Skeletal muscle contraction stimulates multiple signaling cascades that govern a variety of metab... more Skeletal muscle contraction stimulates multiple signaling cascades that govern a variety of metabolic and transcriptional events. Akt/protein kinase B regulates metabolism and growth/muscle hypertrophy, but contraction effects on this target and its substrates are varied and may depend on the mode of the contractile stimulus. Accordingly, we determined the effects of endurance or resistance exercise on phosphorylation of Akt and downstream substrates in six trained cyclists who performed a single bout of endurance or resistance exercise separated by ∼7 days. Muscle biopsies were taken from the vastus lateralis at rest and immediately after exercise. Akt Ser473 phosphorylation was increased (1.8-fold; P = 0.011) after endurance but was unchanged after resistance exercise. Conversely, Akt Thr308 phosphorylation was unaltered after either bout of exercise. Several exercise-responsive phosphoproteins were detected by immunoblot analysis with a phospho-Akt substrate antibody. pp160 and p...
The phosphorylation of the alpha-subunit of Na+/K(+)-transporting ATPase (Na,K-ATPase) by cAMP-de... more The phosphorylation of the alpha-subunit of Na+/K(+)-transporting ATPase (Na,K-ATPase) by cAMP-dependent protein kinase (PKA) and protein kinase C (PKC) was characterized in purified enzyme preparations of Bufo marinus kidney and duck salt gland and in microsomes of Xenopus oocytes. In addition, we have examined cAMP and phorbol esters, which are stimulators of PKA and PKC, respectively, for their ability to provoke the phosphorylation of alpha-subunits of Na,K-ATPase in homogenates of Xenopus oocytes. In the enzyme from the duct salt gland, phosphorylation by PKA and PKC occurs on serine and threonine residues, whereas in the enzyme from B. marinus kidney and Xenopus oocytes, phosphorylation by PKA occurs only on serine residues. Phosphopeptide analysis indicates that a site phosphorylated by PKA resides in a 12-kDa fragment comprising the C terminus of the polypeptide. Studies of phosphorylation performed on homogenates of Xenopus oocytes show that not only endogenous oocyte Na,K-ATPase but also exogenous Xenopus Na,K-ATPase expressed in the oocyte by microinjection of cRNA can be phosphorylated in response to stimulation of oocyte PKA and PKC. In conclusion, these data are consistent with the possibility that the alpha-subunit of Na,K-ATPase can serve as a substrate for PKA and PKC in vivo.
Endurance training represents one extreme in the continuum of skeletal muscle plasticity. The mol... more Endurance training represents one extreme in the continuum of skeletal muscle plasticity. The molecular signals elicited in response to acute and chronic exercise and the integration of multiple intracellular pathways are incompletely understood. We determined the effect of 10 days of intensified cycle training on signal transduction in nine inactive males in response to a 1-h acute bout of cycling at the same absolute workload (164 ± 9 W). Muscle biopsies were taken at rest and immediately and 3 h after the acute exercise. The metabolic signaling pathways, including AMP-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR), demonstrated divergent regulation by exercise after training. AMPK phosphorylation increased in response to exercise (∼16-fold; P < 0.05), which was abrogated posttraining ( P < 0.01). In contrast, mTOR phosphorylation increased in response to exercise (∼2-fold; P < 0.01), which was augmented posttraining ( P < 0.01) in the presenc...
Muscle contraction and metabolic stress are potent activators of AMP-activated protein kinase (AM... more Muscle contraction and metabolic stress are potent activators of AMP-activated protein kinase (AMPK). AMPK restores energy balance by activating processes that produce energy while inhibiting those that consume energy. The role of AMPK in the regulation of active ion transport is unclear. Our aim was to determine the effect of the AMPK activator A-769662 on Na+-K+-ATPase function in skeletal muscle cells. Short-term incubation of differentiated rat L6 myotubes with 100 μM A-769662 increased AMPK and acetyl-CoA carboxylase (ACC) phosphorylation in parallel with decreased Na+-K+-ATPase α1-subunit abundance at the plasma membrane and ouabain-sensitive 86Rb+ uptake. Notably, the effect of A-769662 on Na+-K+-ATPase was similar in muscle cells that do not express AMPK α1- and α2-catalytic subunits. A-769662 directly inhibits the α1-isoform of the Na+-K+-ATPase, purified from rat and human kidney cells in vitro with IC50 57 μM and 220 μM, respectively. Inhibition of the Na+-K+-ATPase by 10...
