Ketone bodies affect the enzymatic activity of macrophage migration inhibitory factor
Introduction
Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine secreted by several cell types of the immune defense including lymphocytes, eosinophils, neutrophils and monocyte/macrophages (Lolis and Bucala, 2003). It is also secreted alongside with ACTH by the pituitary gland in response to stressors like endotoxinaemia (Bernhagen et al., 1993). Therefore MIF could qualify as an endocrine hormone, if a bona fide receptor mediating its effects was known. MIF exhibits perplexing catalytic activities: tautomerisation of d-dopachrome (Rosengren et al., 1996) and of phenylpyruvate (Rosengren et al., 1997), as well as redox modification of protein sulphhydryl moieties (Kleemann et al., 1998) hence it could also be considered an enzyme.
Novel functions of MIF have been reported recently in diverse processes, such as cell proliferation, angiogenesis, atherosclerosis and wound healing (Nishihira, 2000, Burger-Kentischer et al., 2002). The relevance of the enzyme activities regarding MIF's multifaceted roles have not been delineated in detail yet. Whether and how its enzymatic activity contributes to the cytokine function is under debate (Swope et al., 1998, Hermanowsky-Vosatka et al., 1999). The putative “endogenous substrates” have not been identified despite the known topology of the catalytic centers within the crystal structure of the “active” trimeric form of the protein (Taylor et al., 1999). Computer modeling has been used to find “designer” inhibitors of promising potential (Dios et al., 2002).
While screening experimental phase anti-inflammatory agents' effects on MIF phenylpyruvate tautomerase we have identified acidic CH groups as markers of inhibitor potency toward this particular enzymatic activity. Guided by this lead we have sought simple model molecules with strong acidic CH groups to test their potential inhibitor activity. Among these substances acetylacetone was of particular interest because it is known to undergo tautomerisation (Watarai and Suzuki, 1974). Akin to acetylacetone the ketone bodies – acetoacetate, β-hydroxibutyrate and acetone – are also CH acidic. Ketone bodies reportedly counteract certain inflammatory processes (Sato et al., 1992, Sjogren et al., 1999). We have studied the effects of ketone bodies on MIF's enzymatic activity. Here we report, that ketone bodies differentially inhibit MIF phenylpyruvate tautomerase in vitro.
Section snippets
Materials and methods
Chemicals were of the highest available purity from Sigma except the benzylidenacetone – purchased from Aldrich – and the 2-propionyl cyclohexanone. The latter compound was prepared according to literature methods (Hunig et al., 1957).
The enol–keto tautomeric conversion of phenylpyruvate (ketonase reaction) was monitored by decrease of absorbance at 288 nm on a dual path Shimadzu 2100 UV spectrophotometer at room temperature according to Taylor et al. (1999) with minor modifications. Briefly:
Results
Against phenylpyruvate ketonase activity of MIF, acetylacetone and 2-propionyl cyclohexanone exhibited the strongest inhibitor potency, benzylideneacetone, acetoacetate, β-hydroxibutyrate showed moderate, while acetone and ethyl acetoacetate showed magnitudes weaker inhibitor activity (Fig. 1).
The phenylpyruvate enolase activity of MIF has not been affected up to 10 mM of the ketone bodies. The IC50 values obtained against the enolase were 9.2 mM, 154 μM and 137 μM for acetylacetone,
Discussion
We report in this paper that ketone bodies interfere with MIF phenylpyruvate tautomerase in vitro. Due to the raised fatty acid supply ketone body production of the liver is increased in fasting or in uncorrected diabetes. Recently elevated serum MIF levels have been reported in type 2 diabetic patients (Yabunaka et al., 2000) and elevated MIF levels of obese patients have been found suppressed by the antidiabetic drug, metformin (Dandona et al., 2004). Ketone bodies affect migration of bovine
Conclusions
MIF has emerged lately as a cytokine that counteracts glucocorticoids' anti-inflammatory action, and as a stress hormone cosecreted with ACTH from the pituitary. Glucocorticoids, nevertheless, also exert characteristic effects on fuel metabolism affecting among others ketogenesis. According to our data MIF's enzymatic action in turn seems to be altered by metabolic factors like the ketone bodies. Hence a link between metabolism and the immuno-endocrine roles of MIF might be postulated.
Acknowledgement
Hereby the authors express their gratitude in remembrance of the late Professor Gyula Kispál, who has initiated their cooperation.
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