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Am J Physiol Endocrinol Metab 294: E52-E60, 2008. First published October 16, 2007; doi:10.1152/ajpendo.00506.2007
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Cardiotrophin-1 is expressed in adipose tissue and upregulated in the metabolic syndrome

Cristina Natal,1 María Antonia Fortuño,1 Patricia Restituto,2 Antonio Bazán,3 Immaculada Colina,4 Javier Díez,1,5 and Nerea Varo1,2

1Division of Cardiovascular Sciences, Centre for Applied Medical Research, University of Navarra, and Departments of 2Clinical Chemistry, 3Department of Plastic Surgery, University Clinic of Navarra, Pamplona, Spain, 4Internal Medicine, and 5Cardiology and Cardiovascular Surgery (JD), University Clinic of Navarra, Pamplona, Spain

Submitted 3 August 2007 ; accepted in final form 9 October 2007


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Adipose tissue is a target for cardiotrophin-1 (CT-1), a cytokine member of the IL-6 family of cytokines that is involved in cardiac growth and dysfunction. However, it is unknown whether adipocytes are a source of CT-1 and whether CT-1 is overexpressed in diseases characterized by increased fat depots [i.e., the metabolic syndrome (MS)]. Thus this work aimed 1) to test whether adipose tissue expresses CT-1 and whether CT-1 expression can be modulated and 2) to compare serum CT-1 levels in subjects with and without MS diagnosed by National Cholesterol Education Program Adult Treatment Panel III criteria. Gene and protein expression of CT-1 was determined by real-time RT-PCR, ELISA, and Western blotting. CT-1 expression progressively increased, along with differentiation time from preadipocyte to mature adipocyte in 3T3-L1 cells. CT-1 expression was enhanced by glucose in a dose-dependent manner in these cells. mRNA and protein CT-1 expression was also demonstrated in human adipose biopsies. Immunostaining showed positive staining in adipocytes. Finally, increased CT-1 serum levels were observed in patients with MS compared with control subjects (127 ± 9 vs. 106 ± 4 ng/ml, P < 0.05). Circulating levels of CT-1 were associated with glucose levels (r = 0.2, P < 0.05). Taken together, our data suggest that adipose tissue can be recognized as a source of CT-1, which could account for the high circulating levels of CT-1 in patients with MS.

cytokines; obesity; diabetes

CARDIOTROPHIN-1 (CT-1), a naturally occurring protein member of the IL-6 family of cytokines with a molecular mass of ~21.5 kDa, exerts its cellular effects by interacting with the glycoprotein 130 (gp130)/leukemia inhibitory factor receptor-β (LIFR) heterodimer (30). CT-1 is able to induce in vitro hypertrophy and survival signals in neonatal (29, 36) and adult (26) cardiomyocytes.

CT-1 mRNA expression has been detected at high levels in heart, skeletal muscle, prostate, ovaries, and liver, as well as fetal heart, lung, and kidney, and in lower amounts in other tissues (31). Adipose tissue has emerged as a key secretory organ, with the ability to secrete different adipokines, including IL-6, but there is no information about whether adipose tissue also expresses CT-1. However, some studies have shown that adipocytes express gp130 (43) and LIFR (4) and respond to IL-6 family cytokines, including CT-1 (42). Moreover, CT-1 appears to be a mediator of impaired insulin sensitivity, as chronic administration of CT-1 to 3T3-L1 adipocytes resulted in a decrease in insulin-stimulated glucose uptake (44).

Adipose tissue in excess plays a fundamental role in the pathogenesis of the metabolic syndrome (MS) and contributes to the hyperglycemia, hypertension, elevated serum triglycerides (TG), low HDL cholesterol (HDL-C), and insulin resistance that characterize the syndrome (19). MS is associated with higher risk of cardiovascular disease (CVD), and its individual components interact synergistically, causing or accelerating the progression of atherosclerosis (18). In this context, we hypothesized that adipose tissue is not only a target but also a potential source of CT-1 and that this cytokine is overexpressed in MS. More precisely, the objectives of this study were 1) to test whether human adipocytes express CT-1, 2) to analyze whether CT-1 expression can be modulated in this cell type, and 3) to compare serum CT-1 levels in patients with and without MS and its association with clinical and biochemical parameters.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
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 REFERENCES
 
