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.

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].

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 Methionine metabolism by rat muscle and other tissues. Occurrence of a new carnitine intermediate

1987 ◽  
Vol 247 (1) ◽  
pp. 35-40 ◽  
Author(s):  
P W D Scislowski ◽  
B M Hokland ◽  
W I A Davis-van Thienen ◽  
J Bremer ◽  
E J Davis
Keyword(s):  
Skeletal Muscle ◽  
Methionine Metabolism ◽  
Rat Skeletal Muscle ◽  
Citrate Cycle ◽  
Kidney Mitochondria ◽  
Rat Muscle ◽  
Mammalian Tissues ◽  
Liver And Kidney ◽  
Oxidation Of Methionine

Perfused rat hindquarter preparations were shown to incorporate radioactivity from [U-14C]methionine into citrate-cycle intermediates, lactate, alanine, glutamate, glutamine and CO2. During perfusion, large amounts of methionine were also oxidized to methionine sulphoxide. The capacity for transamination of methionine or its oxo analogue, 4-methylthio-2-oxobutyrate, by muscle extracts was demonstrated. Rat skeletal muscle, heart, liver and kidney mitochondria, when incubated with the latter plus radiolabelled carnitine, formed a newly identified carnitine derivative, 3-methylthiopropionylcarnitine. It is concluded that the capacity for oxidation of methionine by a trans-sulphuration-independent pathway occurs in several mammalian tissues. The extent of inter-organ handling of intermediates in this pathway(s) is discussed.

scholarly journals Measurement of the acyl-CoA intermediates of β-oxidation by h.p.l.c. with on-line radiochemical and photodiode-array detection. Application to the study of [U-14C]hexadecanoate oxidation by intact rat liver mitochondria

1989 ◽  
Vol 262 (1) ◽  
pp. 261-269 ◽  
Author(s):  
N J Watmough ◽  
D M Turnbull ◽  
H S A Sherratt ◽  
K Bartlett
Keyword(s):  
Rat Liver ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria ◽  
Beta Oxidation ◽  
Reverse Phase ◽  
Photodiode Array Detection ◽  
Mitochondrial Fractions ◽  
On Line ◽  
Photodiode Array ◽  
Array Detection

The quantitative isolation of acyl-CoA esters of chain length C2-C17 from mitochondrial incubations and their analysis by reverse-phase radio-h.p.l.c. is described. Photodiode-array detection was used to characterize 2-enoyl-CoA esters. The chromatographic behaviour of all 27 intermediates of the beta-oxidation of hexadecanoyl-CoA is documented. Only C16, C14 and C12 intermediates were detected in uncoupled mitochondria oxidizing [U-14C]hexadecanoyl-CoA in the presence of fluorocitrate and carnitine, providing evidence for some organization of the enzymes of beta-oxidation [Garland, Shepherd & Yates (1965) Biochem. J. 97, 587-594; Sumegi & Srere (1984) J. Biol. Chem. 259, 8748-8752]. Rotenone increased concentrations of 3-hydroxyacyl-CoA and 2-enoyl-CoA esters and inhibited flux. These experiments provide the first direct unambiguous measurements of acyl-CoA esters in intact respiring rat liver mitochondrial fractions.

scholarly journals Increased muscle calcium A possible cause of mitochondrial dysfunction and cellular necrosis in denervated rat skeletal muscle

1981 ◽  
Vol 196 (3) ◽  
pp. 663-667 ◽  
Author(s):  
M Joffe ◽  
N Savage ◽  
H Isaacs
Keyword(s):  
Skeletal Muscle ◽  
Mitochondrial Dysfunction ◽  
Causal Relationship ◽  
Cell Necrosis ◽  
Rat Skeletal Muscle ◽  
Mitochondrial Fractions ◽  
Cellular Necrosis ◽  
And Control ◽  
Muscle Calcium ◽  
Possible Cause

Mitochondrial preparations derived from denervated rat skeletal muscle and paired controls were characterized with respect to their ability to take up externally added Ca2+. The denervated and control muscle homogenates and mitochondrial [Ca2+] were also determined. Our data indicate that the denervated mitochondria are able to take up less Ca2+ than the controls before uncoupling occurs. This defect is associated with elevated [Ca2+] in homogenate and mitochondrial fractions in the denervated state. The causal relationship between Ca2+ overload, mitochondrial functional damage and cell necrosis is discussed.

The ubiquitin–proteasome system and skeletal muscle wasting

2005 ◽  
Vol 41 ◽  
pp. 173-186 ◽  
Author(s):  
Didier Attaix ◽  
Sophie Ventadour ◽  
Audrey Codran ◽  
Daniel Béchet ◽  
Daniel Taillandier ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Muscle Wasting ◽  
Proteolytic Enzymes ◽  
Cathepsin L ◽  
Ubiquitin Proteasome System ◽  
Complete Breakdown ◽  
Skeletal Muscle Proteins ◽  
Ubiquitin Proteasome ◽  
Tripeptidyl Peptidase Ii ◽  
F Box Protein

The ubiquitin–proteasome system (UPS) is believed to degrade the major contractile skeletal muscle proteins and plays a major role in muscle wasting. Different and multiple events in the ubiquitination, deubiquitination and proteolytic machineries are responsible for the activation of the system and subsequent muscle wasting. However, other proteolytic enzymes act upstream (possibly m-calpain, cathepsin L, and/or caspase 3) and downstream (tripeptidyl-peptidase II and aminopeptidases) of the UPS, for the complete breakdown of the myofibrillar proteins into free amino acids. Recent studies have identified a few critical proteins that seem necessary for muscle wasting {i.e. the MAFbx (muscle atrophy F-box protein, also called atrogin-1) and MuRF-1 [muscle-specific RING (really interesting new gene) finger 1] ubiquitin–protein ligases}. The characterization of their signalling pathways is leading to new pharmacological approaches that can be useful to block or partially prevent muscle wasting in human patients.

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