Cultured Rat Skeletal Muscle Cells Research Articles

Rutaecarpine ameliorates hyperlipidemia and hyperglycemia in fat-fed, streptozotocin-treated rats via regulating the IRS-1/PI3K/Akt and AMPK/ACC2 signaling pathways.

We have shown that rutaecarpine extracted from the dried fruit of Chinese herb Evodia rutaecarpa (Juss) Benth (Wu Zhu Yu) promotes glucose consumption and anti-inflammatory cytokine expression in insulin-resistant primary skeletal muscle cells. In this study we investigated whether rutaecarpine ameliorated the obesity profiles, lipid abnormality, glucose metabolism and insulin resistance in rat model of hyperlipidemia and hyperglycemia. Rats fed on a high-fat diet for 8 weeks, followed by injection of streptozotocin (30 mg/kg, ip) to induce hyperlipidemia and hyperglycemia. One week after streptozotocin injection, the fat-fed, streptozotocin-treated rats were orally treated with rutaecarpine (25 mg·kg(-1)·d(-1)) or a positive control drug metformin (250 mg·kg(-1)·d(-1)) for 7 weeks. The body weight, visceral fat, blood lipid profiles and glucose levels, insulin sensitivity were measured. Serum levels of inflammatory cytokines were analyzed. IRS-1 and Akt/PKB phosphorylation, PI3K and NF-κB protein levels in liver tissues were assessed; pathological changes of livers and pancreases were examined. Glucose uptake and AMPK/ACC2 phosphorylation were studied in cultured rat skeletal muscle cells in vitro. Administration of rutaecarpine or metformin significantly decreased obesity, visceral fat accumulation, water consumption, and serum TC, TG and LDL-cholesterol levels in fat-fed, streptozotocin-treated rats. The two drugs also attenuated hyperglycemia and enhanced insulin sensitivity. Moreover, the two drugs significantly decreased NF-κB protein levels in liver tissues and plasma TNF-α, IL-6, CRP and MCP-1 levels, and ameliorated the pathological changes in livers and pancreases. In addition, the two drugs increased PI3K p85 subunit levels and Akt/PKB phosphorylation, but decreased IRS-1 phosphorylation in liver tissues. Treatment of cultured skeletal muscle cells with rutaecarpine (20-180 μmol/L) or metformin (20 μmol/L) promoted the phosphorylation of AMPK and ACC2, and increased glucose uptake. Rutaecarpine ameliorates hyperlipidemia and hyperglycemia in fat-fed, streptozotocin-treated rats via regulating IRS-1/PI3K/Akt signaling pathway in liver and AMPK/ACC2 signaling pathway in skeletal muscles.

The Effects of Palmitic Acid on Nitric Oxide Production by Rat Skeletal Muscle: Mechanism via Superoxide and iNOS Activation

Background: Increased plasma concentrations of free fatty acids (FFA) can lead to insulin resistance in skeletal muscle, impaired effects on mitochondrial function, including uncoupling of oxidative phosphorylation and decrease of endogenous antioxidant defenses. Nitric oxide (NO) is a highly diffusible gas that presents a half-life of 5-10 seconds and is involved in several physiological and pathological conditions. The effects of palmitic acid on nitric oxide (NO) production by rat skeletal muscle cells and the possible mechanism involved were investigated. Methods: Primary cultured rat skeletal muscle cells were treated with palmitic acid and NO production was assessed by nitrite measurement (Griess method) and 4,5-diaminofluorescein diacetate (DAF-2-DA) assay. Nuclear factor-kappa B (NF-ĸB) activation was evaluated by electrophoretic mobility shift assay and iNOS protein content by western blotting. Results: Palmitic acid treatment increased nitric oxide production. This effect was abolished by treatment with NOS inhibitors, L-nitro-arginine (LNA) and L-nitro-arginine methyl esther (L-NAME). NF-ĸB activation and iNOS content were increased due to palmitic acid treatment. The participation of superoxide on nitric oxide production was investigated by incubating the cells with DAF-2-DA in the presence or absence of palmitic acid, a superoxide generator system (X-XO), a mixture of NOS inhibitors and SOD-PEG (superoxide dismutase linked to polyethylene glycol). Palmitic acid and X-XO system increased NO production and this effect was abolished when cells were treated with NOS inhibitors and also with SOD-PEG. Conclusions: In summary, palmitic acid stimulates NO production in cultured skeletal muscle cells through production of superoxide, nuclear factor-kappa B activation and increase of iNOS protein content.

