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Endocrinology and Metabolism Unit (A.M.D., J.-C.J., I.B.B., S.M.B.) and Orthopaedic Research Laboratory (O.C.), University of Louvain, Faculty of Medicine, 1200 Brussels, Belgium
Address all correspondence and requests for reprints to: S. M. Brichard, Unité d’Endocrinologie et Métabolisme, UCL/ENDO 5530Avenue Hippocrate, 55, B-1200 Brussels, Belgium. E-mail: brichard{at}endo.ucl.ac.be.
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Abstract |
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+ TNF
. This effect occurred in a time- and dose-dependent manner. Several pieces of evidence suggest that nitric oxide (NO) mediates this up-regulation by cytokines in myotubes or muscle. First, ApN was induced in vitro exclusively in the experimental conditions that stimulated NO production. Second, inducible NO synthase mRNA induction or NO production clearly preceded ApN mRNA induction. Third, preventing NO production by inhibitors of the NO synthases, nitro-L-arginine methyl ester or NG-methyl-L-arginine, suppressed the inductive effect of the cytokines in vitro and in vivo. Finally, ApN mRNA induction by cytokines was reproduced in cultured human myotubes. In conclusion, our data provide evidence that adiponectin is up-regulated in vivo and in vitro in human and rodent myotubes in response to inflammatory stimuli. The underlying mechanisms seem to involve a NO-dependent pathway. This overexpression may be viewed as a local antiinflammatory protection and a way to deliver extra energy supplies during inflammation. |
Introduction |
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ApN exerts insulin-sensitizing properties on liver and muscle in vivo and in vitro. It enhances insulin-dependent suppression of hepatic gluconeogenesis, thereby lowering plasma glucose concentrations (5, 6). It also increases glucose uptake by C2C12 myocytes or isolated mouse muscles (3, 7). ApN alters lipid metabolism as well by increasing fatty-acid oxidation in several tissues including liver and muscle, thereby accelerating the clearance of plasma free fatty acids (2, 3, 7, 8). Most of these effects are mediated by stimulation of AMP kinase and peroxisomal proliferator-activated receptor (PPAR)- ligand activities (4, 9). Eventually, ApN exhibits antiatherogenic and antiinflammatory effects. It inhibits endothelial inflammatory response by abrogating TNF-induced expression of adhesion molecules [through cross-talks between protein kinase A and nuclear factor-
B signaling pathways] (10, 11) and cellular superoxide generation (12). It also suppresses mature macrophage functions (phagocytic activity and TNF production) (13). Further support for the metabolic and antiatherogenic effects of ApN comes from clinical and genetic studies. Thus, plasma ApN levels are decreased in human subjects with obesity (14), type 2 diabetes (15), or cardiovascular disease (10). Recent genome-wide scans have mapped a susceptibility locus for type 2 diabetes and metabolic syndrome to chromosome 3q27, in which the ApN gene is located (16).
Due to the potential beneficial effects of ApN, the regulation of its gene is currently being investigated. To date, this regulation has been nearly exclusively studied in the adipocyte, unique site of ApN production under normal conditions (17). However, in one study (18), it has been reported that the adiponectin gene could be induced in human myotubes exposed to an adiponectin-containing human embryonic kidney 293 cell culture supernatant.
The aim of the present work was first to examine whether adiponectin could be induced in muscle in vivo and cultured myotubes in response to lipopolysaccharides (LPS) or proinflammatory cytokines. We next explored the underlying mechanisms.
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Materials and Methods |
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LPS of Escherichia coli (serotype 0127:B8; Sigma-Aldrich, Bornem, Belgium) was dissolved in saline at a concentration of 0.1 mg/ml. At 0700 h, mice were injected ip with LPS (25 µg/animal). This dose is similar to that used by other investigators (19). In some experiments, inhibitors of the nitric oxide (NO) synthases [nitro-L-arginine methyl ester (L-NAME) or NG-methyl-L-arginine (L-NMMA), both from Sigma-Aldrich] were dissolved in saline and injected ip at a dose of 10 mg/kg (L-NAME) or 20 mg/kg (L-NMMA) 30 min before LPS. Control mice received equivalent volumes of saline. Because LPS induces anorexia, both groups of mice were fasted throughout the experiments.
