Effects of pro-inflammatory cytokines and chemokines on leptin production in human adipose tissue in vitro
Introduction
Leptin, the 16-kDa protein product of the ob gene, is known to be an important regulator of appetite and energy expenditure in rodents and humans, probably through central pathways (Zhang et al., 1994, Campfield et al., 1995, Havel, 2000). Leptin is primarily synthesized and secreted from the adipose tissue and the expression of the leptin (ob) gene as well as the secretion of leptin from the adipose tissue are significantly higher in obese subjects as compared to normal weight subjects (Lonnqvist et al., 1995, Considine et al., 1996, Bastard et al., 2000). The elevated levels of leptin decrease after weight loss (Considine et al., 1996, Bastard et al., 2000), and correlations are found between the amount of body fat and the circulating levels of leptin, suggesting that leptin might act as a part of a feedback mechanism signalling to the brain about the size of the fat stores in the body (Considine et al., 1996, Van Gaal et al., 1999). Although leptin primarily has been reported to have central effects, two recent rodent-studies have suggested that leptin might also have peripheral effects and be involved in insulin-action through an increase in insulin sensitivity in rat adipose tissue and skeletal muscle (Muller et al., 1997, Shimomura et al., 1999, Yaspelkis et al., 1999).
Leptin production and secretion are mainly determined by the size of adipose tissue depot, however, numerous hormones, cytokines and cytokine-like proteins may also influence the leptin production (Kristensen et al., 1999, Trayhurn et al., 1999). Of special interest are the cytokines tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and interleukin-1β (IL-1β) as well as the chemokine, interleukin-8 (IL-8). In rodent studies it has been found that intra-peritoneal injection of lipopolysaccharide (LPS), TNF-α or IL-1 were able to increase both leptin protein levels and leptin gene expression in adipose tissue harvested 5–8 h later (Grunfeld et al., 1996, Sarraf et al., 1997). Furthermore, Sarraf and coworkers performed time course studies where they observed an increase in leptin levels in blood samples obtained 7–10 h after injection of TNF-α or IL-1 in mice (Sarraf et al., 1997). The elevated circulating levels of leptin returned toward baseline after a further 8–10 h for both of the pro-inflammatory cytokines (Sarraf et al., 1997). The pro-inflammatory cytokines TNF-α and IL-1β are well-known to be involved in the activation of the transcription factors such as CCAAT/enhancer binding protein α (C/EBPα), which is located proximal to the ob gene promoter and has been demonstrated to be necessary for ob gene transcription (Miller et al., 1996) and nuclear factor-κB (NF-κB), which has been shown to be involved in various inflammatory disease states including atherosclerosis (Barnes and Karin, 1997, Chen et al., 1999, Baldwin, 2001). Furthermore, TNF-α has been associated with insulin resistance in animal-models as well as in obese humans, and weight loss in obese subjects are reported to decrease the concentration of TNF-α and to partly reverse the insulin resistance (Hotamisligil et al., 1993, Dandona et al., 1998). IL-6 has been found to be positively correlated with the amount of adipose tissue as well as the development of cardiovascular disease (Vgontzas et al., 1997, Yudkin et al., 1999). Recently, IL-8 has been suggested to be involved in atherogenesis (Terkeltaub et al., 1998, Gerszten et al., 1999) as well as in fat accumulation in mature adipocytes and leptin secretion (Gerhardt et al., 2001). Finally, reports have shown that IL-1β is able to induce changes in several of the pathways involved in the pathogenesis of atherosclerosis and cardiovascular disease, probably through an activation of the NF-κB pathway (Libby et al., 1995, Dewberry et al., 2000). As with leptin, IL-6, TNF-α and IL-8 are found to be produced and released from the human adipose tissue itself (Hotamisligil et al., 1993, Mohamed-Ali et al., 1997, Bruun et al., 2001), suggesting that they could have implications on leptin signalling and thereby the obese state through autocrine/paracrine or endocrine pathways.
Since prior investigations concerning cytokine effects on leptin expression and leptin secretion from human adipose tissue primarily have been conducted using TNF-α (Kirchgessner et al., 1997, Gottschling-Zeller et al., 1999, Zhang et al., 2000, Fawcett et al., 2000), we found it of interest, in more detail, to investigate whether IL-1β, IL-6 and the chemokine IL-8 as well as TNF-α could affect the secretion and gene expression of leptin from human adipose tissue fragments in vitro.
Section snippets
Subjects
Subcutaneous adipose tissue fragments were obtained from the abdominal region from 14 healthy normal to overweight women undergoing liposuction for cosmetic reasons. The subjects had an age range of 27–52 years, with a mean weight of 72.9±2.3 kg and a mean body mass index (BMI) of 25.7±0.9 kg/m2 (range: 22–30 kg/m2). None of the subjects received any kind of medication. All subjects were fasted overnight before tissue removal. The adipose tissue was transported to the laboratory in a sterile
Regulation of the leptin production (Fig. 1A+B)
Abdominal subcutaneous adipose tissue fragments from females were incubated for 48 h with various hormones and cytokines. Dexamethasone, which is known to be a potent stimulus for leptin expression and secretion in adipose tissue was used as a control on the responsiveness of the cells (Kristensen et al., 1999). At maximal effective concentrations TNF-α (10 ng/ml) and IL-1β (50 ng/ml) decreased the non-stimulated leptin secretion by 50% (13.9±1.9 ng/ml vs. 27.6±3.1 ng/ml, P<0.01) and 30%
Discussion
The experiments described in this paper demonstrate the effects of three cytokines and the chemokine, IL-8, on leptin secretion and leptin gene expression in human adipose tissue fragments in vitro. We demonstrate that pro-inflammatory cytokines e.g. TNF-α and IL-1β are able to decrease the production and release of leptin from human adipose tissue fragments in vitro in a dose- and time-dependent manner after incubation for up to 48 h. Earlier work has shown that TNF-α was able to increase both
Acknowledgements
The technical assistance of L. Pedersen, D. Phillip and P. Hornbek is gratefully appreciated. The study has been supported by the Novo Nordic Foundation, Aarhus University, Aarhus Amtssygehus Research-Foundation, The Danish Medical Council and The Aarhus University–Novo Nordic Centre for Research in Growth and Regeneration.
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