Biphasic effects of chronic ethanol exposure on insulin‐stimulated glucose uptake in primary cultured rat skeletal muscle cells: role of the Akt pathway and GLUT4

mild or moderate chronic alcohol intake has been shown to be associated with increased insulin sensitivity, while chronic alcohol abuse demonstrates a contrary effect. The mechanism underlying this biphasic effect has not yet been clarified. We investigated whether chronic ethanol exposure mediates biphasic changes on insulin sensitivity and whether the phosphatidylinositol 3-kinase/Akt pathway is involved in vitro. primary cultured rat skeletal muscle cells were exposed to ethanol (0-400 mM) for 24 h. Insulin sensitivity was assessed by the (3) H-labelled 2-deoxyglucose uptake assay. Phosphatidylinositol 3-kinase, cytosol and cell membrane glucose transporter-4 (GLUT4), as well as the Akt phosphorylated form, were analyzed by Western blots. biphasic effects of ethanol on insulin sensitivity were observed in primary cultured skeletal muscle cells in a dose-dependent manner. Compared with the untreated group, 50 and 100 mM concentrations of ethanol resulted in a significant increase in 2-deoxyglucose uptake by 29 and 28%, respectively, while higher concentrations of ethanol (200, 400 mM) showed a significant decrease in 2-deoxyglucose uptake by 28 and 47%, respectively. The changes in glucose transport activity were in line with the changes in Akt Ser473 phosphorylation and GLUT4 expression in an ethanol dose-dependent biphasic manner. The phosphorylation of Akt and GLUT4 protein contents were up-regulated after treatment with low concentrations of ethanol (50, 100 mM) and down-regulated with high concentrations of ethanol (200, 400 mM) for 24 h. ethanol mediates biphasic changes on insulin sensitivity at least in part via the Akt pathway and GLUT4 expression.

EFFECTS OF FATTY ACIDS ON PRODUCTION OF REACTIVE OXYGEN SPECIES (ROS) BY SKELETAL MUSCLE CELLS

Reactive Oxygen Species (ROS) are associated to muscle cell survival. These species in excess cause muscle cell damage and apoptosis. A recent study has shown that palmitic acid (PA) increases superoxide production by skeletal muscle cells in culture. Pathological conditions such as obesity and diabetes mellitus, lead to an increase in plasma levels of free fatty acids (FFA). In spite of these observations, however, the effect of other FFAs on production of ROS by skeletal muscle has not been investigated yet. In this study, the effects of oleic (OL), linoleic (LI), PA and stearic (ST) acids on ROS production by primary cultured rat skeletal muscle cells was investigated. The cells were treated with the fatty acids (25 M) during 60 minutes in the presence of the fluorescent probe dihydroethidium. Production of O2•‐ by the cells treated with ST, OL, LI and PA acids was increased by 55 ± 8%, 59 ± 6%, 62 ± 9% and 73 ± 10%, respectively. We observed that the NF‐κB activation is involved in the increased production of ROS induced by FFAs. Our results revealed that the increase in plasma FFAs in various pathological conditions, can lead to an increase of ROS production by skeletal muscle.

