| Early weaning of piglets will have adverse effects such as immune suppression,lower growth performance and lower liver metabolic disorders.The liver is an important effector organ of the stress system,as well as an important organ for energy supply and carbohydrate metabolism regulation.It is closely related to growth,metabolism and anti-stress ability.After weaning,on the one hand,the body’s anaerobic glycolysis is accelerated,the lactate in the circulatory system enters the liver through the Cori cycle and to be cleared,resulting in a substantial increase of lactate in the liver;on the other hand,the accumulation of lactate in the liver can cause liver damage,affecting the health of piglets and further aggravating the slow growth caused by weaning stress.Therefore,controlling hepatic glucose metabolism disorder and lactate deposition,and accelerating hepatic lactate clearance may be one of the key measures to alleviate the stress of weaning in piglets.The process of liver nutrient metabolism is finely regulated by epigenetic modifications,and protein acetylation modification has been shown to exist in almost all liver glucose and lipid metabolism processes.Acetyltransferase P300/CBP associated factor（P300/CBP associated factor,PCAF）is an important protein that induces glucose and lipid metabolism disorders in the liver.It participates in modifying and regulating many key enzymes in nutrient metabolism pathways,including the key glycolysis pathway and key transcription factors for enzymes and gluconeogenesis pathways.Recent studies have found that under the conditions of inflammation and stress,lactate will accumulate in the liver of humans and rodents,and the expression of PCAF will increase significantly.Therefore,is PCAF a key target for regulating liver lactate metabolism in weaned piglets?What’s the regulatory mechanism?These important issues are worthy of in-depth study.On the one hand,this study took weaned piglets as the research object,explored the acetylation profile of lactate metabolism in the liver of weaned piglets,and deeply explored the key enzymes of lactate metabolism modified by PCAF;on the other hand,by using the adeno-associated virus mouse experiment and the weaned piglet group feeding garcinol experiment,we revealed the regulation mechanism of PCAF on liver lactate metabolism and the nutritional intervention measures.Part one Dissection of acetylation profile of hepatic glucose and lipid metabolism in weaned piglets.In this study,3 litters of 21-day-old ternary healthy piglets were selected,and 2piglets in each litter,6 piglets in total,were weaned（weaning group）;the remaining piglets in each litter continued to be suckled by the sow（Breastfeeding group）.From day0 to day 7 after weaning,the piglets were weighed,the blood was collected,and the liver samples were collected after being slaughtered for laboratory testing.The main results are as follows:1.The results of growth performance and blood biochemical indicators showed that the ADFI and ADG of piglets in the weaning group were significantly lower than those in the suckling group（P<0.05）,while F/G was significantly higher than that of the suckling pigs on day 0-3 and day 4-7 of weaning（P<0.05）.Compared with the suckling piglets,the blood urine nitrogen level of piglets in the weaning group was significantly increased（P<0.05）,ALT and AST were both significantly increased（P<0.05）,and the content of MDA was significantly increased（P<0.05）,T-AOC,SOD and GSH-Px activities were significantly reduced（P<0.05）,the content of free fatty acid,lactate and pyruvate were significantly increased（P<0.05）,and the glucose content was significantly reduced（P<0.05）.2.The results of liver histology and inflammatory response indicators showed that compared with the suckling group,the liver tissue structure of the weaning group was damaged,and the contents of TNF-α,IL-1β,and IL-6 in the liver and blood increased significantly（P<0.05）.The m RNA expression of inflammatory factors TNF-α,IL-1βand IL-6 increased significantly（P<0.05）.3.The results of liver glucose and lipid metabolites,metabolic enzyme activity and protein expression indicators showed that compared with the suckling piglets,the content of acetyl-Co A in the liver of weaned piglets increased significantly（P<0.05）,and the content of total cholesterol decreased significantly（P<0.05）;the content of lactate increased significantly（P<0.05）,the