Additionally, fasted mice were injected ip with two cytokines administered simultaneously [recombinant murine interferon (IFN) (Peprotech, London, UK; 100 ng/animal) and TNF (R&D Systems, Abingdon, UK; 250 ng/animal)]. The cytokines were previously dissolved in saline, each at 1 µg/ml. Control mice received the vehicle only.
Animals were killed by decapitation at various times after the injections. Blood samples were saved and tibialis anterior muscles were dissected, weighted, frozen in liquid nitrogen, and stored at –80 C until RNA extraction.
Muscles used for determination of ApN protein content were first flushed with saline to avoid tissue contamination by circulating ApN. To this end, mice were anesthetized [ketamine (Ketalar, Parke Davis division of Warner-Lambert, Zaventem, Belgium), 50 mg/kg, ip; medetomidin (Domitor, Pfizer, Brussels, Belgium), 1 mg/kg, ip], and a dilute heparin-saline solution (500 IU/ml) was injected in a backward fashion (i.e. into hind limbs) through a 26G catheter inserted into the posterior vena cava, the aorta being transected. Tibialis anterior muscles were then dissected and stored, as described above.
The University Animal Care Committee has approved all procedures.
Cell culture
Myoblasts from the muscle-derived C2C12 cell line were obtained from American Type Culture Collection (Manassas, VA). The seeding density used throughout the experiments was 8 x 104 cells/plate of 35 mm diameter. Undifferentiated cells were grown at 37 C in the presence of 5% CO2 in DMEM supplemented with 10% fetal calf serum (FCS), 1% penicillin-streptomycin, 1% nonessential amino acids, and 2% L-glutamine (all from Life Technologies, Inc., Merelbeke, Belgium). When cells reached 90–100% confluence (after 3 d), 10% heat-inactivated FCS was replaced by 2% heat-inactivated horse serum to induce myogenic differentiation. This medium will be referred to as basal medium. Muscle cells were examined for evidence of myotube formation and growth by use of an inverted IMT microscope (Olympus Optical, Hamburg, Germany). To preserve the characteristics of the C2C12 cell line, the splitting of cells was done up to a maximum of seven times. The medium was changed every 48 h, and differentiation was allowed to continue for 96 h (4 d) before the experimentation period.
Primary cultures of human skeletal muscle cells were initiated from satellite cells of quadriceps samples obtained from organ donors (three men, 39 ± 9 yr). The study had the approval of the local ethical committee. Cultures were performed as described (20). Briefly, muscle biopsies were trimmed of fat and connective tissue, and then minced into 1-mm3 fragments in a calcium- and magnesium-free saline solution. Satellite cells were isolated by trypsin digestion at 37 C. The supernatants of successive dissociations were centrifuged (200 x g for 5 min) and the pellet resuspended in growth medium containing Ham’s F10 supplemented with 10% FCS, 10% heat-inactivated horse serum, and 100 µg/ml Primocin (Invitrogen, San Diego, CA). After filtration of the cell suspension through nylon netting (pore size, 40 µm), the satellite cells were seeded in 35-mm plates. After 8–15 d of proliferation (37 C, 5% CO2) at the end of which the cells align, the growth medium was replaced by the fusion medium, DMEM supplemented with 10% FCS, 1 mM L-glutamine, 1% penicillin-streptomycin, and 10 µg/ml insulin to induce the fusion of myoblasts into myotubes. This medium, which will be referred to as basal medium for sake of simplicity, was changed every 2 or 3 d, and differentiation was allowed to continue for 6 d (time required to obtain mature myotubes with characteristic elongated and multinucleated morphology) before the experimentation period.