Auto-Positive Feedback Regulation for Nicotinic Acetylcholine Receptors by Protein Kinase C Activation

Background/Aims: Protein kinase C (PKC) is well-recognized to modify ligand-gated ion channels such as nicotinic ACh (nACh) receptors by phosphorylating the receptors. The aim of the present study was to obtain direct evidence for PKC activation through nACh receptors. Methods: Two-electrode voltage-clamp was made to Xenopus oocytes expressing wild-type and mutant Torpedo nACh receptors. Western blotting using antibodies against phospho-serine and phospho-threonine was carried out in oocytes expressing Torpedo nACh receptors. In situ PKC activation was monitored in cultured rat skeletal muscle cells expressing nACh receptors. Results: In the Xenopus oocyte expression systems, ACh-evoked whole-cell membrane currents through wild-type Torpedo nACh receptors were depressed by GF109203X, a PKC inhibitor, while currents through mutant receptors lacking PKC phosphorylation sites was not affected. In the Western blot analysis, ACh produced immunoreactive bands against an anti-phospho-serine or an anti-phospho-threonine antibody in oocytes expressing wild-type Torpedo nACh receptors, and those signals were attenuated by α-bungarotoxin, an inhibitor of nACh receptors, or GF109203X. In the in situ PKC assay using cultured rat muscle cells that expressed all the mRNAs for muscle nACh receptor subunits such as the α, β, γ, δ, and ε subunit, ACh activated PKC in the presence of atropine, an inhibitor of muscarinic ACh receptors, and the activation was abolished by α-bungarotoxin or GF109203X. Conclusion: The results of the present study show that ACh activates PKC through nACh receptors and that in turn, activated PKC constantly enhances ACh receptor responses by phosphorylating the receptors. This may represent a new auto-positive feedback regulation for nACh receptors by PKC activation.

Palmitate increases superoxide production through mitochondrial electron transport chain and NADPH oxidase activity in skeletal muscle cells

The effect of unbound palmitic acid (PA) at plasma physiological concentration range on reactive oxygen species (ROS) production by cultured rat skeletal muscle cells was investigated. The participation of the main sites of ROS production was also examined. Production of ROS was evaluated by cytochrome c reduction and dihydroethidium oxidation assays. PA increased ROS production after 1 h incubation. A xanthine oxidase inhibitor did not change PA-induced ROS production. However, the treatment with a mitochondrial uncoupler and mitochondrial complex III inhibitor decreased superoxide production induced by PA. The importance of mitochondria was also evaluated in 1 h incubated rat soleus and extensor digitorum longus (EDL) muscles. Soleus muscle, which has a greater number of mitochondria than EDL, showed a higher superoxide production induced by PA. These results indicate that mitochondrial electron transport chain is an important contributor for superoxide formation induced by PA in skeletal muscle. Results obtained with etomoxir and bromopalmitate treatment indicate that PA has to be oxidized to raise ROS production. A partial inhibition of superoxide formation induced by PA was observed by treatment with diphenylene iodonium, an inhibitor of NADPH oxidase. The participation of this enzyme complex was confirmed through an increase of p47(phox) phosphorylation after treatment with PA.

Effects of PPAR-α and -γ Agonists on Fatty Acid Metabolism of Muscle Cells in Hyperlipidemic and Hyperglycemic Conditions

