Skeletal muscle fiber type comparison of pyruvate dehydrogenase phosphatase activity and isoform expression in fed and food-deprived rats

LeBlanc, Paul J.; Harris, Robert A.; Peters, Sandra J.

doi:10.1152/ajpendo.00327.2006

Cited by 18 publications

(25 citation statements)

References 41 publications

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“…Mitochondria were isolated from the first muscle biopsy by differential centrifugation, following a method originally described by Makinen and Lee (22) and modified by Jackman and Willis (13), which has been used previously in our lab (17,19,20,27,28), with the subsarcolemmal mitochondria being primarily targeted for isolation. Briefly, ϳ75 mg muscle was minced and homogenized on ice by hand in 20 volumes of solution 1 (100 mM KCl, 40 mM Tris·HCl, 10 mM Tris base, 5 mM MgSO 4, 5 mM Na2EDTA, 1 mM ATP, pH 7.5).…”

Section: Methodsmentioning

confidence: 99%

“…Pyruvate dehydrogenase phosphatase (PDP) activity was determined using the nonradioactive phosphatase assay system from Promega (Madison, WI) and a synthetic peptide substrate (14 amino acid long segment of the E1␣ subunit surrounding phosphorylation sites 1 and 2) from New England Peptide (Gardner, MA) as previously described (4,12,17). Briefly, mitochondria were diluted with sucrose and mannitol buffer to a final protein concentration of 2 g/ l and a total volume of 30 l, freeze-thawed three times to fracture mitochondria membranes, and centrifuged at 21,000 g for 1 h at 4°C to pellet out large protein and other insoluble materials.…”

Section: Methodsmentioning

confidence: 99%

“…A piece of muscle (ϳ10 mg) was chipped from the frozen biopsy and homogenized on ice in 100 volumes of homogenizing buffer (250 mM sucrose, 100 mM KCl, 2 mM EDTA, pH 6.8), which also contained a protease inhibitor cocktail (Complete Mini EDTA-free, Roche, Laval, QC, Canada). Standard SDS-PAGE electrophoresis was performed, as described previously (17,19,20,26,27), with proteins being transferred to a polyvinylidene fluoride (Immobilon-P, Millipore, Bedford, MA) (0.45 m pore size). Membranes were then incubated overnight at 4°C in 10 ml of blocking solution (TBST with 5% skim milk) with a polyclonal antisera against 53-kDa PDP1 (diluted 1:2,000; a kind gift from Drs.…”

Section: Methodsmentioning

confidence: 99%

“…It has been found that in states of restricted carbohydrate availability there is an increase in skeletal muscle PDK activity (5,6,26,27,28) and PDK4 protein content (26,27,36), while similar nutritional states also cause a decrease in PDP2 protein content in rat heart and kidney (12) and skeletal muscle, with this decrease leading to a decrease in PDP activity (17). As a result of these adaptations there is an overall decrease in the active form of PDH (PDHa; 6,28,30), allowing for the conservation of carbohydrates, which is essential during these carbohydratedeficient perturbations.…”

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confidence: 99%

“…Many models have been used to perturb this partitioning, such as starvation (12,17,26,36), and a high-fat diet (27,28,30). One such perturbation previously used has been endurance training (17,19,20), which in general causes a shift toward increased fat oxidation and decreased carbohydrate utilization. During submaximal exercise following aerobic training, the body adapts to rely more heavily on fat as a fuel for the production of ATP, thereby decreasing the need for pyruvate production and carbohydrate oxidation.…”

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The relationship between human skeletal muscle pyruvate dehydrogenase phosphatase activity and muscle aerobic capacity

