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TNF up-regulates apelin expression in human and mouse adipose tissue

Danièle Daviaud^*, Jérémie Boucher^*, Stéphane Gesta^*, Cédric Dray^*, Charlotte Guigne^*, Didier Quilliot, Ahmet Ayav, Olivier Ziegler, Christian Carpene^*, Jean-Sébastien Saulnier-Blache^*, Philippe Valet^* and Isabelle Castan-Laurell^*,1

^* INSERM, U586, Unité de Recherches sur les Obésités, Université Paul Sabatier, Institut Louis Bugnard IFR31, Toulouse, France; and

Service de Diabétologie, Maladies Métaboliques et Nutrition, CHU de Nancy, Hôpital J. d’Arc, Nancy, France

¹Correspondence: IFR 31, Institut Louis Bugnard, BP 84 225 INSERM U586, Toulouse F-31432, Cedex 4, France. Email: isabelle.castan{at}toulouse.inserm.fr

SPECIFIC AIMS

Apelin is a novel peptide identified as the endogenous ligand for the orphan receptor APJ. Apelin mRNA has been detected in various tissues but little is known about apelin production and regulation in apelin-expressing cells. During the past decade, the secreted factors by adipose tissue (or adipokines) have gained much attention. Numerous adipokines are altered with obesity and could affect insulin-sensitivity or obesity-associated disorders. These adipokines represent possible drug targets for clinical investigations. Recently, we demonstrated that apelin is secreted by adipocytes, increased with obesity (plasma apelin levels were significantly higher in obese than in lean subjects) and regulated by insulin. Since obesity and insulin resistance are associated with chronically elevated levels of both insulin and TNF, the present study was performed to investigate a putative regulation of apelin expression by TNF in human and mouse adipocytes. This relation was further studied in primary culture of human subcutaneous (s.c.) adipose tissue explants, in C57Bl6/J mice and the signaling pathways by which TNF increased apelin expression was evaluated in 3T3F442A adipocytes.

PRINCIPAL FINDINGS

1. The expression of TNF and apelin are tightly correlated in human adipose tissue from lean to morbide obese subjects
In human adipose tissue, there is a very tight correlation between the expression of TNF and apelin, whatever the anatomical location of fat pad: in s.c. adipose tissue, r = 0.790, slope 0.085, P < 0.01; in intra-abdominal adipose tissue: r = 0.994, slope 0.097, P < 0.01 (Fig. 1 A, B). This correlation occurs in moderately obese subjects (BMI ranged from 20.5 to 36.5 kg/m², n=20) and in morbidly obese subjects (BMI ranged from 39.2 to 59.3 kg/m², n=23). This strong correlation between TNF and apelin was not observed between apelin and other adipokines such as leptin, adiponectin, or PAI-1. To further study apelin regulation by TNF in human adipose tissue in vitro, explants of s.c. adipose tissue were prepared and maintained in primary culture for 48 h. Adipocytes were then isolated from explants, and both TNF and apelin mRNA were quantified. The rise in TNF expression started 3 h after the beginning of primary culture and was significantly different from control cells after 6 h culture. During the same period, apelin expression dramatically increased in isolated adipocytes only beyond the 12th h after the beginning of culture. Furthermore, explants were cultured in the presence of 100 µM isobutylmethylxanthine (IBMX), a phosphodiesterase inhibitor shown to inhibit TNF production in adipocytes from cultured explants. By inhibiting the in situ expression of TNF with IBMX, the up-regulation of apelin was prevented (Fig. 1C ). This suggests a paracrine effect of endogenously produced TNF on apelin expression in human adipose tissue.

View larger version (14K): [in this window] [in a new window]   Figure 1. Correlation between apelin and TNF expression in adipose tissue from both lean and obese humans. A) Human s.c. adipose tissue samples were obtained from 20 patients (40.3±2.2 yr old, BMI: 27.3±1.2 kg/m2) undergoing abdominal lipectomy for plastic surgery. B) Human intra-abdominal adipose tissue samples were obtained from 23 morbidly (grade III) obese subjects (41.3±2.2 yr old, BMI: 45.7±2.9 kg/m2) before a bariatric surgery. Correlations were analyzed using the nonparametric Spearman rank test. C) Primary culture of explants was prepared from human s.c. tissue. Adipocytes were isolated from explants before (time 0) and after 3, 6, 24, and 48 h primary culture (left panel) without (–) or in the presence (+) of 100 nM IBMX (right panel). Results are mean values ± SEM from four separate experiments, *P < 0.05; **P < 0.01 when compared with controls.

