L6 Skeletal Muscle Cells Research Articles

Synthesis of heteroaryl/aryl kojic acid conjugates as stimulators of glucose uptake by GLUT4 translocation

Insulin exerts its metabolic actions through the insulin receptor (IR) and plays an essential role in treatment of diabetes. The inconvenience of daily injections and the undesirable side-effects associated with insulin injections demand novel drugs for the disease. To search for bioactive insulin mimetic, we synthesized a chemical library of small molecules (2a–3f) based on the indolylkojic acid scaffold (B). An In vitro screening assay was performed to stimulate glucose transport in rat L6 skeletal muscle cells, post treatment of the compounds (2a–3f) for the time period incubation of 16 h. Compounds 2f, 2g, 2l, 3a, 3b, 3c and 3d have shown significant glucose uptake stimulation as compared to the controls at micromolar concentrations. In mechanistic studies, we observed that these compounds exert their biological action by enhancing GLUT4 translocation to cell surface via PI3K-dependent signalling pathway in agreement to the insulin mode of action. Hence, these promising conjugates should be useful for further drug development in diabetes treatment.

One-dimensional ordered growth of magneto-crystalline and biocompatible cobalt ferrite nano-needles

In this letter, we have reported a novel synthesis of CoFe2O4 nano-needles by a co-precipitation method using precursors of ferric chloride and cobalt nitrate at 80°C. The structural and magnetic properties of as-grown needle-like CoFe2O4 nanostructures exhibit cubic spinel structure. CoFe2O4 nano-needles have an average diameter of 15nm with an aspect ratio of 30:50, which was depicted by High-resolution transmission electron microscopy (HRTEM). Superconducting Quantum Interference Device (SQUID) confirmed the property of superparamagnetism. The magnetic measurement illustrated that the coercivity (Hc) of CoFe2O4 nano-needles increased from −145.84Oe at 5K to −25.38Oe at 312K. The cell viability studies of CoFe2O4 nano-needles were performed in the concentration range of 5–1000µg/ml on L6 (Skeletal muscle cell lines) and Hep-2 (Larynx carcinoma) cells using 3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. There was more than 50% cell viability for both the cell lines, but Hep-2 cells were more prone to killing as compared to L6 cells. Such biocompatible nanostructures can be used as drug-delivery systems for cancer therapeutics.

Anti-diabetic potential of a Sasa quelpaertensis Nakai extract in L6 skeletal muscle cells

The anti-diabetic potential of a Sasa quelpaertensis Nakai extract (SQE) in L6 muscle cells was evaluated. The SQE stimulated glucose uptake via the AMP-activated protein kinase-activating pathway, but not via the insulinstimulating pathway. The SQE increased the expression of fatty acid translocase and peroxisome proliferator-activated receptor γ, and induced phosphorylation of acetyl-CoA carboxylase. The SQE attenuated oleic acid-induced intracellular triglyceride contents by decreasing the expression of sterol regulatory element-binding protein-1c and fatty acid synthase. These results indicate that the SQE probably has an anti-diabetic potential by enhancing glucose uptake and reducing triglyceride content in L6 skeletal muscle cells.

Mitofusin 2 deficiency leads to oxidative stress that contributes to insulin resistance in rat skeletal muscle cells

Mitofusin 2 (Mfn2) is a dynamin-like protein anchored in the outer mitochondrial membrane that plays a crucial role in ensuring optimal mitochondrial morphological homeostasis. It has been shown that reduced expression of Mfn2 is associated with insulin resistance, but the mechanism is still unclear. We investigated whether Mfn2 deficiency leads to impaired insulin sensitivity via elevated oxidative stress. L6 skeletal muscle cells were treated with palmitate and Mfn2 expression was repressed by transfection with antisense Mfn2. Levels of antioxidant enzymes, reactive oxygen species (ROS), the phosphorylation of c-Jun N-terminal Kinase (JNK) and nuclear factor-κB (NF-κB) and the mitochondrial membrane potential (Δψm) were measured. The results showed palmitate-induced insulin resistance of skeletal muscle cells was accompanied by Mfn2 repression. Meanwhile, the cells had decreased Δψm and activity of antioxidant enzymes which could increase production of ROS, phosphorylation of JNK and NF-κB. When Mfn2 was up-regulated in palmitate-treated cells, oxidative stress and insulin resistance was alleviated. Furthermore, knock-down of Mfn2 in control cells enhanced oxidative stress. Mfn2 deficiency led to increased superoxide concentration and activation of JNK as well as NF-κB associated with insulin signaling. In conclusion, Mfn2 is a potent repressor for oxidative stress and regulation of Mfn2 expression may prove to be a potential method to circumvent insulin resistance.

