| Type 2 diabetes mellitus（T2DM）is a disorder of glucose and lipid metabolism characterized by hyperglycemia,and is the main type of diabetes.The liver tissue of patients usually has lipid deposition dominated by triglycerides,which aggravates the damage of diabetic liver[1].However,the detailed mechanism of lipid deposition is still unclear.The free fatty acids entering hepatocytes are converted into triglycerides undergo a series of reactions in the endoplasmic reticulum and stored as lipid droplets.When energy is needed,the lipid droplets are decomposed into fatty acids,which are then oxidized to provide energy,and then oxidized for energy supply.There are two pathways for the oxidation of fatty acids in eukaryotic cells.One is the mitochondrialβ-oxidation pathway,which mainly decomposes short-,medium-and long-chain fatty acids;the another one is peroxisomeβ-oxidation,which mainly decomposes very long-chain fatty acids（>C22）and branched-chain fatty acids into short-chain fatty acids.The latter is transferred to the mitochondria,which is further decomposed into acetyl-Co A via the mitochondrialβ-oxidation pathway and then oxidized for energy[2].Although the mitochondrialβ-oxidation pathway plays a central role in reducing lipid accumulation in liver,recent studies have found that the peroxisomeβ-oxidation pathway can also decompose medium-and long-chain fatty acids[3],thereby offsetting the accumulation of lipids in hepatocytes during metabolic stress[4].In the different stages of nonalcoholic fatty liver disease（NAFLD）,peroxisomes and mitochondria also play a role in the decomposition of fatty acids[5].In type 2diabetes,due to insulin resistance,the ability of cells to decompose and use glucose to provide energy decreases,and liver cells change to use fatty acid oxidation to provide energy,while synthetic fat stores energy in the form of lipid droplets.When lipolysis of lipid droplets is reduced,type 2 diabetes mellitus complicated with fatty liver.Under such condition,is any alteration in peroxisome function of liver?Is the lipid deposition in the liver related to the alteration of peroxisome function?To this end,in this experiment,a rat model of type 2 diabetes with fatty liver and HepG2 cells treated with sodium oleate were used as the experimental object,and the expression levels of peroxisome membrane proteins,peroxisomal membrane protein 70 k Da（PMP70）and Peroxin 12（PEX12）,and the expression of peroxisome matrix protein catalase（CAT）were used as indicators of changes in peroxisome function,to explore the role of peroxisomes in lipid deposition in the liver of type 2 diabetes at the animal and cellular levels.Our study will provide theoretical basis and new treatment ideas for the prevention and treatment of type 2 diabetes and non-alcoholic fatty live disease.Objective:1.To determine whether there is any change in the function of peroxisomes in type 2 diabetes with non-alcoholic fatty liver.2.To determine the effect of peroxisome dysfunction on lipid deposition in cells.Methods:1.Refering to the method in the reference（M.J.Reed）to a model of type 2diabetes with fatty liver was established by a high-sugar and high-fat diet plus a small dose of STZ injection.Blood glucose and blood lipids in serum of rats were detected by biochemical automatic analyzer.The inflammation infiltration and lipid deposition in the rat liver were observed by HE staining and oil red O staining.MRNA and protein expression levels of peroxisome genes were detected by q RT-PCR and Western Blot.Triglycerides in tissue were detected by spectrophotometry.The correlation between triglycerides and m RNA of peroxisome genes PMP70 and PEX12 in the liver was analyzed.2.HepG2 cells were treated with sodium oleate at different concentrations for same time and at the same concentration for different times.The lipid deposition in the cells was detected to determine the effect of oleic acid on the lipid deposition in the cells under different conditions.3.HepG2 cells were given sodium oleate for 6 hours and then transfected with si-PEX12 or si-PMP70 for 24 hours,respectively.The lipid deposits,triglycerides