At the time zero point, recombinant murine or human cytokines [all from Peprotech, except for murine TNF (R&D Systems)] and/or other agents [L-NAME, N-acetylcysteine (NAC; Merck, VWR International, Zaventem, Belgium), rosiglitazone (Ro; a kind gift from GlaxoSmithKline Pharmaceuticals, Worthing, UK)] were added to the basal medium for up to 48 h. The cytokines were added to the medium every 24 h; the concentrations used were similar to those reported by others (21) and devoid of overt cytotoxicity. At the end of the culture, aliquots of medium were saved and stored at –80 C for subsequent nitrite assay, and the cells rinsed twice in PBS before RNA isolation.
RNA extraction and real-time quantitative PCR (RTQ-PCR)
RNA was isolated from muscles and cultured cells by using an acid guanidinium-thiocyanate-phenol-chloroform mixture (22) and TriPure isolation reagent (Roche Diagnostics, Vilvoorde, Belgium), respectively. Two micrograms of total RNA were reverse transcripted using oligo (dT) primers and Superscript II Rnase H– reverse transcriptase (Invitrogen, Life Technologies). RTQ-PCR primers were designed (Primer Express software, Applied Biosystems, Foster City, CA) for mouse ApN, GLUT4, heme oxygenase-1 (HO-1), inducible NO synthase (iNOS), cyclophilin (Cyclo), human ApN, and hypoxanthine guanine phosphoribosyl transferase (HPRT) (Table 1
). Then 120 ng total RNA equivalents were amplified with iQ SYBR green supermix (Bio-Rad Laboratories, Nazareth, Belgium) containing 300 nM of each specific primer using iCycler iQ real-time PCR detection system (Bio-Rad). The threshold cycles (Ct) were measured in separate tubes and in duplicate. The identity and purity of the amplified product was checked by electrophoresis on agarose minigels and analysis of the melting curve carried out at the end of amplification. To ensure the quality of the measurements, each plate included a negative control for each gene.
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Ct values were calculated in every sample for each gene of interest as followed: Ctgene of interest – Ctreporter gene with Cyclo or HPRT as the reporter gene (mRNA of reporters remained stable throughout the experiments). Relative changes in the expression level of one specific gene (Ct) were calculated as Ct of the test group minus Ct of the control group and then presented as 2–Ct (23). The PCR efficiency equaled 1 for each gene studied.
Quantification of ApN
ApN concentrations were determined by a commercially available kit (RIA mouse adiponectin kit; Linco Research, St. Charles, MO) in mouse tibialis anterior muscles or myotubes homogenized in 1 ml NaHCO3 buffer [20 mM (pH 7.0)] containing saccharose (250 mM), NaN3 (5 mM), phenylmethylsulfonyl fluoride (100 µM), aprotinin (10 µg/ml), and leupeptin (10 µg/ml) (24). Samples (50 µl) were run in duplicate. The intraassay variability was 3.73 and 4.11% at concentrations of 3 and 8 ng/ml, respectively. The interassay variability was 8.24 and 6.56% at the same concentrations. Protein concentrations were measured in each sample by the Bradford method, thereby allowing determination of total protein content in muscle or cultured myotubes.
Nitrite determination
Nitrite (NO2–) is a stable end product used extensively as an indicator of NO production. In our experimental conditions, NO2– accumulation was assayed by the Griess reaction (25). For plasma samples, proteins were first precipitated and nitrate reduced to NO2– as described (26). NO production was thus measured as nitrate plus nitrite (NOx) concentrations. Protein-free plasma samples (250 µl) or culture medium (400 µl) was mixed with four times the amount of Griess reagent (1% wt/vol sulfanilamide, 0.1% wt/vol naphthylethylenediamine, 2.5% vol/vol H3PO4). Samples were incubated at room temperature for 10 min and absorbance was subsequently read at 543 nm using a spectrophotometer. NO2– concentrations were calculated in comparison with a sodium nitrite (NaNO2) standard curve.
