Use of methionine sulfoximine to dissect the role of glutamine synthetase and glutamine in progression of acute liver failure

Methionine sulfoximine (MSO) is a modified amino acid and a well characterized irreversible inhibitor of glutamine synthetase (GS) enzyme. Glutamine is synthesized by GS enzyme and it is the most abundant amino acid in the body. Glutamine is required by immune cells for generation, propagation and maintenance of an immune response. To induce acute liver failure (ALF) in mice, animals were given intraperitoneal injections of E. coli lipopolysaccharides (LPS) and sugar D-galactosamine (D-GalN). When these animals were pretreated with MSO, 80% of the animals were completely rescued from liver failure. Moreover, when we characterized the immune response generated during ALF using cytokine antibody microarrays, we observed 1) that the macrophages and neutrophils appeared to be responsible for the cytokine storm generated during ALF, and 2) MSO pretreatment reduced the entire measured cytokine response. Using ELISA assays we observed that MSO pretreatment reduced plasma cytokine values of TNF-α, IFN-γ and IL-6 by more than 50%. MSO inhibited GS by more than 90% in liver extracts and reduced plasma glutamine by around 75%. MSO pretreatment had no effect on GS protein levels and on kupffer cell (liver macrophages) infiltration in liver. GS and TNF-α transcript levels were unaffected by MSO pretreatment. Also, preliminary results show that MSO does not inhibit cellular and secreted protein synthesis globally in LPS stimulated peritoneal macrophage cell culture. However, MSO treatment reduces TNF-α production in the LPS stimulated peritoneal macrophages. Thus, it seems that inhibiting GS during stress conditions such that low levels of plasma glutamine are maintained can lead to a reduction in inflammatory cytokines. Also, MSO might have targets other than glutamine synthetase. These results show a novel use for MSO in attenuating the overall immune inflammatory response in liver

In vitro suppression of inflammatory cytokine response by methionine sulfoximine

Abstract Background The glutamine synthetase inhibitor methionine sulfoximine (MSO), shown previously to prevent death caused by an inflammatory liver response in mice, was tested on in vitro production of cytokines by mouse peritoneal macrophages triggered with lipopolysaccharide (LPS). Results MSO significantly reduced the production of Interleukin 6 (IL-6) and Tumor Necrosis Factor Alpha (TNFα) at 4 and 6 h after LPS-treatment. This reduction did not result from decreased transcription of IL-6 and TNFα genes, and therefore appeared to result from post-transcriptional inhibition of synthesis of these cytokines. MSO treatment did not inhibit total protein synthesis and did not reduce the production of a third LPS-triggered cytokine CXCL1, so the effect was not a toxic or global downregulation of the LPS response. The anti-inflammatory effects of a glutamine synthetase inhibitor were seen even though the medium contained abundant (2 mM) glutamine, suggesting that the target for this activity was not glutamine synthetase. In agreement with this hypothesis, the L,R isomer of MSO, which does not inhibit glutamine synthetase and was previously thought to be inert, both significantly reduced IL-6 secretion in isolated macrophages and increased survival in a mouse model for inflammatory liver failure. Conclusions Our findings provide evidence for a novel target of MSO. Future attempts to identify the additional target would therefore also provide a target for therapies to treat diseases involving damaging cytokine responses