| Obesity is a chronic metabolic disease caused by increased fat deposition and/or abnormal distribution of adipose tissue in the body.Diabetes,hypertension,cardiovascular diseases and other diseases caused by obesity are increasing year by year and showing a younger trend.Lutein(LUT),a phytochemical rich in vegetables and fruits in the diet,has a potential effect on weight loss,but the relevant molecular mechanism needs to be further studied.Therefore,this study intends to conduct lutein intervention in obese rats,including SD rats fed with high fat and spontaneously obese rats(Zuker Diabetic Fatty,ZDF),and explore the ameliorate effects of lutein on lipid deposition,taking liver and abdominal adipose tissue as the entry point.At the same time,combined with the experiment of 3T3-L1 mature adipocytes cultured in vitro,the mechanism of lutein's role in lipid synthesis and lipolysis based on the SIRT1-mediated lipid metabolism pathway was explored,providing a powerful theoretical basis for the prevention and treatment of obesity and obesity-related diseases in the future.Part 1: Preventive effects of lutein on lipid deposition in obese ratsObjective: To investigate the amelioration effect of dietary lutein supplementation on lipid deposition in SD rats induced by high-fat diet and spontaneously obese ZDF rats.Methods:Animal experiment of SD rats: Forty male SD rats are divided into two groups after adaptive feeding,respectively fed normal diet or high-fat diet after12 days.Based on total cholesterol,the rats fed the ND or HFD were then divided into2 groups respectively,including four groups: ND,ND+LUT,HFD,and HFD+LUT,administered by 25 mg/kg·bw/day lutein for the next 5 weeks.At the end of experiment,all rats were euthanized.The liver,abdominal,perinephric and epididymal adipose tissues of the rats were isolated and weighed,which were used to detect the expression of genes and proteins related to the lipid synthesis and lipolysis.Animal experiment of ZDF rats: Fifty-one male ZDF rats were divided into 6groups according to fasting blood glucose and body weight,namely blank control group(CON,n=10),ZDF/ fa/+;model group(ZDF,n=10),ZDF/fa/fa;lutein intervention group(LUT,n=9),ZDF/fa/fa;resveratrol intervention group(RES,n=9),ZDF/fa/fa;lutein and resveratrol combined action group(LUT+RES,n=6),ZDF/ fa/fa;nicotinamide intervention group(NAM,n=7),ZDF/fa/fa.LUT group,RES group and combined group were given lutein of 50 mg/kg.bw and/or resveratrol of 100 mg/kg.bw respectively,while NAM group was given nicotinamide of 60 mg/kg.bw.OGTT experiment was conducted at the end of intervention 22 weeks.Serum,liver,adipose tissue and other organs were collected at the end of intervention 20 weeks to detect the expression of genes and proteins related to lipid metabolism pathway.Result: High fat feeding significantly increased the final body weight,visceral fat index,FPG,HOMA-IR,ALT and liver TG levels in SD rats.After lutein intervention,the final body weight and abdominal fat index of HFD+LUT group(P < 0.05;P < 0.05),FPG,FINS and HOMA-IR(P < 0.05;P < 0.01;P < 0.001),ALT and liver TG were significantly decreased(P < 0.01;P < 0.001);At the same time,high fat feeding significantly increased liver steatosis,fat droplets stained with oil red,abdominal fat cell damage and the frequency and average area of large abdominal adipocytes per 100 cells.After lutein intervention,liver steatosis and balloon-like change scores were significantly reduced in HFD+LUT group,lipid droplets were significantly decreased in oil red staining,the abdominal fat cell damage was relieved,and the frequency and mean area of larger abdominal adipocytes per 100 cells were significantly decreased(P< 0.001).In the experiment of ZDF rats,compared with the ZDF group,only in LUT+RES group,the final body weight and body weight gain were significantly decreased(P<0.05;P <0.05),and liver and abdominal fat index in LUT group,RES group and LUT+RES group were significantly decreased(P < 0.05);Serum lipid metabolism indexes(TC/TG/LDL-C/HDL-C)and insulin sensitivity indexes(FPG/FINS/HOMA-IR)were significantly increased in ZDF group.Compared with ZDF group,serum TG in LUT group,RES group and LUT+RES group were significantly decreased by 22%,26%and 31%(P < 0.05;P < 0.01;P < 0.01),AUC was significantly decreased,and liver glycogen was significantly increased;As for liver injury indexes,compared with CON group,the contents of serum AST/ALT,liver TG,MDA,GSH and ROS were significantly increased in ZDF group.Compared with the ZDF group,the serum ALT level,liver TG,MDA and red fluorescence intensity of ROS content were significantly decreased in LUT group,RES group and LUT+RES group,and the antioxidant enzyme GSH was significantly increased.In terms of morphological changes of liver and abdominal fat,after the intervention of lutein and resveratrol,the frequency and average area of larger abdominal adipocytes were significantly reduced(P <0.001),the steatosis of liver cells was significantly alleviated,and the lipid droplets stained with oil red were significantly reduced,and the improvement effect of the combined intervention group was more obvious(P < 0.01).Conclusions: Lutein is a phytochemical that can effectively improve liver and abdominal fat deposition in obese rats,providing new preventive measures for preventing obesity and obesity-related diseases.Part 2: Study on the mechanism of lutein improving abdominal fat deposition based on SIRT1-lipid metabolism pathwayObjective: To investigate the potential molecular mechanism of lutein supplementation in improving lipid deposition in abdominal adipose tissues of SD rats fed with high fat and mature 3T3-L1 adipocytes.Methods:Animals: Grouping and feeding of