Increase In Mitochondrial Enzymes Research Articles

PPARβ Is Essential for Maintaining Normal Levels of PGC-1α and Mitochondria and for the Increase in Muscle Mitochondria Induced by Exercise

The objective of this study was to evaluate the specific mechanism(s) by which PPARβ regulates mitochondrial content in skeletal muscle. We discovered that PPARβ increases PGC-1α by protecting it from degradation by binding to PGC-1α and limiting ubiquitination. PPARβ also induces an increase in nuclear respiratory factor 1 (NRF-1) expression, resulting in increases in mitochondrial respiratory chain proteins and MEF2A, for which NRF-1 is a transcription factor. There was also an increase in AMP kinase phosphorylation mediated by an NRF-1-induced increase in CAM kinase kinase-β (CaMKKβ). Knockdown of PPARβ resulted in large decreases in the levels of PGC-1α and mitochondrial proteins and a marked attenuation of the exercise-induced increase in mitochondrial biogenesis. In conclusion, PPARβ induces an increase in PGC-1α protein, and PPARβ is a transcription factor for NRF-1. Thus, PPARβ plays essential roles in the maintenance and adaptive increase in mitochondrial enzymes in skeletal muscle by exercise.

Sensing and responding to energetic stress: The role of the AMPK-PGC1α-NRF1 axis in control of mitochondrial biogenesis in fish

Remodeling the muscle metabolic machinery in mammals in response to energetic challenges depends on the energy sensor AMP-activated protein kinase (AMPK) and its ability to phosphorylate PPAR γ coactivator 1 α (PGC1α), which in turn coactivates metabolic genes through direct and indirect association with DNA-binding proteins such as the nuclear respiratory factor 1 (NRF1) (Wu et al., 1999). The integrity of this axis in fish is uncertain because PGC1α i) lacks the critical Thr177 targeted by AMPK and ii) has mutations that may preclude binding NRF1. In this study we found no evidence that AMPK regulates mitochondrial gene expression through PGC1α in zebrafish and goldfish. AICAR treatment of zebrafish blastula cells increased phosphorylation of AMPK and led to changes in transcript levels of the AMPK targets mTOR and hexokinase 2. However, we saw no increases in mRNA levels for genes associated with mitochondrial biogenesis, including PGC1α, NRF1, and COX7C, a cytochrome c oxidase subunit. Further, AMPK phosphorylated mammalian peptides of PGC1α but not the corresponding region of zebrafish or goldfish in vitro. In vivo cold acclimation of goldfish caused an increase in mitochondrial enzymes, AMP and ADP levels, however AMPK phosphorylation decreased. In fish, the NRF1-PGC1α axis may be disrupted due to insertions in fish PGC1α orthologs within the region that serves as NRF1 binding domain in mammals. Immunocopurification showed that recombinant NRF1 protein binds mammalian but not fish PGC1α. Collectively, our studies suggest that fish have a disruption in the AMPK-PGC1α-NRF1 pathway due to structural differences between fish and mammalian PGC1α.

Physiology and pathophysiology of musculoskeletal aging: current research trends and future priorities

EDITORIAL article Front. Physiol., 09 April 2013Sec. Striated Muscle Physiology Volume 4 - 2013 | https://doi.org/10.3389/fphys.2013.00073

Proteomic Profiling and Its Applications to Muscle Aging and Sarcopenia

Proteomic Profiling and Its Applications to Muscle Aging and Sarcopenia

Variability in the magnitude of response of metabolic enzymes reveals patterns of co-ordinated expression following endurance training in women

