- Protein acetylation in mitochondria plays critical functions in the pathogenesis of fatty liver disease. - Background: Fatty liver is a high incidence of perinatal disease in dairy cows caused by negative energy balance, which seriously threatens the postpartum health and milk production. - Predictably, most metabolic processes in the liver, as a vital metabolic organ, are subjected to acetylation. - Comparative acetylome study were used to quantify the hepatic tissues from the severe fatty liver group and normal group. - Combined with bioinformatics analysis, this study provides new insights for the role of acetylation modification in fatty liver disease of dairy cows.. - Furthermore, this study identified potential important proteins, such as HADHA, ACAT1, and EHHADH, which may be important regulatory factors through modification of acetylation in the development of fatty liver disease in dairy cows and possible therapeutic targets for NAFLD in human beings.. - Conclusion: This study provided a comprehensive acetylome profile of fatty liver of dairy cows, and revealed important biological pathways associated with protein acetylation occurred in mitochondria, which were involved in the regulation of the pathogenesis of fatty liver disease. - Furthermore, potential important proteins, such as HADHA, ACAT1, EHHADH, were predicted to be essential regulators during the pathogenesis of fatty liver disease.. - The work would contribute to the understanding the pathogenesis of NAFLD, and inspire in the development of new therapeutic strategies for NAFLD.. - The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. - More than 60% of dairy cows develop fatty liver during the transition period from dry milk to lactation due to negative energy imbalance [1], resulting in weakened liver function and decreased milk production [2]. - The fatty liver disease in dairy cows is a typical type of non- alcoholic fatty liver disease (NAFLD), mainly caused by obesity and stress response. - In the first month after delivery, 5–10% of dairy cows had a severe form of fatty liver, and 30–40% had mild or moderate fatty liver [3].. - The occurrence of fatty liver in dairy cows leads to huge economic losses, not only because of decreased milk production but also because of prolonged calving inter- vals and weakened reproductive performance [4, 5], and therefore shortened their service life.. - Fatty liver disease in dairy cows is a type of metabolic disorder. - Little is known about the pathogenesis of peri- natal fatty liver in dairy cows [15]. - This study reveals a com- prehensive acetylome profiling of fatty liver disease in dairy cattle and identifies potential biomarkers based on protein acetylation level. - These results provide a strong foundation for further understanding of important pro- tein regulatory targets in the development of NALFD in human beings and/or animals.. - Oil red O staining results of the liver tis- sue samples showed that there was a significant difference between the fatty liver group (86.75. - These results suggest that the metabolites transport in the liver tissue with fat deposition signifi- cantly inhibited. - Although energy metabolism and fatty acid oxidation were enhanced, the accumulation of fat in the liver was unavoidable. - (310/335) of them were again localized in the mito- chondria, with of these sites higher acetylated (Fig. - This suggests that proteins that were associated with mitochondrial function were critical for the liver metabolism, and protein acetyl- ation played an essential role during the development of fatty liver disease in dairy cattle.. - The right panel is a high- powered magnification of the black dashed area in the left panel. - Structural analysis of proteins containing lysine was per- formed using NETSURFP software, so as to understand the locations of acetylated and/or non-acetylated lysine in the secondary structures of proteins (alpha-helix, beta- strand and coil). - However, for lysine located in the alpha-helix and/or coil region, there was no statistical difference be- tween acetylated and/or non-acetylated lysine.. - Mitochondria and cytoplasm are the main distrib- uted areas in the cell for the Kac proteins (Fig. - In another word, the pathogenesis of fatty liver disease in dairy cows were presumed to be closely related to the. - d Among the identified differentially Kac sites, the up-regulated Kac sites located in the mitochondria, account for a large proportion (310/335) of all identified Kac sites. - 5c) and participate in the regulation of. - These acetylated proteins are significantly involved in the PPAR signaling pathway and the amino acid metabolism pathway, such as the tyrosine and trypto- phan metabolism pathway. - a Probable sequence motifs of acetylation sites in fatty liver tissues identified using Motif-X. - c Heat map showing the relative frequencies of amino acids in specific positions, including enrichment (red) or depletion (green) of amino acids flanking the acetylated lysine in fatty liver proteins. - EHHADH is a dehydrogenase involved in the fatty acid β oxidation and is one of the four essential enzymes in the