Roles Of PPARα Signaling Pathway In The Development Of Fatty Liver In Mice

Nonalcoholic fatty liver disease(NAFLD), one common chronic metabolic disease in the liver, is characterized by non-alcohol abuse, hepatic steatosis and fat accumulation. In the situation of oxidative stress, endoplasmic reticulum stress, inflammetation or lipid peroxidation, NAFLD will progress toward the development of nonalcoholic steatohepatitis(NASH), hepatic fibrosis, cirrhosis and liver cancer, which thresten human health. Thus identification of the regulatory mechanisms that influence lipid metabolism and energy homeostasis is one of the hot and difficult issues in the management of fatty liver disease.Researches have shown that peroxisome proliferator-activated receptors(PPARs) play a very important role in the regulation of lipid homeostasis. PPARs contain three members, named PPARα, PPARβ/δ and PPARγ, and PPARα is mainly involved in the regulation of fatty acid oxidation in the liver. Leptin mutant(ob/ob) mice manifested obesity and fatty liver, but showed no liver tumor development. Acyl-coenzyme A oxidase 1(Acox1) mutation in ob/ob mice induced PPARα and PPARα-regulated fatty acid oxidation through endogeous ligands,resulting in attenuation of obesity and hepatic steatosis, but higher risk of liver cancer. To futher evaluate the role of PPARα and its regulated signaling pathways in the development and progression of fatty liver disease, we generated PPARα-deficient ob/ob(PPARα?ob/ob)mice. Wild type(WT), PPARα-/-, ob/ob and PPARα?ob/ob mice were treated with starvation and PPARα ligand Wy-14,643, biotechnologies we used in this study included H&E staining,Oil O Red staining, immunohistochemistry, glucose and insulin tolerance test, biochemical assays of triglyceride and cholesterol, Real-time PCR, Northern Blot, Western Blot and microarray analysis.The results disclosured the important roles of PPARα in the progression of fatty liver disease in ob/ob mice. The specific results are as follows:1. PPARα deficiency aggravated obesity in ob/ob micePPARα-/- mice were mated with OB/ob mice to generate PPARα+/-/OB/ob, which were intercrossed to yield PPARα?ob/ob mice. From weaning until approximately the age of 6weeks, somatic growth of PPARα?ob/ob mice did not significantly differ from that of ob/ob mice, but the body weight of the former was a little lower than that of the latter. From then on,PPARα?ob/ob mice gained more weight than ob/ob mice and were distinctly more obese(super obese) than ob/ob mice at the age of 24 weeks. The food consumption of ob/ob mice was obviously more than that of PPARα?ob/ob mice, suggesting increased obesity observed in PPARα?ob/ob mice was not due to increased food intake. There were no significant differences in body weight and food intake between WT and PPARα-/- mice. The amount ofinguinal white adipose tissue(WAT) and interscapular brown adipose tissue(BAT) in PPARα-/- and PPARα?ob/ob mice was respectively more than that in WT and ob/ob mice.Histological examination of BAT revealed that the adipocyte size in PPARα?ob/ob mice was bigger than that in ob/ob mice, but no significant difference was found in the white adipocyte size. Moreover, PPARα deficiency improved glucose and insulin tolerance in ob/ob mice.2. PPARα deficiency aggravated hepatic steatosis in ob/ob miceAt 3 and 6 months of age, the liver weight was higher in PPARα?ob/ob mice than that in ob/ob mice. According to the results of H&E and Oil O Red staining, ob/ob and PPARα?ob/ob mouse livers revealed prominent macrovesicular steatosis, and the later was much more severe than the former. Hepatic triglyceride and cholesterol contents were higher in PPARα?ob/ob mice as compared with ob/ob mice. And no fat accumulation was found in WT and PPARα-/- mouse livers. However, aging attenuated hepatic steatosis in ob/ob and PPARα?ob/ob mice and induced mild pericellular fibrosis and hepatic oval cell proliferation in PPARα?ob/ob mouse liver. These oval cells occurred either singly or in clusters around partal vein.3. PPARα deficiency aggravated hepatic steatosis in ob/ob mice under starvationAfter 24 hours