and cholesterol in the cells were detected to determine the effect of knocking down PEX12 or PMP70 on lipid deposition in oleic acid-treated cells.Results:1.The liver tissue of model rats has abnormal structure and lipid deposition.Compared to the control group,the blood glucose concentration of the model group was significantly increased（5.76±1.52 vs 31.2±7.93 mmol/L）,and triglyceride TG（0.53±0.11 vs 2.23±0.89 mmol/L）,total cholesterol TCh(2.09±0.23 vs 12.73±3.94 mmol/L,and low-density lipoprotein cholesterol LDL-C（0.82±0.07 vs 7.05±1.18 mmol/L）,the content of high-density lipoprotein cholesterol HDL-C（1.52±0.11 vs 0.54±0.22 mmol/L）,were all significantly reduced.（P<0.001）.Compared to the control group,obvious inflammation and lipid deposition can be seen.Triglyceride content in liver of the model group was significantly higher than that of control group（302.13±100.22 vs 123.93±38.52 nmol/mg prot.,P<0.01）.It indicated that the rat model of type 2 diabetes combined with fatty liver was successfully established.2.The deposition of lipids in the liver of model rats is accompanied by a decrease in the expression of peroxisome-related genes.Compared to control group,the m RNA and protein levels of peroxisome-related genes PMP70,CAT,and PEX12 in the liver of the model group were significantly down-regulated.（P<0.001,P<0.01）.The immunofluorescence staining results showed that the red fluorescence of PMP70 labeled by liver tissue in the model group was also significantly lower than that in the control group.The m RNA levels of PMP70 or PEX12 were significantly negatively correlated with triglyceride content in rat liver tissue（R=-0.8426,P<0.01;R=-0.7819,P<0.01）.Above results showed that the expression of peroxisome-related genes is reduced in fatty liver of type 2 diabetic rats indicating that the alteration of peroxisome function may be related to the deposition of triglycerides in the liver.3.Oleic acid increases the lipid content in HepG2 cells in a concentration and time dependent manner.HepG2 was treated with different concentrations of oleic acid（31.25,62.5,125,250,500μmol/L）for 24 h,or treated with 125μmol/L oleic acid for different times（0 h,3 h,6 h,9h,12 h,24 h）.It was found that lipids increased in a concentration-dependent and time-dependent manner in the cells.It indicated that oleic acid can deposit lipids in HepG2 cells at the cellular level.4.Knockdown of PEX12 aggravated the increase in lipids caused by oleic acid in HepG2 cells.HepG2 cells were stimulated with 62.5μmol/L oleic acid for 6 hours and then transfected with si-PEX12 for 24 hours.It was found that knocking down PEX12 increased the lipid deposition and triglyceride content in the cells treated with sodium oleate（P<0.05）,while the amount of cholesterol was not changed（P>0.05）.However,knocking down PEX12 in non-oleic oil-treated cells,has no effect on the deposition of lipid and amount of triglyceride and cholesterol content（P>0.05）.It indicated that the decreased PEX12 expression of can aggravate the mainly triglyceride lipid deposition induced by oleic acid.5.Knockdown of PMP70 aggravated the increase in lipids induced by oleic acid in HepG2 cells.HepG2 cells were stimulated with 62.5μmol/L sodium oleate for 6 hours and then transfected with si-PMP70 for 24 hours.It was found that knocking down PMP70 increased the lipid deposition and triglyceride content in oleic acid-treated cells（P<0.05）,while the amount of intracellular cholesterol was not changed（P>0.05）.However,knockdown of PMP70 in non-oleic oil treated cells has no effect on the deposition of lipid and the amount of triglyceride and cholesterol content（P>0.05）.It indicated that the decreased PMP70 expression of can aggravate the mainly triglyceride lipid deposition induced by oleic acid.Above results suggest:1.The function of peroxisome in fatty liver of type 2 diabetic rats is reduced.2.Down-regulation of peroxisome function aggravates high fat induced triglyceride deposition in hepatocytes.Conclusions:Dysfunction of peroxisome may be one of the causes of fatty liver in type 2diabetes. |
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