Pathway In Skeletal Muscle Cells Research Articles

Manganese influx and expression of ZIP8 is essential in primary myoblasts and contributes to activation of SOD2.

Trace elements such as copper (Cu), zinc (Zn), iron (Fe), and manganese (Mn) function as enzyme cofactors and second messengers in cell signaling. Trace elements are emerging as key regulators of differentiation and development of mammalian tissues including blood, brain, and skeletal muscle. We previously reported an influx of Cu and dynamic expression of metal transporters during differentiation of skeletal muscle cells. Here, we demonstrate that during differentiation of skeletal myoblasts an increase of Mn, Fe and Zn also occurs. Interestingly the Mn increase is concomitant with increased Mn-dependent SOD2 levels. To better understand the Mn import pathway in skeletal muscle cells, we probed the functional relevance of the closely related proteins ZIP8 and ZIP14, which are implicated in Zn, Mn, and Fe transport. Partial depletion of ZIP8 severely impaired growth of myoblasts and led to cell death under differentiation conditions, indicating that ZIP8-mediated metal transport is essential in skeletal muscle cells. Moreover, knockdown of Zip8 impaired activity of the Mn-dependent SOD2. Growth defects were partially rescued only by Mn supplementation to the medium, suggesting additional functions for ZIP8 in the skeletal muscle lineage. Restoring wild type Zip8 into the knockdown cells rescued the proliferation and differentiation phenotypes. On the other hand, knockdown of Zip14, had only a mild effect on myotube size, consistent with a role for ZIP14 in muscle hypertrophy. Simultaneous knockdown of both Zip8 and Zip14 further impaired differentiation and led cell death. This is the first report on the functional relevance of two members of the ZIP family of metal transporters in the skeletal muscle lineage, and further supports the paradigm that trace metal transporters are important modulators of mammalian tissue development.

Quercetin and its metabolite isorhamnetin promote glucose uptake through different signalling pathways in myotubes

Quercetin and its metabolite isorhamnetin elicit various beneficial effects on human health. However, their bioavailability is low. In this study, we investigated whether low concentrations in the physiological range could promote glucose uptake in L6 myotubes, as well as the underlying molecular mechanisms. We found that 0.1 nM and 1 nM quercetin or 1 nM isorhamnetin significantly increased glucose uptake via translocation of glucose transporter type 4 (GLUT4) to the plasma membrane of L6 myotubes. Quercetin principally activated the CaMKKβ/AMPK signalling pathway at these concentrations, but also activated IRS1/PI3K/Akt signalling at 10 nM. In contrast, 1 nM and 10 nM isorhamnetin principally activated the JAK/STAT pathway. Treatment with siAMPKα and siJAK2 abolished quercetin- and isorhamnetin-induced GLUT4 translocation, respectively. However, treatment with siJAK3 did not affect isorhamnetin-induced GLUT4 translocation, indicating that isorhamnetin induced GLUT4 translocation mainly through JAK2, but not JAK3, signalling. Thus, quercetin preferably activated the AMPK pathway and, accordingly, stimulated IRS1/PI3K/Akt signalling, while isorhamnetin activated the JAK2/STAT pathway. Furthermore, after oral administration of quercetin glycoside at 10 and 100 mg/kg body weight significantly induced GLUT4 translocation to the plasma membrane of skeletal muscles in mice. In the same animals, plasma concentrations of quercetin aglycone form were 4.95 and 6.80 nM, respectively. In conclusion, at low-concentration ranges, quercetin and isorhamnetin promote glucose uptake by increasing GLUT4 translocation via different signalling pathways in skeletal muscle cells; thus, these compounds may possess beneficial functions for maintaining glucose homeostasis by preventing hyperglycaemia at physiological concentrations.

Stabilized β-Hairpin Peptide Inhibits Insulin Degrading Enzyme.

Insulin-degrading enzyme (IDE) plays a critical role in both the proteolytic degradation and inactivation of insulin. The exploration of novel IDE inhibitors could aid in the study of novel therapeutics for type-2 diabetes. Herein, we report a hypothesized stabilized β-hairpin peptide that can efficiently inhibit the enzymatic activity of IDE. The resulting stabilized peptide B35 is demonstrated to activate the AKT phosphorylation pathway in skeletal muscle cells and is shown to slow insulin degradation. An 80 mg kg-1 intraperitoneal (i.p.) injection of the stabilized β-hairpin peptide B35 is demonstrated to improve glucose tolerance during an oral glucose tolerance test in obese mouse model. We note that this stabilized peptide exhibited negligible cytotoxicity in both in vitro and in vivo assays, even at high concentrations (300 μM). This study suggests that IDE peptide inhibitors could function as potentially meaningful candidates for the development of type-2 diabetes therapeutics.