Reagents. RPMI 1640 medium and DMEM, penicillin-streptomycin, calf and fetal bovine sera (FBS), and PBS were purchased from Invitrogen (Carlsbad, CA); dexamethasone, insulin, IBMX, sodium citrate, recombinant TNF-{alpha}, recombinant IL-6, recombinant angiotensin II (ANG II), D-glucose, and D-mannitol from Sigma-Aldrich (St. Louis, MO); recombinant transforming growth factor-β (TGF-β) and CD40 ligand (CD40L) from R & D Systems (Minneapolis, MN); and aldosterone from Fluka (St. Gallen, Switzerland).

3T3-L1 cell culture and stimulation. Murine 3T3-L1 preadipocytes were plated in DMEM containing 4.5 g/l D-glucose (high-glucose DMEM), 10% heat-inactivated calf serum, 100 U/ml penicillin, and 100 µg/ml streptomycin at 37°C in a humidified atmosphere containing 5% CO2. Cells were incubated to confluence and then induced to differentiate in the same culture medium supplemented with 0.5 mM IBMX, 1 µM dexamethasone, and 10 µg/ml insulin for 48 h. Medium was then changed to insulin only, and adipocytes were grown for 2 additional days. After this period, adipocytes were kept in high-glucose DMEM containing only FBS and antibiotics for another 4 days until experimentation. To minimize sample variability, only wells in which >90% of the cells showed fat accumulation and where cell coverage was >90% were employed.

Differentiated 3T3-L1 adipocytes were incubated in the presence or absence of different stimuli for 4 h (gene expression) or 24 h (protein expression) as follows: 100 ng/ml TNF-{alpha}, 50 ng/ml IL-6, 2 ng/ml TGF-β, 1 µg/ml CD40L, 10–7 M ANG II, 10–7 M aldosterone, 400 mg/dl glucose, and 100 nM insulin. Cells were also stimulated with increasing concentrations of glucose (100–400 mg/dl) or mannitol for 4 h (gene expression) or 24 h (protein expression). Cells were starved overnight before experiments.

Monocyte isolation. Lymphomononuclear cells were obtained from peripheral blood of healthy donors following the technique described by Bøyum (5). Cells were incubated at 2.5 x 106/ml in RPMI 1640 medium supplemented with 10% heat-decomplemented FCS, 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin at 37°C in a humidified atmosphere containing 5% CO2. Human monocytes were isolated by adherence to a plastic surface. After 2 h at 37°C, nonadherent cells were removed with medium without FCS. Monocytes isolated in this fashion were >90% pure, as determined by expression of the specific monocyte marker CD14.

Tissue analysis. Epididymal adipose tissue, along with heart, thymus, spleen, kidney, pancreas, and liver, was obtained from mice (n = 3).

Abdominal adipose tissue was obtained from three patients who underwent open abdominal surgery (abdominoplasty) and one patient who underwent laparoscopic gastric bypass with Roux-en-Y gastroenterostomy surgery for treatment of morbid obesity. Samples of adipose tissue were immediately transported to the laboratory. The tissue was washed with 0.9% NaCl, cut with scissors into small (5- to 15-mg) pieces, and collected in TRIzol or lysis buffer for RNA and protein isolation.

Quantitative real-time RT-PCR. Total RNA was extracted from 3T3-L1 adipocytes, adipose tissue, or monocytes using TRIzol (Invitrogen; 1 ml/100 mg of tissue or 1 ml per 10-cm2 dish) and subsequently purified using an RNeasy total RNA isolation kit (Qiagen). RT was performed with 250 ng of total RNA. Real-time RT-PCR was performed with 10 ng of cDNA in an ABI PRISM 7000 Sequence Detection System using specific TaqMan minor groove binder fluorescent probes (Applied BioSystems, Foster City, CA). Constitutive 18S ribosomal RNA (18S) was used as endogenous control (Applied BioSystems). For the relative quantitative analysis of unknown samples, calibration curves were prepared for the target and the endogenous reference representing cycle threshold values as the logarithmic amount of starting material. For each experimental condition, the mean quantity of the target gene and endogenous control was obtained from the standard curve in triplicate. The standard deviation among triplicates was always <0.2. The mean value of the target was divided by the mean value of the endogenous control to obtain a normalized mean quantity per sample.