The anti-diabetic activity of Bifidobacterium lactis HY8101 in vitro and in vivo.

The aim of this study was to evaluate the effects of Bifidobacterium lactis HY8101 on insulin resistance induced using tumour necrosis factor-α (TNF-α) in rat L6 skeletal muscle cells and on the KK-A(Y) mouse noninsulin-dependent diabetes mellitus (NIDDM) model. The treatment using HY8101 improved the insulin-stimulated glucose uptake and translocation of GLUT4 via the insulin signalling pathways AKT and IRS-1(Tyr) in TNF-α-treated L6 cells. HY8101 increased the mRNA levels of GLUT4 and several insulin sensitivity-related genes (PPAR-γ) in TNF-α-treated L6 cells. In KK-A(Y) mice, HY8101 decreased fasting insulin and blood glucose and significantly improved insulin tolerance. HY8101 improved diabetes-induced plasma total cholesterol and triglyceride (TG) levels and increased the muscle glycogen content. We observed concurrent transcriptional changes in the skeletal muscle tissue and the liver. In the skeletal muscle tissue, the glycogen synthesis-related gene pp-1 and GLUT4 were up-regulated in mice receiving HY8101 treatment. In the liver, the hepatic gluconeogenesis-regulated genes (PCK1 and G6PC) were down-regulated in mice receiving HY8101 treatment. Bifidobacterium lactis HY8101 can be used to moderate glucose metabolism, lipid metabolism and insulin sensitivity in mice and in cells. Bifidobacterium lactis HY8101 might have potential as a probiotic candidate for alleviating metabolic syndromes such as diabetes.

In vitro toxic effects of puff adder (Bitis arietans) venom, and their neutralization by antivenom.

This study investigated the in vitro toxic effects of Bitis arietans venom and the ability of antivenom produced by the South African Institute of Medical Research (SAIMR) to neutralize these effects. The venom (50 µg/mL) reduced nerve-mediated twitches of the chick biventer muscle to 19% ± 2% of initial magnitude (n = 4) within 2 h. This inhibitory effect of the venom was significantly attenuated by prior incubation of tissues with SAIMR antivenom (0.864 µg/µL; 67% ± 4%; P < 0.05; n = 3–5, unpaired t-test). Addition of antivenom at t50 failed to prevent further inhibition or reverse the inhibition of twitches and responses to agonists. The myotoxic action of the venom (50 µg/mL) was evidenced by a decrease in direct twitches (30% ± 6% of the initial twitch magnitude) and increase in baseline tension (by 0.7 ± 0.3 g within 3 h) of the chick biventer. Antivenom failed to block these effects. Antivenom however prevented the venom induced cytotoxic effects on L6 skeletal muscle cells. Venom induced a marginal but significant reduction in plasma clotting times at concentrations above 7.8 µg/100 µL of plasma, indicating poor procoagulant effects. In addition, the results of western immunoblotting indicate strong immunoreactivity with venom proteins, thus warranting further detailed studies on the neutralization of the effects of individual venom toxins by antivenom.

