Palmitate increases sphingosine-1-phosphate in C2C12 myotubes via upregulation of sphingosine kinase message and activity

Studies in skeletal muscle demonstrate that elevation of plasma FFAs increases the sphingolipid ceramide. We aimed to determine the impact of FFA oversupply on total sphingolipid profiles in a skeletal muscle model. C2C12 myotubes were treated with palmitate (PAL). Lipidomics analysis revealed pleiotropic effects of PAL on cell sphingolipids not limited to ceramides. ¹³C labeling demonstrated that PAL activated several branches of sphingolipid synthesis by distinct mechanisms. Intriguingly, PAL increased sphingosine-1-phosphate independently of de novo synthesis. Quantitative real-time PCR demonstrated that PAL increased sphingosine kinase 1 (SK1) mRNA by approximately 4-fold. This was accompanied by a 2.3-fold increase in sphingosine kinase enzyme activity. This upregulation did not occur upon treatment with oleate, suggesting some level of specificity for PAL. These findings were recapitulated in the diet-induced obesity mouse model, in which high-fat feeding increased SK1 message in skeletal muscle over 2.3-fold. These data suggest that the impact of elevated FFA on sphingolipids reaches beyond ceramides and de novo sphingolipid synthesis. Moreover, these findings identify PAL as a novel regulatory stimulus for SK1.     

Effects of long-term administration of N-3 polyunsaturated fatty acids (PUFA) and selective estrogen receptor modulator (SERM) derivatives in ovariectomized (OVX) mice

We studied the beneficial effects of dietary consumption of n-3 polyunsaturated fatty acids (PUFA) and two selective estrogen receptor modulator (SERM) derivatives (SERM-I and SERM-II) and their combined effect on serum lipids, skin dermis and adipose layers, bone marrow adipogenesis, and cytokine secretion in mice. Two different ovariectomized (OVX) models were studied: treatment began immediately post-OVX in one and 3 months post-OVX in the other. Our results showed that n-3 PUFA and both SERMs decreased triglyceride levels in the serum, and that SERMs also decreased serum cholesterol levels while n-3 PUFA had no similar effect. SERMs had no effect on IL-6, IL-1 beta, or IL-10 levels, but they decreased ex vivo tumor necrosis factor (TNF-alpha). N-3 PUFA decreased secretion of non-induced IL-6 and TNF-alpha from cultured BMC and IL-1 beta levels in vivo (i.e., in bone marrow plasma), but its main effect was a significant elevation in the secretion of IL-10, a known anti-inflammatory cytokine. OVX-induced B-lymphopoiesis was not affected by LY-139481 (SERM-I) while LY-353381 (SERM-II) exhibited an estrogen-antagonistic effect in sham and OVX mice and elevated the amount of B-cells in bone marrow. Fish oil consumption prevented the elevation in B-lymphopoiesis caused by OVX, but had no curative effect on established augmented B-lymphopoiesis. This activity could be mediated via the elevation of IL-10 which was shown to suppress B-lymphopoiesis. Both SERMs and n-3 PUFA inhibited the increase in adipose tissue thickness caused by OVX in mice. Our results showed that n-3 PUFA, could prevent some of the deleterious outcomes of estrogen deficiency that were not affected by SERMs. We observed no significant beneficial effects of the combined administration of SERM-I, SERM-II, and PUFA on the studied parameters.The exact mechanism by which polyunsaturated fatty acids exert their activities is still not clear, but peroxisome proliferator-activated receptors (PPARs) might be involved in processes which are modulated by n-3 PUFA.     

Proliferation of mitochondria and gene expression of carnitine palmitoyltransferase and fatty acyl-CoA oxidase in rat skeletal muscle, heart and liver by hypolipidemic fatty acids

