Muscle fibres: applications for the study of the metabolic consequences of enzyme deficiencies in skeletal muscle

2000 ◽  
Vol 28 (2) ◽  
pp. 159-164 ◽  
Author(s):  
S. Vielhaber ◽  
A. Kudin ◽  
R. Schröder ◽  
C. E. Elger ◽  
W. S. Kunz
Keyword(s):  
Skeletal Muscle ◽  
Large Scale ◽  
Citrate Synthase ◽  
Quantitative Estimation ◽  
Phenotypic Expression ◽  
Metabolic Effects ◽  
Progressive External Ophthalmoplegia ◽  
Muscle Fibres ◽  
Control Analysis ◽  
Mitochondrial Enzymes

Mitochondrial function in saponin-permeabilized muscle fibres can be studied by high-resolution respirometry, laser-excited fluorescence spectroscopy and fluorescence microscopy. We applied these techniques to study metabolic effects of changes in the pattern of mitochondrial enzymes in skeletal muscle of patients with chronic progressive external ophthalmoplegia or Kearns-Sayre syndrome harbouring large-scale deletions of mitchondrial DNA (mtDNA). In all patients combined deficiencies of respiratory chain enzymes containing mitochondrially encoded subunits were observed. The citrate synthase-normalized activity ratios of these enzymes decreased linearly with increasing mtDNA heteroplasmy. This indicates the absence of any well-defined mutation thresholds for mitochondrial enzyme activities in the entire skeletal muscle. We applied metabolic control analysis to perform a quantitative estimation of the metabolic influence of the observed enzyme deficiencies. For patients with degrees of mtDNA heteroplasmy below about 60% we observed at almost normal maximal rates of respiration an increase in flux control coefficients of complexes I and IV. Permeabilized skeletal-muscle fibres of patients with higher degrees of mtDNA heteroplasmy and severe enzyme deficiencies exhibited additionally decreased maximal rates of respiration. This finding indicates the presence of a ‘metabolic threshold’ which can be assessed by functional studies of muscle fibres providing the link to the phenotypic expression of the mtDNA mutation in skeletal muscle.

scholarly journals Concerted action of two novel tRNA mtDNA point mutations in chronic progressive external ophthalmoplegia

2008 ◽  
Vol 28 (2) ◽  
pp. 89-96 ◽  
Author(s):  
Cornelia Kornblum ◽  
Gábor Zsurka ◽  
Rudolf J. Wiesner ◽  
Rolf Schröder ◽  
Wolfram S. Kunz
Keyword(s):  
Skeletal Muscle ◽  
Northern Blot ◽  
Phenotypic Expression ◽  
Point Mutations ◽  
Skeletal Muscle Fibre ◽  
Progressive External Ophthalmoplegia ◽  
Chronic Progressive External Ophthalmoplegia ◽  
Mtdna Mutations ◽  
Mtdna Sequence ◽  
Functional Consequences

CPEO (chronic progressive external ophthalmoplegia) is a common mitochondrial disease phenotype in adults which is due to mtDNA (mitochondrial DNA) point mutations in a subset of patients. Attributing pathogenicity to novel tRNA mtDNA mutations still poses a challenge, particularly when several mtDNA sequence variants are present. In the present study we report a CPEO patient for whom sequencing of the mitochondrial genome revealed three novel tRNA mtDNA mutations: G5835A, del4315A, T1658C in tRNATyr, tRNAIle and tRNAVal genes. In skeletal muscle, the tRNAVal and tRNAIle mutations were homoplasmic, whereas the tRNATyr mutation was heteroplasmic. To address the pathogenic relevance, we performed two types of functional tests: (i) single skeletal muscle fibre analysis comparing G5835A mutation loads and biochemical phenotypes of corresponding fibres, and (ii) Northern-blot analyses of mitochondrial tRNATyr, tRNAIle and tRNAVal. We demonstrated that both the G5835A tRNATyr and del4315A tRNAIle mutation have serious functional consequences. Single-fibre analyses displayed a high threshold of the tRNATyr mutation load for biochemical phenotypic expression at the single-cell level, indicating a rather mild pathogenic effect. In contrast, skeletal muscle tissue showed a severe decrease in respiratory-chain activities, a reduced overall COX (cytochrome c oxidase) staining intensity and abundant COX-negative fibres. Northern-blot analyses showed a dramatic reduction of tRNATyr and tRNAIle levels in muscle, with impaired charging of tRNAIle, whereas tRNAVal levels were only slightly decreased, with amino-acylation unaffected. Our findings suggest that the heteroplasmic tRNATyr and homoplasmic tRNAIle mutation act together, resulting in a concerted effect on the biochemical and histological phenotype. Thus homoplasmic mutations may influence the functional consequences of pathogenic heteroplasmic mtDNA mutations.