Egg white hydrolysate and peptide reverse insulin resistance associated with tumor necrosis factor-\u03b1 (TNF-\u03b1) stimulated mitogen-activated protein kinase (MAPK) pathway in skeletal muscle cells

PurposeExcessive formation of tumor necrosis factor-α (TNF-α), a pro-inflammatory cytokine, has been implicated in the development of insulin resistance in obesity and type-2 diabetes. In skeletal muscle, chronic exposure to TNF-α impairs insulin-stimulated glucose uptake and insulin signaling. The aim of this study is to investigate the effects of enzymatic egg white hydrolysate (EWH) and its responsible peptide, IRW, on TNF-α-induced insulin resistance and the underlying molecular mechanisms using rat skeletal muscle cells (L6 cells).MethodsInsulin resistance was induced by treating L6 cells with 5 ng/ml TNF-α for 24 h. Effects of EWH and IRW on glucose uptake were detected by glucose uptake assay, glucose transporter 4 (GLUT4) translocation by immunofluorescence, and western blot, while insulin-signaling pathway and mitogen-activated protein kinase (MAPK) pathway were investigated using western blot.ResultsAdding both EWH and IRW significantly improved glucose uptake in TNF-α-treated cells, increased activation of insulin receptor substrate (IRS-1) tyrosine residue and protein kinase B (Akt), whereas decreased activation of IRS-1 serine residue. In addition, TNF-α-induced activation of p38-mitogen-activated protein kinase (p38) and c-Jun N-terminal kinases (JNK) 1/2 were decreased by either EWH or IRW treatment.ConclusionEWH and IRW improve impaired insulin sensitivity by down-regulating the activation of p38 and JNK1/2 in TNF-α-treated skeletal muscle cells.

Mitochondria-Mediated Pathway Regulates C2C12 Cell Apoptosis Induced by Fluoride.

This study was designed to investigate the mechanisms of excessive fluoride-induced apoptosis via mitochondria-mediated pathway in skeletal muscle cells (C2C12 cells). C2C12 cells were cultured with the fluoride concentrations (0, 1, and 2.5mmol/L) for 48h. The morphology and ultrastructural changes of C2C12 cells were observed using a light microscope and transmission electron microscope (TEM). The protein expression levels of apoptosis factors, including Bax, Bcl-2, cytochrome c (Cyt c), caspase-3, and caspase-9, were measured using real-time polymerase chain reaction (real-time PCR) and immunocytofluorescence. The morphology and ultrastructure of C2C12 cells were seriously damaged by fluoride at 1 and 2.5mmol/L doses, including swollen mitochondria, vacuolization, ridge breakage, and disappearance of the nuclear membrane. Simultaneously, compared with the control group, the expression levels of Bax, Bcl-2, Cyt c, caspase-3, and caspase-9 were up-regulated after fluoride treatment. Excessive fluoride damages the ultrastructure in mitochondria, leading to the release of Cyt c from the mitochondria to cytoplasm in C2C12 cells; thereby, activated caspases cascade apoptosis process through a mitochondria-mediated pathway.

Adenosine isolated from Grifola gargal promotes glucose uptake via PI3K and AMPK signalling pathways in skeletal muscle cells

Grifola gargal, an edible mushroom originating in southern Argentina and Chile, is known to have such beneficial health effects as antioxidant and anti-inflammatory activities. A low molecular weight fraction from Grifola gargal (GLF) decreased fasting blood glucose levels in high-fat diet-induced diabetic mice. This implicated Grifola gargal as a functional food to prevent type 2 diabetes mellitus (T2DM). To address the unknown underlying molecular mechanisms, we investigated, using L6 myotubes, whether GLF promoted GLUT4 translocation to the plasma membrane and its related signalling pathways. In addition, adenosine was isolated and identified the active compound in GLF to increase glucose uptake and promote GLUT4 translocation in L6 myotubes. Adenosine activated PI3K/Akt signalling and phosphorylation of AMPK, by binding to the adenosine receptor A1. Thus, Grifola gargal is a promising functional food to prevent post-prandial hyperglycaemia and T2DM by promoting glucose uptake in muscle.

