Study On The Mechanism Of The Inhibition Of BRL-3A Cells Sirtuin 1 Activity Induced By Methylglyoxal

Insulin resistance(IR) plays a critical role in the development of non-alcoholic fatty liver disease(NAFLD) and metabolic syndrome, and Sirtuin1(SIRT1) is an key factor of regulating IR. SIRT1 is a nicotinamide adenine dinucleotide(NAD+)-dependent protein deacetylase, regulating cell metabolism. In cells, much of NAD+ comes from the salvage pathway, and nicotinamide phosphoribosyltransferase(NAMPT) is the rate-limiting enzyme, affecting SIRT1 activity. CLOCK-BMAL1 heterodimer regulate Nampt transcription, and Nampt activation mediated by the complex is related to histone modification on promoter of gene. CLOCK has histone acetyltransferase(HATs) activity, and BMAL1 is transcriptional coactivator of CLOCK, enhancing CLOCK function. The site of histone H3 lysine 14(H3K14) of Nampt is regulated by CLOCK-BMAL1 heterodimer, and the site is much higher than H3K9 in extent. So, change of H3K14 may affect SIRT1 activity. Methylglyoxal(MGO), a kind of reactive carbonyl species(RCS), coming from western foods or polyol pathway in the body of human and animal, is already proved to result in IR. MGO can modify lysine residues of protein forming Schiff base. To investigate whether H3K14 acetylation and SIRT1 activity can be affected by MGO, MALDI-TOF MS and ESI MS of MGO-treated peptide, Schiff base formation of nuclear protein, Western blot analysis of H3K14 acetylation of nuclear protein, histone and hepatocytes, the level of NAMPT and activity of SIRT1 were done in this study. Results of this research are as follows:Results of MALDI-TOF MS and ESI MS showed that lysine residues, inculding H3K14, of H3 peptide were adducted by 4.6 mM MGO after incubation for 2 h at 37oC. DNPH method demonstrated that 0.2-0.8 mM MGO could form Schiff base with nuclear protein in 1.5 h in a time- and concentration-dependent manner. In the meantime, levels of H3K14 acetylation of nuclear protein and histone decreased after treated with 0.8 mM MGO, corresponding to the increase of Schiff base at the same time point. In rat hepatocytes, levels of H3K14 acetylation and NAMPT were inhibited by 0.1-0.8 mM MGO after incubation for 24 h. However, MGO-induced decreases in the acetylation of H3K14 and level of NAMPT were significantly attenuated by an effective carbonyl trapping agent amino guanidine(AG). Furthermore, the activity of SIRT1 was significantly decreased by MGO, and the inhibitory effect of MGO was also significantly attenuated by AG.Conclusion: In this study, MALDI-TOF MS and ESI MS were used to directly demonstrate that H3K14 can be adducted with MGO. The level of H3K14 acetylation was decreased induced by MGO and the mechanism was related to the production of Schiff base in the reaction of MGO and lysine residues of histone. Decrease of SIRT1 acticity was induced by MGO and the mechanism may be that H3K14 was blocked with MGO, leading to reduced acetylation.