2. Injection (i.p.) of TNF into C57Bl6/J mice increases apelin expression in adipose tissue and apelin blood levels
To determine whether increased TNF levels could directly up-regulate apelin expression, we investigated the direct effect of i.p. injection of TNF into C57Bl6/J mice on apelin expression in adipocytes. Eight hours after injection, TNF (2 µg/mouse) elicited a significant increase of apelin expression in isolated adipocytes (6.47 ± 0.86 and 20.34 ± 6.62 arbitrary units, n = 6) and in the stroma-vascular fraction (2.34±0.16 and 11.90±3.82 arbitrary units, P<0.05, n=6) compared with control mice. No significant variations of apelin mRNA levels were observed in other tissues such as heart (9.1±0.91 in control and 10.96±1.54 arbitrary units in TNF-treated mice, n=6) and kidney (8.81±1.23 in control and 9.62±0.76 arbitrary units in TNF-treated mice, n=6) known to express apelin. In addition, a significant increase of apelin plasma levels was observed in TNF-treated mice (1.27±0.06 ng/ml vs. 0.50±0.07 ng/ml in control mice, n=6). All together these results suggest that 1) TNF can up-regulate apelin expression in vivo specifically in adipose tissue; 2) the increased apelin expression in adipose tissue by TNF could contribute to apelin plasma levels.

3. Apelin mRNA induction in response to TNF in adipocytes was mediated via the MAPK, c-Jun NH2-terminal kinase (JNK) and PI3-K but not the PKC signaling pathway
To test a direct effect of TNF on fat cells, 10 d-differentiated adipocytes from the murine 3T3F442A cell line were used. Short exposure (8 h) of TNF induced apelin expression (Fig. 2 A)and secretion in the medium (values ranging from 0.04 ng/ml in control cells to 0.35 ng /ml in treated cells). Since TNF is known to signal through various transduction pathways in adipocytes, we tested the potential intracellular targets of TNF by using specific inhibitors. We showed that the induction of apelin expression by TNF on 3T3 F442A adipocytes was mediated by PI3-kinase, MAPK, JNK but not the PKC signaling (Fig. 2B ).

View larger version (10K): [in this window] [in a new window]   Figure 2. Regulation of apelin expression by TNF±. in 3T3F442A adipocytes. A) Dose response effect of 8 h TNF ± treatment on apelin mRNA levels in 10-day-differentiated 3T3F442A adipocytes. Results are mean values ± SEM from 3 separate experiments. B) Apelin mRNA levels in 10-day-differentiated 3T3F442A adipocytes after 24 h serum deprivation and incubated 8 h or not with 10 ng/ml TNF± in the absence (–) or presence (+; added 1 h before TNF± treatment) of LY294002 (10 µM), PD098059 (50 µM), GF109203X (10 µM), or SP 600125, (20 µM). Results are mean values ± SEM from 4 separate experiments, *P < 0.05 when compared with controls; P < 0.05 when compared with TNF±-stimulated cells.

CONCLUSIONS AND SIGNIFICANCE

TNF has been shown to regulate expression of different adipokines like leptin or the plasminogen activator inhibitor type-1 factor (PAI-1). The present work clearly demonstrates for the first time that TNF may act as a direct regulator of apelin expression in both human and mouse adipose tissue. In addition, i.p. injection of TNF in mice induced increased both apelin expression in adipose tissue and blood plasma levels of apelin. Our data bring new open lines of investigation in this field and propose apelin as a potential target in obesity acting either as an endocrine or paracrine factor (Fig. 3 ). It therefore appears of interest to test whether the TNF-induced increase apelin expression in adipose tissue and secretion is implicated in the changes occurring in adipose tissue during the onset of obesity and the emergence of endocrine, metabolic, or cardiovascular dysregulations.

View larger version (22K): [in this window] [in a new window]   Figure 3. Schematic diagram representing the putative roles of apelin secreted by adipose tissue in response to TNF.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-5243fje

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This Article Abstract Full Text Full Text (PDF) All Versions of this Article: fj.05-5243fjev1 20/9/1528    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Daviaud, D. Articles by Castan-Laurell, I. Search for Related Content PubMed PubMed Citation Articles by Daviaud, D. Articles by Castan-Laurell, I.