Background: Studies for the regulation of fatty acid metabolism are deficient relatively in skeletal muscle and heart. The investigations in pathological conditions for malonyl-CoA decarboxylase (MCD) and for the relation of MCD and PPAR-․-γ agonists are insufficient in particular. Methods: In the current study, fully differentiated H9c2 muscle cells were exposed to pathological conditions such as hyperlipidemic (0.1 mM Palmitate) and hyperglycemic (16.5 mM Glucose) condition with 5 uM PPAR-γ agonist (rosiglitazone) and 10 uM PPAR- agonist (WY14,643) and then experiments such as MCD activity assay, MCD real-time RT-PCR, MCD reporter gene assay, MCD Western blotting, PPAR- Western blotting, and palmitate oxidation test were carried out. Results: Only PPAR- agonist increased MCD activity. In the result of real-time RT-PCR, both PPAR- and PPAR-γ agonists elevated MCD mRNA expression in hyperlipidemic condition. MCD protein expression was decreased in hyperlipidemic condition, however, increased in rosiglitazone, or WY14,643 treated conditions. Rosiglitazone, and WY14,643 treated groups showed incresed MCD protein expression in hyperglycemic condition. Hyperlipidemic control group and PPAR-․ﾇ-γ agonists treated groups presented about 3.8 times more increased palmitate oxidation level than normolipidemic control group in hyperlipidemic condition. PPAR- agonist treated group showed 49% more increased palmitate oxidation rate than hyperlipidemic control group in primary cultured rat skeletal muscle cells. The amount of palmitate oxidation from differentiated H9c2 muscle cells that had overexpressed PPAR- structural genes was more increased than control group. Conclusion: This study suggests that PPAR- agonist ameliorates the defects induced by hyperlipidemic condition through the regulation of MCD. In summary, a closely reciprocal relation among PPAR- agonist, MCD, and fatty acid oxidation existed distinctly in hyperlipidemic condition, but not in hyperglycemic condition.

High efficiency transfection of primary skeletal muscle cells with lipid-based reagents.

Lipofection is a convenient method for gene transfer into muscle cells but reportedly is inefficient. We tested the efficacy of commercially available lipid-based and polyamine transfection reagents. Primary rat skeletal muscle cell cultures were transfected at three stages of development and assayed after fusion. Efficiency reached 30% during the proliferation stage and up to 23% when most myoblasts had fused into myotubes. Optimization of transfection conditions with three different vectors yielded efficiencies exceeding 50%. Thus, lipid-based transfection into primary skeletal muscle cells can be several times more efficient than previously reported.

High leptin levels acutely inhibit insulin-stimulated glucose uptake without affecting glucose transporter 4 translocation in l6 rat skeletal muscle cells.

Obesity is a major risk factor for the development of insulin resistance, characterized by impaired stimulation of glucose disposal into muscle. The mechanisms underlying insulin resistance are unknown. Here we examine the direct effect of leptin, the product of the obesity gene, on insulin-stimulated glucose uptake in cultured rat skeletal muscle cells. Preincubation of L6 myotubes with leptin (2 or 100 nM, 30 min) had no effect on basal glucose uptake but reduced insulin-stimulated glucose uptake. However, leptin had no effect on the insulin-induced gain in myc-tagged glucose transporter 4 (GLUT4) appearance at the cell surface of L6 myotubes. Preincubation of cells with leptin also had no effect on insulin-stimulated tyrosine phosphorylation of insulin receptor, IRS-1 and IRS-2, phosphatidylinositol 3-kinase activity, or Akt phosphorylation. We have previously shown that insulin regulates glucose uptake via a signaling pathway sensitive to inhibitors of p38 MAP kinase. Here, leptin pretreatment reduced the extent of insulin-stimulated p38 MAP kinase phosphorylation and phosphorylation of cAMP response element binder, a downstream effector of p38 MAP kinase. These results show that high leptin levels can directly reduce insulin-stimulated glucose uptake in L6 muscle cells despite normal GLUT4 translocation. The mechanism of this effect could involve inhibition of insulin-stimulated p38 MAP kinase and GLUT4 activation.

Viral vector-mediated expression of K+ channels regulates electrical excitability in skeletal muscle.