Skeletal muscle Na+-K+-ATPase plays a central role in the clearance of K+ from the extracellular ... more Skeletal muscle Na+-K+-ATPase plays a central role in the clearance of K+ from the extracellular fluid, therefore maintaining blood [K+]. Na+-K+-ATPase activity in peripheral tissue is impaired in insulin resistant states. We determined effects of high-fat diet (HFD) and exercise training (ET) on skeletal muscle Na+-K+-ATPase subunit expression and insulin-stimulated translocation. Skeletal muscle expression of Na+-K+-ATPase isoforms and transcription factor DNA binding was determined before or after 5 days of swim training in Wistar rats fed chow or HFD for 4 or 12 wk. Skeletal muscle insulin resistance was observed after 12 wk of HFD. Na+-K+-ATPase α1-subunit protein expression was increased 1.6-fold ( P < 0.05), whereas α2- and β1-subunits and protein expression were decreased twofold ( P < 0.01) in parallel with decrease in plasma membrane Na+-K+-ATPase activity after 4 wk of HFD. Exercise training restored α1-, α2-, and β1-subunit expression and Na+-K+-ATPase activity to ...
Na,K-ATPase is a potential target for regulatory phosphorylation by protein kinase A and C (PKA a... more Na,K-ATPase is a potential target for regulatory phosphorylation by protein kinase A and C (PKA and PKC). To identify the phosphorylation sites, we have mutated the alpha 1-subunit of Bufo marinus in a highly conservative PKA and in 20 different PKC consensus sequences. The mutants were expressed in Xenopus oocytes and their phosphorylation capacity tested in homogenates upon stimulation of PKA or PKC. While serine 943 (Ser-943) was identified as a unique target site for PKA, none of the PKC consensus serine or threonine residues are implicated in PKC phosphorylation. Controlled trypsinolysis of phosphorylated alpha-subunits of various purified enzyme preparations and of alpha/beta complexes from oocyte homogenates revealed that PKC phosphorylation was exclusively associated with the N terminus. A fusion protein containing the first 32 amino acids of the Bufo alpha-subunit was phosphorylated in vitro and serine and threonine residues (Thr-15 and Ser-16) in this region were identifie...
The Na,K‐ATPase is involved in a large number of regulatory activities including cSrc‐dependent s... more The Na,K‐ATPase is involved in a large number of regulatory activities including cSrc‐dependent signalling. Upon inhibition of the Na,K‐ATPase with ouabain, cSrc activation is shown to occur in many cell types. This study tests the hypothesis that acute potentiation of agonist‐induced contraction by ouabain is mediated through Na,K‐ATPase‐cSrc signalling‐dependent sensitization of vascular smooth muscle cells to Ca2+.
The mechanism by which methotrexate (MTX) improves glucose homeostasis in patients with rheumatoi... more The mechanism by which methotrexate (MTX) improves glucose homeostasis in patients with rheumatoid (RA) and psoriatic arthritis (PsA) remains undetermined. Animal studies indicate a role for intracellular accumulation of 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranosyl 5&#39;-monophosphate (ZMP) but this has not been directly demonstrated in humans. We explored whether accumulation of ZMP is associated with improvements in glucose homeostasis during MTX therapy. MTX-naïve, non-diabetic RA (n = 16) and PsA (n = 10) patients received uninterrupted MTX treatment for 6 months. To evaluate whether ZMP accumulated during MTX therapy, we measured the concentration of ZMP in erythrocytes and the concentration of its dephosphorylated derivative 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR) in urine using liquid chromatography mass spectrometry (LC-MS/MS). To assess glucose homeostasis, we determined the concentration of glycated haemoglobin (HbA1c) and homeostasis model assessment of insulin resistance [HOMA-IR: fasting glucose (mmol/L) × fasting insulin (μU/mL)/22.5]. Erythrocyte ZMP and urinary AICAR concentrations did not increase during 6 months of MTX therapy. HbA1c concentration was reduced from 5.80 ± 0.29% at baseline to 5.51 ± 0.32% at 6 months (p &lt; 0.001), while HOMA-IR remained unaltered. Reduction in HbA1c concentration was not associated with increased ZMP or AICAR concentrations. MTX therapy probably does not produce a chronic increase in erythrocyte ZMP or urinary AICAR concentrations. Collectively, our data do not support the hypothesis that MTX improves glucose homeostasis through chronic accumulation of ZMP.