Love

LeBlanc

Inglis

et al. 2011

Journal of Applied Physiology

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Love LK, LeBlanc PJ, Inglis JG, Bradley NS, Choptiany J, Heigenhauser GJ, Peters SJ. The relationship between human skeletal muscle pyruvate dehydrogenase phosphatase activity and muscle aerobic capacity. J Appl Physiol 111: 427-434, 2011. First published May 19, 2011 doi:10.1152/japplphysiol.00672.2010.-Pyruvate dehydrogenase (PDH) is a mitochondrial enzyme responsible for regulating the conversion of pyruvate to acetyl-CoA for use in the tricarboxylic acid cycle. PDH is regulated through phosphorylation and inactivation by PDH kinase (PDK) and dephosphorylation and activation by PDH phosphatase (PDP). The effect of endurance training on PDK in humans has been investigated; however, to date no study has examined the effect of endurance training on PDP in humans. Therefore, the purpose of this study was to examine differences in PDP activity and PDP1 protein content in human skeletal muscle across a range of muscle aerobic capacities. This association is important as higher PDP activity and protein content will allow for increased activation of PDH, and carbohydrate oxidation. The main findings of this study were that 1) PDP activity (r 2 ϭ 0.399, P ϭ 0.001) and PDP1 protein expression (r 2 ϭ 0.153, P ϭ 0.039) were positively correlated with citrate synthase (CS) activity as a marker for muscle aerobic capacity; 2) E1␣ (r 2 ϭ 0.310, P ϭ 0.002) and PDK2 protein (r 2 ϭ 0.229, P ϭ0.012) are positively correlated with muscle CS activity; and 3) although it is the most abundant isoform, PDP1 protein content only explained ϳ18% of the variance in PDP activity (r 2 ϭ 0.184, P ϭ 0.033). In addition, PDP1 in combination with E1␣ explained ϳ38% of the variance in PDP activity (r 2 ϭ 0.383, P ϭ 0.005), suggesting that there may be alternative regulatory mechanisms of this enzyme other than protein content. These data suggest that with higher muscle aerobic capacity (CS activity) there is a greater capacity for carbohydrate oxidation (E1␣), in concert with higher potential for PDH activation (PDP activity). carbohydrate oxidation; PDH; PDP1; E1␣; E2; PDK2 PYRUVATE DEHYDROGENASE (PDH) is a multienzyme complex consisting of multiple copies of E1 (␣ and ␤), E2 (the core of the PDH complex), and E3 subunits, along with an E3 binding protein (E3BP), which serves to bind E3 to the complex (as reviewed by 1, 39). Each of these subunits (with the exception of E3BP) is involved in the conversion of pyruvate to acetylCoA in a stepwise manner. The complex is regulated largely via covalent modification by the addition of a phosphate group to at least one of its three serine residues located on the E1␣ subunit of the complex (15,31,33,34,37). Phosphorylation and inactivation are accomplished by a group of specific PDH kinases (PDK1-4), while dephosphorylation and activation are accomplished by a pair of PDH phosphatases (PDP1 and -2; Refs. 21, 34, 37). Each of these regulatory enzyme isoforms has different specificities and tissue expressions, with PDK2 and PDP1 being the most abundant isoforms in skeletal muscle (3, 11). Both isoforms...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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The relationship between human skeletal muscle pyruvate dehydrogenase phosphatase activity and muscle aerobic capacity

Love

LeBlanc

Inglis

et al. 2011

Journal of Applied Physiology

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Training state and fasting-induced PDH regulation in human skeletal muscle

Gudiksen

Bertholdt

Stankiewicz

et al. 2018

Pflugers Arch - Eur J Physiol

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The aim of the present study was to examine the influence of training state on fasting-induced skeletal muscle pyruvate dehydrogenase (PDH) regulation, including PDH phosphorylation. Trained and untrained subjects, matched for skeletal muscle CS activity and OXPHOS protein, fasted for 36 h after receiving a standardized meal. Respiratory exchange ratio (RER) was measured and blood as well as vastus lateralis muscle biopsies were obtained 2, 12, 24, and 36 h after the meal. RER decreased with fasting only in untrained individuals, while PDHa activity decreased from 12 h after the meal in untrained, but only tended to decrease at 36 h in trained. PDH-E1α, PDP1 protein, PDH phosphorylation, and PDH acetylation in skeletal muscle was higher in trained than untrained subjects, but did not change with fasting, while PDK4 protein was higher at 36 h than at 2 h after the meal in both groups. In conclusion, the present results suggest that endurance exercise training modifies the fasting-induced regulation of PDHa activity in skeletal muscle and the substrate switch towards fat oxidation. PDH phosphorylation could not explain the fasting-induced regulation of PDHa activity suggesting other post translational modifications.

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Isolation of Sarcolemmal Plasma Membranes by Mechanically Skinning Rat Skeletal Muscle Fibers for Phospholipid Analysis

Fajardo

McMeekin

Basic

et al. 2013

Lipids

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Membrane phospholipid (PL) composition has been shown to affect cellular function by altering membrane physical structure. The sarcolemma plasma membrane (SLpm) is integral to skeletal muscle function and health. Previous studies assessing SLpm PL composition have demonstrated contamination from transverse (t)-tubule, sarcoplasmic reticulum, and nuclear membranes. This study assessed the possibility of isolating SL by mechanically skinning skeletal muscle fiber segments for the analysis of SLpm PL composition. Mechanically skinned SLpm from rat extensor digitorum longus (EDL) muscle fibers underwent Western blot analysis to assess contamination from t-tubule, sarcoplasmic reticulum, nuclear and mitochondrial membranes. The results indicate that isolated SLpm had minimal nuclear and mitochondrial membrane contamination and was void of contamination from sarcoplasmic reticulum and t-tubule membranes. After performing both high-performance thin layer chromatography and gas chromatography, we found that the SLpm obtained by mechanical skinning had higher sphingomyelin and total fatty acid saturation and lower phosphatidylcholine when compared to previous literature. Thus, by avoiding the use of various chemical treatments and membrane fractionation, we present data that may truly represent the SLpm and future studies can use this technique to assess potential changes under various perturbations and disease conditions such as insulin resistance and muscular dystrophy.

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Skeletal muscle fiber type comparison of pyruvate dehydrogenase phosphatase activity and isoform expression in fed and food-deprived rats

Cited by 18 publications

References 41 publications

The relationship between human skeletal muscle pyruvate dehydrogenase phosphatase activity and muscle aerobic capacity

The relationship between human skeletal muscle pyruvate dehydrogenase phosphatase activity and muscle aerobic capacity

Training state and fasting-induced PDH regulation in human skeletal muscle

Isolation of Sarcolemmal Plasma Membranes by Mechanically Skinning Rat Skeletal Muscle Fibers for Phospholipid Analysis

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