Modification of K+ currents by exogenous gene expression may lead to therapeutic interventions in skeletal muscle diseases characterized by alterations in electrical excitability. In order to study the specific effects of increasing outward K+ currents, we expressed a modified voltage-dependent K+ channel in primary cultured rat skeletal muscle cells. The rat Kv1.4 channel was expressed as an N-terminal fusion protein containing a bioluminescent marker (green fluorescent protein). Transgene expression was carried out using the helper-dependent herpes simplex 1 amplicon system. Transduced myoballs, identified using fluorescein optics and studied electrophysiologically with single-cell patch clamp, exhibited a greater than two-fold increase in K+ conductance by 20-30 h after infection. This increase in K+ current led to a decrease in membrane resistance and a 10-fold increase in the current threshold for action potential generation. Electrical hyperexcitability induced by the Na+ channel toxin anemone toxin II (1 microM) was effectively counteracted by overexpression of Kv1.4 at 30-32 h after transduction. Thus, virally induced overexpression of a voltage-gated K+ channel in skeletal muscle has a powerful effect in reducing electrical excitability.

Effect of electrical stimulation on beta-adrenergic receptor population and cyclic amp production in chicken and rat skeletal muscle cell cultures.

Expression of the beta-adrenergic receptor (betaAR) and its coupling to cyclic AMP (cAMP) synthesis are important components of the signaling system that controls muscle atrophy and hypertrophy, and the goal of this study was to determine if electrical stimulation in a pattern simulating slow muscle contraction would alter the betaAR response in primary cultures of avian and mammalian skeletal muscle cells. Specifically, chicken skeletal muscle cells and rat skeletal muscle cells that had been grown for 7 d in culture were subjected to electrical stimulation for an additional 2 d at a pulse frequency of 0.5 pulses/sec and a pulse duration of 200 msec. In chicken skeletal muscle cells, the betaAR population was not significantly affected by electrical stimulation; however, the ability of these cells to synthesize cyclic AMP was reduced by approximately one-half. In contrast, the betaAR population in rat muscle cells was increased slightly but not significantly by electrical stimulation, and the ability of these cells to synthesize cyclic AMP was increased by almost twofold. The basal levels of intracellular cyclic AMP in neither rat muscle cells nor chicken muscle cells were affected by electrical stimulation.

The effects of Securidaca longepedunculata root extract on ionic currents and contraction of cultured rat skeletal muscle cells

The effects of the primary extract roots of Securidaca longepedunculata were tested on sodium, calcium and potassium currents in rat skeletal muscle cells developed in culture. In addition, they were tested on depolarisation-induced contraction and resting intracellular calcium levels. S. longepedunculata extract (10 −6 g/l) increases sodium current at all potentials. No clear effect was observed on calcium current except for a slight increase at negative potentials (−30, −10 mV) revealing a 5 mV shift towards negative potentials of the I Ca/ V curve, as with potassium current. In contrast, at the same concentration, S. longepedunculata enhanced the contractile response elicited by durable depolarisation. This was not attributable to the slight increase in resting intracellular free calcium concentration which did not change during and following S. longepedunculata application. These results strongly suggest that S. longepedunculata root extract contains one or more components acting on the voltage-sensor of excitation–contraction coupling (dihydropyridine receptors), regardless of its implication as a calcium channel.

Role of Serine/Threonine Protein Phosphatases in Insulin Regulation of Na+/K+-ATPase Activity in Cultured Rat Skeletal Muscle Cells

