The Role Of SREBP-1c In Dietary Fatty Acids Regulating Lipid Metabolism In Hepatic Cells

Nonalcoholic fatty liver disease (NAFLD) is a clinical syndrome with the features ofsteatosis and deposition of fat in hepatocytes, which is not due to excessive alcohol use. Itaffects30%of the general Western adult population and has become one of the most commoncauses of elevated liver enzymes and chronic liver disease in the world. Its incidence in adultsand children is rising rapidly due to the ongoing epidemics of obesity and type2diabetes.Although a benign disorder in the majority of instances, up to20%of patients with NAFLDhave nonalcoholic steatohepatitis (NASH), and can progress to cirrhosis, liver failure andhepatocellular carcinoma. Several risk factors for NAFLD have been identified, includingelevated body mass index (BMI), type2diabetes mellitus, advancing age andhypertriglyceridemia. The pathophysiologic basis of NAFLD is thought to be insulinresistance. Most experts consider NAFLD to be the hepatic manifestation of the metabolicsyndrome, which includes persons with some combination of insulin resistance, obesity,hypertension and dyslipidemia. However, the effective drugs to treatment NAFLD so far havenot been found. The study of the relationship between dietary, nutritional factors and NAFLDwas concerned. The excessive energy or fat intake and the imbalance of dietary fatty acid indiet, especially the excessive intake of saturated fatty acids and the insufficient of n-3polyunsaturated fatty acids (n-3PUFA) may be the major risk factors that lead to fatty liver.The epidemiological survey showed that the PUFA content of blood and liver tissue wasdecreased in NAFLD patients, and the prevalence of NAFLD was negative correlated to theamount of n-3PUFA intake.Sterol regulatory element-binding proteins(SREBPs) are the key nuclear transcriptionfactors, which regulate the expression of lipid metabolism genes. SREBPs have threesubtypes. They play different roles in lipid synthesis. SREBP-1c is a key regulator of hepaticlipid metabolism, almost involved in the transcription of all liver fatty acid and triglyceridesynthesis gene. Some scholars have found that the fatty acid synthesis markedly accelerated,the triglyceride accumulation of liver increased in SREBP-1c transgenic mice, and it can naturally progress to NASH at20weeks of age. Excessive intake or imbalance of dietary fattyacids can lead to the exaltation of saturated fatty acids in plasma which can activate theSREBP-1c of liver accordingly. The SREBP-1c can promote the expression of fatty acid andtriglyceride synthesis gene, resulting in the increase of liver triglyceride synthesis and thereduction of outflow. Ishii found that n-3PUFA played a role in alleviating the situation offatty liver disease by inhibiting the synthesis and storage of the lipid in liver by animalexperiments, but its potential molecular mechanisms is not enough understand. Therefore, wehypothesized that dietary fatty acids may affect the occurrence and the development ofNAFLD by regulating the expression and activity of SREBP-1c, and n-3PUFA may play animportant role in prevention and treatment of NAFLD.This study explored the effect of SREBP-1c in dietary fatty acids regulating lipidmetabolism in hepatic cells. Firstly, we observed the expression of SREBP-1c in HepG2cellstreated by T0901317with RT-PCR. Then, the effect of different fatty acids on the growth ofHepG2cells was detected by CCK-8test, and the storage of lipid was investigated indifferent fatty acid groups and different fatty acids+T0901317mixture groups by the totallipids and triglyceride content of hepatic cells testing, and oil red staining. Finally, theexpression of lipid metabolism-related genes(PPARα, FAS and SREBP-1c) was detected byRT-PCR and Western blot.The main results and conclusions were summarized as the following:1．The expression of SREBP-1c mRNA was significant increase in HepG2cells treatedwith different concentrations of LXR ligand(T0901317). The expression of SREBP-1c wasthe strongest in10μM T0901317group, which had no significant effect on HepG2cellproliferation. So we ultimately selected the10μM T0901317to treat HepG2cells according tothe literature reports.2．After treated by150μmol/L of different fatty acids for24h, the proliferation of HepG2cells were determined by CCK-8test. The results showed that PA and1:1PA+OA mixtureinhibited the growth of cells significantly (P＜0.05), while EPA and1:1EPA+LA mixturehad no significant effect on cell growth compared with control.3．Oil red staining showed that a small number of cells had fatty degeneration incontrol group, a small amount of lipid droplets accumulated in EPA group and1:1EPA+LAmixture group and a large number of the lipid droplets accumulated in PA group and1:1PA+ OA mixture group. The situation of fatty degeneration aggravated in T0901317groupcompared with the control group. The steatosis in T0901317+fatty acids groups were moreobvious than the groups with fatty acids alone, espesically in T0+PA+OA mixture group andT0+PA mixture group.4．The total lipids and triglyceride content of hepatic cells test showed that thetriglyceride and the total lipids amount in cells were significantly lower in EPA group and1:1EPA+LA mixture group, and significantly higher in PA group and1:1PA+OA mixture group(P＜0.05) compared with the control group. The triglyceride and the total lipids amount inHepG2cells treated by T0901317were significantly higher than that in control group(P＜0.05). The triglyceride and the total lipids amount in T0+EPA+LA group and T0+EPA groupwere significantly lower, while significantly higher in T0+PA+OA group and T0+PA groupthan that in T0901317group(P＜0.05).5．Western blot and RT-PCR showed that the expression level of SREBP-1c and FasmRNA and protein were significantly down-regulated and the expression level of PPARα wasup-regulated by EPA or EPA+LA mixture with or without T0901317(P＜0.05), and theexpression level of SREBP-1c and Fas were significantly up-regulated and the expressionlevel of PPARα was down-regulated by PA or PA+OA mixture with or without T0901317(P＜0.05).We concluded that SREBP-1c plays an important role in the process of fattydegeneration of hepatic cells in vitro. Dietary fatty acids may affect the deposition of fat inhepatocytes by regulating the expression and transcriptional activity of SREBP-1c, conduce tothe occurrence and development of NAFLD.