Skeletal muscles improve their oxidative fatty acid and glucose metabolism following endurance training, but the magnitude of response varies considerably from person to person. In 20 untrained young women we examined interindividual variability in training responses of metabolic enzymes following 6 weeks of endurance training, sufficient to increase maximal oxygen uptake by 10 ± 8% (mean ± SD). Training led to increases in mitochondrial enzymes [succinate dehydrogenase (SDH; 47 ± 78%), cytochrome c oxidase (52 ± 70%) and ATP synthase (63 ± 69%)] and proteins involved in fatty acid metabolism [3-hydroxyacyl CoA dehydrogenase (69 ± 92%) and fatty acid transporter CD36 (86 ± 31%)]. Increases in enzymes of glucose metabolism [phosphofructokinase (29 ± 94%) and glucose transporter 4 (18 ± 65%)] were not significant. There was no relationship between changes in maximal oxygen uptake and the changes in the metabolic proteins. Considerable interindividual variability was seen in the magnitude of responses. The response of each enzyme was proportional to the change in SDH; individuals with a large increase in SDH also showed high gains in all other enzymes, and vice versa. Peroxisome proliferator-activated receptor γ coactivator 1α protein content increased after training, but was not correlated with changes in the metabolic proteins. In conclusion, the results revealed co-ordinated adaptation of several metabolic enzymes following endurance training, despite differences between people in the magnitude of response. Differences between individuals in the magnitude of response might reflect the influence of environmental and genetic factors that govern training adaptations.

Reductions in RIP140 are not required for exercise and high fat diet mediated increases in mitochondrial enzymes

The over-expression of RIP140 reduces the expression of mitochondrial enzymes whereas the deletion of RIP140 increases mitochondrial content. Despite the importance of RIP140 in the control of mitochondrial biogenesis the effects of physiological perturbations on regulating RIP140 in skeletal muscle has not been determined. To this end, rats were exercised by 2 hours of daily swim training for 14 consecutive days or fed a high fat diet (HFD) for 6 weeks. In an additional study 6 healthy college aged males completed 6 sessions of high intensity exercise training consisting of 8–12 X 1 min sprints on a stationary bicycle at 100% of peak power. Exercise training increased the protein contents of CORE1 (48% increase), COXI (91% increase) and COXIV (30% increase) and citrate synthase activity (45% increase) while having no effect on RIP140 mRNA levels and protein content in rat triceps. Similarly, high intensity exercise training in humans did not cause reductions in the nuclear protein content of RIP140. The consumption of a HFD increased the protein contents of CORE1 (72% increase) and COXIV (53% increase) and the enzyme activities of citrate synthase (26% increase) and beta HAD (71% increase). RIP140 mRNA and protein content was not reduced by the consumption of a HFD. Collectively our results demonstrate that reductions in RIP140 are not required for the induction of mitochondrial biogenesis in skeletal muscle.

Regulating the regulators: the role of transcriptional regulatory proteins in the adaptive response to exercise in human skeletal muscle

Regulating the regulators: the role of transcriptional regulatory proteins in the adaptive response to exercise in human skeletal muscle

Comparative studies on carbofuran-induced changes in some cytoplasmic and mitochondrial enzymes and proteins of epigeic, anecic and endogeic earthworms

Exposure of epigeic ( Perionyx sansibaricus), anecic ( Lampito mauritii) and endogeic ( Metaphire posthuma) earthworms to four different concentrations (10 ppm, 20 ppm, 40 ppm and 80 ppm) of carbofuran for 16 days induced specific activities of cytoplasmic malate dehydrogenase (cMDH), mitochondrial malate dehydrogenase (mMDH) and lactate dehydrogenase (LDH). P. sansibaricus showed 33–52% increase in specific activities of aerobic (cMDH, mMDH) and anaerobic (LDH) enzymes and proteins (cytoplasmic and mitochondrial). In L. mauritii, the enhancement in enzyme and protein level was 30–46%. Similarly, M. posthuma indicated 29–43% increase in profile of enzymes and proteins. The significant increase was observed on 2nd or 3rd day and it was maximum on 16th day of carbofuran exposure. Maximum effect of carbofuran was on epigeic earthworm. The sensitivity of mitochondrial dehydrogenase and protein of different earthworm species to carbofuran appears to be associated with oxygen availability in different strata of soil habitats. Inductions in enzyme specific activity and protein content were concentration and time-dependent. The effect was more pronounced on aerobic enzymes (cMDH, mMDH) as compared to anaerobic (LDH) one. The increases in mitochondrial enzyme (47%) and protein (43%) were more than the cytoplasmic enzymes (35%) and protein (31%), which suggest a greater effect of carbofuran on respiratory metabolism of earthworms. The carbofuran-dependent increase in cytoplasmic and mitochondrial enzymes and proteins might be due to increased synthesis of metabolic enzymes and stress proteins.