Morphological and biochemical effects were induced at the subcellular level in the skeletal muscle, heart and liver of male rats as a result of feeding with EPA, DHA, and 3-thia fatty acids. The 3-thia fatty acid, tetradecylthioacetic acid (TTA) and EPA induced mitochondrial growth in type I muscle fibers in both the diaphragm and soleus muscle, and the size distribution of mitochondrial areas followed a similar pattern. Only the 3-thia fatty acid induced mitochondrial growth in type II muscle fibers. The mean area occupied by the mitochondria and the size distribution of mitochondrial areas in both fiber types were highly similar in DHA-treated and control animals. Only the 3-thia fatty acid increased the gene-expression of carnitine palmitoyltransferase (CPT)-II in the diaphragm. In the heart, however, the gene expression decreased. In hepatocytes an increase in the mean size of mitochondria was observed after EPA treatment, concomitant with an increase in mitochondrial CPT-II gene expression. Administration of 2-methyl-substituted EPA (methyl-EPA) induced a higher rate of growth of mitochondria than EPA. At the peroxisomal level in the hepatocytes a 3-thia fatty acid, EPA, and DHA increased the areal fraction concomitant with the induction of gene expression of peroxisomal fatty acyl-CoA oxidase (FAO). In the diaphragm, mRNA levels of FAO were not affected by EPA or DHA treatment, whereas gene expression was significantly increased after 3-thia fatty acid treatment. In the heart, both 3-thia fatty acid, EPA and DHA tended to decrease the levels of FAO mRNA. The areal fraction of fat droplets in all three tissue types was significantly lower in the groups treated with 3-thia fatty acid. In the group treated with EPA a lower areal fraction of fat droplets was observed, while the DHA group was similar to the control. This indicates that EPA and DHA have different effects on mitochondrial biogenesis.     

Relative roles of TGF‐β1 and Wnt in the systemic regulation and aging of satellite cell responses

Muscle stem (satellite) cells are relatively resistant to cell‐autonomous aging. Instead, their endogenous signaling profile and regenerative capacity is strongly influenced by the aged P‐Smad3, differentiated niche, and by the aged circulation. With respect to muscle fibers, we previously established that a shift from active Notch to excessive transforming growth factor‐beta (TGF‐β) induces CDK inhibitors in satellite cells, thereby interfering with productive myogenic responses. In contrast, the systemic inhibitor of muscle repair, elevated in old sera, was suggested to be Wnt. Here, we examined the age‐dependent myogenic activity of sera TGF‐β1, and its potential cross‐talk with systemic Wnt. We found that sera TGF‐β1 becomes elevated within aged humans and mice, while systemic Wnt remained undetectable in these species. Wnt also failed to inhibit satellite cell myogenicity, while TGF‐β1 suppressed regenerative potential in a biphasic fashion. Intriguingly, young levels of TGF‐β1 were inhibitory and young sera suppressed myogenesis if TGF‐β1 was activated. Our data suggest that platelet‐derived sera TGF‐β1 levels, or endocrine TGF‐β1 levels, do not explain the age‐dependent inhibition of muscle regeneration by this cytokine. In vivo, TGF‐β neutralizing antibody, or a soluble decoy, failed to reduce systemic TGF‐β1 and rescue myogenesis in old mice. However, muscle regeneration was improved by the systemic delivery of a TGF‐β receptor kinase inhibitor, which attenuated TGF‐β signaling in skeletal muscle. Summarily, these findings argue against the endocrine path of a TGF‐β1‐dependent block on muscle regeneration, identify physiological modalities of age‐imposed changes in TGF‐β1, and introduce new therapeutic strategies for the broad restoration of aged organ repair.     

C1q/TNF-related protein-3 (CTRP-3) attenuates lipopolysaccharide (LPS)-induced systemic inflammation and adipose tissue Erk-1/-2 phosphorylation in mice in vivo

Background

The C1q/TNF-related proteins comprise a growing family of adiponectin paralogous proteins. CTRP-3 represents a novel adipokine with strong expression in adipose tissue and was shown to inhibit chemokine and cytokine release in adipocytes and monocytes in vitro. The aim of the study was to gain the proof of principle that CTRP-3 is a potent anti-inflammatory adipokine in vivo.

Methods

C57BL/6N mice were treated intraperitoneally (i.p.) with bacterial lipopolysaccharide (LPS) for 2 h. The effects of a 30 min pre-treatment with CTRP-3 i.p. or intravenously (i.v.) on systemic and on epididymal, perirenal and subcutaneous adipose tissue inflammation was analyzed via real-time RT-PCR, ELISA and Western blot analysis.

Results

LPS (1 μg i.p.) significantly increased serum IL-6 and MIP-2 levels as well as epididymal adipose tissue expression of IL-6 and MIP-2 in mice, whereas CTRP-3 (10 μg i.p.) alone or PBS (i.p.) had no effect. Pre-treatment of mice by CTRP-3 i.p. prior to LPS application significantly attenuated LPS-induced cytokine levels but had no effect on adipose tissue cytokine mRNA expression. In contrast to i.p. application of CTRP-3, systemic i.v. application was not sufficient to inhibit LPS-induced cytokine levels or mRNA tissue expression. CTRP-3 given i.p. significantly attenuated LPS-induced phosphorylation of Erk-1/-2 in inguinal adipose tissue.