Suppression of Rho-kinase 1 is responsible for insulin regulation of the AMPK/SREBP-1c pathway in skeletal muscle cells exposed to palmitate.

Clinical and experimental data suggest that early insulin therapy could reduce lipotoxicity in subjects and animal models with type 2 diabetes mellitus. However, the underlying mechanisms need to be clarified. Sterol regulatory element-binding protein 1c (SREBP-1c), which is negatively regulated by AMP-activated protein kinase (AMPK), plays a critical role in lipotoxicity and insulin resistance in skeletal muscle cells. Here, we investigated the effect and molecular mechanism of insulin intervention on the AMPK/SREBP-1c pathway in skeletal muscle cells with chronic exposure to palmitic acid (PA). Male C57BL/6 mice were fed with a high-fat diet for 12weeks and were then treated with insulin, AMPK inhibitor, or metformin. L6 myotubes incubated with palmitic acid (PA) were treated with insulin or metformin. Dominant-negative AMPKα2 (DN-AMPKα2) lentivirus, AMPKα2 siRNA, or Rho-kinase 1 (ROCK1) siRNA were transfected into PA-treated L6 myotubes. We found that the ability of PA to stimulate SREBP-1c and inhibit AMPK was reversed by insulin in L6 cells. Moreover, DN-AMPKα2 lentivirus and AMPKα2 siRNA were transfected into PA-treated L6 myotubes, and the decrease in SREBP-1c expression caused by insulin was blocked by AMPK inhibition independent of the phosphatidylinositol-4,5-biphosphate-3-kinase (PI3K)/AKT pathway. The serine/threonine kinase Rho-kinase (ROCK) 1, a downstream effector of the small G protein RhoA, was activated by PA. Interestingly, knockdown of ROCK1 by siRNA blocked the downregulation of AMPK phosphorylation under PA-treated L6 myotubes, which indicated that ROCK1 mediated the effect of insulin action on AMPK. Our study indicated that insulin reduced lipotoxicity via ROCK1 and then improved AMPK/SREBP-1c signaling in skeletal muscle under PA-induced insulin resistance.

AMPK activation by prolonged stimulation with interleukin-1β contributes to the promotion of GLUT4 translocation in skeletal muscle cells.

Impaired insulin signaling in skeletal muscle cells causes insulin resistance associated with the onset of type 2 diabetes. Although interleukin (IL)-1β has been considered to be implicated in the pathogenesis of type 2 diabetes, the action of prolonged stimulation with IL-1β on the insulin signaling pathway in skeletal muscle cells remains poorly understood. In the current study, we investigated the effect of IL-1β stimulation on insulin signal transduction from the insulin receptor (IR), resulting in glucose transporter 4 (GLUT4) translocation in skeletal muscle cells. In L6-GLUT4myc cells, stimulation with IL-1β for 24 h promoted GLUT4 translocation to the plasma membrane and increased glucose uptake in a concentration-dependent manner, whereas short-term stimulation with IL-1 for up to 6 h did not affect that. In addition, stimulation with IL-1β for 24 h further increased insulin-stimulated GLUT4 translocation. Interestingly, stimulation with IL-1β for 24 h did not cause any change in the phosphorylation of insulin signal molecules IR, insulin receptor substrate (IRS)-1, Akt, and p21-activated kinase (PAK1). Stimulation with IL-1β for 24 h significantly increased AMP-activated protein kinase (AMPK) phosphorylation and GLUT4 protein expression. Small interfering RNA (siRNA) targeting AMPK1/2 significantly inhibited IL-1β-stimulated GLUT4 translocation. These results suggest that prolonged stimulation with IL-1β positively regulates GLUT4 translocation in skeletal muscle cells. IL-1β may have a beneficial effect on maintaining glucose homeostasis in skeletal muscle cells in patients with type 2 diabetes. .