Response of mitochondria of different types of skeletal muscle to thyrotoxicosis

1977 ◽  
Vol 232 (5) ◽  
pp. C180-C184 ◽  
Author(s):  
W. W. Winder ◽  
J. O. Holloszy
Keyword(s):  
Skeletal Muscle ◽  
Citrate Synthase ◽  
Differential Sensitivity ◽  
Mitochondrial Enzymes ◽  
Muscle Mitochondria ◽  
Glycerophosphate Dehydrogenase ◽  
Transport Chain ◽  
Different Types ◽  
Fast Twitch

To determine the effect of long-term thyrotoxicosis on muscle mitochondria, we measured representative mitochondrial enzymes from three different types of skeletal muscle (fast-twitch red and fast-twitch white from the quadriceps, and slow-twitch red from the soleus) in rats given 3 mg L-thyroxine and 1 mg triiodo-L-thyronine per kilogram of diet for 12 wk. Marker enzymes of the electron transport chain and citric acid cycle (cytochrome oxidase, cytochrome c, and citrate synthase) increase approximately twofold in soleus muscle in response to this treatment. The fast-twitch muscles exhibit no more than 44% increases in these enzymes in response to the same treatment. Relative to initial concentration, 3-hydroxybutyrate dehydrogenase increased to the same extent in fast-twitch red muscle as it did in the soleus (70%). Mitochondrial alpha-glycerophosphate dehydrogenase increased 76% in red quadriceps and 170% in soleus, but did not change in white muscle in the thyrotoxic rats. This differential sensitivity of the three types of muscle provides a tool for studying the mechanisms underlying the action of thyroid hormones on muscle mitochondria.

High-intensity interval training increases SIRT1 activity in human skeletal muscle

2010 ◽  
Vol 35 (3) ◽  
pp. 350-357 ◽  
Author(s):  
Brendon J. Gurd ◽  
Christopher G.R. Perry ◽  
George J.F. Heigenhauser ◽  
Lawrence L. Spriet ◽  
Arend Bonen
Keyword(s):  
Skeletal Muscle ◽  
High Intensity ◽  
Human Skeletal Muscle ◽  
Citrate Synthase ◽  
Interval Training ◽  
Peak Oxygen Consumption ◽  
Peroxisome Proliferator ◽  
Mitochondrial Enzymes ◽  
High Intensity Interval Training ◽  
High Intensity Interval

The effects of training on silent mating-type information regulator 2 homolog 1 (SIRT1) activity and protein in relationship to peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) and mitochondrial content were determined in human skeletal muscle. Six weeks of high-intensity interval training (∼1 h of 10 × 4 min intervals at 90% peak oxygen consumption separated by 2 min rest, 3 days per week) increased maximal activities of mitochondrial enzymes in skeletal muscle by 28% to 36% (citrate synthase, β-hydroxyacyl-coenzyme A dehydrogenase, and cytochrome c oxidase subunit IV) and PGC-1α protein (16%) when measured 4 days after training. Interestingly, total muscle SIRT1 activity (31%) and activity per SIRT1 protein (58%) increased despite decreased SIRT1 protein (20%). The present data demonstrate that exercise-induced mitochondrial biogenesis is accompanied by elevated SIRT1 activity in human skeletal muscle.

scholarly journals IL-15 Mediates Mitochondrial Activity through a PPARδ-Dependent-PPARα-Independent Mechanism in Skeletal Muscle Cells

PPAR Research ◽  
2016 ◽  
Vol 2016 ◽  
pp. 1-12 ◽  
Author(s):  
Shantaé M. Thornton ◽  
James E. Krolopp ◽  
Marcia J. Abbott
Keyword(s):  
Skeletal Muscle ◽  
Citrate Synthase ◽  
Metabolic Effects ◽  
Mitochondrial Activity ◽  
Mrna Levels ◽  
Metabolic Processes ◽  
Atp Production ◽  
Positive Effects ◽  
Increase Energy Expenditure ◽  
Interleukin 15