Design and synthesis of lupeol analogues and their glucose uptake stimulatory effect in L6 skeletal muscle cells

Structure modifications of lupeol at the isopropylene moiety have been described via allylic oxidation using selenium dioxide. The antidiabetic efficacy of lupeol analogues were evaluated in vitro as glucose uptake stimulatory effect in L6 skeletal muscle cells. From all tested compounds, 2, 3, 4b and 6b showed significant stimulation of glucose uptake with respective percent stimulation of 173.1 (p <0.001), 114.1 (p <0.001), 98.3 (p <0.001) and 107.3 (p <0.001) at 10μM concentration. Stimulation of glucose uptake by these compounds is associated with enhanced translocation of glucose transporter 4 (GLUT4) and activation of IRS-1/PI3-K/AKT-dependent signaling pathway in L6 cells. Structure–activity relationship analysis of these analogues demonstrated that the integrity of α,β-unsaturated carbonyl and acetyl moieties were important in the retention of glucose uptake stimulatory effect. It is therefore proposed that naturally occurring lupeol and their analogues might reduce blood glucose, at least in part, through stimulating glucose utilization by skeletal muscles.

Metformin ameliorates insulin resistance in L6 rat skeletal muscle cells through upregulation of SIRT3

Background SIRT3 is an important regulator in cell metabolism, and recent studies have shown that it may be involved in the pharmacological effects of metformin. However, the molecular mechanisms underlying this process are unclear. Methods The effects of SIRT3 on the regulation of oxidative stress and insulin resistance in skeletal muscle were evaluated in vitro. Differentiated L6 skeletal muscle cells were treated with 750 μmol/L palmitic acid to induce insulin resistance. SIRT3 was knocked down and overexpressed in L6 cells. SIRT3, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) p65, c-Jun N-terminal kinase 1 (JNK1), and superoxide dismutase 2 (SOD2) were evaluated by Western blotting. Results Over expression of SIRT3 increased glucose uptake and decreased ROS production in L6-IR cells as well as in L6 cells. Knock-down of SIRT3 induced increased production of ROS while decreased glucose uptake in both L6 and L6-IR cells, and these effects were reversed by N-acetyl-L-cysteine (NAC). Metformin increased the expression of SIRT3 (1.5-fold) and SOD2 (2-fold) while down regulating NF-κB p65 (1.5-fold) and JNK1 (1.5-fold). Knockdown of SIRT3 (P&lt;0.05) reversed the metformin-induced decreases in NF-κB p65 and JNK1 and the metformin-induced increase in SOD2 (P&lt;0.05). Conclusions Upregulated SIRT3 is involved in the pharmacological mechanism by which metformin promotes glucose uptake. Additionally, SIRT3 may function as an important regulator of oxidative stress and a new alternative approach for targeting insulin resistance-related diseases.

Preventative effect of Zingiber officinale on insulin resistance in a high-fat high-carbohydrate diet-fed rat model and its mechanism of action.

Insulin resistance is a core component of metabolic syndrome and usually precedes the development of type 2 diabetes mellitus. We have examined the preventative effect of an ethanol extract of ginger (Zingiber officinale, Zingiberaceae) on insulin resistance in a high-fat high-carbohydrate (HFHC) diet-fed rat model of metabolic syndrome. The HFHC control rats displayed severe insulin resistance, whilst rats treated with ginger extract (200 mg/kg) during HFHC diet feeding showed a significant improvement of insulin sensitivity using the homeostatic model assessment of insulin resistance (HOMA-IR) after 10 weeks (p < 0.01). An in vitro mechanistic study showed that (S)-[6]-gingerol, the major pungent phenolic principle in ginger, dose-dependently (from 50 to 150 μM) increased AMPK α-subunit phosphorylation in L6 skeletal muscle cells. This was accompanied by a time-dependent marked increment of PGC-1α mRNA expression and mitochondrial content in L6 skeletal muscle cells. These results suggest that the protection from HFHC diet-induced insulin resistance by ginger is likely associated with the increased capacity of energy metabolism by its major active component (S)-[6]-gingerol.