Conclusion

The present study shows the proof of principle that the novel adipokine CTRP-3 is a potent inhibitor of LPS-induced systemic inflammation and LPS-induced signaling in adipose tissue in vivo.     

Characterizing the effects of saturated fatty acids on insulin signaling and ceramide and diacylglycerol accumulation in 3T3-L1 adipocytes and C2C12 myotubes

A strong correlation between intramyocellular lipid concentrations and the severity of insulin resistance has fueled speculation that lipid oversupply to skeletal muscle, fat, or liver may desensitize these tissues to the anabolic effects of insulin. To identify free fatty acids (FFAs) capable of inhibiting insulin action, we treated 3T3-L1 adipocytes or C2C12 myotubes with either the saturated FFA palmitate (C16:0) or the monounsaturated FFA oleate (C18:1), which were shown previously to be the most prevalent FFAs in rat soleus and gastrocnemius muscles. In C2C12 myotubes, palmitate, but not oleate, inhibited insulin-stimulation of glycogen synthesis, as well as its activation of Akt/Protein Kinase B (PKB), an obligate intermediate in the regulation of anabolic metabolism. Palmitate also induced the accrual of ceramide and diacylglycerol (DAG), two lipid metabolites that have been shown to inhibit insulin signaling in cultured cells and to accumulate in insulin resistant tissues. Interestingly, in 3T3-L1 adipocytes, neither palmitate nor oleate inhibited glycogen synthesis or Akt/PKB activation, nor did they induce ceramide or DAG synthesis. Using myotubes, we also tested whether other saturated fatty acids blocked insulin signaling while promoting ceramide and DAG accumulation. The long-chain fatty acids stearate (18:0), arachidate (20:0), and lignocerate (24:0) reproduced palmitate's effects on these events, while saturated fatty acids with shorter hydrocarbon chains [i.e., laurate (12:0) and myristate (14:0)] failed to induce ceramide accumulation or inhibit Akt/PKB activation. Collectively these findings implicate excess delivery of long-chain fatty acids in the development of insulin resistance resulting from lipid oversupply to skeletal muscle.     

Expression and localization of estrogen receptor alpha in the C2C12 murine skeletal muscle cell line

The classical model of 17beta-estradiol action has been traditionally described to be mediated by the estrogen receptor (ER) localized exclusively in the nucleus. However, there is increasing functional evidence for extra nuclear localization of ER. We present biochemical, immunological and molecular data supporting mitochondrial-microsomal localization of ER alpha in the C2C12 skeletal muscle cell line. We first established [(3)H]17beta estradiol binding characteristics in whole cells in culture. Specific and saturable [(3)H]17beta estradiol binding sites of high affinity were then detected in mitochondrial fractions (K(d) = 0.43 nM; B(max) = 572 fmol/mg protein). Immunocytological studies revealed that estrogen receptors mainly localize at the mitochondrial and perinuclear level. These results were also confirmed using fluorescent 17beta estradiol-BSA conjugates. The immunoreactivity did not translocate into the nucleus by 17beta-estradiol treatment. Western and Ligand blot approaches corroborated the non-classical localization. Expression and subcellular distribution of ER alpha proteins were confirmed in C2C12 cells transfected with ER alpha siRNA and by RT-PCR employing specific primers. The non-classical distribution of native pools of ER alpha in skeletal muscle cells suggests an alternative mode of ER localization/function.     

Conditioned media from human palatine tonsil mesenchymal stem cells regulates the interaction between myotubes and fibroblasts by IL‐1Ra activity