Raising plasma fatty acid concentration induces increased biogenesis of mitochondria in skeletal muscle.

A number of studies have reported that a high-fat diet induces increases in mitochondrial fatty acid oxidation enzymes in muscle. In contrast, in two recent studies raising plasma free fatty acids (FFA) resulted in a decrease in mitochondria. In this work, we reevaluated the effects of raising FFA on muscle mitochondrial biogenesis and capacity for fat oxidation. Rats were fed a high-fat diet and given daily injections of heparin to raise FFA. This treatment induced an increase in mitochondrial biogenesis in muscle, as evidenced by increases in mitochondrial enzymes of the fatty acid oxidation pathway, citrate cycle, and respiratory chain, with an increase in the capacity to oxidize fat, as well as an increase in mitochondrial DNA copy number. Raising FFA also resulted in an increase in binding of peroxisome proliferator-activated receptor (PPAR) delta to the PPAR response element on the carnitine palmitoyltransferase 1 promoter. We interpret our results as evidence that raising FFA induces an increase in mitochondrial biogenesis in muscle by activating PPARdelta.

UP-REGULATION OF ANTIOXIDANT ENZYME ACTIVITY FOLLOWING UNILATERAL RESISTANCE EXERCISE TRAINING IN OLDER ADULTS

A prominent theory for aging, the free radical theory, states that reactive oxygen species (ROS), formed as a by-product of normal energy metabolism, may cause cellular damage ultimately lead to a loss of cell function. Resistance exercise training leads to many benefits for the older adults, however, exercise has also been shown to induce oxidative stress. Whether resistance exercise augments or attenuates oxidative stress in older adults remains unknown. PURPOSE To examine the effects of resistance exercise training on markers of oxidative stress, antioxidant enzymes, and mitochondrial enzymes in older adults. METHODS Elderly men (n=12) were recruited to take part in a 10 week unilateral resistance exercise training protocol. Muscle biopsies were taken from the vastus lateralis of the training leg (T) and the non-training (NT) leg before and 48hrs following the last exercise bout. RESULTS Muscle protein carbonyls did not change significantly in the T or NT leg. There was no increase in mitochondrial enzymes (citrate synthase, complex II+III, complex IV) in either the T or NT leg. Catalase activity increased significantly in the trained leg (Pre-8.19 ±2.3, Post-14.88 ±7.6, P=0.02), and did not change in the NT leg. There was a trend towards an increase in Total Superoxide dismutase (SOD) following training (P=0.13), whereas there was no change in Total SOD in the NT leg. CuZn SOD (cytosolic) increased significantly in the T leg (Pre-7.2 ±2.7, Post-12.6 ±5.5, P=0.002), and remained unchanged in the NT leg, and MnSOD (mitochondrial) did not change in either leg. CONCLUSION These results suggest that, in older adults, resistance exercise training does not lead to an increase in oxidative stress, potentially due to significant increases in, predominantly cytosolic, antioxidant enzyme activity. Furthermore, 10 weeks of resistance training in older adults does not induce adaptations at the mitochondrial level. This work was supported by NSERC.

Adaptations of skeletal muscle to exercise: rapid increase in the transcriptional coactivator PGC-1.