expression of PDH protein and enzyme activity were significantly reduced（P<0.05）,the expression of LCAD,HADH,L-CPT-1,GK and PK was significantly increased（P<0.05）,the enzyme activity of LDHB was significantly decreased（P<0.05）,the enzyme activity of GK was significantly increased（P<0.05）,and the enzyme activity of L-CPT-1 was significantly increased（P<0.05）.4.The results of differential acetylation proteomics in the liver showed that a total of160 proteins with 251 sites were found to have significant changes in the acetylation level（weaning group/lactating group）,of which 58 protein acetylation levels were significantly up-regulated,102 protein acetylation levels were significantly down-regulated;KEGG results showed that most up-regulated acetylated proteins are involved in glycolysis and gluconeogenesis pathway metabolism,most down-regulated branched-chain amino acid degradation and peroxisome and fatty acid metabolism;The protein with the largest increase of acetylation levels is lactate dehydrogenase B chain（LDHB）.The results of the motif probability graph of the acetylation site indicate that the acetylation of LDHB is very likely to be highly acetylated at the K82 site.5.The results of liver acetyltransferase/deacetyltransferase expression results showed that,compared with the suckling group,the expression of PCAF and P300 in the liver of the weaning group increased significantly（P<0.05）,and SIRT5 expression decreased significantly.The above results indicate that weaning can cause disorders of glucose and lipid metabolism in the liver of piglets,and accumulation of lactate.The LDHB-K82 will be highly acetylated and accompanied by abnormal expression of PCAF in the liver of weaned piglets.Part two The molecular mechanism of PCAF-induced hepatocyte lactate clearance obstacle.In this study,the hepatocytes of the lactating and weaning piglets were isolated and cultured,combined with mouse liver cell experiment,by using Co-IP,targeted acetylation site mutations（K→R to simulate deacetylation;K→Q to simulate acetylation）,Western blot,and RNA stem related genes experimental techniques,such as overexpression,have verified the modification and regulation relationship of PCAF and LDHB.The main results are as follows:1.The results of the isotope tracer cell experiment of 13C-labeled lactate showed that compared with the lactation group,the abundance of 13C-labeled pyruvate in the weaned group hepatocytes decreased by 35-40%,while the abundance of 13C-labeled glucose decreased by 25%-30%.2.The results of two-way activity analysis of two types of lactate dehydrogenase（LDH）isoenzymes showed that the LDHB activity（conversion of lactate to pyruvate）of weaned piglet liver cells was significantly lower than that of suckling piglet liver cells（P<0.05）,while LDHA activity（conversion of pyruvate to lactate）had no significant difference between two groups（P>0.05）.3.The results of the Co-IP experiment show that PCAF and SIRT5 can interact with LDHB,but there is no interaction between P300 and LDHB;the scanning results of the confocal microscope show that PCAF,SIRT5 and LDHB are mainly co-localized in the cytoplasm with low amount in the nucleus.4.It showed that overexpression of PCAF in mice hepatocytes would significantly increase the acetylation level of LDHB（P<0.05）,while overexpression of SIRT5 would significantly inhibit the acetylation level of LDHB;knocking down PCAF would significantly decrease the acetylation level of LDHB,while knocking down SIRT5 will increase the acetylation level of LDHB.The results of targeted site mutation experiments show that the mutation of lysine at K82 to arginine will significantly reduce the acetylation level of LDHB;overexpression and knocking down of PCAF will significantly affect the acetylation level of LDHB（P<0.05）,while SIRT5 has no significant regulatory effect on it.5.It was found that after knocking out PCAF,the activity of LDHB（from lactate to pyruvate）was significantly increased（P<0.05）,and the ratio of NAD+/NADH in liver cells was significantly reduced（P<0.05）;PCAF overexpression significantly reduced the activity of LDHB（P<0.05）,and significantly increased the ratio of NAD+/NADH（P<0.05）,the treatment of PCAF inhibitor—embelin significantly alleviated the inhibition of PCAF on LDHB activity（P<0.05）.The treatment of embelin significantly reduced the ratio of NAD+/NADH（P<0.05）.The K82 site of LDHB was mutated in vitro