American journal of physiology. Endocrinology and metabolism, Jan 12, 2016
Mechanisms regulating skeletal muscle growth involve a balance between the activity of serine/thr... more Mechanisms regulating skeletal muscle growth involve a balance between the activity of serine/threonine protein kinases including the mammalian target of rapamycin (mTOR) and 5'-AMP-activated protein kinase (AMPK). The contribution of different AMPK subunits to the regulation of cell growth size remains inadequately characterized. Using AMPKγ3 mutant-overexpressing transgenic Tg-Prkag3(225Q) and AMPKγ3 knockout (Prkag3(-/-)) mice, we investigated the requirement for the AMPKγ3 isoform in functional overload-induced muscle hypertrophy. While genetic disruption of the γ3 isoform did not impair muscle growth, control (sham-operated) AMPKγ3 transgenic mice displayed heavier plantaris muscles despite lower Igf1 expression, but in response to overload hypertrophy, underwent smaller mass gain compared to wild-type littermates. mTOR signaling was upregulated with functional overload, but unchanged between genetically modified animals and wild-type littermates. Differences in AMPK-relate...
Tyrosine protein kinase present in the membrane fraction of bovine cerebral cortex were extracted... more Tyrosine protein kinase present in the membrane fraction of bovine cerebral cortex were extracted and chromatographically fractionated. The activity associated with tyrosine protein kinases was fully extracted from the membranes by 1% sodium cholate and eluted in two peaks (I and II) during chromatography of protein extracts on DEAE-Toyopearl in the presence of sodium cholate. The predominant in cerebral cortex membrane tyrosine protein kinase of peak I (about 75% of the total activity) was purified 1930-fold by gel filtration on Sephacryl S-300, chromatography on hexyl- and phenyl-Sepharose and by rechromatography on DEAE-Toyopearl. The amount of the enzyme prepared from 250 g of bovine brain was 20 micrograms, the enzyme yield and specific activity being 3.8% and 3.9 nmol/mg protein/min, respectively. The purified protein kinase of peak I represents a protein with Mr of 62-63,000 (p62) capable of being autophosphorylated in the presence of [gamma-32P]. Protein kinase p62 phosphory...
A common polymorphism (R577X) in the α-actinin (ACTN) 3 gene, which leads to complete deficiency ... more A common polymorphism (R577X) in the α-actinin (ACTN) 3 gene, which leads to complete deficiency of a functional protein in skeletal muscle, could directly influence metabolism in the context of health and disease. Therefore, we tested the hypothesis that states of glucose tolerance are associated with the ACTN3 R577X genotype. We analyzed the prevalence of the ACTN3 R577X polymorphism in people with normal glucose tolerance (NGT) and type 2 diabetes (T2D) and measured muscle-specific α-actinin 2 and 3 mRNA and protein abundance in skeletal muscle biopsies. Furthermore, we investigated the protein abundance of the myosin heavy chain isoforms and the components of the mitochondrial electron transport chain in skeletal muscle from people with NGT or T2D. mRNA of selected sarcomeric z-disk proteins was also assessed. Although the prevalence of the ACTN3 577XX genotype was higher in T2D patients, genotype distribution was unrelated to metabolic control or obesity. ACTN2 and ACTN3 mRNA e...