Syringaresinol induces mitochondrial biogenesis through activation of PPARβ pathway in skeletal muscle cells

Activation of peroxisome proliferator-activated receptors (PPARs) plays a crucial role in cellular energy metabolism that directly impacts mitochondrial biogenesis. In this study, we demonstrate that syringaresinol, a pharmacological lignan extracted from Panax ginseng berry, moderately binds to and activates PPARβ with KD and EC50 values of 27.62±15.76μM and 18.11±4.77μM, respectively. Subsequently, the expression of peroxisome proliferator-activated receptor γ coactivator-1α together with PPARβ transcriptional targets, mitochondrial carnitine palmitoyltransferase 1 and uncoupling protein 2, was also enhanced in terms of both mRNA and protein levels. The activation of these proteins induced mitochondrial biogenesis by enrichment of mitochondrial replication and density within C2C12 myotubes. Importantly, knockdown of PPARβ reduced the syringaresinol-induced protein expression followed by the significant reduction of mitochondrial biogenesis. Taken together, our results indicate that syringaresinol induces mitochondrial biogenesis by activating PPARβ pathway.

DJ-1 protects against undernutrition-induced atrophy through inhibition of the MAPK-ubiquitin ligase pathway in myoblasts.

The purpose of this study is to explore whether antioxidant DJ-1 protein affects the atrophy of skeletal muscle cell induced by undernutrition. To determine cell atrophic responses, L6 cell line and skeletal primary cells from mouse hind limbs were cultivated under condition of FBS-free and low glucose. Changes of protein expression were analyzed using Western blot. Overexpression and knockdown of DJ-1 was performed in cells to assess its influence on cell atrophic responses. Undernutrition decreased cell size and increased the abundance of oxidized form and total form of DJ-1 protein in L6 myoblasts. The undernourished cells revealed an elevation in the expression of muscle-specific RING finger-1 (MuRF-1) and atrogin-1, and in the phosphorylations of p38 mitogen-activated protein kinase (MAPK) and stress-activated protein kinase/c-Jun N-terminal kinase compared with control groups. Moreover, DJ-1-knockout mice showed a decrease in cell size and an enhancement in the expression of MuRF-1 and atrogin-1, as well as in the phosphorylation of MAPKs in gastrocnemius muscles; these changes were also observed in L6 cells transfected with siRNA of DJ-1. On the other hand, L6 cells overexpressing full-length DJ-1 did not exhibit the alterations in cell size and ubiquitin ligases seen after undernourished states of control cells. Myotubes differentiated from L6 cells also showed elevated expression of MuRF-1 and atrogin-1 in response to undernutrition. These results suggest that DJ-1 protein may contribute to undernutrition-induced atrophy via MAPKs/ubiquitin ligase pathway in skeletal muscle cells.

Mechanical stretch activates mammalian target of rapamycin and AMP-activated protein kinase pathways in skeletal muscle cells.

Cellular protein synthesis is believed to be antagonistically regulated by mammalian target of rapamycin (mTOR) and AMP-activated protein kinase (AMPK) signaling pathways. In the present study, we examined the relationship between mTOR/p70 S6 kinase (p70S6K) and AMPK in response to mechanical stretch. C2C12 myoblasts were grown on a silicone elastomer chamber to confluence and further cultured in differentiation medium for 4 days to form multinucleated myotubes. Cells were subjected to 15% cyclic uniaxial stretch for 4 h at a frequency of 1 Hz. Phosphorylation of p70S6K at threonine 389 and AMPK at threonine 172 of the catalytic α subunit were concomitantly increased by mechanical stretch. Stimulation of the mTOR pathway by adding leucine and insulin increased the phosphorylation of p70S6K without inactivation of AMPK. In contrast, addition of compound C, a pharmacological inhibitor of AMPK, increased the phosphorylation of p70S6K in stretched cells. Activation of AMPK by the addition of 5-amino-4-imidazolecarboxamide ribonucleoside reduced the phosphorylation of p70S6K in response to mechanical stretch. In conclusion, crosstalk between mTOR and AMPK signaling was not tightly regulated in response to physiological stimuli, such as mechanical stress and/or nutrients. However, pharmacological modulation of AMPK influenced the mTOR/p70S6K signaling pathway.

Irisin, a Novel Myokine, Regulates Glucose Uptake in Skeletal Muscle Cells via AMPK.