and then implanted into hepatocytes for expression.It was found that overexpression and interference of PCAF had a significant effect on the activity of wild-type LDHB（from lactate to pyruvate）（P<0.05）,while it had no efffect on the LDHB enzyme activity in K82Q and K82R groups（P>0.05）.6.Compared with hepatocytes expressing wild-type LDHB,the ratio of lactate to pyruvate was significantly reduced by nearly 50-60%at the intracellular and extracellular levels of cells expressing K82Q mutant or wild-type LDHB+Flag-PCAF（P<0.05）,while the cells expressing K82R mutant or wild-type LDHB+si PCAF,the ratio of lactate to pyruvate significantly increased by nearly 50%（P<0.05）at the intracellular and extracellular levels.7.PCAF overexpression significantly reduced the content of mitochondrial DNA and the activity of mitochondrial complexⅠ,Ⅱ,andⅢ（P<0.05）.PCAF knockdown significantly increased the content of DNA and the activity of mitochondrial complexⅠ,Ⅱ,andⅢ（P<0.05）.The above results indicate that PCAF is an important protein that regulates the acetylation of LDHB-K82.PCAF acetylation of LDHB significantly reduces the conversion of liver lactate to pyruvate,hinders the liver’s lactate clearance,and leads to mitochondrial dysfunction.Part three Dissection of LDHB（K82）acetylation on liver inflammation and liver injury.In this study,AAV8 was selected as the liver-selective serotype,and LDHB-K82targeted site mutants were constructed in vitro（LDHB-K82R and LDHB-K82Q;R and Q mimic the deacetylation and acetylation sites of K82,respectively）.The AAV8-LDHB（K82Q/R）weaned mouse model was established by continuously injecting the tail vein of weaned mice.The experimental components were as follows:control group（weaned mice untreated）,K82Q group（injected AAV8-LDHB-K82Q）and K82R group（injection of AAV8-LDHB-K82R）.We further analyzed and tested the indicators of liver and blood.The main results are as follows:1.The contents of ALT and AST in the blood of mice in the K82R group were significantly lower than the control group（P<0.05）,while the levels of ALT and AST in the K82Q group were significantly higher than the control and K82R groups（P<0.05）.In terms of liver lactate metabolism,the blood lactate content and the liver lactate content showed the same trend.The lactate content in the liver and blood of the K82R group was significantly lower than that of the control group（P<0.05）.The content of pyruvate in the liver of mice in the K82R group was significantly higher than that of the control group and the K82Q group（P<0.05）,and the free fatty acids in the liver were significantly lower than the control group and the K82Q group（P<0.05）.2.The results of hepatic antioxidant index showed that compared with the control group,the K82 group mice significantly reduced the liver ROS level and MDA content,and significantly increased the SOD activity（P<0.05）,while the K82Q group mice increased the ROS level and MDA content,significantly reduced SOD activity（P<0.05）.3.The results of liver histology and inflammatory response indicators showed that the liver of the mice in the control group had obvious pathological changes,the liver damage in the K82R group was alleviated,and the liver of the K82Q group was further aggravated;the contents of TNF-ɑ,IL-1β,IL-6 and IL-10 in the K82Q group were significantly higher than those in the control group and the K82R group（P<0.05）.The relative expressions of TNF-ɑ,IL-1βand IL-6 m RNA in the liver of the K82R group were significantly lower than those in the control group.The content and expression of TNF-ɑand IL-1βin the liver of the K82Q group was extremely significantly higher than that of the control group and K82R group（P<0.001）.4.The content of mitochondrial DNA in the liver of mice in the K82R group was significantly higher than that in the control group（P<0.05）,and the content of mitochondrial DNA in the K82Q group was significantly lower than that in the control and K82R groups（P<0.05）.The activity of liver mitochondrial complexes showed the same trend（P<0.05）.5.The content of lactate in the nucleus of liver cells in the K82R group was significantly decreased,while the content of lactate in the nucleus of the K82Q was significantly increased（P<0.05）.The HDAC activity in the nucleus of the K82R group was significantly higher than that of the control group,and the activity of the acetylase HDAC in the K82Q group was significantly lower than that of