Ketone bodies affect the enzymatic activity of macrophage migration inhibitory factor

Abstract

Macrophage migration inhibitory factor (MIF), a long known proinflammatory cytokine exhibits perplexing enzymatic activities: tautomeric conversion of d-dopachrome and phenylpyruvate. Whether these catalytic activities bear functional relevance regarding MIF's multifaceted roles is under current scrutiny. Nevertheless, intense search has already started for pharmacological agents that target MIF's tautomerase activity. We have probed several antiinflammatory compounds against keto–enol (enolase) and enol–keto (ketonase) conversion of phenylpyruvate by MIF with spectrophotometry. We have identified acidic CH groups as markers of inhibitor potency toward MIF phenylpyruvate tautomerase. Among simple model molecules with strong acidic CH groups we found acetylacetone the best inhibitor particularly against the ketonase activity. Ketones of physiological importance – ketone bodies – also feature acidic CH groups and have been reported to exert certain anti-inflammatory effects. In this paper we report that ketone bodies inhibit preferentially the ketonase activity of MIF in vitro. Future studies should address whether such an interaction might operate in vivo and delineate its possible relevance concerning cytokine and non-cytokine roles of MIF.

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.