starvation, serum glucose levels in PPARα-/- and PPARα?ob/ob mice aged2-month were reducd rapidly, which were significantly lower than that in WT and ob/ob mice.H&E and Oil O Red staining results showed severe fatty livers in PPARα-/- and PPARα?ob/ob mice after starvation, and hepatic steatosis was exaggerated with prolonged fasting. In contrast, hepatic steatosis in ob/ob mice was alleviated gradually. Northern Blot, Western Blot and Q-PCR results showed that genes associated with lipid metabolism and starvation stress were induced significantly in WT and ob/ob mice after starvation, but no changes were found in PPARα-/- and PPARα?ob/ob mice. Oppositely, expression levels of PPARγ, Sirt1 and PIMT increased obviously in fasted PPARα-/- and PPARα?ob/ob mice.4. PPARα deficiency showed no influence on liver tumor risk in ob/ob miceWy-14,643, one of PPARα ligands, induced hepatocyte and peroxisome proliferation and upregulated genes involved in lipid metabolism and endoplasmic reticulum stress in WT and ob/ob mice, wich resulted in liver tumor in these two kinds of mice. In contrast, PPARα-/- and PPARα?ob/ob mice failed to respond to Wy-14,643, thus no liver tumor was found in these mice.5. Influence of PPARα deficiency on global transcriptional profiling in ob/ob miceCompared with ob/ob mice, microarray analysis showed that genes involved in lipid metabolism, autophagy, oxidative stress, peroxisome, cell growth and proliferation and angiogenesis were significantly downregulated in 3-month-old PPARα?ob/ob mice, while lipogenesis gene Thrsp and genes related with apoptosis were significantly upregulated.What’s more, we fed WT, Acox1-/-, ob/ob and Acox1?ob/ob mice with normal and 60%high fat diet(HFD), and biotechnologies we used in this study included H&E staining, Oil ORed staining, immunohistochemistry and Real-time PCR. The results disclosured the significant roles of PPARα in the progression of fatty liver induced by leptin deficiency and HFD. The specific results are as follows:1. Acox1 deficiency-induced PPARα hyperactivation was resistant to HFD induced obesityForty weeks after HFD administered, body weight, WAT and BAT weight, brown adipocyte size increased significantly in WT mice compared with the control group. At the beginning of HFD feeding, ob/ob mice got more weight than the control group, but less in the end, and no significant difference was found in the body weight of ob/ob mice between control and HFD group. After HFD, ob/ob mice got more WAT and BAT weight, but the adipocyte size showed no obvious changes. Acox1-/- mice did not get more body weight after HFD, but the amount of WAT and BAT, and brown adipocyte size increased significantly,which were still much lower than that in WT mice. Acox1?ob/ob mice showed no obvious changes in body weight, WAT and BAT amount and adipocyte size.2. Acox1 deficiency-induced PPARα hyperactivation was resistant to HFD induced hepatic steatosisForty weeks after HFD administered, liver weight in WT, Acox1-/-, ob/ob and Acox1?ob/ob mice increased, and WT mice developed severe fatty liver, but hepatic steatosis in Acox1-/-, ob/ob and Acox1?ob/ob mice did not change significantly.3. HFD increased liver tumor risk in Acox1?ob/ob miceForty weeks after HFD administered, no liver tumors were found in WT and ob/ob mice,however, the incidence of liver cancer in Acox1-/- and Acox1?ob/ob mice increased compared with the control group. Q-PCR results showed that genes associated with lipid metabolism and endoplasmic reticulum stress in Acox1-/- and Acox1?ob/ob mice were induced significantly, but no changes were found in WT and ob/ob mice.We conclude that PPARα deficiency aggrevated obesity and fatty liver, but showed no influence on the liver tumor risk in ob/ob mice. On the contrary, PPARα activation was resistant to leptin deficiency and HFD induced obesity and fatty liver, but increased liver tumor risk. All of this predicts that PPARα and its regulated pathway play a very important role in the progression of fatty liver disease, and PPARα maybe considered as a potential regulatory target for transformation from NAFLD to liver tumor.