Molecular mediators of metabolic processes, to increase energy expenditure, have become a focus for therapies of obesity. The discovery of cytokines secreted from the skeletal muscle (SKM), termed “myokines,” has garnered attention due to their positive effects on metabolic processes. Interleukin-15 (IL-15) is a myokine that has numerous positive metabolic effects and is linked to the PPAR family of mitochondrial regulators. Here, we aimed to determine the importance of PPARαand/or PPARδas targets of IL-15 signaling. C2C12 SKM cells were differentiated for 6 days and treated every other day with IL-15 (100 ng/mL), a PPARαinhibitor (GW-6471), a PPARδinhibitor (GSK-3787), or both IL-15 and the inhibitors. IL-15 increased mitochondrial activity and induced PPARα, PPARδ, PGC1α, PGC1β, UCP2, and Nrf1 expression. There was no effect of inhibiting PPARα, in combination with IL-15, on the aforementioned mRNA levels except for PGC1βand Nrf1. However, with PPARδinhibition, IL-15 failed to induce the expression levels of PGC1α, PGC1β, UCP2, and Nrf1. Further, inhibition of PPARδabolished IL-15 induced increases in citrate synthase activity, ATP production, and overall mitochondrial activity. IL-15 had no effects on mitochondrial biogenesis. Our data indicates that PPARδactivity is required for the beneficial metabolic effects of IL-15 signaling in SKM.

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

2012 ◽  
Vol 302 (12) ◽  
pp. E1541-E1549 ◽  
Author(s):  
Erin J. Stephenson ◽  
Donny M. Camera ◽  
Trisha A. Jenkins ◽  
Sepideh Kosari ◽  
Jong Sam Lee ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Energy Demand ◽  
Citrate Synthase ◽  
Blood Glucose Concentration ◽  
Control Diet ◽  
Fasting Blood Glucose ◽  
Western Diet ◽  
Mitochondrial Enzymes ◽  
Serum Triglycerides ◽  
Respiratory Capacity

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.

scholarly journals Methodological considerations when assessing mitochondrial respiration and biomarkers for mitochondrial content in human skeletal muscle

2021 ◽  
Author(s):  
Jujiao Kuang ◽  
Nicholas J Saner ◽  
Javier Botella ◽  
Matthew J-C Lee ◽  
Cesare Granata ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Mitochondrial Respiration ◽  
Coefficient Of Variation ◽  
Human Skeletal Muscle ◽  
Citrate Synthase ◽  
Membrane Integrity ◽  
Mitochondrial Outer Membrane ◽  
Muscle Fibres ◽  
Biological Variability ◽  
Mitochondrial Content

Background: The assessment of mitochondrial respiration and mitochondrial content are two common measurements in the fields of skeletal muscle research and exercise science. However, to verify the validity of the observed changes in both mitochondrial respiration and mitochondrial content following an intervention such as exercise training, it is important to determine the reliability and reproducibility of the experimental design and/or techniques employed. We examined the repeatability of widely used methodologies for assessing mitochondrial respiration and mitochondrial content, respectively; the measurement of maximal mitochondrial oxidative phosphorylation in permeabilized muscle fibres using high-resolution respirometry, and the measurement of citrate synthase activity as a biomarker for mitochondrial content in a microplate with spectrophotometer. Result: For mitochondrial respiration, the coefficient of variation for repeated measurements using muscle sampled from same biopsy decreased from 12.7% to 11% when measured in triplicate with outliers excluded, rather than in duplicate. The coefficient of variation was 9.7% for repeated muscle biopsies sampled across two separated days. For measurements of citrate synthase activity, the coefficient of variation was 3.5% of three technical repeats on the same plate, 10.2% for duplicate analyses using the same muscle lysate when conducted in the same day, and 30.5% when conducted four weeks apart. Conclusion: We have provided evidence for important technical considerations when measuring mitochondrial respiration with human skeletal muscle: 1) the relatively large technical variability can be reduced by increasing technical repeats and excluding outliers; 2) the biological variability and absolute mitochondrial respiration value of the participants should be considered when estimating the required sample size; 3) a new threshold of 15% for the increase in respiration rate after the addition of cytochrome c test for testing mitochondrial outer membrane integrity. When analysing citrate synthase activity, our evidence suggests it is important to consider the following: 1) all samples from the same study should be homogenized and measured at the same time using the same batch of freshly made chemical reagents; 2) biological variability should be considered when detecting small change in mitochondrial content; 3) the relative change should be used to compare the outcomes from different studies.

