scholarly journals Intramitochondrial control of the oxidation of hexadecanoate in skeletal muscle. A study of the acyl-CoA esters which accumulate during rat skeletal-muscle mitochondrial β-oxidation of [U-14C]hexadecanoate and [U-14C]hexadecanoyl-carnitine

1993 ◽  
Vol 289 (1) ◽  
pp. 161-168 ◽  
Author(s):  
S Eaton ◽  
A K M J Bhuiyan ◽  
R S Kler ◽  
D M Turnbull ◽  
K Bartlett
Keyword(s):  
Skeletal Muscle ◽  
Steady State ◽  
Complex I ◽  
Rat Skeletal Muscle ◽  
Beta Oxidation ◽  
Mitochondrial Inner Membrane ◽  
Mitochondrial Fractions ◽  
Nadh Ratio ◽  
14Co2 Production ◽  
Small Pool

1. We describe the acyl-CoA and acyl-carnitine esters which arise from the incubation of well-coupled State 3 rat skeletal-muscle mitochondrial fractions with [U-14C]hexadecanoate and [U-14C]hexadecanoyl-carnitine. 2. Acyl-CoA ester intermediates of chain length 16, 14, 12, 10 and 8 carbons were detected. 3. Although incubations were in steady state in respect of oxygen consumption, 14CO2 production and generation of acid-soluble radioactivity, quantitative analysis of acyl-CoA esters showed that steady state was not achieved in respect of all intermediates. 4. 3-Hydroxyacyl- and 2-enoyl-CoA and -carnitine esters were found under normoxic conditions. 5. Direct measurement of NAD+ and NADH shows that under identical incubation conditions our observations cannot be explained by gross perturbation of the [NAD+]/[NADH] ratio. 6. We hypothesize that there is a small pool of rapidly recycling NAD+ channelled between complex I of the respiratory chain and the newly described mitochondrial-inner-membrane-associated beta-oxidation trifunctional enzyme [Uchida, Izai, Orii and Hashimoto (1992) J. Biol. Chem. 267, 1034-1041].

scholarly journals Skeletal muscle mitochondrial β-oxidation. A study of the products of oxidation of [U-14C]hexadecanoate by h.p.l.c. using continuous on-line radiochemical detection

1988 ◽  
Vol 253 (2) ◽  
pp. 541-547 ◽  
Author(s):  
N J Watmough ◽  
A K Bhuiyan ◽  
K Bartlett ◽  
H S Sherratt ◽  
D M Turnbull
Keyword(s):  
Skeletal Muscle ◽  
Proteolytic Enzymes ◽  
Rat Skeletal Muscle ◽  
Beta Oxidation ◽  
Citrate Cycle ◽  
Mitochondrial Fractions ◽  
On Line ◽  
14Co2 Release

Well-coupled mitochondrial fractions were prepared from rat skeletal muscle without the use of proteolytic enzymes. The products of [U-14C]hexadecanoate oxidation by rat skeletal muscle mitochondrial fractions were analysed by h.p.l.c. with on-line radiochemical detection. In the presence of 1 mM-carnitine, 70% of the products is acetylcarnitine. In agreement with Veerkamp et al. [Veerkamp, van Moerkerk, Glatz, Zuurveld, Jacobs & Wagenmakers (1986) Biochem. Med. Metab. Biol. 35, 248-259] 14CO2 release is shown to be an unreliable estimate of flux through beta-oxidation in skeletal muscle mitochondrial fractions. The flux through beta-oxidation is recorded unambiguously polarographically in the presence of 1 mM-carnitine and the absence of citrate cycle intermediates.

Regulation of CPT I activity in intermyofibrillar and subsarcolemmal mitochondria from human and rat skeletal muscle

2004 ◽  
Vol 286 (1) ◽  
pp. E85-E91 ◽  
Author(s):  
Veronic Bezaire ◽  
George J. F. Heigenhauser ◽  
Lawrence L. Spriet
Keyword(s):  
Skeletal Muscle ◽  
Vastus Lateralis ◽  
Moderate Intensity ◽  
Rat Skeletal Muscle ◽  
Moderate Intensity Exercise ◽  
Mitochondrial Fractions ◽  
Decreasing Ph ◽  
Subsarcolemmal Mitochondria ◽  
Cpt I