Irisin is a novel myokine produced by skeletal muscle. However, its metabolic role is poorly understood. In the present study, irisin induced glucose uptake in differentiated skeletal muscle cells. It increased AMP-activated protein kinase (AMPK) phosphorylation and the inhibition of AMPK blocked glucose uptake. It also increased reactive oxygen species (ROS) generation. N-acetyl cysteine, a ROS scavenger, blocked irisin-induced AMPK phosphorylation. Moreover, irisin activated p38 MAPK in an AMPK-dependent manner. The inhibition and knockdown of p38 MAPK blocked irisin-induced glucose uptake. A colorimetric absorbance assay showed that irisin stimulated the translocation of glucose transporter type 4 to the plasma membrane and that this effect was suppressed in cells pretreated with a p38 MAPK inhibitor or p38 MAPK small interfering RNA. In primary cultured myoblast cells, irisin increased the concentration of intracellular calcium. STO-609, a calcium/calmodulin-dependent protein kinase kinase inhibitor, blocked irisin-induced AMPK phosphorylation, implying that calcium is involved in irisin-mediated signaling. Our results suggest that irisin plays an important role in glucose metabolism via the ROS-mediated AMPK pathway in skeletal muscle cells.

Black Tea Polyphenols Promotes GLUT4 Translocation through Both PI3K-and AMPK-dependent Pathways in Skeletal Muscle Cells

We previously reported that the intake of black tea promotes translocation of the insulin-sensitive glucose transporter (GLUT) 4 in skeletal muscle. In this study, we investigated whether black tea polyphenols (BTP) promote GLUT4 translocation in L6 myotubes. BTP promoted glucose uptake accompanied by GLUT4 translocation in L6 myotubes. As the molecular mechanism, BTP induced the phosphorylation of insulin receptor substrate-1, atypical protein kinase C, Akt Thr308, Akt substrate 160, and AMP-activated protein kinase (AMPK), but did not affect that of Akt Ser473. BTP increased glycogen accumulation through inactivation of glycogen synthase kinase 3β (GSK-3β). Theaflavin, one of the major components in black tea, also promoted the glucose uptake accompanied by GLUT4 translocation observed with BTP in L6 myotubes. These results indicate that BTP activates both PI3K- and AMPK-dependent pathways to promote GLUT4 translocation and glycogen accumulation in skeletal muscle cells. Moreover, theaflavin is one of the active components in BTP.

P38 MAPK activation upregulates proinflammatory pathways in skeletal muscle cells from insulin-resistant type 2 diabetic patients

Skeletal muscle is the key site of peripheral insulin resistance in type 2 diabetes. Insulin-stimulated glucose uptake is decreased in differentiated diabetic cultured myotubes, which is in keeping with a retained genetic/epigenetic defect of insulin action. We investigated differences in gene expression during differentiation between diabetic and control muscle cell cultures. Microarray analysis was performed using skeletal muscle cell cultures established from type 2 diabetic patients with a family history of type 2 diabetes and clinical evidence of marked insulin resistance and nondiabetic control subjects with no family history of diabetes. Genes and pathways upregulated with differentiation in the diabetic cultures, compared with controls, were identified using Gene Spring and Gene Set Enrichment Analysis. Gene sets upregulated in diabetic myotubes were associated predominantly with inflammation. p38 MAPK was identified as a key regulator of the expression of these proinflammatory gene sets, and p38 MAPK activation was found to be increased in the diabetic vs. control myotubes. Although inhibition of p38 MAPK activity decreased cytokine gene expression from the cultured diabetic myotubes significantly, it did not improve insulin-stimulated glucose uptake. Increased cytokine expression driven by increased p38 MAPK activation is a key feature of cultured myotubes derived from insulin-resistant type 2 diabetic patients. p38 MAPK inhibition decreased cytokine expression but did not affect the retained defect of impaired insulin action in the diabetic muscle cells.