scholarly journals Insulin and 5-Aminoimidazole-4-Carboxamide Ribonucleotide (AICAR) Differentially Regulate the Skeletal Muscle Cell Secretome

Proteomes ◽  
2021 ◽  
Vol 9 (3) ◽  
pp. 37
Author(s):  
Alba Gonzalez-Franquesa ◽  
Lone Peijs ◽  
Daniel T. Cervone ◽  
Ceren Koçana ◽  
Juleen R. Zierath ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Growth Factor ◽  
Large Scale ◽  
Conditioned Media ◽  
Metabolic Effects ◽  
Secreted Proteins ◽  
Whole Body ◽  
C2c12 Myotubes ◽  
Fibroblast Growth Factor 21 ◽  
Insulin Growth Factor

Skeletal muscle is a major contributor to whole-body glucose homeostasis and is an important endocrine organ. To date, few studies have undertaken the large-scale identification of skeletal muscle-derived secreted proteins (myokines), particularly in response to stimuli that activate pathways governing energy metabolism in health and disease. Whereas the AMP-activated protein kinase (AMPK) and insulin-signaling pathways have received notable attention for their ability to independently regulate skeletal muscle substrate metabolism, little work has examined their ability to re-pattern the secretome. The present study coupled the use of high-resolution MS-based proteomics and bioinformatics analysis of conditioned media derived from 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR—an AMPK activator)- and insulin-treated differentiated C2C12 myotubes. We quantified 858 secreted proteins, including cytokines and growth factors such as fibroblast growth factor-21 (Fgf21). We identified 377 and 118 proteins that were significantly altered by insulin and AICAR treatment, respectively. Notably, the family of insulin growth factor binding-proteins (Igfbp) was differentially regulated by each treatment. Insulin- but not AICAR-induced conditioned media increased the mitochondrial respiratory capacity of myotubes, potentially via secreted factors. These findings may serve as an important resource to elucidate secondary metabolic effects of insulin and AICAR stimulation in skeletal muscle.

Activation of AMP-activated protein kinase increases mitochondrial enzymes in skeletal muscle

2000 ◽  
Vol 88 (6) ◽  
pp. 2219-2226 ◽  
Author(s):  
W. W. Winder ◽  
B. F. Holmes ◽  
D. S. Rubink ◽  
E. B. Jensen ◽  
M. Chen ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Protein Kinase ◽  
Muscle Contraction ◽  
Citrate Synthase ◽  
Aminolevulinic Acid ◽  
Chemical Activation ◽  
Quadriceps Muscle ◽  
Mitochondrial Enzymes ◽  
Glut 4 ◽  
Amp Activated Protein Kinase

Muscle contraction causes an increase in activity of 5′-AMP-activated protein kinase (AMPK). This study was designed to determine whether chronic chemical activation of AMPK will increase mitochondrial enzymes, GLUT-4, and hexokinase in different types of skeletal muscle of resting rats. In acute studies, rats were subcutaneously injected with either 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR; 1 mg/g body wt) in 0.9% NaCl or with 0.9% NaCl alone and were then anesthetized for collection and freezing of tissues. AMPK activity increased in the superficial, white region of the quadriceps and in soleus muscles but not in the deep, red region of the quadriceps muscle. Acetyl-CoA carboxylase (ACC) activity, a target for AMPK, decreased in all three muscle types in response to AICAR injection but was lowest in the white quadriceps. In rats given daily, 1 mg/g body wt, subcutaneous injections of AICAR for 4 wk, activities of citrate synthase, succinate dehydrogenase, and malate dehydrogenase were increased in white quadriceps and soleus but not in red quadriceps. Cytochrome c and δ-aminolevulinic acid synthase levels were increased in white, but not red, quadriceps. Carnitine palmitoyl-transferase and hydroxy-acyl-CoA dehydrogenase were not significantly increased. Hexokinase was markedly increased in all three muscles, and GLUT-4 was increased in red and white quadriceps. These results suggest that chronic AMPK activation may mediate the effects of muscle contraction on some, but not all, biochemical adaptations of muscle to endurance exercise training.

scholarly journals PO-048 Effects of royal jelly administration on endurance training-induced mitochondrial adaptations in skeletal muscle