Cellular and molecular biology (Noisy-le-Grand, France), 2006
The cardiac glycoside ouabain initiates a cascade of signaling events through Na+,K+-ATPase, lead... more The cardiac glycoside ouabain initiates a cascade of signaling events through Na+,K+-ATPase, leading to an increase in cell growth and proliferation in different cell types. We explored the effects of ouabain on glucose metabolism in skeletal muscle and clarified the mechanisms of ouabain signal transduction. In rat soleus muscle 200 microM ouabain decreased basal glucose uptake without effect on insulin-stimulated glucose uptake. Ouabain increased glycogen synthesis additively to insulin and this effect was abolished in the presence of a MEK1/2 inhibitor (PD98059) or a c-Src inhibitor (PP2). Ouabain exposure reduced glucose oxidation, and this effect was reversed in the presence of PP2. Incubation with ouabain did not affect intramuscular ATP and its metabolites; however acetyl-CoA carboxylase phosphorylation was reduced, with no effect on AMPK phosphorylation. Insulin-stimulated Akt phosphorylation was not affected by ouabain. Ouabain reduced basal and insulin-stimulated phosphory...
In mature human skeletal muscle, insulin-stimulated glucose transport is mediated primarily via t... more In mature human skeletal muscle, insulin-stimulated glucose transport is mediated primarily via the GLUT4 glucose transporter. However, in contrast to mature skeletal muscle, cultured muscle expresses significant levels of the GLUT1 glucose transporter. To assess the relative contribution of these two glucose transporters, we used a novel photolabelling techniques to assess the cell surface abundance of GLUT1 and GLUT4 specifically in primary cultures of human skeletal muscle. We demonstrate that insulin-stimulated glucose transport in cultured human skeletal muscle is mediated by GLUT4, as no effect on GLUT1 appearance at the plasma membrane was noted. Furthermore, GLUT4 mRNA and protein increased twofold (p < 0.05), after differentiation, whereas GLUT1 mRNA and protein decreased 55% (p < 0.005). Incubation of differentiated human skeletal muscle cells with a non-peptide insulin mimetic significantly (p < 0.05) increased glucose uptake and glycogen synthesis. Thus, culture...
Skeletal muscle contraction stimulates multiple signaling cascades that govern a variety of metab... more Skeletal muscle contraction stimulates multiple signaling cascades that govern a variety of metabolic and transcriptional events. Akt/protein kinase B regulates metabolism and growth/muscle hypertrophy, but contraction effects on this target and its substrates are varied and may depend on the mode of the contractile stimulus. Accordingly, we determined the effects of endurance or resistance exercise on phosphorylation of Akt and downstream substrates in six trained cyclists who performed a single bout of endurance or resistance exercise separated by ∼7 days. Muscle biopsies were taken from the vastus lateralis at rest and immediately after exercise. Akt Ser473 phosphorylation was increased (1.8-fold; P = 0.011) after endurance but was unchanged after resistance exercise. Conversely, Akt Thr308 phosphorylation was unaltered after either bout of exercise. Several exercise-responsive phosphoproteins were detected by immunoblot analysis with a phospho-Akt substrate antibody. pp160 and p...
The phosphorylation of the alpha-subunit of Na+/K(+)-transporting ATPase (Na,K-ATPase) by cAMP-de... more The phosphorylation of the alpha-subunit of Na+/K(+)-transporting ATPase (Na,K-ATPase) by cAMP-dependent protein kinase (PKA) and protein kinase C (PKC) was characterized in purified enzyme preparations of Bufo marinus kidney and duck salt gland and in microsomes of Xenopus oocytes. In addition, we have examined cAMP and phorbol esters, which are stimulators of PKA and PKC, respectively, for their ability to provoke the phosphorylation of alpha-subunits of Na,K-ATPase in homogenates of Xenopus oocytes. In the enzyme from the duct salt gland, phosphorylation by PKA and PKC occurs on serine and threonine residues, whereas in the enzyme from B. marinus kidney and Xenopus oocytes, phosphorylation by PKA occurs only on serine residues. Phosphopeptide analysis indicates that a site phosphorylated by PKA resides in a 12-kDa fragment comprising the C terminus of the polypeptide. Studies of phosphorylation performed on homogenates of Xenopus oocytes show that not only endogenous oocyte Na,K-ATPase but also exogenous Xenopus Na,K-ATPase expressed in the oocyte by microinjection of cRNA can be phosphorylated in response to stimulation of oocyte PKA and PKC. In conclusion, these data are consistent with the possibility that the alpha-subunit of Na,K-ATPase can serve as a substrate for PKA and PKC in vivo.