In this study, we examined the potential role of serine/threonine protein phosphatase-1 (PP-1) and PP-2A in the mechanism of Na+/K+-ATPase activation by insulin in the rat skeletal muscle cell line L6. Incubation of L6 cells with insulin caused a time- and dose-dependent stimulation of ouabain-sensitive plasma membrane Na+/K+-ATPase activity. Pretreatment with okadaic acid (OA; 0.1-1 microM) or calyculin A (1 microM) blocked insulin's effect on Na+/K+-ATPase activation. Low concentrations of OA that specifically inhibit PP-2A were ineffective. Immunoprecipitation of the enzyme from 32P-labeled cells with an antibody directed against the alpha-1 subunit of the enzyme revealed a 60% decrease in 110-kDa protein phosphorylation in insulin-treated cells. The presence of calyculin A blocked insulin-mediated dephosphorylation of Na+/K+-ATPase, whereas low concentrations of OA were ineffective. To further confirm the role of PP-1, we used L6 cell lines that overexpress the glycogen/SR-associated regulatory subunit of PP-1, PP-1G. Overexpression of PP-1G resulted in a 3-fold increase in insulin-stimulated PP-1 catalytic activity. This was accompanied by a 30% increase in basal Na+/K+-ATPase activity and a >2-fold increase in insulin's effect on pump activity. Inhibition of phosphatidylinositol-3 kinase with wortmannin blocked insulin-stimulated PP-1 activation as well as the dephosphorylation and activation of Na+/K+-ATPase. We conclude that insulin regulates the activity of Na+/K+-ATPase by promoting dephosphorylation of the alpha subunit via an insulin-stimulated PP-1 and that phosphatidylinositol-3 kinase-generated signals may mediate insulin activation of PP-1 and Na+/K+-ATPase.

The effect of modulating the glycogen-associated regulatory subunit of protein phosphatase-1 on insulin action in rat skeletal muscle cells.

Recent studies from this laboratory have shown that insulin rapidly stimulates a membranous protein phosphatase-1 (PP-1) in cultured rat skeletal muscle cells and isolated rat adipocytes. Stimulation of PP-1 is accompanied by the phosphorylation of a 160-kDa regulatory subunit of PP-1 (PP-1G). To further evaluate the exact role of this subunit in insulin action, L6 rat skeletal muscle cells were stably transfected with a vector containing the gene for PP-1G in the sense and antisense orientations. Transfection with the vector containing the PP-1G gene in the sense orientation yielded three stable clones with a 4- to 6-fold increase in PP-1G protein expression compared to those of wild-type L6 cells and neo control cells harboring an empty expression vector. Compared to the neo control, overexpression of PP-1G resulted in a 3-fold increase in insulin-stimulated PP-1 catalytic activity bound to PP-1G immunoprecipitates. These cell lines were examined for insulin's effect on glucose uptake, glycogen synthase activity, and glycogen synthesis. Insulin treatment resulted in an approximately 2-fold increase in 2-deoxyglucose uptake in recombinant cells compared to control cells (P < 0.05). This increase in 2-deoxyglucose transport was accompanied by an approximately 2-fold increase in insulin-stimulated glycogen synthase fractional activity (P < 0.05) and a 2- to 4-fold increase in insulin-stimulated glycogen synthesis compared to control cells. In conjunction with these observations, we found that an 85% depletion of endogenous PP-1G, using antisense constructs, resulted in a complete lack of PP-1 activation and an inhibition of basal and insulin-stimulated glucose transport. We conclude that the PP-1G holoenzyme is the major phosphatase regulated by insulin in vivo and plays an important role in insulin-stimulated glycogen synthesis by regulating the catalytic activity of bound PP-1.

Interferon-γ Alters Nerve-Induced Redistribution of Acetylcholine Receptors in Cultured Rat Skeletal Muscle Cells

The influence of recombinant interferon-γ (rIFN-γ) on the development of acetylcholine receptor (AChR) aggregates in cocultures of rat embryonic muscle cells and spinal cord neurons was studied by counting the number of AChR aggregates in relation to cholinergic nerve fibers coming to the muscle fibers. rIFN-γ caused no decrease in the number of cholinergic nerve fibers, but inhibited the increase in the number of AChR aggregates that occurs early during cocultivation and is an early sign in the development of neuromuscular junctions. rIFN-γ stimulated release of nitric oxide, but no effects on aggregation of AChRs occurred after exposure to a nitric oxide synthase inhibitor, l-NG-monomethylarginine, or by the addition of nitroprusside, a generator of nitric oxide. No effect was seen on the number of AChR aggregates when the cultures were exposed to rIFN-γ at later time points of cocultivation, when the increase in number of AChRs had already occurred. These studies indicate that the key immunoregulatory cytokine IFN-γ can cause alterations in the early process of synapse formation and that these effects are independent of the nitric oxide release caused by the cytokine.