SD rats were the same as the Part 1.The expression of lipid metabolism-related genes and proteins were detected by immunohistochemistry,RT-PCR and Western blot assay.The culture of differentiated 3T3-L1 cells: The 3T3-L1 preadipocytes were induced to differentiate for 10-14 days using the “classical cocktail” method.After 90%of the cells showed mature adipocyte phenotype,they were divided into 4 groups:control group(Con),lutein intervention group(Lut,40 ?M),inhibitor group(Ex527,10?M),and combined action group(Lut+Ex527).After 24 h intervention,the morphology of lipid droplets was monitored by oil red O staining,the glycerol release of supernatant and intracellular TG content were determined,and the expression of genes related to TG decomposition and synthesis in adipocytes was detected.Result: High fat feeding significantly reduced the m RNA and protein levels of SIRT1,Fox O1 and ATGL,as well as the phosphorylation levels of HSL and ACC in the abdominal adipose tissue,and increased the m RNA and protein expression levels of SREBP-1 and FAS,which were involved in TG synthesis.After the intervention of lutein,the expression of SIRT1 protein in the abdominal adipose tissue in HFD+LUT group was significantly increased,and the m RNA and protein levels of Fox O1 and ATGL,as well as the phosphorylation levels of HSL and ACC were significantly up-regulated,but the m RNA and protein levels of SREBP-1 and FAS were significantly down-regulated,thus promoting the lipolysis of TG and inhibiting the synthesis of TG.At the same time,the results of SIRT1,ATGL and p-HSL immunohistochemical staining in the abdominal adipose tissue were consistent with the results of western blot.Lutein at a dose of 40 ?M significantly decreased the TG content and oil red lipid droplets,notably increased glycerol release in comparison with the untreated differentiated control.Meanwhile,we discovered significantly elevated SIRT1(P <0.001)and a similar trend in differentiated 3T3-L1 cells incubated with lutein,which meant downregulation of FAS,SREBP-1,and the p-ACC(Ser79)/ACC ratio and upregulation of Fox O1,the p-HSL(Ser660)/HSL ratio and ATGL protein levels.Thereby,the blocked synthesis and accelerated lipolysis of triglycerides collectively alleviate the accumulation of lipid droplets in differentiated 3T3-L1 cells.Nevertheless,after coincubation with Ex527 and lutein,the TG content prominently increased,and glycerol release dramatically decreased in contrast with the Lut group.The positive effect of lutein on alleviating lipid accumulation disappeared,and then the protein levels of SIRT1,SREBP-1,the p-ACC(Ser 79)/ACC ratio and FAS were upregulated,while Fox O1,ATGL and the p-HSL(Ser 660)/HSL ratio were downregulated in comparison with the Lut group.Conclusions: Lutein plays the role of improving lipid deposition by activating the expression of SIRT1 protein in the abdominal adipose tissue and differentiated 3T3-L1 cells.Upregulation of SIRT1,on the one hand,inhibits the SREBP-1/FAS/ACC pathway to reduce triglyceride synthesis and thus weakening the lipid synthesis of the abdominal adipose tissue and differentiated 3T3-L1 cells;and on the other hand,it upregulates the Fox O1/ATGL/HSL pathway and then increasing the lipolysis of triglycerides,thereby enhancing the lipid lipolysis of the abdominal adipose tissue and differentiated 3T3-L1 cells.Part 3: Study on the mechanism of lutein improving liver fat deposition based on SIRT1-lipid metabolism pathwayObjective: This part was designed to explore the potential mechanism by which lutein ameliorated the excessive lipid accumulation in liver of SD and ZDF rats.Methods: Grouping and feeding of SD and ZDF rats were the same as the Part 1.The expression of lipid metabolism-related proteins was detected by Western blot assay.Result: At the same time,high fat diet can significantly reduce the expression levels of SIRT1(48%,P < 0.01)and fatty acid ?-oxidation related protein PPAR??CPT1A?ACOX1,and inhibited the expression levels of SREBP-1?FAS?ACC,which were involved in de novo synthesis of liver fat.After Lutein intervention,liver SIRT1 protein expression in HFD+LUT group was significantly increased,and PPAR??CPT1A? ACOX1? ATGL protein expression and HSL phosphorylation level were significantly up-regulated,which promoted liver fatty acid ?-oxidation,and significantly down-regulated the protein levels of SREBP-1?FAS?ACC,and thus inhibiting de novo synthesis of liver fatty acid.Meanwhile,compared with the CON group,the expressions of liver SIRT1?PPAR??CPT1A?ACOX1? ATGL? the phosphorylation levels of HSL and ACC were significantly down-regulated,and the protein levels of SREBP-1 and FAS were significantly increased in the ZDF group.Compared with ZDF group,liver SIRT1 and the expression of PPAR??CPT1A?ACOX1?ATGL?the phosphorylation levels of HSL was significantly increased in LUT group,RES group and LUT+RES group,and thus promoting liver fatty acid ?-oxidation,but the expression levels of SREBP-1,PPAR?,FAS,ACC decreased significantly,inhibiting the liver de novo synthesis of fatty acid.In addition,compared with ZDF group,the phosphorylation levels of liver AKT and PI3 K protein in LUT group,RES group and LUT+RES group were significantly increased.Conclusions: By upregulating the expression of SIRT1 in liver,lutein,on the one hand,inhibits the expression of SREBP-1/ FAS /ACC to weaken de novo fatty acid synthesis of liver fat;on the other hand,it can up-regulate the expression of PPAR?/CPT1A/ACOX1 and ATGL/HSL to enhance ?-oxidation of fatty acids,thus improving lipid deposition in liver.Besides,lutein alleviates insulin resistance in ZDF rats by activating SIRT1-PI3K/AKT pathway. |
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