Murraya koenigii(L.) Spreng. ameliorates insulin resistance in dexamethasone-treated mice by enhancing peripheral insulin sensitivity

Murraya koenigii (L.) Spreng. is an important medicinal plant used traditionally as an antiemetic, antidiarrhoeal agent and blood purifier and as a medicine for a variety of ailments. This study investigated the effects of ethanolic extract of M. koenigii (MK) on diabetes-associated insulin resistance induced in mice by chronic low-dose injection of dexamethasone. Mice treated with dexamethasone exhibited hyperglycaemia and impaired glucose tolerance. Treatment with MK reduced the extent of dexamethasone-induced hyperglycaemia and decreased insulin resistance as indicated by improved glucose tolerance and increased insulin-stimulated AKT phosphorylation in skeletal muscle tissue. Further evaluation in clonal skeletal muscle cell lines suggested that MK increased glucose uptake in L6 skeletal muscle cells by increasing cell surface GLUT4 density via an AKT-mediated pathway. MK can ameliorate dexamethasone-induced hyperglycaemia and insulin resistance in part by increasing glucose disposal into skeletal muscle.

Metformin and Resveratrol Induce Beneficial Metabolic Adaptations in L6 Muscle Cells Treated with HIV Protease Inhibitors: Evidence for Inhibition of JNK1/2 Signaling

C-Jun-N-terminal kinase (JNK1/2) pro-inflammatory signaling has been shown to play a role in highly active antiretroviral therapy (HAART)-induced insulin resistance in skeletal muscle cells. To determine the effectiveness of metformin and resveratrol in blunting HAART-induced insulin resistance, L6 skeletal muscle cells were treated ± (with or without) HIV protease inhibitors (ritonavir+atazanavir sulfate; RA) and either metformin, resveratrol, or the JNK1/2 inhibitor SP600125 and incubated ± insulin (100 nM). SP600125 as well as metformin and resveratrol reduced (P<0.05) JNK1/2 phosphorylation in RA-treated cells but not in healthy cells. In basal and insulin-stimulated cells, RA treatment significantly increased JNK1/2 phosophorylation (P<0.05). Additionally, when RA-stimulated cells were treated with metformin, resveratrol or SP6000125, JNK1/2 phosphorylation was reduced (P<0.05) indicating reduced inflammation. Basal and insulin-stimulated glucose uptake was increased in RA-treated cells (P<0.05). The presence of insulin resistance in RA-treated cells was partially abrogated (P>0.05) by metformin and SP600125 treatment. RA-treatment increased FA (fatty acid) uptake and oxidation in both the basal and insulin-stimulated state. Increased percent change of AKTSer473 phosphorylation indicated increased insulin sensitivity in RA+metformin (P=0.08) and RA+resveratrol (P<0.05) treated cells. Treatment with metformin, resveratrol or SP600125 had very little impact on p38 phosphorylation in basal and insulin-stimulated cells made insulin-resistant by incubation with RA. Our data indicate that metformin and resveratrol induce beneficial metabolic changes in cells made insulin resistant by treatment with protease inhibitors in part via inhibition of JNK1/2 pro-inflammatory signaling and stimulation of insulin-mediated AKTSer473 phosphorylation.

Involvement of Amp Kinase in Glucose Uptake and Palmitate Oxidation in L6 Muscle Cell Cultures

AICAR (5-aminoimidazole-4-carboxamide 1-s-D-ribonucleoside) is known to activate the AMP-activated protein kinase (AMPK) in many cell types, including skeletal muscle. We showed that AICAR activated glucose uptake, fatty acid oxidation, and lactate formation by L6 skeletal muscle cell cultures. All of the actions were duplicated by adiponectin, another known activator of AMPK, demonstrating selectivity of AICAR. Moreover, AICAR and adiponectin similarly caused phosphorylation of AMPK, and its target proteins acetyl CoA carboxylase and S6Kinase. While creatine treatment of cells is known to greatly enhance creatine and creatine phosphate levels, it was proposed to paradoxically activate AMPK, and yet not affect glucose transport. However, we found creatine treatment to be without effect on glucose transport, fatty acid oxidation, or phosphorylation of AMPK or its target proteins. Unlike AICAR and adiponectin, creatine treatment stimulated glycogen formation from glucose and inhibited lactate formation. Consistent with this, we observed a diminution in phosphofructokinase expression. Thus, we conclude that creatine and creatine phosphate are not significant allosteric modulators of AMPK in intact cells. We investigated the role of AMPK in glycogen metabolism, using an inhibitor of glycogen phosphorylase. While the inhibitor enhanced glucose incorporation into glycogen, AICAR and adiponectin still diminished glycogen formation, supporting a direct action of AMPK on glycogen synthase. To probe the mechanism for adiponectin, we used siRNA constructs against adenyate kinase and LKB1 (the AMPK kinase). We concluded that adiponectin likely acts through an activation of LKB1, rather than indirectly increasing the turnover of ATP, involving adenylate kinase.

