Fatty liver disease frequently occurs in dairy cows, a clinico-pathologically defined nonalcoholic fatty liver disease (NAFLD), resulting in a high culling rate of dairy cows during their perinatal period, because of limitations of milk production performance and attenuation of reproduction performance in the subsequent serving life. It is estimated that 40–60% of high-yielding dairy cows (daily milk yield > 35 kg) develop moderate to severe fatty liver disease within 2 weeks after calving [5, 15]. Moreover, the perinatal fatty liver disease remain as prevalent now as they were 20 years ago [1]. The biology underlying the transition to lactation of dairy cattle was considered as the “final frontier” in our understanding of dairy cattle [1, 2]. Rather than attempting to define the transition state by using transcriptome or metabolome, the purpose of this study was to focus on the proteomic and its acetylatome of fatty liver as an emerging aspect of transition cow biology, because protein is the ultimate realization agent of the life.
Mitochondrial dysfunction and inflammation activation were the important biological processes that accompanied with the pathogenesis of fatty liver disease.
Proteome-wide analysis revealed that the pathogenesis of fatty liver disease in dairy cattle was accompanied with energy metabolism suppression and immune response activation. Majority of DEPs were enriched in the biological processes that closely associated with mitochrondrial functions, such as mitochrondrial respiratory chain complexes, fatty acid metabolism, glycolysis/gluconeogenesis, carbon metabolism, amino acid metabolism, pyruvate metabolism and ketone body synthesis and degradation (Fig. 2D, 2E, 3B and 3C). Especially, many down-regulated DEPs were found to be involved in energy metabolism occurred in mitochondria (such as tricarboxylic acid cycle/krebs cycle) by modifying their oxidase/dehydrogenase and/or transferase activities (Fig. 2E, 3B and 3C). It was presumed that mitochondrial dysfunction was one of the underlying reason for the onset of fatty liver disease in dairy cattle (Fig. 8)[11].
Mitochondria are crucial for energy metabolism processes in a cell, including the production of more than 90% of cellular ATP, β-oxidation, apoptosis, cell-cycle progression, proliferation and aging, and their dysfunction has been implicated in a wide range of metabolic diseases [16]. The role of mitochondrial dysfunction was already well-established in NAFLD and non-alcoholic steatohepatitis (NASH), with structural mitochondrial abnormalities, a reduction in mitochondrial respiratory chain activity and overwhelmed β-oxidation (oxidative stress) induced by excessive reactive oxygen species (ROS) production because of increased free fatty acid (FFA) load, therefore resulting in subsequent activation of inflammatory pathways (Fig. 8)[16, 17]. Hepatic lipid accumulation may be initiated by repression of mitochondrial fatty acid oxidation, which consequently causes the imbalance between lipid synthesis and lipid expenditure in liver. Associations between mitochondrial injuries and NAFLD development had been confirmed, mitochondrial dysfunction can inhibit mitochondrial β-oxidation and thereby cause fatty liver and increase lipid toxic metabolites, which might in turn exert adverse effects on mitochondrial function, resulting in a vicious cycle [18–20]. Lipotoxicity and/or oxidative stress caused by fatty acids can result in the swelling of mitochondria (mitochondrial ultrastructural damage), activity reduction of enzymes in mitochondria (impaired respiratory chain function), dysfunction of mitochondria in hepatocytes and eventually apoptosis of hepatocytes. The metabolic disorder caused by mitochondria in hepatocytes is an important cause of liver metabolic disorder and abnormal deposition of fat [16, 17, 19]. One of a recent study on early lactation Holstein-Friesian cows indicated that mitochondrial function was impaired during early lactation in pasture-fed cows, but not in total mixed rotations-fed cows [10]. Interestingly, acetylome analysis also revealed that the TCA cycle occurred in mitochondria was significantly inhibited and the immunoregulatory pathways were activated in the fatty liver tissues of dairy cows [11]. Our findings suggest that mitochondrial dysfunction might play a critical role in the development of fatty liver disease in dairy cattle, and mitochondria could be a target to improve liver function. In addition, it is worthy to mention that increasing experimental evidence points to mitochondrial alterations and/or dysfunction contribute to tumorigenesis and the development of drug resistance, suggesting that mitochondrial targeting might be an effective strategy for chemotherapy [21].
As for the up-regulated DEPs identified by proteome analysis, most of them were significantly enriched into pathways either associated with DNA stability and/or transcription activity or immune defense responses (Fig. 2D and 3D). The conversion of free fatty acid (FFA) to hepatic TAG may serve as a protective measure to prevent direct hepatic lipotoxicity. However, excessive hepatic FFA supply can contribute to inflammation, with elevated hepatic expression of inflammatory cytokines and/or adipokines, which had been endorsed by murine models of NAFLD [17, 22]. It was proposed that the abnormality in lipid and lipoprotein metabolism accompanied by chronic inflammation is the central pathway for the development of NAFLD [23]. Actually, inflammation activation was considered as an emerging aspect of transition cow biology [1], studies had clearly shown that essentially all cows would experience systemic subacute inflammation in the several days after parturition, even in the absence of disease, with increased haptoglobin and globulin, exaggerated cytokine response and reduced levels of liver hydrolase paraoxonase [8, 24].