Western blotting. 3T3-L1 adipocytes, adipose explants, and monocyte extracts were harvested in 1 ml of ice-cold lysis buffer [20 mM Tris (pH 8), 150 mM NaCl, 10% glycerol, 1% Triton X-100, 2 mM EDTA, and protease inhibitor cocktail (Boehringer Mannheim, Ingelheim, Germany)]. After centrifugation, protein concentration was assessed in the supernatants by the Bradford method (Bio-Rad, Hercules, CA). Proteins (15 µg) were resuspended in loading buffer [20% β-mercaptoethanol, 8% SDS, 40% glycerol, 0.05% bromophenol blue, and 0.250 mM Tris (pH 6.4)], boiled for 5 min, size fractionated on 12% polyacrylamide gels by electrophoresis, and transferred onto nitrocellulose membranes. Membranes were blocked in 5% nonfat dry milk in 0.05% Tween-PBS and incubated with a monoclonal anti-human CT-1 (1:500 dilution; Abcam, Cambridge, UK) or a polyclonal anti-mouse CT-1 (1:200 dilution; Santa Cruz Biotechnology, Santa Cruz, CA) antibody. Bound antibody was detected by peroxidase-conjugated secondary antibody (Amersham Biosciences, Piscataway, NJ) and visualized using the ECL-Plus chemiluminescence detection system (Amersham Biosciences). Negative control included incubation of membranes with blocking solution in the absence of the primary antibody.

Immunohistochemistry. Biopsies were fixed for 24 h in 4% paraformaldehyde (Panreac Quimica, Barcelona, Spain), stored in 70% ethanol (Sigma-Aldrich), and then embedded in paraffin. Sections (6 µm thick) were cut with a Leica microtome and mounted onto slides. After they were dewaxed and rehydrated, the slides were heated for 10 min in a solution containing 10 mM sodium citrate (pH 6.0) to maximize antigen retrieval, incubated in 1% H2O2 for 10 min to inactivate endogenous peroxidases, and blocked with 5% normal goat serum (NGS) in PBS for 1 h. For CT-1 detection, slides were incubated overnight in PBS containing 1% NGS and a mouse anti-human CT-1 antibody (1:100 dilution; Abcam), washed three times, and then incubated for 30 min with the horseradish peroxidase-labeled polymer conjugated to secondary antibodies (Dako Cytomation, Carpentaria, CA). The signal was revealed by using diaminobenzidine chromagen substrate (Dako Cytomation) in the presence of 0.01% H2O2 for 5 min. The slides were counterstained with hematoxylin-eosin (Sigma-Aldrich), and bright-field images were observed with a Nikon microscope and digitized using a charge-coupled device camera. Control sections were incubated with NGS in the absence of the primary antibody.

Study population. We performed a pilot study on a population of 137 apparently healthy subjects (79% men, 55 ± 12 yr old) seen in the Cardiovascular Risk Area of the University Clinic of Navarra for a general check-up. Subjects were free from clinically apparent atherosclerotic disease on the basis of absence of history of coronary disease, stroke, or peripheral artery disease and normal electrocardiogram. Exclusion criteria included impaired renal or liver function, arteritis, connective tissue diseases, or chronic inflammatory diseases.

All participants underwent a complete medical examination, and anthropometric measurements, including weight and height, were obtained using standardized techniques. The body mass index (BMI) was calculated using the following formula: weight (kg)/height2 (m). Blood pressure was measured on the right arm, with the subjects in a seated position and after a 5-min rest, via a mercury sphygmomanometer. The average of two measurements, at the beginning and end of the visit, was considered.