Des-aspartate-angiotensin I attenuates ICAM-1 formation in hydrogen peroxide-treated L6 skeletal muscle cells and soleus muscle of mice subjected to eccentric exercise

L6 skeletal muscle cells overexpressed ICAM-1 when treated with H2O2. Maximum effect was observed at 200μM H2O2. Des-aspartate-angiotensin I (DAA-I) concentration-dependently attenuated the overexpression. Maximum attenuation occurred at 10−10M DAA-I. H2O2 activated NFκB and its translocation into the nucleus of L6 muscle cells suggesting that NFκB mediates the H2O2-induced overexpression of ICAM-1. DAA-I inhibited the activation and translocation of NFκB. H2O2 is a major oxidant formed during skeletal muscle contraction and is implicated in oxidative stress and skeletal muscle damage in excessive unaccustomed exercise. The data show that DAA-I has antioxidant action, and its action was further investigated in the soleus muscle of mice subjected to 240min of eccentric exercise on a rodent treadmill. The eccentric exercise induced superoxide formation and overexpression of ICAM-1 in the soleus muscle of the mice at 3days post exercise. DAA-I (0.2nmole/kg/day) administered orally on day 1 (pre-exercise) and 2days post-exercise attenuated both the ROS formation and ICAM-1 overexpression. Earlier studies show that DAA-I acts as an agonist on the angiotensin AT1 receptor and elicits responses opposing those of angiotensin II. The present and earlier findings support the recent suggestion that angiotensin II is involved in skeletal muscle damage, and curtailment of its actions via ACE inhibitors and losartan protects and improves skeletal muscle damage. These findings open up new avenues for treatment and management of skeletal muscle damage via the interventions of the renin angiotensin system.

Insulin stimulates mitochondrial fusion and function in cardiomyocytes via the Akt-mTOR-NFκB-Opa-1 signaling pathway.

Insulin regulates heart metabolism through the regulation of insulin-stimulated glucose uptake. Studies have indicated that insulin can also regulate mitochondrial function. Relevant to this idea, mitochondrial function is impaired in diabetic individuals. Furthermore, the expression of Opa-1 and mitofusins, proteins of the mitochondrial fusion machinery, is dramatically altered in obese and insulin-resistant patients. Given the role of insulin in the control of cardiac energetics, the goal of this study was to investigate whether insulin affects mitochondrial dynamics in cardiomyocytes. Confocal microscopy and the mitochondrial dye MitoTracker Green were used to obtain three-dimensional images of the mitochondrial network in cardiomyocytes and L6 skeletal muscle cells in culture. Three hours of insulin treatment increased Opa-1 protein levels, promoted mitochondrial fusion, increased mitochondrial membrane potential, and elevated both intracellular ATP levels and oxygen consumption in cardiomyocytes in vitro and in vivo. Consequently, the silencing of Opa-1 or Mfn2 prevented all the metabolic effects triggered by insulin. We also provide evidence indicating that insulin increases mitochondrial function in cardiomyocytes through the Akt-mTOR-NFκB signaling pathway. These data demonstrate for the first time in our knowledge that insulin acutely regulates mitochondrial metabolism in cardiomyocytes through a mechanism that depends on increased mitochondrial fusion, Opa-1, and the Akt-mTOR-NFκB pathway.