In current study, crucial pathways closely associated with mitochondrial function and/or immune response were identified to play essential regulatory roles in the development of fatty liver disease in dairy cows (Fig. 8), such as metabolic pathways, cyclochrome P450 related metabolism, amino acid metabolism and/or biosynthesis, retinol metabolism and PPAR signaling pathway (Fig. 2D, 2E and 4A). The possible important dysregulated proteins causing the abnormal metabolism in liver were indicated by protein-protein interaction networks, such as ACACA, CYP1A1, ALDH1A1 and AOX1, displaying vigorous connections with other proteins (Fig. 4B).
Protein acetylation was the vitally important manner regulating the pathogenesis of fatty liver disease.
Comparison analysis of the proteome and acetylome data indicated that DEPs obtained from proteome were highly consistent with the annotation of DAPs identified from acetylome, with similar GO enrichment results (Fig. 2C, Fig. S2A)[11]. Intriguingly, our previous acetylome data indicated that most of the proteins with higher acetylation level were significantly enriched in energy and amino acid metabolic pathways, while proteins with lower acetylation level were significantly enriched in immune response and/or disease-related pathways [11]. This was exactly opposite to the enrichment results obtained from expression level down-regulated proteins (Fig. 2E, 3C and 3D) and up-regulated proteins (Fig. 2D and 3D). This suggests that lysine acetylation might be a vitally important post-transcriptional modification manner on these metabolism modulating proteins. In another word, the effect of the acetylation on the functional modification of the target protein could play critical roles in the development of fatty liver disease in dairy cows.
Moreover, the enriched pathways from proteome analysis got high consistence with that of acetylome analysis [11], with plenty of pathways and/or biological processes commonly enriched, such as pyruvate metabolism, cyclochrome P450 related metabolism, stereoid hormone biosynthesis, retino metabolism and glycolysis/gluconegesis (Fig. S2A), suggesting these acetylated proteins play essential regulatory roles in the pathogenesis of fatty liver disease via participating in these mitochondrial metabolism pathways. Amazingly, a considerable portion of DEPs are located in mitochondria, and their acetylation levels have been significantly modified, such as ACADVL, ALDH1L1, LDHB (Fig. 6E and 6F)[11]. The acetylation of these proteins were confirmed to be involved in different aspects of energy metabolism in mitochondria, such as TCA cycle, β-oxidation (fatty acid oxidation), lipid metabolism, therefore being associated with NAFLD [16, 25–29]. Protein acetylation has a crucial role in energy metabolism. Changes in cellular nutrient availability or energy status can induce global changes in mitochondrial protein acetylation [16, 30]. For example, insulin inhibits β-oxidation in the mitochondria through modifying the protein acetylation [10, 31–33], resulting in the accumulation of lipids in hepatocytes. Lysine acetylation, as a common post-transcriptional modification of proteins, is vitally important in both immunological and metabolic pathways and regulates the balance between energy storage and expenditure [32]. Additionally, acetyl-CoA, as the core product of TCA cycle in mitochondria, is an indicator of cellular energy status [16, 32]. Protein acetylation could be a convergence point for carbohydrate, amino acid and fat metabolism, clearly emerging as a common post-transcriptional modification in mitochondria and fluctuating the metabolic enzyme activity. Mitochondrial protein acetylation is sensitive to metabolic perturbations, sensing the energy/nutrient deprivation or excess and even ethanol or environmental exposure [10, 16, 20, 30, 31, 34].
Histone acetyltransferses (HATs) and histone deacetylase (HDACs) mediate acetylation and deacetylation of histone proteins and transcription factors, as a modulator to modify the acetylation level of target proteins. For example, mammalian sirtuins (SIRTs), NAD-dependent histone deacetylase such as SIRT1 and SIRT3, were involved in oxidative stress and lipid metabolism regulation [33], and had been proposed as a reliable biomarker and/or therapeutic target for fatty liver disease [10, 19, 33, 35, 36]. Especially in dairy cows, a significant increase in protein lysine acetylation was found in pasture-fed Holstein-Friesian dairy cows during their early lactation, accounting for impaired hepatic mitochrondrial function in this period [10].