Results presentation and statistical analysis
Results are the means ± SEM for indicated numbers of individual mice (in vivo study) or separate experiments (in vitro studies). Ranges for gene expression levels were presented in each figure as 2–(Ct± SEM), where SEM is calculated from the Ct values (23; user bulletin no. 2 Applied Biosystems, http://www.appliedbiosystems.com/search/ and search for 777802–001).
Comparisons between two conditions were made using two-tailed unpaired Student’s t test. Comparisons of at least three conditions were carried out by ordinary or repeated ANOVA followed by the Newman Keuls (comparison of all pairs) or Dunnett’s tests (all vs. control) as appropriate. Statistical analysis for gene expression levels was performed on the Ct values. Differences were considered statistically significant at P < 0.05.
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Results |
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27) in tibialis anterior muscle of saline-injected control mice (Fig. 1A and data not shown). Injection of a small dose of LPS caused an approximately 10-fold rise in ApN mRNAs at 24 h (Fig. 1A). Concomitantly, ApN levels increased by approximately 70% in muscle of LPS-injected mice, compared with control animals (Fig. 1B, data in nanograms per milligram protein). Quantitatively similar results were obtained when data were expressed as nanograms per milligram muscle (not shown).
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combined with TNF tended to elevate ApN mRNAs in mouse tibialis anterior at 24 h (2.2-fold vs. control mice; n = 5 animals per group, P = 0.10).
Induction of ApN mRNA in C2C12 cells
To further investigate the mechanisms by which LPS inflammation induced ApN gene expression in muscle, we used an in vitro approach. We first attempted to reproduce ApN induction in C2C12 myotubes cultured for 48 h with various proinflammatory cytokines (IL-1ß, IL-6, IFN, or TNF). None of the cytokines used alone was able to modify ApN gene expression. Some combinations were tested. IFN together with IL-1ß failed to induce ApN mRNAs. However, in agreement with the in vivo study, the combination of IFN and TNF was effective. This combination markedly increased ApN mRNAs, suggesting a synergistic rather than an additive action of both cytokines (Fig. 2A). ApN protein levels also rose significantly in treated myocytes (Fig. 2B).
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. Under basal (control) conditions, ApN mRNA levels remained stable and low in C2C12 myotubes cultured for up to 48 h. In contrast, IFN combined with TNF (both used at 5 ng/ml concentrations) doubled ApN mRNA after 24 h, and this stimulation was further amplified after 48 h.
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(10 ng/ml) and increasing concentrations of TNF for 48 h. Under these conditions, the stimulation was detectable at TNF concentrations above 1 ng/ml, maximal at 2.5 ng/ml, and then plateaued (Fig. 3B
). Likewise, in the presence of TNF at a fixed concentration (2.5 ng/ml), ApN mRNAs rose dose-dependently with increasing concentrations of IFN (Fig. 3C).
Mechanisms of ApN induction
We first hypothesized that excessive production of reactive oxygen species by cytokines could mediate ApN gene induction (27). To test this assumption, we examined whether NAC, an antioxidant, was able to inhibit the effect of the cytokine mix. Figure 4A clearly shows that NAC failed to prevent ApN mRNA induction by cytokines. Yet in our system, NAC exerted its expected antioxidant action as shown by its inhibitory effect on HO-1 mRNA, an enzyme induced in response to oxidative stress (28) (Fig. 4B). Thus, NAC largely prevented cytokine-induced accumulation of HO-1 mRNA levels. Moreover, NAC used at the same concentration (10 mM) fully reversed the potent induction of HO-1 expression produced by 100 µM H2O2 (data not shown).