Leucine activation of mTORC1 in skeletal muscle cells is inhibited by the lysosomotropic agent chloroquine (820.2)

Recent work in human embryonic kidney cells has uncovered many of the cellular mechanisms underlying amino acid activation of the mTORC1 signaling pathway. It is now known that mTORC1 translocation to the lysosomal surface is required for the amino acid activation of mTORC1 signaling. It is not known if the lysosome is required for amino acid activation of mTORC1 in skeletal muscle cells. To test this hypothesis, we exposed C2C12 muscle cells (that were plated and differentiated into myotubes for 7‐8 days) to both leucine and a potent lysosomal inhibitor (chloroquine). Cells were placed into three groups: control (1), leucine only (2) and leucine+chloroquine (3). The first two groups were taken out of media and starved in a Hepes buffer for 9hrs. Group 3 was starved for 8hrs followed by a 1hr starve in 2000ug/ml chloroquine in the same Hepes buffer. Groups 2 and 3 were given 1mM leucine for ten minutes before collection. Western blotting was used to determine phosphorylation of mTOR and its downstream targets. The leucine only group showed a significant increase in mTOR and p70s6k phosphorylation compared to control and leucine+chloroquine groups (p<0.05). It appears that disruption of lysosomal function by chloroquine blocks leucine activation of the mTORC1 signaling pathway in skeletal muscle cells. Future work will determine the role of the lysosome for amino acid activation of mTORC1 in vivo.Grant Funding Source: Supported by NIH AR049877

AICAR-induced activation of AMPK negatively regulates myotube hypertrophy through the HSP72-mediated pathway in C2C12skeletal muscle cells

5'-AMP-activated protein kinase (AMPK) plays an important role as a negative regulator of skeletal muscle mass. However, the precise mechanism of AMPK-mediated regulation of muscle mass is not fully clarified. Heat shock proteins (HSPs), stress-induced molecular chaperones, are related with skeletal muscle adaptation, but the association between AMPK and HSPs in skeletal muscle hypertrophy is unknown. Thus, we investigated whether AMPK regulates hypertrophy by mediating HSPs in C2C12 cells. The treatment with AICAR, a potent stimulator of AMPK, decreased 72-kDa HSP (HSP72) expression, whereas there were no changes in the expressions of 25-kDa HSP, 70-kDa heat shock cognate, and heat shock transcription factor 1 in myotubes. Protein content and diameter were less in the AICAR-treated myotubes in those without treatment. AICAR-induced suppression of myotube hypertrophy and HSP72 expression was attenuated in the siRNA-mediated AMPKα knockdown myotubes. AICAR increased microRNA (miR)-1, a modulator of HSP72, and the increase of miR-1 was not induced in AMPKα knockdown condition. Furthermore, siRNA-mediated HSP72 knockdown blocked AICAR-induced inhibition of myotube hypertrophy. AICAR upregulated the gene expression of muscle Ring-finger 1, and this alteration was suppressed in either AMPKα or HSP72 knockdown myotubes. The phosphorylation of p70 S6 kinase Thr(389) was downregulated by AICAR, whereas this was attenuated in AMPKα, but not in HSP72, knockdown myotubes. These results suggest that AMPK inhibits hypertrophy through, in part, an HSP72-associated mechanism via miR-1 and protein degradation pathways in skeletal muscle cells.

Paraoxonase1 (PON1) reduces insulin resistance in mice fed a high-fat diet, and promotes GLUT4 overexpression in myocytes, via the IRS-1/Akt pathway

ObjectiveTo analyze Paraoxonase1 (PON1) impact on GLUT4 expression, glucose metabolism, and the insulin signaling pathway in skeletal muscle cells. Methods and resultsWe analyzed the effect of PON1 in high-fat-diet-induced insulin resistance in C57BL/6J and in PON1KO mice. Mice were fed normal diet (ND) or high Fat Diet (HFD) for 8 weeks. PON1 deficiency caused enhanced insulin resistance in both ND and HFD mice. PON1 deficiency was associated with increased oxidative stress (OS), increased p38MAPK activity and attenuated insulin-mediated tyrosine phosphorylation of muscle insulin receptor substrate-1 (IRS-1), with a corresponding increase in serine phosphorylation. These effects resulted in decreased glucose uptake in whole-body level, as reflected by glucose tolerance test (GTT), by insulin tolerance test (ITT) and by cellular glycogen accumulation in the liver and in the muscles. PON1 addition to cultured C2 muscle cells enhanced GLUT4 mRNA expression, in a time and concentration dependent manner, increased GLUT4 protein and cellular glycogen accumulation. These effects were mediated via inhibition of p38MAPK activity, resulting in reduced IRS-1 serine phosphorylation and in enhanced IRS-1 tyrosine phosphorylation. The ability of PON1 to increase myocytes GLUT4 expression was partially inhibited upon blocking PON1 SH group, and completely abolished upon PON1 mutation in HIS115 of its catalytic site. ConclusionPON1 plays a beneficial role in glucose regulation and metabolism and may serve as an important tool in diabetes control.