Endurance training represents one extreme in the continuum of skeletal muscle plasticity. The mol... more Endurance training represents one extreme in the continuum of skeletal muscle plasticity. The molecular signals elicited in response to acute and chronic exercise and the integration of multiple intracellular pathways are incompletely understood. We determined the effect of 10 days of intensified cycle training on signal transduction in nine inactive males in response to a 1-h acute bout of cycling at the same absolute workload (164 ± 9 W). Muscle biopsies were taken at rest and immediately and 3 h after the acute exercise. The metabolic signaling pathways, including AMP-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR), demonstrated divergent regulation by exercise after training. AMPK phosphorylation increased in response to exercise (∼16-fold; P < 0.05), which was abrogated posttraining ( P < 0.01). In contrast, mTOR phosphorylation increased in response to exercise (∼2-fold; P < 0.01), which was augmented posttraining ( P < 0.01) in the presenc...
Muscle contraction and metabolic stress are potent activators of AMP-activated protein kinase (AM... more Muscle contraction and metabolic stress are potent activators of AMP-activated protein kinase (AMPK). AMPK restores energy balance by activating processes that produce energy while inhibiting those that consume energy. The role of AMPK in the regulation of active ion transport is unclear. Our aim was to determine the effect of the AMPK activator A-769662 on Na+-K+-ATPase function in skeletal muscle cells. Short-term incubation of differentiated rat L6 myotubes with 100 μM A-769662 increased AMPK and acetyl-CoA carboxylase (ACC) phosphorylation in parallel with decreased Na+-K+-ATPase α1-subunit abundance at the plasma membrane and ouabain-sensitive 86Rb+ uptake. Notably, the effect of A-769662 on Na+-K+-ATPase was similar in muscle cells that do not express AMPK α1- and α2-catalytic subunits. A-769662 directly inhibits the α1-isoform of the Na+-K+-ATPase, purified from rat and human kidney cells in vitro with IC50 57 μM and 220 μM, respectively. Inhibition of the Na+-K+-ATPase by 10...
Skeletal muscle Na+-K+-ATPase plays a central role in the clearance of K+ from the extracellular ... more Skeletal muscle Na+-K+-ATPase plays a central role in the clearance of K+ from the extracellular fluid, therefore maintaining blood [K+]. Na+-K+-ATPase activity in peripheral tissue is impaired in insulin resistant states. We determined effects of high-fat diet (HFD) and exercise training (ET) on skeletal muscle Na+-K+-ATPase subunit expression and insulin-stimulated translocation. Skeletal muscle expression of Na+-K+-ATPase isoforms and transcription factor DNA binding was determined before or after 5 days of swim training in Wistar rats fed chow or HFD for 4 or 12 wk. Skeletal muscle insulin resistance was observed after 12 wk of HFD. Na+-K+-ATPase α1-subunit protein expression was increased 1.6-fold ( P < 0.05), whereas α2- and β1-subunits and protein expression were decreased twofold ( P < 0.01) in parallel with decrease in plasma membrane Na+-K+-ATPase activity after 4 wk of HFD. Exercise training restored α1-, α2-, and β1-subunit expression and Na+-K+-ATPase activity to ...
Na,K-ATPase is a potential target for regulatory phosphorylation by protein kinase A and C (PKA a... more Na,K-ATPase is a potential target for regulatory phosphorylation by protein kinase A and C (PKA and PKC). To identify the phosphorylation sites, we have mutated the alpha 1-subunit of Bufo marinus in a highly conservative PKA and in 20 different PKC consensus sequences. The mutants were expressed in Xenopus oocytes and their phosphorylation capacity tested in homogenates upon stimulation of PKA or PKC. While serine 943 (Ser-943) was identified as a unique target site for PKA, none of the PKC consensus serine or threonine residues are implicated in PKC phosphorylation. Controlled trypsinolysis of phosphorylated alpha-subunits of various purified enzyme preparations and of alpha/beta complexes from oocyte homogenates revealed that PKC phosphorylation was exclusively associated with the N terminus. A fusion protein containing the first 32 amino acids of the Bufo alpha-subunit was phosphorylated in vitro and serine and threonine residues (Thr-15 and Ser-16) in this region were identifie...