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combined with TNF for 48 h potently enhanced NO release in the medium of C2C12 cells, through marked (300-fold) up-regulation of iNOS mRNA (Fig. 5, A and E) (29). L-NAME, a competitive inhibitor of NO synthases, abrogated both cytokine-induced NO production and ApN mRNA induction without affecting iNOS mRNA induction (Fig. 5, C–E). The time course of iNOS up-regulation further supports a role for NO in ApN induction (Fig. 5B). iNOS mRNA levels were already significantly increased in myotubes after 8 h of culture in the presence of the cytokine mix and reached a maximum at approximately 24 h, whereas ApN induction was detectable only from 24 h onward. Hence, iNOS induction clearly preceded ApN induction. In this context, we measured plasma NOx concentrations in mice injected with LPS or saline and observed a similar finding. As expected (26), NOx levels were significantly higher in LPS mice from 2 h onward, reached a peak at 6 h [28 ± 0.1 µM (LPS mice) vs. 8 ± 0.1 µM (saline mice), n = 3 at each point as in Fig. 1A; P < 0.01], and then returned to control levels at 24 h (not shown). Accordingly, the in vivo peak of NO production also preceded ApN up-regulation in tibialis anterior muscle. Eventually, in the light of the results obtained in C2C12 cells, we treated mice with inhibitors of the NO synthases (either L-NAME or L-NMMA) 30 min before LPS injection. As expected, this treatment fully prevented LPS-induced ApN mRNA up-regulation (Fig. 1A).
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-target genes in a PPAR-dependent manner in macrophages (30), we tested whether this antidiabetic drug could exert similar properties in C2C12 cells. As shown in Fig. 6A, 10 µM Ro for 48 h did not affect ApN mRNA levels when tested either alone or in combination with the cytokine mix. Thus, unlike in macrophages, Ro failed to inhibit induction of cytokine target genes (i.e. ApN) in myotubes. However, Ro exerted its expected stimulatory effect on the insulin-sensitive glucose transporter gene whether the cytokine mix was present or not, as shown by the 2- to 3-fold increase of GLUT4 mRNAs in C2C12 cells (Fig. 6B) (31).
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, the combination of IFN and TNF enhanced ApN mRNA abundance in primary cultures of human skeletal muscle cells.
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Discussion |
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, IL-1ß, IL-6, or TNF in classical immune tissues as well as nonimmune tissues such as skeletal muscle (32, 33). Admittedly, this tissue is composed of several cell types, and we did not prove that ApN was specifically expressed within myocytes. However, we then demonstrated that a combination of cytokines was able to reproduce ApN induction in cultured murine and human myotubes. Importantly, the concentrations of cytokines used in vitro were similar to those reached in vivo in the plasma of LPS-injected mice (19, 34, 35).
We next characterized the mechanisms responsible for ApN induction mostly in vitro but also in vivo. Several pieces of evidence suggest that NO mediates ApN induction by cytokines in myotubes or muscle. First, ApN was induced in vitro exclusively in the experimental conditions that stimulated NO production. In this respect, only the combination IFN + TNF was efficient in up-regulating both ApN expression and NO synthesis. Second, iNOS mRNA induction clearly preceded ApN induction in myotubes. This is consistent with the in vivo data. Plasma NOx concentrations were higher in mice injected with LPS than in saline mice and reached a peak 6 h after treatment, thereby also preceding ApN up-regulation in tibialis anterior muscle. Third, preventing NO production by using inhibitors of the NO synthases, L-NAME or L-NNMA, suppressed the inductive effect of the cytokine mix or LPS both in vitro and in vivo.