Endurance exercise induces increases in mitochondria and the GLUT4 isoform of the glucose transporter in muscle. Although little is known about the mechanisms underlying these adaptations, new information has accumulated regarding how mitochondrial biogenesis and GLUT4 expression are regulated. This includes the findings that the transcriptional coactivator PGC-1 promotes mitochondrial biogenesis and that NRF-1 and NRF-2 act as transcriptional activators of genes encoding mitochondrial enzymes. We tested the hypothesis that increases in PGC-1, NRF-1, and NRF-2 are involved in the initial adaptive response of muscle to exercise. Five daily bouts of swimming induced increases in mitochondrial enzymes and GLUT4 in skeletal muscle in rats. One exercise bout resulted in approximately twofold increases in full-length muscle PGC-1 mRNA and PGC-1 protein, which were evident 18 h after exercise. A smaller form of PGC-1 increased after exercise. The exercise induced increases in muscle NRF-1 and NRF-2 that were evident 12 to 18 h after one exercise bout. These findings suggest that increases in PGC-1, NRF-1, and NRF-2 represent key regulatory components of the stimulation of mitochondrial biogenesis by exercise and that PGC-1 mediates the coordinated increases in GLUT4 and mitochondria.

Intermittent increases in cytosolic Ca2+ stimulate mitochondrial biogenesis in muscle cells.

Muscle contractions cause numerous disturbances in intracellular homeostasis. This makes it impossible to use contracting muscle to identify which of the many signals generated by contractions are responsible for stimulating mitochondrial biogenesis. One purpose of this study was to evaluate the usefulness of L6 myotubes, which do not contract, for studying mitochondrial biogenesis. A second purpose was to evaluate further the possibility that increases in cytosolic Ca2+ can stimulate mitochondrial biogenesis. Continuous exposure to 1 microM ionomycin, a Ca2+ ionophore, for 5 days induced an increase in mitochondrial enzymes but also caused a loss of myotubes, as reflected in an approximately 40% decrease in protein per dish. However, intermittent (5 h/day) exposure to ionomycin, or to caffeine or W7, which release Ca2+ from the sarcoplasmic reticulum, did not cause a decrease in protein per dish. Raising cytosolic Ca2+ intermittently with these agents induced significant increases in mitochondrial enzymes. EGTA blocked most of this effect of ionomycin, whereas dantrolene, which blocks Ca2+ release from the sarcoplasmic reticulum, largely prevented the increases in mitochondrial enzymes induced by W7 and caffeine. These findings provide evidence that intermittently raising cytosolic Ca2+ stimulates mitochondrial biogenesis in muscle cells.

Effect of physical activity on thiamine, riboflavin, and vitamin B-6 requirements

Because exercise stresses metabolic pathways that depend on thiamine, riboflavin, and vitamin B-6, the requirements for these vitamins may be increased in athletes and active individuals. Theoretically, exercise could increase the need for these micronutrients in several ways: through decreased absorption of the nutrients; by increased turnover, metabolism, or loss of the nutrients; through biochemical adaptation as a result of training that increases nutrient needs; by an increase in mitochondrial enzymes that require the nutrients; or through an increased need for the nutrients for tissue maintenance and repair. Biochemical evidence of deficiencies in some of these vitamins in active individuals has been reported, but studies examining these issues are limited and equivocal. On the basis of metabolic studies, the riboflavin status of young and older women who exercise moderately (2.5-5 h/wk) appears to be poorer in periods of exercise, dieting, and dieting plus exercise than during control periods. Exercise also increases the loss of vitamin B-6 as 4-pyridoxic acid. These losses are small and concomitant decreases in blood vitamin B-6 measures have not been documented. There are no metabolic studies that have compared thiamine status in active and sedentary persons. Exercise appears to decrease nutrient status even further in active individuals with preexisting marginal vitamin intakes or marginal body stores. Thus, active individuals who restrict their energy intake or make poor dietary choices are at greatest risk for poor thiamine, riboflavin, and vitamin B-6 status.

Identical responses of fast muscle to sustained activity by low-frequency stimulation in young and aging rats.