Carnitine palmitoyltransferase I (CPT I) is considered the rate-limiting enzyme in the transfer of long-chain fatty acids (LCFA) into the mitochondria and is reversibly inhibited by malonyl-CoA (M-CoA) in vitro. In rat skeletal muscle, M-CoA levels decrease during exercise, releasing the inhibition of CPT I and increasing LCFA oxidation. However, in human skeletal muscle, M-CoA levels do not change during moderate-intensity exercise despite large increases in fat oxidation, suggesting that M-CoA is not the sole regulator of increased CPT I activity during exercise. In the present study, we measured CPT I activity in intermyofibrillar (IMF) and subsarcolemmal (SS) mitochondria isolated from human vastus lateralis (VL), rat soleus (Sol), and red gastrocnemius (RG) muscles. We tested whether exercise-related levels (∼65% maximal O2 uptake) of calcium and adenylate charge metabolites (free AMP, ADP, and Pi) could override the M-CoA-induced inhibition of CPT I activity and explain the increased CPT I flux during exercise. Protein content was ∼25-40% higher in IMF than in SS mitochondria in all muscles. Maximal CPT I activity was similar in IMF and SS mitochondria in all muscles (VL: 282 ± 46 vs. 280 ± 51; Sol: 390 ± 81 vs. 368 ± 82; RG: 252 ± 71 vs. 278 ± 44 nmol·min-1·mg protein-1). Sensitivity to M-CoA did not differ between IMF and SS mitochondria in all muscles (25-31% inhibition in VL, 52-70% in Sol and RG). Calcium and adenylate charge metabolites did not override the M-CoA-induced inhibition of CPT I activity in mitochondria isolated from VL, Sol, and RG muscles. Decreasing pH from 7.1 to 6.8 reduced CPT I activity by ∼34-40% in both VL mitochondrial fractions. In summary, this study reports no differences in CPT I activity or sensitivity to M-CoA between IMF and SS mitochondria isolated from human and rat skeletal muscles. Exercise-induced increases in calcium and adenylate charge metabolites do not appear responsible for upregulating CPT I activity in human or rat skeletal muscle during moderate aerobic exercise.

scholarly journals An Isolated Complex V Inefficiency and Dysregulated Mitochondrial Function in Immortalized Lymphocytes from ME/CFS Patients

2020 ◽  
Author(s):  
Daniel Missailidis ◽  
Sarah Annesley ◽  
Claire Allan ◽  
Oana Sanislav ◽  
Brett Lidbury ◽  
...  
Keyword(s):  
Steady State ◽  
Fatty Acid ◽  
Mitochondrial Function ◽  
Complex I ◽  
Atp Synthesis ◽  
Beta Oxidation ◽  
Respiratory Capacity ◽  
Respiratory Complexes ◽  
Stress Sensing ◽  
Mitochondrial Respiratory

Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is an enigmatic condition characterized by exacerbation of symptoms after exertion (post-exertional malaise or “PEM”), and by fatigue whose severity and associated requirement for rest are excessive and disproportionate to the fatigue-inducing activity. There is no definitive molecular marker or known underlying pathological mechanism for the condition. Increasing evidence for aberrant energy metabolism suggests a role for mitochondrial dysfunction in ME/CFS. Our objective was therefore to measure mitochondrial function and cellular stress sensing in actively metabolising patient blood cells. We immortalized lymphoblasts isolated from 51 ME/CFS patients diagnosed according to the Canadian Consensus Criteria and an age- and gender-matched control group. Parameters of mitochondrial function and energy stress sensing were assessed by Seahorse extracellular flux analysis, proteomics, and an array of additional biochemical assays. As a proportion of the basal oxygen consumption rate (OCR), the rate of ATP synthesis by Complex V was significantly reduced in ME/CFS lymphoblasts, while significant elevations were observed in Complex I OCR, maximum OCR, spare respiratory capacity, nonmitochondrial OCR and “proton leak” as a proportion of the basal OCR. This was accompanied by a reduction of mitochondrial membrane potential, chronically hyperactivated TOR Complex I stress signalling and upregulated expression of mitochondrial respiratory complexes, fatty acid transporters and enzymes of the β-oxidation and TCA cycles. By contrast, mitochondrial mass and genome copy number, as well as glycolytic rates and steady state ATP levels were unchanged. Our results suggest a model in which ME/CFS lymphoblasts have a Complex V defect accompanied by compensatory upregulation of their respiratory capacity that includes the mitochondrial respiratory complexes, membrane transporters and enzymes involved in fatty acid β-oxidation. This homeostatically returns ATP synthesis and steady state levels to “normal” in the resting cells, but may leave them unable to adequately respond to acute increases in energy demand as the relevant homeostatic pathways are already activated.

scholarly journals Effects of magnesium and nucleotides on the proton conductance of rat skeletal-muscle mitochondria