To date, the adipocyte has been considered the almost exclusive site of ApN production (17). Our study is one of the very few that deals with ApN regulation in nonadipose cells or tissues. In mouse liver, ApN was found to be elevated after carbon tetrachloride administration, a finding that is not surprising because hepatic injury may result in steatosis (36). More recently it has been reported that ApN could be induced in human myotubes exposed to an adiponectin-containing human embryonic kidney 293 cell culture supernatant (18). We demonstrate here that ApN can be up-regulated both in muscle in vivo and myotubes in vitro in response to proinflammatory cytokines. In many respects, ApN regulation in muscle seems to be fully distinct, if not opposite, from that described in adipose tissue. First, in contrast to the up-regulation found in muscle, LPS injection down-regulated ApN mRNA levels of mouse adipose tissue (Bauche, I., and S. M. Brichard, unpublished data). Second, TNF also inhibited ApN production in cultured adipocytes (37, 38, 39), whereas this cytokine induced ApN in C2C12 cells incubated in the presence of IFN. Third, ApN regulation by PPAR agonists is also quite different in the two tissues. Thiazolidinediones, potent adipogenic agents used as antidiabetic drugs, are strong inducers of ApN gene expression in adipose tissue (38, 40, 41) but had no effect on ApN production in myotubes. This lack of effect also enables us to ensure that ApN expression in myotubes did not result from contamination of the myotube cultures with adipocytes or from transdifferentiation of myotubes into adipocytes.
It is worth noting that the maximum ApN mRNA content reached in muscle remained lower than that obtained in adipose tissue. In our in vivo experiment, 24 h after LPS injection, ApN mRNA levels in tibialis anterior muscle were 2 orders of magnitude lower than those measured in epididymal adipose tissue (our unpublished data). Yet the local efficacy of ApN on myocytes might still be high because this adipocytokine could directly act on these cells via autocrine or paracrine mechanisms. In addition, ApN may undergo some posttranslational modifications (e.g. a proteolytic cleavage leading to the globular form) that could further raise its local potency (4).
The physiological relevance of this overexpression of ApN in response to inflammatory injury in muscle is still under investigation. On one hand, ApN exerts antiinflammatory properties on several cell types or tissues (endothelium, macrophages, and pancreatic ß-cells) (10, 11, 13, 31). In these cells, it controls early events of inflammation but also late events by preventing immune responses from continuing chronically (13). Thus, induction of ApN in muscle may be viewed as a protective mechanism against excessive and deleterious inflammatory reactions. On the other hand, ApN up-regulation in muscle could also be a useful means to meet the extra energy needs of inflammatory processes. This may be achieved from the ability of ApN to stimulate fatty acid oxidation and glucose transport in skeletal muscle (2, 3, 7).
In conclusion, our data provide evidence that adiponectin is up-regulated in vivo and in vitro in human and rodent myotubes in response to inflammatory stimuli. The underlying mechanisms seem to involve a NO-dependent pathway. This overexpression may be viewed as a local antiinflammatory protection and a way to deliver extra energy supplies during inflammation.
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Acknowledgments |
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Footnotes |
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Abbreviations: ApN, Adiponectin; Ct, cycle threshold; Cyclo, cyclophilin; FCS, fetal calf serum; HO-1, heme oxygenase-1; HPRT, hypoxanthine guanine phosphoribosyl transferase; IFN, interferon; iNOS, inducible NO synthase; L-NAME, nitro-L-arginine methyl ester; L-NMMA, NG-methyl-L-arginine; LPS, lipopolysaccharides, NAC, N-acetylcysteine; NO, nitric oxide; NO2–, nitrite; NOx, nitrate plus nitrite; PPAR, peroxisome proliferator-activated receptor; Ro, rosiglitazone; RTQ-PCR, real-time quantitative PCR.
Received April 19, 2004.
Accepted for publication August 12, 2004.
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and PPAR negatively regulate specific subsets of lipopolysaccharide and IFN- target genes in macrophages. Proc Natl Acad Sci USA 100:6712–6717 agonist, GW501516, regulates the expression of genes involved in lipid catabolism and energy uncoupling in skeletal muscle cells. Mol Endocrinol 17:2477–2493 ligands increase expression and plasma concentrations of adiponectin, an adipose-derived protein. Diabetes 50:2094–2099 agonists: a potential mechanism of insulin sensitization. Endocrinology 143:998–1007This article has been cited by other articles:
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) or with (
) IFN
, 
) in C2C12 cells cultured for up to 48 h without (