MS was diagnosed according to the criteria of the National Cholesterol Education Program Adult Treatment Panel III guidelines (2, 3) with modification of the waist criterion to BMI >30 kg/m2.

Serum and plasma were collected by venipuncture in Vacutainer tubes. Fasting plasma glucose, serum cholesterol, serum TG, and HDL-C were measured using the Modular Autoanalyzer (Roche Diagnostics, Indianapolis, IN).

The local committee on human research approved the study, which was performed in accordance with the Declaration of Helsinki, and all participants gave written informed consent.

Measurement of CT-1 concentration. CT-1 concentration was measured by ELISA according to the manufacturer's instructions (R & D Systems for mouse CT-1 and Antigenix America for human CT-1). Briefly, adipocyte protein extracts (10 µg) or human serum samples were applied in triplicates to 96-well plates precoated with rat polyclonal anti-mouse CT-1 or rabbit polyclonal anti-human CT-1 antibody and blocked in 1% BSA in PBS or buffer (coating stabilizer; Antigenix America, New York, NY), respectively. Then a sandwich complex was formed with a specific goat anti-mouse CT-1 or rabbit anti-human CT-1 antibody that was labeled with biotin. After incubation, the unbound material was washed off, and a rabbit peroxidase conjugate was added for detection of the bound CT-1. Subsequently, plates were washed, and antibody binding was determined using 3,3',5,5'-tetramethylbenzidine substrate. Absorbance was read at 650 nm, and cell extracts or serum CT-1 concentrations were determined by comparison with serial dilutions of recombinant murine or human CT-1, respectively. Intra-assay variation among the triplicates for all samples was <15%.

Statistical analysis. The statistical analysis was performed with SPSS for Windows version 13.0. The sample size was calculated on the basis of the differences observed in a previous pilot study of 30 patients with and without MS to detect differences of 2 ng/ml with a significance of 0.05 and a statistical power of 80%. The normal distribution of variables was tested with the Shapiro-Wilks test. The unpaired Mann-Whitney U test was used to assess statistical differences between groups of patients or experimental conditions. Spearman's correlation coefficients for continuous variables were also used to assess univariate correlations of CT-1 levels with all variables. Analysis of linear trend was used to evaluate the effect of increasing glucose concentrations. Multivariate linear regression analysis with stepwise selection was performed to assess the independent relationship between CT-1 levels and glucose after adjustment for other confounding factors. Values are means ± SE computed from the average measurements obtained from each group or experimental condition. P < 0.05 was considered significant.


   RESULTS
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 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Culture adipocytes. The 3T3-L1 preadipocyte cell line was employed to study whether CT-1 is expressed in adipocytes. Cultured mouse 3T3-L1 fibroblasts were grown and differentiated for 10 days to adipocytes. RNA was isolated from cells in both states, preadipocyte (day 0) and adipocyte (day 10), and assayed by real-time RT-PCR. As shown in Fig. 1A, mature adipocytes express higher levels (3-fold) of CT-1 than undifferentiated cells (CT-1-to-18S ratio = 124 ± 81 vs. 41 ± 22 arbitrary units, P < 0.05).


Figure 1
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  		Fig. 1. Comparative expression of cardiotrophin-1 (CT-1) in 3T3-L1 preadipocytes and differentiated adipocytes. A: total RNA was isolated from both cell types, and CT-1 mRNA was assayed by real-time RT-PCR, with 18S ribosomal RNA (18S) used as endogenous control. Mean value of the target was divided by the mean value of the endogenous control to obtain a normalized mean quantity per sample. Values are means ± SE (n = 3). *P < 0.05. B and C: 10 µg of whole extracts obtained from 3T3-L1 cells at different states of differentiation were assessed by ELISA and Western blot, respectively, to quantify CT-1 content. Results are representative of 2 different experiments.

 
Total protein was extracted from cells at different stages of the differentiation process, and CT-1 expression was determined by ELISA and Western blot. Levels of CT-1 progressively increased, along with the differentiation time (Fig. 1, B and C).