Enhancement of glucose uptake in skeletal muscle L6 cells and insulin secretion in pancreatic hamster-insulinoma-transfected cells by application of non-thermal plasma jet

Type-II diabetes Mellitus is characterized by defects in insulin action on peripheral tissues, such as skeletal muscle, adipose tissue, and liver and pancreatic beta cells. Since the skeletal muscle accounts for approximately 75% of insulin-stimulated glucose-uptake in our body, impaired insulin secretion from defected beta cell plays a major role in the afflicted glucose homoeostasis. It was shown that the intracellular reactive oxygen species and nitric oxide level was increased by non-thermal-plasma treatment in ambient air. These increased intracellular reactive species may enhance glucose uptake and insulin secretion through the activation of intracellular calcium (Ca+) and cAMP production.

Molecular Effects of Diphenyl Diselenide on Cholesterol and Glucose Cell Metabolism

This study was designed to investigate the molecular effects of diphenyl diselenide ((PhSe)₂) on cholesterol metabolism in HepG2 cell line in a dose-dependent manner. The protein levels of both total and phosphorylated 3- hydroxy-3-methylglutaryl coenzyme A reductase (HMGR and P-HMGR), low-density lipoprotein receptors (LDLr) and the proteins involved in their regulatory network were analyzed by Western blotting, and the effect of (PhSe)₂ on HMGR activity was measured. Additionally, we also evaluated the effects of this compound on glucose transporter type 4 (GLUT4) translocation using fluorescence microscopy in L6 skeletal muscle cell line. Results demonstrated that (PhSe)₂ increased P-HMGR, HMGR, and LDLr protein levels as well as simvastatin treatment, which was used as positive control, without directly affecting HMGR activity. We observed that both long- and short-term HMGR regulation mechanisms are involved in the effects of (PhSe)₂, as this compound was able to augment Sterol regulatory element binding proteins (SREBP)-1 and Insulin induced gene (Insig)1 protein levels, and to increase AMP activated kinase (AMPK) activation state. We also found that, in L6 skeletal myotubes, 10 μM (PhSe)₂ increases GLUT4 translocation through AMPK activation. Taken together, these findings suggest that (PhSe)₂ can modulate the expression of some proteins involved in cholesterol and glucose cell metabolism.

Dietary nitrite improves insulin signaling through GLUT4 translocation

Diabetes mellitus type 2 is a syndrome of disordered metabolism with inappropriate hyperglycemia owing to a reduction in the biological effectiveness of insulin. Type 2 diabetes is associated with an impaired nitric oxide (NO) pathway that probably serves as the key link between metabolic disorders and cardiovascular disease. Insulin-mediated translocation of GLUT4 involves the PI3K/Akt kinase signal cascade that results in activation of endothelial NO synthase (eNOS). eNOS is dysfunctional during diabetes. We hypothesize that loss of eNOS-derived NO terminates the signaling cascade and therefore cannot activate GLUT4 translocation and that dietary nitrite may repair this pathway. In this study, we administered 50mg/L sodium nitrite to db/db diabetic mice for 4 weeks. After 4 weeks treatment, the db/db mice experienced less weight gain, improved fasting glucose levels, and reduced insulin levels. Cell culture experiments using CHO-HIRc-myc-GLUT4eGFP cell lines stably expressing insulin receptor and myc-GLUT4eGFP protein, as well as L6 skeletal muscle cells stably expressing rat GLUT4 with a Myc epitope (L6-GLUT4myc), showed that NO, nitrite, and GSNO stimulate GLUT4 translocation independent of insulin, which is inhibited by NEM. Collectively our data suggest that nitrite improves insulin signaling through restoration of NO-dependent nitrosation of GLUT4 signaling translocation. These data suggest that NO-mediated nitrosation of GLUT4 by nitrite or other nitrosating agents is necessary and sufficient for GLUT4 translocation in target tissue. Description of this pathway may justify a high-nitrate/nitrite diet along with the glycemic index to provide a safe and nutritional regimen for the management and treatment of diabetes.

