Regulation Of Chromium And Magnesium On The Lipid Metabolism Of NAFLD

As the incidence of non-alcoholic fatty liver disease(NAFLD)increases year by year,it has become one of the important worldwide public health problems in the 21st century,and exploration of the pathological mechanism and discovery of potential therapeutic targets are the focus of current research.Metal ions play important roles in cell function,signal transduction and energy metabolism for instance,and participate in multiple physiological activities and disease development as a key enzyme cofactor in lipid metabolism,while yet there are few studies on their regulation in lipid metabolism of NAFLD.Trivalent chromium ion is one of the essential trace elements in mammals.It plays an important role in maintaining blood glucose and lipid metabolism,and has been widely used in health care products,feed additives and pharmaceutical industries.It has been reported to have a potential effect on the prevention and alleviation of NAFLD,however the molecular mechanism of lipid metabolism has not yet been elucidated.NAFLD cell and mouse models were constructed in SMMC-7721 cells and BALB/c mice treated by oleic acid(OA)as the single exogenous fatty acid,respectively.The supplementation of trivalent chromium ions to NAFLD cells and mouse models showed that chromium ions can effectively relieve NAFLD.Chromium ions could reduce excessive accumulation of triglyceride and fatty acids in liver,reduced liver weight,body weight and transaminase ALT,AST,reduced overexpression of CD36 and DGAT2,and stabilized inflammatory factors in mouse serum.The combination experiment of chromium ions and DGAT2 inhibitor or its agonist showed that chromium ions still attenuated triglyceride synthesis by decreasing the expression of DGAT2,indicating that DGAT2 may be an important target molecule involved in the regulation of NAFLD lipid metabolism by trivalent chromium ions.Metallomics analysis of the OA-induced NAFLD cell model and mouse model revealed that lipid degeneration was accompanied by magnesium deficiency,whether the magnesium deficiency and lipid metabolism disorder of NAFLD model could be improved after supplementation of exogenous magnesium.Magnesium ions are the second largest cation in human body.It participates in more than 600 enzyme systems in cells as a coenzyme factor,and plays an important role in energy metabolism,cell proliferation and nerve conduction.Magnesium deficiency can cause many diseases such as paralysis,depression,hyperlipemia and obesity.Our results showed that the supplementation of magnesium ions can effectively alleviate the lipid degeneration of the NAFLD model:using Oil red O staining,TLC and GCMS to detect lipid changes,it was found that supplementation with magnesium ions can alleviate excessive accumulation of triglyceride and fatty acids in the NAFLD model,excessive transaminase ALT and AST levels in NAFLD mice serum were also inhibited after magnesium ions supplementation.We speculated that magnesium ions could reduce the overexpression of DGAT2 and CD36 induced by OA.indicating that magnesium ions can reduce liver uptake of OA by decreasing the expression of fatty acid transporter CD36,and reduce triglyceride synthesis in liver by reducing the expression of triglyceride synthesizing rate-limiting enzyme DGAT2.Previous study has shown that inflammatory factor disorder can lead to and aggravate the degree of NAFLD,and the balance between pro-inflammatory and anti-inflammatory factors may determine the progress of NAFLD.ELISA analyzed the changes of serum inflammatory factors in NAFLD mice,and the results showed that magnesium ions could reduce the levels of pro-inflammatory factors(IL-1β,TNF-a and IL-12),increase the level of anti-inflammatory factors(IL-10),and improve the inflammatory response in NAFLD mice.In addition,the composition and content of metal ions in NAFLD cells and mouse models were determined by ICP-AES.The results showed that magnesium ions could effectively relieve the symptoms of iron overload in NAFLD model.The mechanism of relieving effects of magnesium ions on NAFLD showed that exogenous OA down-regulated the expression levels of magnesium ions transporter genes PRL3 and CNNM3 in NAFLD cell model,and decreased the concentration of magnesium ions in hepatocytes;In contrast,overexpression of PRL3 and CNNM3 by recombinant plasmids increased the content of intracellular magnesium ions,and PRL3 responded to to the regulation of magnesium ions on DGAT2.Speculations whether the magnesium ions responsed-PRL3 affect triglyceride synthesis by regulating the expression of DGAT2?In this study,recombinant plasmid transfection and siRNA interference technology were used to overexpress or silence the signaling key proteins in the pathway.The results indicated that PRL3 acts as an upstream gene on the PI3K/AKT/mTOR and mTOR/Lipinl/SREBPl signaling pathway,and SREBP1 regulates the expression of DGAT2.PRL3 inhibitor and mTOR inhibitor were added to verify the effect of magnesium ion on lipid accumulation in NAFLD cell model.Western blot results indicated that magnesium ions acted on DGAT2 via PRL3/AKT/mTOR and mTOR/Lipinl/SREBP1 and affected the synthesis of triglycerides through this pathway.SREBP1 acts as a lipid metabolism gene-regulated transcription factor,and its transcriptional regulatory region cloning overexpression vector can down-regulate DGAT2 expression,while siRNA-SREBP1 up-regulated expression of DGAT2.The SREBP1 binding site(5’-ATCACCCCAC-3’)was found in the promoter region of DGAT2.The luciferase reporter gene assay revealed that SREBP1 negatively regulates the transcriptional activity of the DGAT2 gene promoter.The results of EMSA and ChIP experiments confirmed that SREBP1 protein and DGAT2 promoter DNA bind to each other both in vitro and in vivo.At the same time,magnesium ions acts as a cofactor to participate in the binding process of SREBP1 protein and DGAT2 promoter.In conclusion,Our data show that both chromium and magnesium ions can reduce lipid accumulation,liver weight,body weight and transaminase,and stabilize the disordered lipid metabolism genes and inflammatory factors;Magnesium ions play a role in relieving iron loading and magnesium deficiency.The cellular lipid metabolic mechanism of magnesium involved in NAFLD suggest that magnesium ions act on the expression of DGAT2 via PRL3/AKT/mTOR and mTOR/Lipinl/SREBP1,and participate in the transcriptional regulation of DGAT2 by SREBP1.All in all,the results indicate the important regulatory role of metal ions in lipid metabolism of NAFLD,and demonstrate the importance of DGAT2 as a potential therapeutic target for NAFLD.