Glucose and some cytokines and neurohumoral factors implicated in obesity and inflammation were screened for their ability to induce CT-1 expression. As shown in Fig. 2A, TNF-{alpha}, CD40L, ANG II, and glucose significantly increased CT-1 mRNA levels (P < 0.05). CT-1 protein expression was determined by ELISA in whole cells extracts, and only glucose induced a statistically significant increase of CT-1 (Fig. 2B).


Figure 2
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  		Fig. 2. Regulation of CT-1 expression in 3T3-L1 adipocytes. Differentiated adipocytes were incubated in the presence or absence of different stimuli, and CT-1 expression was determined by real-time RT-PCR for 4 h (A) or ELISA for 24 h (B). Values are means ± SE (n = 3). *P < 0.05.

 
To evaluate more precisely the effect of glucose on CT-1 expression, we next treated differentiated adipocytes with increasing concentrations of glucose (100, 200, or 400 mg/dl) for 4 h (gene expression) or 24 h (protein expression). Real-time RT-PCR showed that glucose significantly increases CT-1 mRNA expression in a dose-dependent manner [P < 0.001 (for trend); Fig. 3A ]. The analysis of protein expression following stimulation with glucose showed a significant increase in CT-1 expression [P < 0.001 (for trend); Fig. 3B]. Mannitol served as an osmotic control, and no significant effect was produced by any of the concentrations tested (data not shown).


Figure 3
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  		Fig. 3. Regulation of CT-1 expression by glucose in 3T3-L1 adipocytes. Differentiated 3T3-L1 adipocytes were incubated in the presence or absence of increasing glucose concentrations (100, 200, or 400 mg/dl), and CT-1 expression was determined by real-time RT-PCR for 4 h (A) or ELISA for 24 h (B). Values are means ± SE (n = 7). **P < 0.001 (for trend).

 
CT-1 expression in adipose tissue. We next explored whether not only culture lines of adipocytes, but also whole murine or human adipose tissue, could be a source of CT-1. We first compared CT-1 mRNA expression in mouse tissue from different locations (Fig. 4). There was a broad range of transcript abundance in the various tissues. Interestingly, CT-1 expression levels were substantially (3-fold) higher in adipose tissue than in heart or liver (CT-1-to-18S ratio = 44 ± 2 vs. 12 ± 2 arbitrary units, P < 0.05) or liver (CT-1-to-18S ratio = 44 ± 2 vs. 13 ± 6 arbitrary units, P < 0.05).


Figure 4
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  		Fig. 4. CT-1 expression in murine tissue. Total RNA was isolated from mouse tissue obtained from different locations. CT-1 mRNA was assayed by real-time RT-PCR, with 18S ribosomal RNA (18S) used as endogenous control for normalization. Mean value of the target was divided by mean value of the endogenous control to obtain a normalized mean quantity per sample. Values are means ± SE. *P < 0.05 vs. heart, thymus, kidney, and liver.

 
To confirm whether human adipose tissue also expressed CT-1, total RNA or whole protein extracts were isolated from human abdominal adipose biopsies. Real-time RT-PCR amplified cDNA for CT-1 from RNA obtained from the human biopsies (Fig. 5A). The number of PCR cycles for adipose tissue was 29–31. Proper RT was confirmed in all cases by amplification of 18S.


Figure 5
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  		Fig. 5. CT-1 expression in human adipose tissue. Total RNA or protein was isolated from human abdominal adipose biopsies (n = 4) and human monocytes isolated from peripheral blood (n = 2). A: real-time RT-PCR, with 18S ribosomal RNA (18S) used as endogenous control, shows CT-1 mRNA amplification at cycle threshold of 29 for adipose tissue and no amplification for monocytes. B: 15 µg of each extract were separated by SDS-PAGE, transferred to nitrocellulose, and subjected to Western blot analysis, which shows a positive band in the adipose tissue and no expression in monocytes. C: representative microscopic views of CT-1 staining in human adipocytes. CT-1 stained brown within cytoplasm of adipocytes. Top: cells incubated with antibody for human CT-1 (at 1:100 dilution). Bottom: corresponding control sections incubated in blocking serum without primary antibody. Sections are representative of 4 different biopsies.