Docosahexaenoic acid (DHA) prevents atrophy-related signaling in palmitate-treated C2C12 MUSCLE CELLS

Hypertriglyceridemia is a comorbidity of chronic kidney disease (CKD) that contributes to insulin resistance and dysfunctional protein metabolism in skeletal muscle, resulting in debilitating muscle atrophy. The saturated fatty acid palmitate (PA) induces muscle insulin resistance and promotes apoptosis and atrophic signaling pathways. In contrast, omega-3 polyunsaturated fatty acids like docosahexaenoic acid (DHA) improve insulin sensitivity and may attenuate activation of pathways that lead to muscle atrophy. The purpose of this study was to examine the effects of saturated and omega-3 fatty acids on atrophy-related signaling pathways in skeletal muscle cells. Our hypothesis was that DHA prevents the detrimental effects of PA. C2C12 myotubes were treated with 0.1mM DHA and/or 0.5mM PA for 48 h. Insulin (100nM; 15 min) was added to cells to activate signaling pathways. The expression of proteins involved in atrophic and apoptotic signaling pathways was examined by Western analysis. PA reduced myotube size and number. PA also reduced insulin-stimulated Akt and Foxo3a phosphorylation plus induced caspase-3 and actin-fragmentation. All of these responses were blocked by the addition of DHA; DHA alone had minimal effects on these pathways. In conclusion, these data suggest that DHA protects muscle cells from activation of atrophic signaling pathways by high levels of saturated fatty acids. Therefore, protein-energy wasting during CKD may be improved by DHA supplements.Support: NIH DK007734

Docosahexaenoic acid prevents atrophy‐related signaling in palmitate‐treated myotubes

Lipid accumulation promotes insulin resistance and dysfunctional protein metabolism in skeletal muscle, resulting in debilitating muscle atrophy. While the saturated fatty acid palmitate (PA) induces muscle insulin resistance and promotes apoptosis and atrophic signaling pathways, omega‐3 polyunsaturated fatty acids such as docosahexaenoic acid (DHA) improve insulin sensitivity and may attenuate activation of pathways that lead to atrophy. Therefore, the purpose of this study was to examine the effects of saturated and omega‐3 fatty acids on atrophy‐related signaling pathways in skeletal muscle cells with the hypothesis that DHA prevents the detrimental effects of PA. C2C12 myotubes were treated with 0.1mM DHA and/or 0.5mM PA for 48 h. Insulin (100nM; 15 min) was added to cells to activate signaling pathways. The expression of proteins involved in atrophic and apoptotic signaling pathways was examined by Western analysis. PA reduced myotube size and number. PA also reduced insulin‐stimulated Akt, MEK1/2, and Foxo3a phosphorylation plus induced caspase‐3 and actin‐fragmentation. All of these responses were blocked by the addition of DHA; DHA alone had minimal effects on these pathways. In conclusion, these data suggest that DHA protects C2C12 myotubes from activation of atrophic signaling pathways by high levels of saturated fatty acids by maintaining insulin survival signaling.Support: NIH DK007734

Myostatin regulates glucose metabolism via the AMP-activated protein kinase pathway in skeletal muscle cells

Myostatin (Mstn) is a secreted growth factor predominately expressed in skeletal muscle that negatively regulates skeletal muscle mass. Recent studies have indicated that loss function of myostatin not only increases muscle mass but also improves insulin sensitivity in vivo. In the present report, we demonstrated that myostatin regulates glucose metabolism by promoting glucose consumption and glucose uptake, increasing glycolysis, and inhibiting glycogen synthesis in skeletal muscle cells. Microarray analysis revealed that myostatin upregulates several genes involved in regulating glucose metabolism such as Glut1, Glut4, Hk2, and IL-6. Further investigation of the molecular basis of these phenomena revealed that AMP-activated protein kinase (AMPK), a key component for maintaining energy homeostasis, was activated by myostatin for promotion of glycolysis. Taken together, these findings provide the first experimental evidence that myostatin regulates glucose metabolism through the AMPK signal pathway in muscle cells. Importantly, our findings highlight that continued investigation of the metabolic function of myostatin is necessary for a comprehensive understanding of its active role in the regulation of skeletal muscle energy metabolism.