This study investigated the expression of leptin both – in vitro – within an inflammatory model of human cultured adipocytes and – in vivo – in patients suffering from severe sepsis and additionally evaluated the specific treatment effect of drotrecogin alpha (activated) (DAA).
Leptin mRNA and protein expression was down-regulated by TNF alpha after 6 and 24
hours of incubation in human adipocytes. But this effect seems not to be due to a reduced number of viable cells because of TNF alpha treatment: Treatment with DAA (50
ng/ml or 5
μg/ml) and TNF alpha (1
ng/ml) did not affect adipocyte viability, as assessed by trypan blue exclusion. Moreover, administration of DAA (50
ng/ml or 5
μg/ml) in the presence of TNF alpha (1
ng/ml) up-regulated leptin mRNA and protein expression in a dose dependent way at 24
hours. In patients suffering from severe sepsis leptin serum levels remained at the same level from days 1 to 5 in those patients without treatment of DAA. However, in patients treated with DAA leptin serum levels increased from day 1 to day 3
day of severe sepsis during the infusion of DAA over 96
hours. Leptin serum levels were inversely correlated with adiponectin and positively correlated with APACHE II score, body temperature, white blood cell count and interleukin 6.
The present analysis confirmed preliminary reports demonstrating decreased mRNA expression of leptin following TNF alpha stimulation within an in-vitro
inflammatory model [30
]. However, conflicting results have been obtained from cell cultures demonstrating an up-regulation of leptin mRNA expression after stimulation with TNF alpha [35
]. These varieties might in part be explained by the use of different cell species, anatomical localization of the fat tissue and the duration of exposure to the stimulating cytokines [32
]. Hence, mRNA transcription was even more down-regulated after longer incubation of 24
hours compared to 6
hours (59% vs. 46%).
In accordance with recently published data we could demonstrate increasing levels of leptin in patients suffering from severe sepsis compared to healthy controls [11
] and showed several correlations with established clinical markers of sepsis. Therefore our observations support evidence that leptin appears to be involved in the pathogenesis and the course of a systemic inflammatory response during sepsis. Interestingly, with regard to the body of literature it was shown that leptin itself increases the production of TNF alpha or Interleukin 6 from macrophages [42
DAA has been shown to reveal antithrombotic, profibrinolytic, anti-inflammatory and antiapoptotic properties [27
]. However, no data currently exist, whether DAA influences the release of the adipokine leptin. To the best of our knowledge this study is the first to investigate the influence of DAA on leptin expression. It could have been demonstrated that administration of DAA increases the amount of leptin mRNA and protein expression over time in human adipocytes and furthermore significantly increases leptin serum levels in patients suffering from severe sepsis after 96
hours administration of DAA.
Therefore, the present findings might reveal another effect of DAA during sepsis, i.e. the up-regulation of the hormone leptin under circumstances of ongoing systemic inflammation. Several in-vitro studies already demonstrated specific anti-inflammatory effects of DAA, e.g. inhibition of transcription factor NF-kappa B in isolated mononuclear cells or the inhibition of macrophage inflammatory protein-1 (MIP-1-alpha), monocyte chemoattractant protein-1 (MCP-1), neopterin [19
] or up-regulation of adiponectin [45
]. However, the role of specifically leptin during severe sepsis is not yet completely understood. It could have been demonstrated that patients surviving an acute sepsis revealed increased levels of leptin, whereas leptin correlated with the disease severity and is an independent predictor of death [37
]. Shapiro et al. [46
] postulated that leptin might be able to pronounce endothelial dysfunction during sepsis and therefore worsens outcome of the disease. However, increases of the soluble leptin receptor (sLR) were explained as a compensatory anti-inflammatory mechanism.
The exact mechanisms contributing to increased leptin mRNA and protein expressions after administration of DAA in human adipocytes and patients suffering from severe sepsis were not determined in this study. However, we performed cell culture experiments with adipocytes in order to confirm our clinical findings in septic patients with a more straightforward system and proving the involvement of adipocytes. Instead of LPS we used TNF alpha as a more selective and efficient pro-inflammatory stimulus for sepsis according to our established protocols, respectively in an inflammatory model with endothelial cells having been published already [20
]. It was our intention to apply the same conditions as in the above mentioned inflammatory models in order to guarantee comparability of our own experimental approach. However, TNF alpha serum levels were not measured in our septic patients. Regarding clinical benefits of septic patients increased leptin levels could not show a survival benefit over 28-days follow-up in our study. Nevertheless, our results need to be confirmed by ongoing basic research analyses and larger prospective clinical studies to explain the more detailed mechanisms beyond our findings.