Effects of pro-inflammatory cytokines and chemokines on leptin production in human adipose tissue in vitro

Abstract

Leptin is synthesized in adipocytes and acts primarily through central pathways suppressing appetite and increasing the metabolic rate in rodents as well as in humans. Recently leptin has also been suggested to have peripheral effects and be involved in insulin action. Since cytokines and chemokines may have effects on appetite regulation as well as on some of the obesity-related complications e.g. insulin resistance and cardiovascular disease, we investigated the effects of various cytokines and chemokines on leptin production in human adipose tissue fragments in vitro. Abdominal subcutaneous adipose tissue from healthy normal to overweight females was incubated for up to 48 h with the cytokines: tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and interleukin-1β (IL-1β) and the chemokine: interleukin-8 (IL-8). IL-1β (50 ng/ml) and TNF-α (10 ng/ml) decreased leptin production by 30–50% (P<0.05) and gene expression by 80–90% (P<0.05). In contrast, IL-6 and IL-8 had no effect on either leptin production or leptin gene expression. Interestingly, IL-1β elicited a biphasic effect on leptin release with an incremental phase observed within 4 h with no concomitant change in leptin gene expression, followed by a long-lasting inhibition of leptin release and leptin gene expression. This could suggest that IL-1β through a post-translational pathway induced an acute increase in leptin-secretion, perhaps through the release of leptin from a pre-formed pool within the adipose tissue. The long-term decrease in both leptin secretion and transcription could indicate that pro-inflammatory cytokines such as IL-1β and TNF-α might influence the circulating leptin levels and thereby influence the adipose tissue to brain signalling, which could be of importance in relation to the obesity-associated diseases such as insulin resistance and cardiovascular disease.

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.