To investigate effects of sustained activity on major phenotypic properties, the left extensor digitorum longus muscle of young (15 wk) and aging (101 wk) male Brown Norway rats was subjected to 50 days of chronic low-frequency stimulation (CLFS; 10 Hz, 10 h/day). The contralateral muscle served as control. Changes in metabolic enzymes were analyzed by using glyceraldehyde-3-phosphate dehydrogenase and lactate dehydrogenase as reference enzymes of glycolysis and by using citrate synthase and 3-hydroxyacyl-CoA dehydrogenase as mitochondrial enzymes representative of aerobic-oxidative metabolism. Myosin heavy chain (MHC) isoforms were analyzed by SDS-PAGE. No differences existed between the enzyme activity profiles of control muscles from young and aging rats. CLFS induced similar increases in mitochondrial enzymes, as well as similar decreases in glycolytic enzymes. Although the MHC composition of the control muscles in the aging rats displayed a shift toward slower isoforms, the ultimate changes induced by CLFS led to nearly identical MHC phenotypes in both young and aging rats. These results demonstrate an unaltered adaptability of skeletal muscle to increased neuromuscular activity in the aging rat.

Sequential increases in capillarization and mitochondrial enzymes in low-frequency-stimulated rabbit muscle.

To investigate temporal changes in capillarization and increases in mitochondrial enzyme activity, rabbit tibialis anterior muscles underwent chronic low-frequency stimulation for up to 50 days. Capillary density (CD), capillary-to-fiber ratio (C/F), intercapillary distance (ICD), and mean capillary area (MCA), as well as several other parameters of capillarization, were examined. In addition, tissue levels of mRNA specific to vascular endothelial growth factor (VEGF) were assessed by reverse transcriptase-polymerase chain reaction. Citrate synthase (CS) activity, a marker of aerobic-oxidative metabolic potential, was measured in the same muscles. Significant increases in CD and C/F, respectively, and decreases in ICD and MCA were observed after 2 days. These changes reached stable maxima by 14 days. The increases in capillarization occurred in a fiber-type-specific manner, affecting type IId fibers before types IIda and IIa. VEGF mRNA levels increased in a bimodal time pattern with a first elevation (2.5-fold) after 1 day and a second (9-fold) after 6-8 days. Increases in CS were first noted after 8 days. Obviously, increases in capillarization as induced by enhanced contractile activity precede increases in the aerobic-oxidative potential of energy metabolism.

Mitochondrial enzymes increase in muscle in response to 7-10 days of cycle exercise.

Endurance exercise training induces a significant increase in the respiratory capacity of skeletal muscle. This is reflected by a training-induced increase in mitochondrial enzyme activity. One consequence of this adaptation is that there is a decreased reliance on carbohydrate utilization with a concomitant increase in fat utilization, resulting in an improvement in endurance capacity. Recently it has been reported that 7-14 days of cycle ergometer exercise training does not induce an increase in mitochondrial enzyme levels in skeletal muscle but, nevertheless, results in smaller decreases in phosphocreatine and glycogen and smaller increases in Pi and lactate in muscle in response to the same exercise after compared with before training. However, previous studies in rats have shown that an adaptive increase in mitochondrial enzymes is already evident after only 2 days of exercise training. In view of this discrepency, the present study was performed to reevaluate the effect of short-term training (7-10 days) on mitochondrial enzymes in skeletal muscle of humans. Twelve subjects [6 men and 6 women, 27 +/- 5 (SE) yr old] underwent 7 (n = 5) or 10 days (n = 7) of cycle ergometer exercise for 2h/day at 60-70% of peak O2 consumption. Peak O2 consumption was increased by 9% (from 2.97 +/- 0.16 to 3.24 +/- 0.17 l/min) in response to training. Blood lactate levels were lower at the same absolute work rates after than before training. The activities of citrate synthase, beta-hydroxyacyl-CoA dehydrogenase, mitochondrial thiolase, and carnitine acetyltransferase were increased by approximately 30% in response to training. The results of the present study provide evidence that in humans, as in rats, the adaptive increase in mitochondrial enzymes in skeletal muscle occurs fairly rapidly in response to exercise training. They provide no support for the claim that this adaptive response is delayed for > 2 wk after the onset of training.

Muscle lipoprotein lipase activity in voluntarily exercising rats.