The Na,K‐ATPase is involved in a large number of regulatory activities including cSrc‐dependent s... more The Na,K‐ATPase is involved in a large number of regulatory activities including cSrc‐dependent signalling. Upon inhibition of the Na,K‐ATPase with ouabain, cSrc activation is shown to occur in many cell types. This study tests the hypothesis that acute potentiation of agonist‐induced contraction by ouabain is mediated through Na,K‐ATPase‐cSrc signalling‐dependent sensitization of vascular smooth muscle cells to Ca2+.
The mechanism by which methotrexate (MTX) improves glucose homeostasis in patients with rheumatoi... more The mechanism by which methotrexate (MTX) improves glucose homeostasis in patients with rheumatoid (RA) and psoriatic arthritis (PsA) remains undetermined. Animal studies indicate a role for intracellular accumulation of 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranosyl 5&#39;-monophosphate (ZMP) but this has not been directly demonstrated in humans. We explored whether accumulation of ZMP is associated with improvements in glucose homeostasis during MTX therapy. MTX-naïve, non-diabetic RA (n = 16) and PsA (n = 10) patients received uninterrupted MTX treatment for 6 months. To evaluate whether ZMP accumulated during MTX therapy, we measured the concentration of ZMP in erythrocytes and the concentration of its dephosphorylated derivative 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR) in urine using liquid chromatography mass spectrometry (LC-MS/MS). To assess glucose homeostasis, we determined the concentration of glycated haemoglobin (HbA1c) and homeostasis model assessment of insulin resistance [HOMA-IR: fasting glucose (mmol/L) × fasting insulin (μU/mL)/22.5]. Erythrocyte ZMP and urinary AICAR concentrations did not increase during 6 months of MTX therapy. HbA1c concentration was reduced from 5.80 ± 0.29% at baseline to 5.51 ± 0.32% at 6 months (p &lt; 0.001), while HOMA-IR remained unaltered. Reduction in HbA1c concentration was not associated with increased ZMP or AICAR concentrations. MTX therapy probably does not produce a chronic increase in erythrocyte ZMP or urinary AICAR concentrations. Collectively, our data do not support the hypothesis that MTX improves glucose homeostasis through chronic accumulation of ZMP.
American journal of physiology. Endocrinology and metabolism, Jan 12, 2016
Mechanisms regulating skeletal muscle growth involve a balance between the activity of serine/thr... more Mechanisms regulating skeletal muscle growth involve a balance between the activity of serine/threonine protein kinases including the mammalian target of rapamycin (mTOR) and 5'-AMP-activated protein kinase (AMPK). The contribution of different AMPK subunits to the regulation of cell growth size remains inadequately characterized. Using AMPKγ3 mutant-overexpressing transgenic Tg-Prkag3(225Q) and AMPKγ3 knockout (Prkag3(-/-)) mice, we investigated the requirement for the AMPKγ3 isoform in functional overload-induced muscle hypertrophy. While genetic disruption of the γ3 isoform did not impair muscle growth, control (sham-operated) AMPKγ3 transgenic mice displayed heavier plantaris muscles despite lower Igf1 expression, but in response to overload hypertrophy, underwent smaller mass gain compared to wild-type littermates. mTOR signaling was upregulated with functional overload, but unchanged between genetically modified animals and wild-type littermates. Differences in AMPK-relate...