Cytotoxic and non-cytotoxic effects of the MEIC reference chemicals on spontaneously contracting primary cultured rat skeletal muscle cells

This study was designed to evaluate the suitability of a multi-endpoint test system using primary cultured spontaneously contracting rat skeletal muscle cells to indicate an acute neuro- and/or cardiotoxic potential of chemicals. The concentration-dependent effects of the 50 MEIC (Multicenter Evaluation of In Vitro Cytotoxicity) reference chemicals on contractility, indicative of the functional integrity of the electrically active muscle cell membrane, were determined. Additionally, effects on two other endpoints, glucose consumption and viability, were monitored to reveal whether alterations in contractility were associated with cytotoxicity. In total, 30 of the tested compounds inhibited contractility at non-cytotoxic concentrations. The contractility-inhibiting and cytotoxic potencies differed by factors of more than 10 in the case of diazepam, phenol, propranolol, phenobarbital, mercuric chloride, thioridazine, verapamil, chloroquine, quinidine, phenytoin and atropine, of more than 100 in the case of amitriptyline, dextropropoxyphene, orphenadrine and amphetamine, and even more than 1000 for nicotine. On the basis of the available knowledge of the acute toxic effects and modes of acute toxic action of the test compounds, this characteristic response pattern is shown to be highly predictive for compounds reported to be cardio- and/or neurotoxic owing to interference with excitable membrane functions and/or neurotransmission.

Effect of tumor necrosis factor-alpha on insulin action in cultured rat skeletal muscle cells.

In this study, the acute effects of tumor necrosis factor (TNF)-alpha on insulin-stimulated glucose uptake, glycogen synthesis, and protein phosphatase-1 (PP-1) activation were examined in cultured rat skeletal muscle cell line, L6. Exposure of L6 cells to low concentrations of TNF-alpha (10 ng/ml for 60 min) inhibited basal and insulin stimulated 2-deoxyglucose uptake (40-50% decrease in basal and insulin stimulated glucose uptake respectively, when compared with controls, P < 0.05). The effect of TNF-alpha was more pronounced when the incubation period was extended to 6 and 12 h. TNF-alpha also blocked insulin activation of glycogen synthase (GS) and inhibited glycogen synthesis (measured as [14C]-glucose incorporated into glycogen). Because GS is activated by dephosphorylation via protein phosphatase-1 (PP-1), we examined the effect of TNF- alpha on PP-1 activation. As reported by us earlier (Srinivasan, M., and N. Begum, J Biol Chem 269:16662-16667, 1994), insulin rapidly stimulated PP-1 and concomitantly inhibited PP-2A activities in L6 cells. Pretreatment with TNF- alpha for 10-60 min blocked subsequent insulin-induced activation of PP-1. The impaired activation of PP-1 was accompanied by a reduction in insulin-stimulated phosphorylation of the regulatory subunit of PP-1. cAMP-Rp diastereomer, a cAMP antagonist failed to prevent the detrimental effects of TNF-alpha on PP-1. Cell permeable ceramide analogs, C2, C6, and Sphingomyelinase mimicked the effects of TNF-alpha on PP-1 inhibition. Furthermore, TNF-alpha treatment was accompanied by an increase in cellular ceramide levels, with concomitant reductions in sphingomyelin. We conclude that TNF-alpha blocks insulin-stimulated glycogen synthesis by inhibiting PP-1 activation via ceramide release.

Effect of tumor necrosis factor-alpha on insulin action in cultured rat skeletal muscle cells

Effect of tumor necrosis factor-alpha on insulin action in cultured rat skeletal muscle cells

Effect of tumor necrosis factor-alpha on insulin-stimulated mitogen-activated protein kinase cascade in cultured rat skeletal muscle cells.