Pivotal proteins/genetic factors, such as AOX1 and ALDH1A1, was identified as important regulators in the pathogenesis of fatty liver disease
Since both proteome and acetylome data indicated that mitochondrial energy metabolism suppression and inflammation activation (disease defense) were shown as the underlying reasons for pathogenesis of fatty live disease in dairy cattle, it would be remarkable to identify the corresponding functional proteins. Conjoint analysis of DEPs from proteome and DAPs from acetylome identify 54 proteins that were significantly expressed in fatty liver tissues and their acetylation levels were also widely modified (Fig. 6). Interestingly, protein-protein interaction network analysis of these 54 proteins got similar biological pathway enrichment with that based on all DEPs from proteome analysis, such as metabolic pathways, metabolism associated with cytochrome P450, chemical carcinogenesis, retinol metabolism and pathways associated with amino acid metabolism (Fig. 6G and 4A). Moreover, the 54 proteins kept and recognized most of the core proteins with abundant interactions with other proteins in the DEPs, such as CYP1A1, AOX1, ALDH1A1, DHRS4 (dehydrogenase/reductase SDR family member 4), GSTMs (glutathione S-transferase mu), etc (Fig. 4B and 6G). More importantly, 20 proteins were further confirmed to have significantly different expression level in normal liver and fatty liver tissues (Fig. 5 and Fig. 7). These identified proteins were supposed to be pivotal proteins/genetic factors that regulating the pathogenesis of fatty liver disease in dairy cattle, not only for their significant different expression abundance but also significant different acetylation modification levels.
Some of the 20 proteins had previously been reported to closely associate with the onset of NAFLD and/or its related biological processes. For example, AOX1 is a cytoplasmic enzyme that is highly expressed in the liver and plays a key role in the metabolism of drugs containing aromatic heterocyclic substituents. AOX1 can produce reactive oxygen species, which can promote cell damage and fibrosis. Fatty liver pathogenesis is associated with elevated liver AOX1 [37], while adiponectin can inhibit the expression of AOX1 by activating PPARɑ, therefore enhancing the lipid oxidation, attenuating the inflammation reaction and alleviating the liver injuries [22]. Another example is ALDH1A1, mainly expressed in the cytoplasm, playing important regulatory roles in the development of fatty liver. Both AOX1 and ALDH1A1 are involved in the retinol metabolic pathway. It is well-known that retinoic acid is a key regulator of glucose and lipid metabolism in the liver and adipose tissue. Liver diseases, especially those that cause fibrosis and cirrhosis, are related to impaired vitamin A (retinol) homeostasis and/or vitamin A deficiency [37]. It has been proved that ALDH1A1 can catalyze the oxidation of retinol to retinoic acid. ALDH1A1 knockout mice are resistant to obesity induced by high-fat diet, indicating that ALDH1A1 may be a candidate gene for obesity treatment [38].
In this study, some previously confirmed to be functional important in liver were also identified to be significantly regulated. For example, FABP1 and GSTM2 were down-regulated, PC and LDHB were up-regulated. FABP1, as a transport of fatty acids, can inhibit the cellular lipotoxicity caused from FA and regulate the synthesis and distribution of lipids in cells [39, 40]. The down-regulation of FABP1 in the liver will lead to the impairment of FA uptake and suppression of the production of VLDL, therefore increasing the accumulation of liver lipids [41, 42]. GSTM2, mu subtypes of glutathione-S-transferase, has enzymatic function of eliminating toxic and harmful factors (free radicals, peroxides and electrophilic groups) in vitro and in vivo, playing a role in protecting cells and regulating cell growth [43, 44]. Lactate dehydrogenase (LDH) and pyruvate carboxylase (PC) are important enzyme catalyzing the metabolism in mitochondria. Liver-specific PC knockout mice developed exacerbated oxidative stress and elevated liver inflammation, along with suppressed gluconeogenesis [45], indicating the specific necessity of PC for maintaining oxidation, biosynthesis, and pathways distal to TCA cycle.
These core proteins were recognized based on liver-specific proteome and acetylome analysis, as well as validation by individual proteome data, because of their close associations with immune response and/or energy metabolism, especially with metabolic pathways occurred in mitochondria. These proteins were identified as important candidate genetic factors that regulate the pathogenesis of fatty liver disease in dairy cattle, could potentially be developed into accurate and reproducible biomarkers to diagnose and/or pre-warn the risk of fatty liver dairies and also therapeutic targets for the disease treatment. Of course, it would be of our next interest to elucidate the detailed molecular functions in the pathology and pathogenesis of fatty liver disease in dairy cattle.
Additionally, NAFLD is a clinico-pathologically defined process associated with metabolic syndrome and fundamentally pin-pointed to the pathogenesis of lipid metabolism [26], causing obesity, type II diabetes, liver disease (such as steatohepatitis, fibrosis), threatening the health of humans and animals. Although the research on NAFLD biomarkers has advanced in the last two decades, there are still no reliable biomarkers for the diagnosis or the staging of the disease (NAFLD vs NASH) [46, 47]. NAFLD usually occurred accompanying with increased plasma insulin and fatty acid concentration, elevated fasting aminotransferase (AST/ALT) and/or TAG level, and also abnormal lipid accumulation in the liver [25, 48]. Moreover, another of the most important risk factors is histological evidence of hepatic inflammation [49] caused by acute inflammation and subacute inflammation [1, 15]. Meanwhile, NAFLD is a metabolic disease closely related with the acetylation of histones and non-histones [50, 51]. Dairy cows with fatty liver disease is a typical (non-progressive) NAFLD animal model, with similar fundamental metabolic disorder syndrome. The identification of pivotal proteins in dairy cattle fatty liver disease would be beneficial to inspire and refresh our understanding of the pathology and pathogenesis of NAFLD.