Effect And Mechanism Of Threonine In Alleviating Disorders Of Hepatic Lipid Metabolism In Cadmium-Exposed Mice

Cadmium(Cd),a common contaminant in food,is highly susceptible to accumulation in humans through the food chain and can have adverse health effects.Recent studies have shown that Cd exposure can lead to disturbances in hepatic lipid metabolism in humans.The effective prevention and control of hepatic lipid metabolism disorders due to foodborne Cd exposure is one of the key concerns in the field of food safety.As an essential amino acid,threonine has a significant role in regulating lipid metabolism in the body.Previous studies by our team found that threonine was effective in mitigating the cytotoxicity caused by Cd exposure.Related reports have also found that Thr inhibits fat accumulation and improves lipid metabolism in obese mice.However,the mechanism of action of Thr in alleviating hepatic lipid disorders in Cd-exposed mice is not yet clear.In this study,we investigated the regulatory effects and mechanisms of Thr on the disturbance of liver lipid metabolism in Cd-exposed mice by gavage of different doses of Thr.(1)To investigate the alleviating effects of Thr on liver injury and lipid deposition in Cd-exposed mice.(2)To identify key regulatory pathways and key targets of lipid metabolism through lipidomic and transcriptomic studies.(3)To explore the role of Thr in alleviating Cd-induced disruption of liver lipid metabolism in mice by analyzing the association between lipid content,gene expression and apoptosis.(4)To validate the role and mechanism of Thr in alleviating Cd-induced disorders of lipid metabolism in HepG2 hepatocytes in an in vitro cellular assay.The study found that :(1)After 7 weeks of continuous gavage with 0.04mmol/g/d and 0.08 mmol/g/d doses of threonine,the body weight growth rate of Cdexposed mice was restored and the liver coefficient was significantly reduced.At the same time,Cd exposure reduced the elevated serum activities of liver function enzymes glutamate transaminase(ALT),glutamic oxalacetic transaminase(AST),alkaline phosphatase(ALP)and lactate dehydrogenase(LDH),which protected the liver function of Cd-exposed mice.Thr treatment increased superoxide dismutase(SOD),catalase(CAT)and glutathione peroxidase(GSH-Px)enzyme activities in the livers of Cd-exposed mice,inhibited the elevation of malondialdehyde(MDA)caused by Cd exposure,reduced the levels of inflammatory factors tumour necrosis factor-α(TNF-α)and interleukin-6(IL-6)in the livers of Cd-exposed mice,and inhibited the expression of cyclooxygenase(COX-2)and macrophage inflammatory protein(MIP-2),key enzymes in the hepatic inflammatory response.In addition,Cd exposure resulted in significantly increased serum and liver levels of triglycerides(TG),total cholesterol(TC)and low-density lipoprotein(LDL-C),increased red lipid droplets in Oil Red O staining,and reduced lipid accumulation by reducing blood and liver lipid levels in mice after Thr gavage.These results suggest that Thr is effective in alleviating liver damage and lipid deposition caused by Cd exposure.(2)Analysis of the total liver lipid content revealed that compared to the Control group,the livers of the Cd-exposed mice contained triglycerides(TG),diglycerides(DG),free fatty acids(FFA),phosphatidylcholine(PC),phosphatidylethanolamine(PE),phosphatidylinositol(PI),phosphatidylserine(PS),lysophosphatidylcholine(LPC),cholesterol lipids(CE),sphingomyelin(SM)and ceramide(CER)were significantly increased.In contrast,the levels of all these lipid species,except LPA and CAR,were significantly reduced in the lipid profile after low and high doses of Thr treatment,indicating that Thr alleviated hepatic steatosis due to dyslipidemia and dysregulated lipid metabolism in Cd-exposed mice.The beneficial effects of Thr were associated with a reduction of FFA,DG,TG,CER and PE in the liver,including 9 TG,2 DG 2 FFA,2 PE,2PG,2 Cer,and 1 PC are potential lipid metabolites responsible for the lipid-lowering effect of Thr.(3)Transcriptomic KEGG and GO showed that DEGs were significantly enriched in amino acid biosynthesis,fatty acid biosynthesis,inflammatory signaling pathways and apoptotic cell death pathways after Cd treatment,indicating that Cd exerted toxic effects by inducing amino acid synthesis,fatty acid synthesis,inflammation and apoptosis.In contrast,Thr treatment reduced Cd-induced lipid accumulation in mouse liver by regulating fatty acid synthesis,fatty acid degradation and triglyceride metabolism.Thr mainly inhibited the expression of SREBP-1c,ACC1,FASN,FADS1,FADS2,OXSM and ACSBG2,GPD1 and DAGT2 or promote the expression of ACOX1,fatty acid transporter CD36,ACAD10,PPARα,CPT1 A and and CPT1 B reduced the accumulation of triglycerides and disorders of lipid metabolism in the liver.In addition,three important signalling pathways,including PI3K-AKT-mTOR,JAK-STAT and LKB1/AMPK signalling pathways,were significantly enriched in Thr treatment.(4)In vivo experiments,Thr significantly inhibited the Cd-induced elevation of pro-apoptotic proteins(Bax and caspase-3)and restored the level of anti-apoptotic protein(Blc-2).In addition,Thr activated the upstream regulator of apoptosis LKB1-AMPKmTOR or inhibited the JAK-STAT and PI3K/AKT signaling pathways to down-regulate the expression of fatty acid synthesis genes SREBP1,ACC1,PPARγ,FASN and SCD1 and up-regulate the expression of fatty acid β-oxidation genes ACOX1,CD36,ACAD10,CPT1 and PPARα expression.As a result,fatty acid synthesis was restricted,thereby reducing hepatic lipid synthesis.(5)In vitro,Thr inhibited fatty acid synthesis through the LKB1/AMPK-mTORSREBP-1c-FASN/ACC1 signaling pathway and promoted fatty acid β-oxidation through upregulation of PPARα and CPT1 protein expression.In addition,Thr also inhibits fatty acid synthesis gene expression and promotes fatty acid β-oxidation-related gene expression through inhibition of JAK2-STAT1/3 and PI3K/AKT signaling pathways,thereby inhibiting pro-inflammatory responses and apoptosis and alleviating lipid metabolism disorders.In summary,this study revealed that Thr alleviated apoptosis and lipid metabolism disorders in liver and HepG2 hepatocytes of Cd-exposed mice by inhibiting LKB1/AMPK-mTOR,JAK2-STAT1/3 and PI3K/AKT signaling pathways,as evidenced by the upregulation of fatty acid synthesis genes and downregulation of fatty acid oxidation genes,thereby attenuating Cd-induced lipid accumulation in hepatocytes.