Tumor necrosis factor-alpha (TNF-alpha) is a proposed mediator of insulin resistance in obese/diabetic animals through its effects on tyrosine phosphorylation of the insulin receptor and its substrate, insulin receptor substrate-1. In this study, the acute effects of TNF-alpha on the mitogen-activated protein kinase (MAPK) signalling cascade were examined in cultured rat skeletal muscle cell line, L6. Insulin treatment of L6 cells resulted in a rapid increase in MAPK activity (> twofold in 5 min with 10 nM insulin). Prior treatment with TNF-alpha for 60 min blocked subsequent insulin-induced activation of MAPK in a dose- and time-dependent manner. Metabolic labelling studies with inorganic [32P]phosphate followed by immuno-precipitation of MAPK and its upstream activator, mitogen-activated protein kinase kinase, indicated decreased phosphorylation of MAPK and its kinase in response to insulin in cells exposed to TNF-alpha. This effect of TNF-alpha was not due to inhibition of insulin-stimulated p21ras-GTP loading or Raf-1 phosphorylation. Low concentrations (2 nM) of okadaic acid, a serine/threonine phosphatase inhibitor, prevented TNF-alpha-induced inhibition of MAPK and restored insulin's effect on MAPK activity, while orthovanadate (a tyrosine phosphatase inhibitor), inhibitor 2 (phosphatase-1 inhibitor) and FK506 (phosphatase-2B inhibitor) were ineffective. These results suggested an involvement of an okadaic-acid-sensitive serine/threonine phosphatase in TNF-alpha-induced blockade of insulin's effect on MAPK and/or its kinase. Therefore, we examined the effect of TNF-alpha on protein phosphatase-1 (PP-1) and protein phosphatase-2A (PP-2A) activities. As reported by us earlier, insulin rapidly stimulated PP-1 and concomitantly inhibited PP-2A activities in control cells. TNF-alpha treatment blocked insulin-induced activation of PP-1. In contrast to PP-1, TNF-alpha caused a 60% increase in PP-2A activity and insulin failed to prevent this TNF-alpha effect. The time course of PP-2A activation by TNF-alpha preceded the kinetics of inhibition of MAPK. Cell-permeable ceramide analogs mimicked the TNF-alpha effect on MAPK inhibition and PP-2A activation. We conclude that TNF-alpha abrogates the insulin effect on MAPK activation by increasing dephosphorylation of MAPK kinase via an activated phosphatase.

Effects of HMG-CoA reductase inhibitors on growth and differentiation of cultured rat skeletal muscle cells

HMG-CoA reductase inhibitors have been associated with skeletal muscle myopathy, ranging from asymptomatic elevations of serum creatine kinase (CK) activity to rhabdomyolysis. In this study, we assessed the effects of addition of different concentrations of simvastatin and pravastatin on growth and differentiation of cultured primary rat skeletal muscle cells. Protein concentration, CK activity and percentage CK-MM, which is a parameter for maturation, were determined. Effects were generally stronger if inhibitors were added to both growth and differentiation medium rather than only to differentiation medium. Addition of 25 μM pravastatin caused only a decrease of CK activity. Addition of 1–5 μM simvastatin resulted in a decrease of protein concentration, CK activity and percentage CK-MM, whereas 25 μM simvastatin resulted in cell death. Addition of mevalonic acid or cholesterol could not prevent the effects of 1 μM simvastatin. In addition, 1 μM simvastatin did not influence the cholesterol and phospholipid content of the cells. Superfusion of cultured cells with simvastatin concentrations of 10 μM and higher caused a transient increase of the cytoplasmic calcium concentration followed by an apparent second rise and cell puncture. The results indicate that HMG-CoA reductase inhibitors may affect skeletal muscle cell regeneration in vivo by a direct toxic effect on growth and differentiation.