peroxisome β oxidation pathway. - In the interaction network, each acetylated protein was represented by a circle. - The size of the circle indicates the number of acetylation sites in the protein. - The color of the circle indicates the modified directions of the acetylated sites in the protein, red presents higher-acetylated sites and greed presents lower-acetylated sites. - 6, Table 3), which was the most frequent interactive protein in the network. - This suggests dysfunction of these important proteins via acetylation modification might destroy the metabolic processes, such as tricarboxylic acid cycle, thereby contributing to the development of fatty liver disease in dairy cows.. - a Number of all differentially acetylated sites (All) in the protein, higher acetylated sites (Up) and lower acetylated sites (Down) in the protein, separated by slash. - b Number of the nodes that were showing the reciprocally interactive proteins in the protein-protein interaction (PPI) network obtained by STRING analysis (shown as in Fig. - Fatty liver in dairy cows. - These NEFAs are partly oxidized in the liver and partly re-esterified to synthesize triglycerides (TG), which is difficult to be transported out of the liver by very-low- density lipoprotein (VLDL) [27]. - Especially for dairy cattle, the lack of adequate lipid and liver esterase in the liver limits the hydrolysis of TAG, which leads to the ex- cessive accumulation of TAG in the liver therefore resulting in susceptibility to fatty liver disease.. - While, nonalcoholic fatty liver disease (NAFLD) occur- ring in human beings, metabolic disorder syndromes and obesity are also usually the main causes, with in- creased plasma insulin and fatty acid concentration, ele- vated fasting aminotransferase (AST/ALT) and/or triglycerides level, and also abnormal lipid accumulation in the liver [31, 32]. - Thus, dairy cows with fatty liver disease is a typical NAFLD animal model, good for revealing the pathology and pathogenesis of NAFLD [15]. - The objective of the study is to reveal the molecu- lar mechanism of pathogenesis for NAFLD by taking fatty liver diseased dairy cattle as research model, identifying potential important biological pathways and protein targets, and thereby further discovering the therapeutic targets.. - Liver acetylome profiling identified important biological pathways regulating the pathogenesis of fatty liver in dairy cows. - Enrichment ana- lysis of identified DAPs indicated that the protein modi- fications of the acetylation levels remarkably participated in the regulation of energy metabolic pathways, such as. - Actually, the fatty liver disease occurred in dairy cows during their perinatal period is usually complicated by a high incidence of ketosis [36], with increased ketone body produced in the liver. - Human nonalcoholic fatty liver disease (NAFLD) has four stages: simple stea- tosis, non-alcoholic steatohepatitis (NASH), cirrhosis, and hepatocellular carcinoma. - Simple fatty liver is a be- nign stage of NAFLD and can be reversed by treatment.. - About 10–20% of simple fatty liver develops into NASH [37]. - At present, NASH is an important link in the de- velopment of NAFLD for end-stage liver disease, such as cirrhosis, hepatocellular carcinoma, and liver failure [38].. - Therefore, acetylation modification of proteins that are involved in metabolic pathways may be responsible for the increasing severity of fatty liver disease.. - Acetylation of proteins locates in the mitochondria contributes to the development of fatty liver disease in dairy cows. - Currently, However, little is known about the protein acetylome of fatty liver disease caused by negative energy balance in dairy cows, while there are relatively more studies in human and mouse liver tissues [39]. - suggesting that the acetylation modification of hepatic proteins located in mitochondria plays a crucial role in both lipid and amino acid metabolism disorder in dairy cows, thereby contributes to the pathogenesis of fatty liver disease. - Mammalian sirtuins (SIRTs) is an important type of NAD-dependent histone deacetylase, such as SIRT1 and SIRT3, were involved in oxidative stress and lipid metabolism regulation [47], and had been proposed as a reliable biomarker and/or therapeutic target for fatty liver disease [45–49]. - It suggests that acetylation of mito- chondrial protein was an emerging and fundamental mechanism regulating the development of fatty liver dis- ease in dairy cows, through modifying the activities of mitochondrial proteins and overall mitochondrial func- tion [47–49]. - The mechanism how these acetylated pro- teins to be modified and their molecular function are involved in the pathogenesis of fatty liver disease will be necessarily elucidated in the future.. - Identification of candidate mitochondrial proteins as important regulation targets during the pathogenesis of fatty liver. - From the acetylated proteins, it was observed that some mitochondrial proteins are involved not only in the. - fatty liver-related disorder protein network but also in several lipid metabolism-related pathways (Table 3).. - Therefore, it was speculated that