Effects Of PNPLA3 Gene On Porcine Hepatic Lipid Metabolism Under Stress

PNPLA3 belongs to the patatin-like phospholipase family and can be involved in liver fat metabolism,and the mutation of this gene is closely related to the occurrence of nonalcoholic fatty liver disease.However,under stress conditions,how PNPLA3 gene affects liver fat metabolism is not well understood.In this paper,three studies were conducted to investigate the mechanisms of PNPLA3 gene affecting liver fat metabolism in pigs under heat and endoplasmic stress conditions.Test 1 The expression of PNPLA3 in liver of growing pigs under heat stress and its effect on lipid metabolism.In total,27 healthy pigs(from 9 litters,3 heads / litter)with normal body weight of 40.8±2.7 kg were selected.The single-factor randomized block design was used to randomly divide the pigs into three groups: control group(TN),the heat stress group(HS)and the feed intake paired group(PFTN)with 9 replicates and the pigs of each group from different litter.The test period was 21 d.The results showed that:(1)Compared with the TN,the expression of GRP94 and p-PERK in the liver of HS were both significantly increased(P < 0.05),indicating that heat stress can cause the liver endoplasmic stress.(2)Compared with the TN and the PFTN,the expression of PNPLA3 gene in the liver of the HS was significantly increased(P < 0.05),whereas the activity of fatty acid synthase,acetyl-CoA carboxylase,hormone-sensitive lipase and Lipoprotein lipase was significantly reduced(P < 0.05).(3)The expression of PNPLA3 gene in liver was correlated with the index of fat metabolism under heat stress conditions.Test 2 Preparation of immortalized pig liver stellate cellsNormal pig hepatic stellate cells were isolated from pig liver by density gradient centrifugation,and then transfected with SV40 LT overexpression of lentivirus to obtain immortalized cells.These immortalized cells have no significant difference in cell morphology and characteristics compared with primary pig hepatic stellate cells.The pig hepatic stellate cell line provides stable,transmissible cells for subsequent experiments.Test 3 Effects of PNPLA3 gene mutation on pig liver fat metabolism under endoplasmic stressThe PNPLA3 gene mutant lentivirus was constructed and the virus was used to infect immortalized pig hepatic stellate cells.After 72 h,5 μg/mL tunicamycin or DMSO was added to continue the culture for 24 h.The test had six groups:(1)blank control group;(2)blank control + tunicamycin group;(3)infected control virus group;(4)infected control virus + tunicamycin group;(5)infected PNPLA3 gene Mutant virus group;(6)infected with PNPLA3 gene mutant virus + tunica group.The results showed that:(1)under physiological conditions and endoplasmic stress,the expression of PNPLA3 gene in the PNPLA3 mutant virus group was significantly higher than that in the uninfected groups(P < 0.05).(2)Under physiological conditions,the PNPLA3 gene-mutant virus did not affect the protein expression of GRP78 and GRP94 in pig hepatic stellate cells.However,under the condition of endoplasmic reticulum stress,the infection of PNPLA3 gene could significantly increase the protein expression of GRP94(P < 0.01),but did not have a significant effect on the protein expression of GRP78.(3)Under physiological conditions,the PNPLA3 gene-mutant virus can significantly increase the expression of cell acetylase-CoA carboxylase,fatty acid synthase,stearoyl-CoA desaturase,peroxisome proliferator-activated receptor gamma and cholesterol regulator binding protein 1(P < 0.05).Under the condition of endoplasmic reticulum stress,the infection of PNPLA3 gene mutant virus had no significant effect on the expression of the above genes.(4)Compared with the infected control virus group,under the physiological conditions and endoplasmic reticulum stress conditions,the PNPLA3 gene-mutant virus significantly increased the protein content of peroxisome proliferator-activated receptor γ in the cells(P <0.05).Conclusion(1)Chronic heat stress can cause endoplasmic reticulum stress in pig livers and reduce the activity of fat-metabolizing enzymes,such as ACC、PPARγ and FAS.(2)Chronic Heat stress can increase the expression of PNPLA3 gene in pig liver,and PNPLA3 gene expression is associated with enzyme activity related to fat metabolism.(3)Under physiological conditions,the mutation(expression)of PNPLA3 gene can affect the fat metabolism of the liver;compared with normal physiological conditions,endoplasmic reticulum stress can inhibit the effect of PNPLA3 gene mutation(expression)on fat metabolism.