 
Western blot analysis showed CT-1 expression in human adipose tissue, with a positive band in a slightly higher-than-expected molecular weight (Fig. 5B). To ascertain that the band corresponded to CT-1, it was cut from another gel run simultaneously, digested, and unequivocally identified by mass spectrometry. The corresponding positive and negative controls included recombinant CT-1 or incubation with the blocking buffer and omission of the primary antibody. CT-1 protein expression was also corroborated by ELISA in cell extracts (data not shown).

Inasmuch as different cell types constitute adipose tissue, we further investigated whether specifically adipocytes expressed CT-1. CT-1 expression was visualized by immunohistochemistry to verify that the cytoplasm of human adipocytes showed intense immunoreactivity with anti-CT-1 antibodies (Fig. 5C, top), whereas the corresponding control section did not show any positive reaction (Fig. 5C, bottom). In addition, human monocytes obtained from peripheral blood were also assayed by real time RT-PCR (Fig. 5A) and Western blotting (Fig. 5B). CT-1 mRNA or protein expression was not detected in this cell type compared with adipose tissue samples.

Serum CT-1 levels in patients with MS. After complete clinical examination, subjects were divided into two groups: those with (n = 43) and those without (n = 94) MS. Clinical characteristics of the study population are presented in Table 1. As expected, BMI, SBP, DBP, and glucose and TG levels were higher in patients with MS than in those without MS (P < 0.001). HDL-C levels were lower in patients with MS than in the control group (P < 0.001).


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  		Table 1. Characteristics of study population

 
Interestingly, mean serum CT-1 concentration was significantly higher in the group exposed to MS than in the control group (127 ± 8 vs. 106 ± 4 ng/ml, P < 0.05; Fig. 6A). We further explored the association of CT-1 circulating levels with the parameters that define MS. Interestingly, in the whole population, CT-1 concentrations positively correlated with glucose (Spearman's r = 0.2, P = 0.014). However, we found no association with blood pressure, TG, HDL-C, or BMI.


Figure 6
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  		Fig. 6. Increased CT-1 levels in metabolic syndrome (MS), hyperglycemia, and obesity. A: patients were divided into 2 groups [without (No) and with (Yes) MS]. B and C: patients were divided into 2 groups (glucose >100 mg/dl and <100 mg/dl and body mass index >30 kg/m2 and <30 kg/m2). Serum CT-1 concentrations were determined by ELISA. Values are means ± SE. *P < 0.05. **P < 0.01.

 
Patients were divided into two groups according to having or not each of the criteria of MS. There were no statistically significant differences between hypertensive patients vs. normotensive subjects (119 ± 6 vs. 106 ± 6 ng/ml), subjects with high TG vs. those with low TG (126 ± 10 vs. 108 ± 4 ng/ml), or patients with low HDL-C vs. those with high HDL-C (115 ± 7 vs. 111 ± 5 ng/ml).

CT-1 levels were significantly higher in subjects with hyperglycemia (fasting glucose >100 mg/ml) than in those without hyperglycemia (127 ± 7 vs. 102 ± 5 ng/ml, P < 0.05; Fig. 6B). We also found that CT-1 levels were significantly increased in obese patients (BMI >30 kg/m2) compared with normal-weight subjects (123 ± 9 vs. 110 ± 5 ng/ml, P < 0.05; Fig. 6C).


   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The main findings of the present study can be summarized as follows. 1) Mouse and human adipose tissue, specifically adipocytes, express CT-1. 2) CT-1 expression progressively increases, along with the differentiation time from preadipocyte to mature adipocyte in culture. 3) Glucose dose dependently induces CT-1 expression in adipocytes independently of its osmotic effects. 4) CT-1 circulating levels are increased in patients with MS compared with those without MS and positively correlate with glucose levels. 5) CT-1 levels are significantly higher in subjects with hyperglycemia or obesity than in controls.