acetylation of these proteins plays a key role in the development of fatty liver disease of dairy cows. - For example, carbamoyl phos- phate synthase (CPS1) is the catalytic enzyme of the first step in the urea cycle, which is very important for the removal of excess urea from cells. - Previous prote- omic studies on non-alcoholic fatty liver showed that serum concentration of CPS1 decreases gradually in the order of control steatosis and NASH patient subjects and CPS1 has been confirmed to be serum candidate markers of NAFLD [50]. - The significantly abnormal acetylation level of HMGCS2 in fatty livers (Table 3) indicated a ketone metabolism disorder in dairy cows with fatty liver disease. - In addition, the expression of EHHADH is regulated by PPARα, which is involved in the tricarboxylic acid cycle and peroxisome fatty acid oxidation [61]. - Thus, EHHADH, a fairly new candidate protein, was identi- fied to be potentially associated with the pathogenesis of fatty liver disease, for its abundant interaction networks with other proteins (Table 3).. - In conclusion, this study provides a comprehensive acet- ylome profile of liver in dairy cattle and reveals import- ant mitochondria-associated pathways that are involved in the regulation of the pathogenesis of fatty liver dis- ease, including the TCA cycle, propionate metabolism, glycolysis/gluconeogenesis, pyruvate metabolism, oxi- dative phosphorylation, fatty acid degradation, valine, leucine, and isoleucine degradation, drug metabolism - cytochrome P450, and the PPAR signaling pathway. - Fur- thermore, this study identified potential important pro- teins, such as HADHA, ACAT1, and EHHADH, which may be important regulatory factors being acetylation level modified in the development of fatty liver disease in dairy cows, potentially being therapeutic targets for NAFLD in human beings. - The biopsied dairy cattle had their fatty liver condition diagnosed according to the average percentage of hepatic cells containing lipid droplets in the tissue, and other background information of these cows including produc- tion and serum biochemical indexes [1, 60] was shown in Table S2. - As a result, six samples were diagnosed as normal liver and eight as severe fatty liver . - Peptides were then selected for MS/MS using the NCE setting as 28, and the fragments were detected in the Orbitrap at a resolution of 17,500. - List of differentially expressed acetylated proteins identified and quantified by the TMT analysis in the study.. - Production information of normal (Norm) and fatty liver dairy cows that were liver biopsied and their serum biochemical parameters 1. - We also appreicate Hangzhou Jingjie Biotechnology Co., Ltd., PTM Bio for the assistance in the data analysis. - LZ, CH, and KS participated in the execution of all experiments, analyzed the data, and drafted the manuscript. - CH, LZ, KS, XZ, YZ, SW, ZX, RL and TL participated in the fatty liver disease investigation and liver sample collection. - The funding body did not play a role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript.. - We have obtained written informed consent from the animal owner to use these animals in the study.. - The role of TCA cycle anaplerosis in ketosis and fatty liver in periparturient dairy cows. - Invited review: pathology, etiology, prevention, and treatment of fatty liver in dairy cows. - Identification of crucial genetic factors, such as PPAR γ , that regulate the pathogenesis of fatty liver disease in dairy cows is imperative for the sustainable development of dairy industry. - Nonalcoholic fatty liver disease: molecular mechanisms for the hepatic steatosis. - Relationships between fatty liver and fertility and some periparturient diseases in commercial Dutch dairy herds. - Relevance of apolipoproteins in the development of fatty liver and fatty liver-related peripartum diseases in dairy cows. - Serum paraoxonase-1 as biomarker for improved diagnosis of fatty liver in dairy cows. - Natural history of nonalcoholic fatty liver disease: a prospective follow-up study with serial biopsies.. - Nonalcoholic fatty liver disease - current status and future directions. - The epidemiology of non-alcoholic fatty liver disease. - Impaired hepatic autophagic activity in dairy cows with severe fatty liver is associated with inflammation and reduced liver function. - Elevated apoptosis in the liver of dairy cows with ketosis. - Fatty liver is associated with reduced SIRT3 activity and mitochondrial protein hyperacetylation.. - Low expression of sirtuin 1 in the dairy cows with mild fatty liver alters hepatic lipid metabolism. - Inhibition of mitochondrial fatty acid oxidation contributes to development of nonalcoholic fatty liver disease induced by environmental cadmium exposure. - Acetylation of mitochondrial trifunctional protein alpha- subunit enhances its stability to promote fatty acid oxidation and is decreased in nonalcoholic fatty liver disease. - Non- alcoholic fatty liver disease proteomics. - Reduced AgRP activation in the hypothalamus of cows with high extent of fat mobilization after parturition.
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