the control group（P<0.05）.The degree of acetylation of histone H3K9 in the K82R group was the lowest,while that in the K82Q group was the highest.The expression of phosphorylated IKKβ,IκBαand p65 protein in the pro-inflammatory response NF-κB signaling pathway in the K82R group was the lowest,while the K82Q group was the highest.The above results indicate that the accumulation of lactate mediated by LDHB（K82）acetylation activates the hyperacetylation of H3K9 and initiates the expression of inflammatory response genes,thereby causing liver damage and mitochondrial dysfunction.Part Four Dissection of garcinol on relieving the accumulation of lactate in the liver of weaned pigletsIn this experiment,24 weaned piglets with good health and similar parity were randomly divided into 4 groups,namely the control group,the 200 mg/kg garcinol group,the 400 mg/kg garcinol group and the 600 mg/kg garcinol group.Each group had 6replicates,and each replicate had 1 piglet.The test period was 28 days.After the experiment,the growth performance,antioxidant capacity,liver inflammation and lactate metabolism relative indicators of weaned piglets were measured to explore whether garcinol regulates the accumulation of lactate in weaned piglets by regulating PCAF and relieves weaning stress.The main results are as follows:1.The growth performance results showed that the ADFI and ADG of the diet supplemented with garcinol group were significantly higher than those of the control group（P<0.05）,and the F/G of the 400 mg/kg garcinol group was significantly lower than that of the control group（P<0.05）.The ADG of 400 and 600 mg/kg garcinol group was significantly higher than that of 200 mg/kg garcinol group（P<0.05）.2.The results of blood index showed that compared with the control group,the total protein and albumin of the 200,400,600 mg/kg garcinol group had a tendency to increase,but there was no significant difference（P>0.05）;the blood urine nitrogen level was significant decreased（P<0.05）;ALT and AST were significantly increased（P<0.05）;the content of MDA was significantly decreased（P<0.05）,T-AOC,SOD and GSH-Px activities were significantly increased（P<0.05）;the content of lactate in the blood was significantly reduced（P<0.05）,and the content of glucose was significantly increased（P<0.05）.There was no significant difference in all indexes of 400 mg/kg garcinol group and 600 mg/kg garcinol group（P>0.05）.The the content of MDA and lactate in the 400mg/kg garcinol group and the 600 mg/kg garcinol group were significantly lower than the200 mg/kg garcinol group.3.The results of liver inflammation and lactate metabolism showed that dietary supplementation of garcinol can significantly increase the content of Ig A,Ig G,and Ig M in the liver（P<0.05）,and significantly reduce the content of TNF-α,IL-1β,and IL-6 in the blood and the content of lactate in the liver（P<0.05）,significantly reduce the m RNA expression of these inflammatory factors in the liver（P<0.05）,and significantly alleviate the liver damage.4.Compared with the control group,the liver mitochondrial DNA content and the activity of mitochondrial complexⅠ,ⅡandⅢin the garcinol group were significantly decreased（P<0.05）.5.PCAF,LDHB activity and acetylation results showed that dietary garcinol can significantly reduce PCAF activity（P<0.05）,significantly reduce the level of LDHB acetylation（P<0.05）,and significantly increase LDHB activity（P<0.05）and decrease H3K9 acetylation level.The above results indicated that the addition of garcinol to the diet can improve the growth performance,antioxidant capacity and immune function,and alleviate lactate accumulation,inflammation and mitochondrial dysfunction in the liver of weaned piglets.In summary:（1）Weaning can cause the accumulation of lactate in the liver of piglets,LDHB-K82hyperacetylation and PCAF overexpression.（2）PCAF acetylation of LDHB can significantly reduce the conversion of liver lactate to pyruvate and can hinder the liver’s lactate clearance.（3）The accumulation of lactate induced by acetylation of LDHB-K82 can cause the hyperacetylation of histone H3K9,which aggravating the inflammatory response of the liver and causes liver damage.（4）The addition of garcinol to the diet can improve the growth performance,antioxidant capacity and immune function of the weaned piglet,and alleviate lactate accumulation,inflammation and mitochondrial dysfunction in liver of weaned piglets. |