Saturated free fatty acids (FFAs) act as lipid mediators and induce insulin resistance in skeletal muscle. Specifically, in obesity‐related diseases such as type 2 diabetes, FFAs directly reduce insulin sensitivity and glucose uptake in skeletal muscle. However, the knowledge of how FFAs mediate inflammation and subsequent tissue disorders, including fibrosis in skeletal muscle, is limited. FFAs are a natural ligand for toll‐like receptor 2 (TLR2) and TLR4, and induce chronic low‐grade inflammation that directly stimulates skeletal muscle tissue. However, persistent inflammatory stimulation in tissues could induce pro‐fibrogenic processes that ultimately lead to perturbation of the tissue architecture and dysfunction. Therefore, blocking the link between inflammatory primed skeletal muscle tissue and connective tissue might be an efficient therapeutic option for treating obesity‐induced muscle inactivity. In this study, we investigated the impact of conditioned medium obtained from human palatine tonsil‐derived mesenchymal stem cells (T‐MSCs) on the interaction between skeletal muscle cells stimulated with palmitic acid (PA) and fibroblasts. We found that PA‐treated skeletal muscle cells actively secreted interleukin‐1β (IL‐1β) and augmented the migration, proliferation and expression of fibronectin in L929 fibroblasts. Furthermore, T‐CM inhibited the skeletal muscle cell‐derived pro‐fibrogenic effect via the production of the interleukin‐1 receptor antagonist (IL‐1Ra), which is an inhibitor of IL‐1 signalling. Taken together, our data provide novel insights into the therapeutic potential of T‐MSC‐mediated therapy for the treatment of pathophysiological processes that occur in skeletal muscle tissues under chronic inflammatory conditions.     

Inward relocation of exogenous phosphatidylserine triggered by IGF-1 in non-apoptotic C2C12 cells is concentration dependent

The plasma membrane is composed of two leaflets that are asymmetric with regard to their phospholipid composition with phosphatidylserine (PS) predominantly located within the inner leaflet whereas other phospholipids such as phosphatidylcholine (PC) are preferentially located in the outer leaflet. An intimate relationship between cellular physiology and the composition of the plasma membrane has been demonstrated, with for example apoptosis requiring PS exposure for macrophage recognition. In skeletal muscle development, differentiation also requires PS exposure in myoblasts to create cell-cell contact areas allowing the formation of multinucleate myotubes. Although it is clearly established that membrane composition/asymmetry plays an important role in cellular physiology, the role of cytokines in regulating this asymmetry is still unclear. When incubated with myoblasts, insulin-like growth factor I (IGF-1) has been shown to promote proliferation versus differentiation in a concentration dependent manner and therefore, may be a potential candidate regulating cell membrane asymmetry. We show, in non-apoptotic C2C12 cells, that relocation of an exogenous PS analogue, from the outer into the inner leaflet, is accelerated by IGF-1 in a concentration-dependent manner and that maintenance of membrane asymmetry triggered by IGF-1 is however independent of the PI3K inhibitor wortmannin.     

Transgenic expression of n‐3 fatty acid desaturase (fat‐1) in C57/BL6 mice: Effects on glucose homeostasis and body weight

The fat‐1 gene, derived from Caenorhabditis elegans, encodes for a fatty acid n‐3 desaturase. In order to study the potential metabolic benefits of n‐3 fatty acids, independent of dietary fatty acids, we developed seven lines of fat‐1 transgenic mice (C57/BL6) controlled by the regulatory sequences of the adipocyte protein‐2 (aP2) gene for adipocyte‐specific expression (AP‐lines). We were unable to obtain homozygous fat‐1 transgenic offspring from the two highest expressing lines, suggesting that excessive expression of this enzyme may be lethal during gestation. Serum fatty acid analysis of fat‐1 transgenic mice (AP‐3) fed a high n‐6 unsaturated fat (HUSF) diet had an n‐6/n‐3 fatty acid ratio reduced by 23% (P < 0.025) and the n‐3 fatty acid eicosapentaenoic acid (EPA) concentration increased by 61% (P < 0.020). Docosahexaenoic acid (DHA) was increased by 19% (P < 0.015) in white adipose tissue. Male AP‐3‐fat‐1 line of mice had improved glucose tolerance and reduced body weight with no change in insulin sensitivity when challenged with a high‐carbohydrate (HC) diet. In contrast, the female AP‐3 mice had reduced glucose tolerance and no change in insulin sensitivity or body weight. These findings indicate that male transgenic fat‐1 mice have improved glucose tolerance likely due to increased insulin secretion while female fat‐1 mice have reduced glucose tolerance compared to wild‐type mice. Finally the inability of fat‐1 transgenic mice to generate homozygous offspring suggests that prolonged exposure to increased concentrations of n‐3 fatty acids may be detrimental to reproduction. J. Cell. Biochem. 107: 809–817, 2009. © 2009 Wiley‐Liss, Inc.     

