by the Estrogen-related receptor α (ERRα), also known as an Estrogen-related receptor response element (ERRE). Additional ERREs confirming to the known consensus sequence were also identified in the region upstream of the promoter. When partnered with peroxisome proliferator-activated receptor-gamma co-activator-1alpha (PGC-1α) or beta (PGC-1β), ERRα induces the expression of many genes important for cellular bioenergetics. We therefore hypothesized that PGC-1 and ERRα could also regulate CrT gene expression and creatine uptake in skeletal muscle. Here we show that adenoviral overexpression of PGC-1α or PGC-1β in L6 myotubes increased CrT mRNA (2.1 and 1.7-fold, P<0.0125) and creatine uptake (1.8 and 1.6-fold, P<0.0125), and this effect was inhibited with co-expression of shRNA for ERRα. Overexpression of a constitutively active ERRα (VP16-ERRα) increased CrT mRNA approximately 8-fold (P<0.05), resulting in a 2.2-fold (P<0.05) increase in creatine uptake. Lastly, chromatin immunoprecipitation assays revealed that PGC-1α and ERRα directly interact with the CrT gene and increase CrT gene expression.

Altering the redox state of skeletal muscle by glutathione depletion increases the exercise-activation of PGC-1α

We investigated the relationship between markers of mitochondrial biogenesis, cell signaling, and antioxidant enzymes by depleting skeletal muscle glutathione with diethyl maleate (DEM) which resulted in a demonstrable increase in oxidative stress during exercise. Animals were divided into six groups: (1) sedentary control rats; (2) sedentary rats + DEM; (3) exercise control rats euthanized immediately after exercise; (4) exercise rats + DEM; (5) exercise control rats euthanized 4 h after exercise; and (6) exercise rats + DEM euthanized 4 h after exercise. Exercising animals ran on the treadmill at a 10% gradient at 20 m/min for the first 30 min. The speed was then increased every 10 min by 1.6 m/min until exhaustion. There was a reduction in total glutathione in the skeletal muscle of DEM treated animals compared to the control animals (P < 0.05). Within the control group, total glutathione was higher in the sedentary group compared to after exercise (P < 0.05). DEM treatment also significantly increased oxidative stress, as measured by increased plasma F2-isoprostanes (P < 0.05). Exercising animals given DEM showed a significantly greater increase in peroxisome proliferator activated receptor γ coactivator-1α (PGC-1α) mRNA compared to the control animals that were exercised (P < 0.05). This study provides novel evidence that by lowering the endogenous antioxidant glutathione in skeletal muscle and inducing oxidative stress through exercise, PGC-1α gene expression was augmented. These findings further highlight the important role of exercise induced oxidative stress in the regulation of mitochondrial biogenesis.

New gene targets of PGC-1α and ERRα co-regulation in C2C12 myotubes

As a transcriptional coactivator, PGC-1α contributes to the regulation of a broad range of metabolic processes in skeletal muscle health and disease; however, there is limited information about the genes it transcriptionally regulates. To identify new potential gene targets of PGC-1α regulation, mouse C2C12 myotubes were screened by microarray analysis following PGC-1α overexpression. Genes with an mRNA expression of 2.5-fold or more (P < 0.001) were identified. From these, further genes were singled out if they had no previous connection to PGC-1α regulation or characterization in skeletal muscle, or were unannotated with no known function. Following confirmation of their regulation by PGC-1α using qPCR analysis, eight genes were focused on for further investigation (Akr1b10, Rmnd1, 1110008P14Rik, 1700021F05Rik, Mtfp1, Mrm1, Oxnad1 and Cluh). Bioinformatics indicated a number of the genes were linked to a range of metabolic-related functions including fatty acid oxidation, oxido-reductase activity, and mitochondrial remodeling and transport. Treating C2C12 myotubes for 6 h with AICAR, a known activator of AMP kinase and inducer of Pgc-1α gene expression, increased the mRNA levels of both Pgc-1α (P < 0.001) and of Mtfp1, Mrm1, Oxnad1 and Cluh (P < 0.05). Screening of the promoter and intron 1 regions also revealed all genes to contain either a consensus or near consensus response elements for the estrogen-related receptor α (ERRα), a key transcription factor-binding partner of PGC-1α in skeletal muscle. Furthermore, knockdown of endogenous ERRα levels partially or completely blocked the induction of gene expression of all genes by PGC-1α, while each gene was significantly upregulated in the presence of a constitutively active form of ERRα (P < 0.05) except for Akr1b10. These findings provide preliminary evidence for the novel regulation of these genes by PGC-1α and its signaling pathway in skeletal muscle.

Postexercise muscle cooling enhances gene expression of PGC-1α

This study aimed to investigate the influence of localized muscle cooling on postexercise vascular, metabolic, and mitochondrial-related gene expression.

Treadmill Training Increases SIRT-1 and PGC-1 alpha Protein Levels and AMPK Phosphorylation in Quadriceps of Middle-Aged Rats in an Intensity-Dependent Manner

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)