6-Mercaptopurine augments glucose transport activity in skeletal muscle cells in part via a mechanism dependent upon orphan nuclear receptor NR4A3

The purine anti-metabolite 6-mercaptopurine (6-MP) is widely used for the treatment of leukemia and inflammatory diseases. The cellular effects of 6-MP on metabolism remain unknown; however, 6-MP was recently found to activate the orphan nuclear receptor NR4A3 in skeletal muscle cell lines. We have reported previously that NR4A3 (also known as NOR-1, MINOR) is a positive regulator of insulin sensitivity in adipocytes. To further explore the role of NR4A3 activation in insulin action, we explored whether 6-MP activation of NR4A3 could modulate glucose transport system activity in L6 skeletal muscle cells. We found that 6-MP increased both NR4A3 expression and NR4A3 transcriptional activity and enhanced glucose transport activity via increasing GLUT4 translocation in both basal and insulin-stimulated L6 cells in an NR4A3-dependent manner. Furthermore, 6-MP increased levels of phospho-AS160, although this effect was not modulated by NR4A3 overexpression or knockdown. These primary findings provide a novel proof of principle that 6-MP, a small molecule NR4A3 agonist, can augment glucose uptake in insulin target cells, although this occurs via both NR4A3-dependent and -independent actions; the latter is related to an increase in phospho-AS160. These results establish a novel target for development of new treatments for insulin resistance.

Ceramide accumulation in L6 skeletal muscle cells due to increased activity of ceramide synthase isoforms has opposing effects on insulin action to those caused by palmitate treatment

An accumulation of ceramides has been implicated in the generation of insulin resistance in skeletal muscle upon an oversupply of fatty acid. Different ceramide species are generated through the actions of ceramide synthases (CerSs), which incorporate specific acyl side chains. We tested whether particular CerS isoforms promoted insulin resistance through the generation of more inhibitory ceramide species, thus representing potential targets for intervention. CerS isoforms CerS1, CerS2, CerS4, CerS5 and CerS6 were overexpressed in L6 myotubes using adenovirus, and cells were treated with palmitate and stimulated with insulin. Alternatively, CerS isoforms were knocked down using siRNAs. Sphingolipids were examined by mass spectrometry and tracer incorporation. Phosphorylation of IRS1 and Akt was measured by immunoblotting, while glucose disposal was assessed by measuring GLUT4 translocation and the incorporation of [(14)C]glucose into glycogen. Palmitate treatment increased the levels of several ceramides but reduced the levels of sphingomyelins, while insulin had no effect. The fatty acid also inhibited insulin-stimulated Akt phosphorylation and glycogen synthesis. Overexpression of CerS isoforms increased specific ceramides. Unexpectedly, the overexpression of CerS1 and CerS6 promoted insulin action, while no isoform had inhibitory effects. CerS6 knockdown had effects reciprocal to those of CerS6 overexpression. Palmitate may increase intracellular ceramide levels through sphingomyelin hydrolysis as well as de novo synthesis, but no particular species were implicated in the generation of insulin resistance. The modulation of ceramides through an alteration of CerS expression does not affect the action of insulin in the same way as ceramide generation by palmitate treatment. Conversely, certain isoforms promote insulin action, indicating the importance of ceramides in cell function.

Kiwifruit (Actinidia chinensis) extract annuls chronic insulininduced insulin resistance in l6 skeletal muscle cells

Kiwi fruit has long been believed to be beneficial to human health due to its numerous biological actions; however the pharmacological mechanisms associated with these benefits are incompletely understood. In the present study, the effects of ‘Halla Gold’ (Actinidia chinensis var.), a new kiwifruit cultivar bred by the Jeju Rural Development Administration (RDA), on chronic insulin-induced insulin resistance in L6 skeletal muscle cells was investigated. Chronic insulin reduced phosphorylation of Akt and GSK-3α/β, and increased phosphorylation of glycogen synthase (GS). Furthermore, glucose consumption and glucose transporter 4 (Glut4) translocation to the plasma membrane were inhibited. However, ‘Halla Gold’ extract (HGE) increased phosphorylation of Akt and GSK-3α/β concomitantly with decreased phosphorylation of GS. Moreover, HGE improved insulin-stimulated glucose consumption by facilitating Glut4 translocation and this stimulation was suppressed by inhibition of PI3 kinase. Together, our data suggest that ‘Halla Gold’ extract abolishes chronic insulin-induced insulin resistance through the PI3K/Akt pathway.