2000 ◽  
Vol 348 (1) ◽  
pp. 209-213 ◽  
Author(s):  
Susana CADENAS ◽  
Martin D. BRAND
Keyword(s):  
Skeletal Muscle ◽  
Atp Synthase ◽  
Atp Synthesis ◽  
Proton Conductance ◽  
Rat Skeletal Muscle ◽  
Maximal Inhibition ◽  
Muscle Mitochondria ◽  
Mitochondrial Inner Membrane ◽  
Skeletal Muscle Mitochondria ◽  
The Matrix

During oxidative phosphorylation most of the protons pumped out to the cytosol across the mitochondrial inner membrane return to the matrix through the ATP synthase, driving ATP synthesis. However, some of them leak back to the matrix through a proton-conductance pathway in the membrane. When the ATP synthase is inhibited with oligomycin and ATP is not being synthesized, all of the respiration is used to drive the proton leak. We report here that Mg2+ inhibits the proton conductance in rat skeletal-muscle mitochondria. Addition of Mg2+ inhibited both oligomycin-inhibited respiration and the proton conductance, while removal of Mg2+ using EDTA activated these processes. The proton conductance was inhibited by more than 80% as free Mg2+ was raised from 25 nM to 220 μM. Half-maximal inhibition occurred at about 1 μM free Mg2+, which is close to the contaminating free Mg2+ concentration in our incubations in the absence of added magnesium chelators. ATP, GTP, CTP, TTP or UTP at a concentration of 1 mM increased the oligomycin-inhibited respiration rate by about 50%. However, these NTP effects were abolished by addition of 2 mM Mg2+ and any NTP-stimulated proton conductance was explained completely by chelation of endogenous free Mg2+. The corresponding nucleoside diphosphates (ADP, GDP, CDP, TDP or UDP) at 1 mM had no effect on oligomycin-inhibited respiration. We conclude that proton conductance in rat skeletal-muscle mitochondria is very sensitive to free Mg2+ concentration but is insensitive to NTPs or NDPs at 1 mM.

Contribution of mitochondrial proton leak to skeletal muscle respiration and to standard metabolic rate

1996 ◽  
Vol 271 (4) ◽  
pp. C1380-C1389 ◽  
Author(s):  
D. F. Rolfe ◽  
M. D. Brand
Keyword(s):  
Skeletal Muscle ◽  
Metabolic Rate ◽  
Respiration Rate ◽  
Rat Hepatocytes ◽  
Standard Metabolic Rate ◽  
Proton Leak ◽  
Rat Skeletal Muscle ◽  
Isolated Rat Hepatocytes ◽  
Mitochondrial Inner Membrane

We have tested the hypothesis that the leak of protons across the mitochondrial inner membrane (proton leak) is a significant contributor to standard metabolic rate (SMR). We found that proton leak accounts for around one-half of the resting respiration rate of perfused rat skeletal muscle. Proton leak is known to make a significant (26%) contribution to the resting respiration rate of isolated rat hepatocytes (M. D. Brand, L.-F. Chien, E. K. Ainscow, D. F. S. Rolfe, and R. K. Porter. Biochim. Biophys. Acta 1187: 132-139, 1994). If the importance of proton leak in these isolated and perfused systems is similar to its importance in vivo, then using literature values for the contribution of liver and skeletal muscle to SMR, we can calculate that proton leak in liver and skeletal muscle alone accounts for 11-26% (mean 20%) of the SMR of the rat. If proton leak activity in the other tissues of the rat is similar to that in liver cells, then the contribution of proton leak to rat SMR would be 16-31% (mean 25%).

scholarly journals Noninactivating tension in rat skeletal muscle. Effects of thyroid hormone.

1989 ◽  
Vol 94 (1) ◽  
pp. 183-203 ◽  
Author(s):  
M Chua ◽  
A F Dulhunty
Keyword(s):  
Skeletal Muscle ◽  
Steady State ◽  
Membrane Potential ◽  
Extensor Digitorum Longus ◽  
Voltage Dependence ◽  
Surface Membrane ◽  
Membrane Potentials ◽  
Resting Membrane Potential ◽  
Rat Skeletal Muscle ◽  
Contraction Coupling