Adipose tissue as a source of CT-1. CT-1 was first described in cardiac muscle cells, but different levels of CT-1 expression have also been found in a variety of tissues outside the cardiac compartment (liver, lung, kidney, skeletal muscle, pancreas, and others) (32). Adipose tissue is not only a storage tissue, but it is also an active endocrine organ that secretes numerous proinflammatory hormones and cytokines, such as TNF-{alpha} and IL-6 (21), and multiple proteomic tools have been used to characterize the secretome of preadipocyte cell lines, primary adipocytes, and adipose tissue (1, 8, 23). However, no information is available about whether adipose tissue expresses the cytokine CT-1. Some studies have demonstrated the expression of gp130 and LIFR (the complex receptor for CT-1) in preadipocytes and adipocytes, indicating that both cell types were likely to be activated by CT-1 (4, 43). In fact, CT-1 induces signaling in fat cells (42). Given that adipocytes express gp130 and LIFR, we hypothesized that this cell type could also express CT-1. The difficulty in differentiating human preadipocytes into adipocytes and the lack of adipocyte cell lines of human origin has resulted in most investigators working with preadipocyte murine cell lines and differentiating them over several days into mature cells that accumulate large amounts of lipids. Thus, employing 3T3-L1 preadipocytes, we have demonstrated that CT-1 expression progressively increases along with the differentiation time from preadipocyte to mature adipocyte. Furthermore, the analysis of different murine tissues confirmed this novel finding that adipose tissue is an important source of CT-1, inasmuch as CT-1 expression in fat exceeded by threefold that found in the other tissues tested. Moreover, the analysis of human abdominal adipose tissue samples with different techniques confirmed the presence of this cytokine.

Large numbers of fibroblasts, mast cells, leukocytes, and other cells involved in inflammation are embedded within the adipose tissue framework. Immune cells in adipose tissue, specifically macrophages, have increasingly caught the attention of the scientific community, because they secrete proinflammatory mediators that upregulate the secretory activity of the adipocytes (7). Our immunohistochemical experiments confirm CT-1 expression specifically in human adipocytes; however, these findings do not exclude the possibility that CT-1 release by adipose tissue was due to resident cells other than adipocytes.

At the cellular level, obesity involves two different physiological components: lipid metabolism (lipogenesis and lipolysis) (24) and adipogenesis. Adipogenesis is the discernible cellular transition through which a spindle-shaped fibroblastic cell proceeds, first forming a preadipocyte, then a multilocular adipocyte, and, finally, a mature unilocular adipocyte (11, 17). Obesity is characterized by a massive accumulation of fat with an increased number of differentiated cells that secrete multiple adipokines. Our observation that mature adipocytes express higher levels of CT-1 than preadipocytes suggests that, in pathologies characterized by an excess of visceral fat, CT-1 levels would be enhanced. This hypothesis is supported by our finding of enhanced circulating CT-1 levels in patients with MS and in obese patients.

Regulation of CT-1 expression in adipose tissue. Despite the importance that is attributed to CT-1 at the cardiovascular level, there is little information about the regulation of its expression. There is evidence that CT-1 is induced by some cardiovascular function, such as mechanical stress (28), ANG II (35), endothelin-1 (40), and norepinephrine (15). ANG II is the biologically active component of the renin-angiotensin system, which is present in adipose tissue, and evidence suggests that ANG II is intimately linked to obesity (22). Also, the obese state is a low-grade systemic inflammation characterized by increased blood levels of inflammatory markers such as TNF-{alpha} (6), IL-6 (34), TGF-β (33), and CD40L (10). Thus, to evaluate whether CT-1 expression is inducible in adipose tissue, we tested these cytokines and some of the above-mentioned humoral factors known to be elevated in obesity for their ability to modulate CT-1 expression. Interestingly, all the stimuli tested positively increased CT-1 expression in 3T3-L1 cells, but a crucial finding is that glucose significantly increased adipocyte-derived CT-1 expression in a dose-dependent manner. This effect of glucose on adipocytes could account for the elevated circulating CT-1 concentrations associated with glucose levels observed in patients with MS and the increased CT-1 levels observed in subjects with hyperglycemia. However, this does not exclude the possibility that other hormones or local factors may directly or indirectly mediate the observed effects.