Tyrosine protein kinase present in the membrane fraction of bovine cerebral cortex were extracted... more Tyrosine protein kinase present in the membrane fraction of bovine cerebral cortex were extracted and chromatographically fractionated. The activity associated with tyrosine protein kinases was fully extracted from the membranes by 1% sodium cholate and eluted in two peaks (I and II) during chromatography of protein extracts on DEAE-Toyopearl in the presence of sodium cholate. The predominant in cerebral cortex membrane tyrosine protein kinase of peak I (about 75% of the total activity) was purified 1930-fold by gel filtration on Sephacryl S-300, chromatography on hexyl- and phenyl-Sepharose and by rechromatography on DEAE-Toyopearl. The amount of the enzyme prepared from 250 g of bovine brain was 20 micrograms, the enzyme yield and specific activity being 3.8% and 3.9 nmol/mg protein/min, respectively. The purified protein kinase of peak I represents a protein with Mr of 62-63,000 (p62) capable of being autophosphorylated in the presence of [gamma-32P]. Protein kinase p62 phosphory...
A common polymorphism (R577X) in the α-actinin (ACTN) 3 gene, which leads to complete deficiency ... more A common polymorphism (R577X) in the α-actinin (ACTN) 3 gene, which leads to complete deficiency of a functional protein in skeletal muscle, could directly influence metabolism in the context of health and disease. Therefore, we tested the hypothesis that states of glucose tolerance are associated with the ACTN3 R577X genotype. We analyzed the prevalence of the ACTN3 R577X polymorphism in people with normal glucose tolerance (NGT) and type 2 diabetes (T2D) and measured muscle-specific α-actinin 2 and 3 mRNA and protein abundance in skeletal muscle biopsies. Furthermore, we investigated the protein abundance of the myosin heavy chain isoforms and the components of the mitochondrial electron transport chain in skeletal muscle from people with NGT or T2D. mRNA of selected sarcomeric z-disk proteins was also assessed. Although the prevalence of the ACTN3 577XX genotype was higher in T2D patients, genotype distribution was unrelated to metabolic control or obesity. ACTN2 and ACTN3 mRNA e...
Cellular and molecular biology (Noisy-le-Grand, France), 2006
The cardiac glycoside ouabain initiates a cascade of signaling events through Na+,K+-ATPase, lead... more The cardiac glycoside ouabain initiates a cascade of signaling events through Na+,K+-ATPase, leading to an increase in cell growth and proliferation in different cell types. We explored the effects of ouabain on glucose metabolism in skeletal muscle and clarified the mechanisms of ouabain signal transduction. In rat soleus muscle 200 microM ouabain decreased basal glucose uptake without effect on insulin-stimulated glucose uptake. Ouabain increased glycogen synthesis additively to insulin and this effect was abolished in the presence of a MEK1/2 inhibitor (PD98059) or a c-Src inhibitor (PP2). Ouabain exposure reduced glucose oxidation, and this effect was reversed in the presence of PP2. Incubation with ouabain did not affect intramuscular ATP and its metabolites; however acetyl-CoA carboxylase phosphorylation was reduced, with no effect on AMPK phosphorylation. Insulin-stimulated Akt phosphorylation was not affected by ouabain. Ouabain reduced basal and insulin-stimulated phosphory...
In mature human skeletal muscle, insulin-stimulated glucose transport is mediated primarily via t... more In mature human skeletal muscle, insulin-stimulated glucose transport is mediated primarily via the GLUT4 glucose transporter. However, in contrast to mature skeletal muscle, cultured muscle expresses significant levels of the GLUT1 glucose transporter. To assess the relative contribution of these two glucose transporters, we used a novel photolabelling techniques to assess the cell surface abundance of GLUT1 and GLUT4 specifically in primary cultures of human skeletal muscle. We demonstrate that insulin-stimulated glucose transport in cultured human skeletal muscle is mediated by GLUT4, as no effect on GLUT1 appearance at the plasma membrane was noted. Furthermore, GLUT4 mRNA and protein increased twofold (p < 0.05), after differentiation, whereas GLUT1 mRNA and protein decreased 55% (p < 0.005). Incubation of differentiated human skeletal muscle cells with a non-peptide insulin mimetic significantly (p < 0.05) increased glucose uptake and glycogen synthesis. Thus, culture...
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Papers by A. Chibalin