Inactivation of excitation-contraction coupling was examined in extensor digitorum longus (EDL) and soleus muscle fibers from rats injected daily with tri-iodothyronine (T3, 150 micrograms/kg) for 10-14 d. Steady-state activation and inactivation curves for contraction were obtained from measurements of peak potassium contracture tension at different surface membrane potentials. The experiments tested the hypothesis that noninactivating tension is a "window" tension caused by the overlap of the activation and inactivation curves. Changes in the amplitude and voltage dependence of noninactivating tension should be predicted by the changes in the activation and inactivation curves, if noninactivating tension arises from their overlap. After T3 treatment, the area of overlap increased in EDL fibers and decreased in soleus fibers and the overlap region was shifted to more negative potentials in both muscles. Noninactivating tension also appeared at more negative membrane potentials after T3 treatment in both EDL and soleus fibers. The effects of T3 treatment were confirmed with a two microelectrode voltage-clamp technique: at the resting membrane potential (-80 mV) contraction in response to a brief test pulse required less than normal depolarization in EDL, but more than normal depolarization in soleus fibers. After T3 treatment, the increase in contraction threshold at depolarized holding potentials (attributed to inactivation) occurred at more depolarized holding potentials in EDL, or less depolarized holding potentials in soleus. The changes in contraction threshold could be accounted for by the effects of T3 on the activation and inactivation curves. In conclusion, (a) T3 appeared to affect the expression of both activation and inactivation characteristics, but the activation effects could not be cleanly distinguished from T3 effects on the sarcoplasmic reticulum and contractile proteins, and (b) the experiments provided evidence for the hypothesis that the noninactivating tension is a steady-state "window" tension.

Hypoxia causes glycogenolysis without an increase in percent phosphorylase a in rat skeletal muscle

1992 ◽  
Vol 263 (6) ◽  
pp. E1086-E1091 ◽  
Author(s):  
J. M. Ren ◽  
E. A. Gulve ◽  
G. D. Cartee ◽  
J. O. Holloszy
Keyword(s):  
Skeletal Muscle ◽  
Steady State ◽  
Strenuous Exercise ◽  
Rat Skeletal Muscle ◽  
Glycogen Breakdown ◽  
Stimulation Of

Stimulation of skeletal muscle to contract activates phosphorylase b-to-a conversion and glycogenolysis. Despite reversal of the increase in percentage of phosphorylase a after a few minutes, continued glycogen breakdown can occur during strenuous exercise. Hypoxia causes sustained glycogenolysis in skeletal muscle without an increase in percentage of phosphorylase a. We used this model to obtain insights regarding how glycogenolysis is mediated in the absence of an increase in percentage of phosphorylase a. Hypoxia caused a 70% decrease in glycogen in epitrochlearis muscles during an 80-min incubation despite no increase in percentage of phosphorylase a above the basal level of approximately 10%. Muscle Pi concentration increased from 3.8 to 8.6 mumol/g muscle after 5 min and 15.7 mumol/g after 20 min. AMP concentration doubled, attaining a steady state of 0.23 mumol/g in 5 min. Incubation of oxygenated muscles with 0.1 microM epinephrine induced an approximately sixfold increase in percentage of phosphorylase a but resulted in minimal glycogenolysis. Muscle Pi concentration was not altered by epinephrine. Despite no increase in percentage of phosphorylase a, hypoxia resulted in a fivefold greater depletion of glycogen over 20 min than did epinephrine. To evaluate the role of phosphorylase b, muscles were loaded with 2-deoxyglucose 6-phosphate, which inhibits phosphorylase b. The rate of glycogenolysis during 60 min of hypoxia was reduced by only approximately 14% in 2-deoxyglucose 6-phosphate-loaded muscles.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Activity of a gelsolin-like actin modulator in rat skeletal muscle under protein catabolic conditions

1987 ◽  
Vol 248 (2) ◽  
pp. 397-402 ◽  
Author(s):  
J D'Haese ◽  
M Rutschmann ◽  
B Dahlmann ◽  
H Hinssen
Keyword(s):  
Skeletal Muscle ◽  
Steady State ◽  
Molecular Mass ◽  
Actin Filaments ◽  
Actin Polymerization ◽  
Proteolytic Degradation ◽  
Rat Skeletal Muscle ◽  
Corticosterone Treatment ◽  
Increased Activity ◽  
Stoichiometric Complex

A gelsolin-like actin-modulating protein was isolated from rat skeletal muscle and characterized with respect to its interaction with actin. The protein, with a molecular mass of approx. 85 kDa, forms a stoichiometric complex with two actin molecules and is activated by micromolar concentrations of Ca2+. It effectively severs actin filaments and promotes nucleation of actin polymerization. The activity of this protein is detectable already in crude extracts by its capability to reduce the steady state viscosity of actin. Actin-modulating activities were determined in muscle extracts of rats kept under protein catabolic conditions, i.e. as generated by corticosterone treatment and starvation. In both cases we found a marked increase of modulator activity. The possibility is discussed that the increased activity of actin modulator indicates a fragmentation of actin filaments prior to the proteolytic degradation of actin.

Sign in / Sign up

Export Citation Format

Share Document