TLR2 and TLR4 activation induces p38 MAPK‐dependent phosphorylation of S6 kinase 1 in C2C12 myotubes

Toll‐like receptors 2 (TLR2) and 4 (TLR4) are present in the plasma membrane of skeletal muscle cells where their functions remain incompletely resolved. They can bind various extracellular ligands, such as FSL‐1, lipopolysaccharide (LPS) and/or palmitic acid (PA). We have investigated the link between PA, TLR2/4 and ribosomal S6 kinase 1 (S6K1) in C2C12 myotubes. Incubation with agonists of either TLR2 or TLR4, and with a high concentration of PA, increased S6K1 phosphorylation. Canonical upstream kinases of S6K1, protein kinase B (PKB) and mammalian target of rapamycin complex 1 (mTORC1), were regulated in the opposite way by PA, indicating that these kinases were probably not involved. By using the SB202190 inhibitor, p38 MAPK (mitogen‐activated protein kinase) was found to be a key mediator of PA‐induced phosphorylation of S6K1. Downregulation of either tlr2 or tlr4 gene expression by small interfering RNAs prevented the activation of both p38 MAPK and S6K1 by FSL‐1, LPS or PA. Thus TLR2 and TLR4 agonists can increase the level of S6K1 phosphorylation in a p38 MAPK‐dependent way in C2C12 myotubes. As PA induced the same intracellular signalling, a novel atypical pathway for PA is induced at the cellular membrane level and results in a higher phosphorylation state of S6K1.     

Cortisone induces insulin resistance in C2C12 myotubes through activation of 11beta‐hydroxysteroid dehydrogenase 1 and autocrinal regulation

The enzyme 11β‐hydroxysteroid dehydrogenase 1 (11β‐HSD1) is known to catalyse inactive glucocorticoids into active forms, and its dysregulation in adipose and muscle tissues has been implicated in the development of metabolic syndrome. To delineate the molecular mechanism by which active cortisol has an antagonizing effect against insulin, we optimized the metabolic production of cortisol and its biological functions in myotubes (C2C12). Myotubes supplemented with cortisone actively catalysed its conversion into cortisol, which in turn abolished phosphorylation of Akt in response to insulin treatment. This led to diminished uptake of insulin‐induced glucose. This was corroborated by the application of 11β‐HSD1 inhibitor glycyrrhetinic acid and a glucocorticoid receptor antagonist RU‐486, which reversed completely the antagonizing effects of cortisol on insulin action. Therefore, development of specific inhibitors targeting 11β‐HSD1 might be a promising way to improve impaired insulin‐stimulated glucose uptake. Copyright © 2013 John Wiley & Sons, Ltd.     

MAP kinases p38 and JNK are activated by the steroid hormone 1alpha,25(OH)2-vitamin D3 in the C2C12 muscle cell line

In chick skeletal muscle cell primary cultures, we previously demonstrated that 1alpha,25(OH)2-vitamin D3 [1alpha,25(OH)2D3], the hormonally active form of vitamin D, increases the phosphorylation and activity of the extracellular signal-regulated mitogen-activated protein (MAP) kinase isoforms ERK1 and ERK2, their subsequent translocation to the nucleus and involvement in DNA synthesis stimulation. In this study, we show that other members of the MAP kinase superfamily are also activated by the hormone. Using the muscle cell line C2C12 we found that 1alpha,25(OH)2D3 within 1 min phosphorylates and increases the activity of p38 MAPK. The immediately upstream mitogen-activated protein kinase kinases 3/6 (MKK3/MKK6) were also phosphorylated by the hormone suggesting their participation in p38 activation. 1Alpha,25(OH)2D3 was able to dephosphorylate/activate the ubiquitous cytosolic tyrosine kinase c-Src in C2C12 cells and studies with specific inhibitors imply that Src participates in hormone induced-p38 activation. Of relevance, 1alpha,25(OH)2D3 induced in the C2C12 line the stimulation of mitogen-activated protein kinase activating protein kinase 2 (MAPKAP-kinase 2) and subsequent phosphorylation of heat shock protein 27 (HSP27) in a p38 kinase activation-dependent manner. Treatment with the p38 inhibitor, SB203580, blocked p38 phosphorylation caused by the hormone and inhibited the phosphorylation of its downstrean substrates. 1Alpha,25(OH)2D3 also promotes the phosphorylation of c-jun N-terminal protein kinases (JNK 1/2), the response is fast (0.5-1 min) and maximal phosphorylation of the enzyme is observed at physiological doses of 1alpha,25(OH)2D3 (1 nM). The relative contribution of ERK-1/2, p38, and JNK-1/2 and their interrelationships in hormonal regulation of muscle cell proliferation and differentiation remain to be established.