2018 ◽  
Vol 1 (3) ◽  
Author(s):  
Yumiko Takahashi ◽  
Kamiyu Hijikata ◽  
Hideo Hatta
Keyword(s):  
Skeletal Muscle ◽  
Endurance Training ◽  
Soleus Muscle ◽  
Royal Jelly ◽  
Citrate Synthase ◽  
Treadmill Running ◽  
Type I ◽  
Mitochondrial Enzymes ◽  
Positive Effects ◽  
Hydroxyacyl Coa Dehydrogenase

Objective In this study, we investigated effect of royal jelly (RJ), which is produced by honey bees to feed to developing larvae and contains various ingredients including protein, carbohydrate, lipids and minerals, on endurance training-induced adaptations in skeletal muscle in ICR mice. Methods Male mice received either RJ (1.0 mg/g body weight) or distilled water for 3 weeks. Mice in the training group performed treadmill running at 20 m/min for 60 min from 30 min after the administration five times a week. Results We found a significant positive main effects of RJ treatment on the weight of tibialis anterior (TA) muscle and gastrocnemius muscle. There was a significant positive main effect of endurance training on the maximum activities of citrate synthase and β-hydroxyacyl CoA dehydrogenase, which are mitochondrial enzymes, in TA and plantaris muscle (type IIb/IIx dominant), while no significant effect of RJ treatment was found. In soleus muscle (about 40% fiber consistent with type I), the maximum activities of citrate synthase and β-hydroxyacyl CoA dehydrogenase were significantly increased by endurance training in the RJ treated group, while no significant effect of endurance training was found in the control group. Conclusions Our results suggest that RJ treatment had positive effects on the induction of mitochondrial adaptation by endurance training in soleus muscle.

High-intensity aerobic interval training increases fat and carbohydrate metabolic capacities in human skeletal muscle

2008 ◽  
Vol 33 (6) ◽  
pp. 1112-1123 ◽  
Author(s):  
Christopher G.R. Perry ◽  
George J.F. Heigenhauser ◽  
Arend Bonen ◽  
Lawrence L. Spriet
Keyword(s):  
Skeletal Muscle ◽  
High Intensity ◽  
Citrate Synthase ◽  
Interval Training ◽  
Carbohydrate Oxidation ◽  
Peak Oxygen Consumption ◽  
Whole Body ◽  
Moderate Intensity ◽  
Mitochondrial Enzymes ◽  
Aerobic Interval Training

High-intensity aerobic interval training (HIIT) is a compromise between time-consuming moderate-intensity training and sprint-interval training requiring all-out efforts. However, there are few data regarding the ability of HIIT to increase the capacities of fat and carbohydrate oxidation in skeletal muscle. Using untrained recreationally active individuals, we investigated skeletal muscle and whole-body metabolic adaptations that occurred following 6 weeks of HIIT (~1 h of 10 × 4 min intervals at ~90% of peak oxygen consumption (VO2 peak), separated by 2 min rest, 3 d·week–1). A VO2 peak test, a test to exhaustion (TE) at 90% of pre-training VO2 peak, and a 1 h cycle at 60% of pre-training VO2 peak were performed pre- and post-HIIT. Muscle biopsies were sampled during the TE at rest, after 5 min, and at exhaustion. Training power output increased by 21%, and VO2 peak increased by 9% following HIIT. Muscle adaptations at rest included the following: (i) increased cytochrome c oxidase IV content (18%) and maximal activities of the mitochondrial enzymes citrate synthase (26%), β-hydroxyacyl-CoA dehydrogenase (29%), aspartate-amino transferase (26%), and pyruvate dehydrogenase (PDH; 21%); (ii) increased FAT/CD36, FABPpm, GLUT 4, and MCT 1 and 4 transport proteins (14%–30%); and (iii) increased glycogen content (59%). Major adaptations during exercise included the following: (i) reduced glycogenolysis, lactate accumulation, and substrate phosphorylation (0–5 min of TE); (ii) unchanged PDH activation (carbohydrate oxidation; 0–5 min of TE); (iii) ~2-fold greater time during the TE; and (iv) increased fat oxidation at 60% of pre-training VO2 peak. This study demonstrated that 18 h of repeated high-intensity exercise sessions over 6 weeks (3 d·week–1) is a powerful method to increase whole-body and skeletal muscle capacities to oxidize fat and carbohydrate in previously untrained individuals.

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