Voluntary exercise of rats in freely rotating work wheels has been extensively used, but muscle adaptations that result from such exercise training are poorly documented. The purpose of this study was to determine whether the exercise performed by voluntarily active rats would increase succinate dehydrogenase or lipoprotein lipase activities in the soleus muscle (SM) or the red portion of the vastus lateralis muscle (RV). In SM the activities of these two enzymes were not increased after 7 or 16 wk of voluntary exercise. Succinate dehydrogenase activity in RV was moderately increased after 7 and 16 wk of voluntary activity (P less than 0.05). Substantial increases occurred in RV lipoprotein lipase activity (P less than 0.01). The increase in RV lipoprotein lipase activity was positively related to distance run by the rats. The results indicate that only small muscle-dependent increases in mitochondrial enzymes occur in rats allowed to exercise voluntarily in rodent work wheels. Voluntary exercise training induced a selective increase in lipoprotein lipase activity in a muscle containing a high percentage of fast-twitch red fibers, a response absent in a muscle containing a predominance of slow-twitch red fibers. It is unlikely that this differential response can be explained by exercise-induced changes in plasma hormone concentrations involved in the regulation of lipoprotein lipase.

Superoxide dismutase and catalase in skeletal muscle: adaptive response to exercise.

Cellular damage caused by free radical reactions may play a role in the aging process. A bout of exercise can increase free radical concentration with damage to mitochondria in muscle (Davies et al., 1982). This study was undertaken to determine if muscle adapts to exercise training with an enhancement of enzymatic defenses against free radical damage. A program of running that induced two-fold increases in mitochondrial enzymes in leg muscles of rats resulted in no increase in catalase or cytoplasmic superoxide dismutase (SOD) activities. Mitochondrial SOD activity was increased 37% in fast-twitch red and slow-twitch red types of muscle and 14% in white muscle. Thus, despite an increase in mitochondrial SOD, the ratio of SOD to mitochondrial citrate cycle and respiratory chain enzymes was decreased. It seems unlikely that increased capacity for enzymatic scavenging of superoxide radical is a major protective adaptation against free radical damage in exercise-trained muscle.

Maintenance of the adaptation of skeletal muscle mitochondria to exercise in old rats

The purpose of this study was to determine whether the adaptive increase in mitochondrial enzymes in skeletal muscle induced by a constant exercise program is altered by aging. Male pathogen-free Long-Evans rats were exercised by means of swimming for 3h/d, 5d/wk beginning at age 6 months and studied at ages 9 and 24 months. The levels of activity of the four mitochondrial enzymes used as indicators of the capacity for aerobic metabolism (citrate synthase, succinate DH, 3-hydroxyacyl-CoA-DH, and 3-ketoacid-CoA-transferase) were significantly increased in epitrochlearis muscle in both the 9-month and 24-month-old swimmers. The increases ranged from approximately 20% for 3-hydroxyacyl-CoA-DH to 50-60% for 3-ketoacid-CoA-transferase. Neither the 24-month-old sedentary rats nor the 24-month-old swimmers had significantly lower muscle enzyme levels than the comparable 9-month-old rats. Thus, it appears that aging does not result in a progressive decline in the capacity of muscle for aerobic metabolism or a progressive impairment in the ability to maintain an increase in muscle mitochondrial enzymes in response to chronic exercise in healthy rats.

Adaptation of the mitochondrial systems of Rhodotorula gracilis to low oxygen pressure

The obligate aerobic yeast, Rhodotorula gracilis, was grown in a liquid minimal medium at 1 mm Hg partial pressure of oxygen, conditions under which growth (measured as the increase in total protein) is reduced to 30% of the maximum rate. A significant increase in the ratio between mitochondrial oxidative enzymes and total protein occurs rapidly under these conditions. A concurrent increase in the ratio area of mitochondria/area of cytoplasm was also observed. The relative increase in mitochondrial enzymes, oxidase activities and mitochondrial membranes is due to the inhibition affecting the increase in cytoplasmic structures more significantly than the increase in mitochondrial structures at low pO 2. The difference between mitochondrial and cytoplasmic syntheses cannot be ascribed to changes in the availability of ATP but it might rest with some other oxygen-utilising process (pyrimidine redox coenzymes, synthesis of sterols). The experimental conditions studied appear to offer a valuable tool for the investigation of the relationships between mitochondrial and cytoplasmic structures.