Enhanced CT-1 levels in MS. As previously mentioned, CT-1 was first described as a growth-promoting (29) and cytoprotective (37) agent in cardiomyocytes. In humans, CT-1 provides prognostic information on cardiovascular risk in patients with untreated essential hypertension and associates with the magnitude of left ventricular hypertrophy in these patients (25). Plasma CT-1 may also predict the regression of left ventricular hypertrophy in response to antihypertensive treatment (16). In addition, diverse studies show increased circulating levels of CT-1 in heart failure from different origin (14, 38, 41). The novel observation of the present pilot study is increased levels of CT-1 in the serum of patients with MS, particularly in obese and diabetic patients, which could be explained by overexpression of the cytokine from adipose tissue.

MS and obesity are important risk factors for atherosclerosis, but the mechanisms for increased incidence of cardiovascular events remain unclear (19, 20). One possibility is that the altered release of cytokines by adipose tissue may have negative effects on the cardiovascular tree, explaining the adverse outcomes of MS (9, 12, 39). Our novel observation that adipose tissue expresses CT-1, a cytokine with known actions on cardiovascular and metabolic cells, raises the possibility that CT-1 may play a pathophysiological role in MS, acting as a link between obesity-related complications and CVD. Future studies are required to clarify the exact pathophysiological role of adipose tissue-derived CT-1 in the development of CVD associated with MS and obesity.

Dissociation of obesity and primary insulin resistance in patients with MS is difficult. CT-1 and other gp130 cytokines have been implied as potential mediators of various aspects of obesity/type 2 diabetes (13). For example, previous in vitro studies demonstrated that chronic administration of CT-1 to adipocytes resulted in the development of insulin resistance (44). Our results lead possibly to important clinical implications, since in patients with MS, particularly those with hyperglycemia, CT-1 may increase significantly. Induction of CT-1 by glucose in adipose tissue may explain further the relation between obesity and insulin resistance in MS.

Limitations of the study. The design of the present study in humans is cross-sectional; thus the results cannot be used to establish an association with prognosis. Future studies are required to further evaluate the usefulness of the measurement of CT-1 serum levels in determining the prognosis of patients with MS by analyzing whether CT-1 levels associate with clinical parameters that indicate cardiac and vascular damage.

Also, we show here that adipocytes are able to produce CT-1. Future studies are needed to clarify the mechanisms of release of the cytokine into the bloodstream.

Because waist circumference measurements were not available, we used BMI to determine obesity. This approach was adopted by Malik et al. (27), and BMI was reported to have identification and prognosis values similar to those for waist circumference. Despite the increase of CT-1 levels in obese subjects, we failed to find any significant correlation with the weight or the BMI in our patients. Also it may be possible that the abdominal perimeter or the waist-to-hip ratio associates with CT-1.

Finally, with the experiments shown here, we cannot exclude the possibility that inflammatory or resident cells in the adipose tissue may produce CT-1.

Summary. In this clinical and experimental study, we have shown for the first time that adipose tissue and, more precisely, adipocytes are an important source of the cytokine CT-1, which could account for the elevated circulating levels in patients with MS. We have also demonstrated that glucose triggers CT-1 expression in adipocytes and that elevated circulating CT-1 levels associate with high glucose concentrations. Given that abdominal obesity has been increasingly accepted as an important driving force of cardiometabolic risk, taken together, our results suggest that CT-1 overexpression in adipocytes could be an important link between MS and CVD. Further studies are required to definitively validate this hypothesis.


   GRANTS
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This project was funded through the agreement between the Foundation for Applied Medical Research (FIMA) and UTE project CIMA and Red Temática de Investigatión Cooperativa en Enfermedades Cardiovasculares (RECAVA) Grant RD06/0014/0008 from the Instituto de Salud Carlos III, Ministry of Health, Spain.


   ACKNOWLEDGMENTS
 
We acknowledge the skillful assistance of Sonia Auseré.


   FOOTNOTES
 

Address for reprint requests and other correspondence: N. Varo, Center for Applied Medical Research, Avda Pío XII 55, 31008 Pamplona, Spain (e-mail: nvaro{at}unav.es)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
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