Liver Of Cows Research Articles

Supplementation of Rumen-Protected Glucose Increased the Risk of Disturbance of Hepatic Metabolism in Early Postpartum Holstein Cows.

The dual stress of reduced feed intake and increased milk yield in dairy cows early postpartum results in a negative energy balance. Rumen-protected glucose (RPG) has been reported to replenish energy, increase milk yield, and improve gut health. However, early postpartum cows often develop an insulin resistance, implying that RPG may not be well utilized and increased milk production may increase the liver’s fat oxidization burden. This study aimed to investigate the effects of RPG on the hepatic oxidative/antioxidative status and protein profile. Starting 7 d before expected calving, six pairs of cows were supplemented with rumen-protected glucose (RPG, n = 6) or with an equal amount of rumen-protecting coating fat (CON, n = 6). Liver samples were obtained from 10 cows 14 d after calving (d 14). Concentration of malondialdehyde and activity of glutathione peroxidase were increased and the activities of catalase and superoxide dismutase tended to increase in the livers of the RPG cows compared to the CON cows. The revised quantitative insulin sensitivity check index (RQUICKI) was decreased by RPG, but triacylglycerol concentration in liver was increased by RPG supplementation. The overall profiles of hepatic proteins were similar between CON and RPG. A partial least square regression was conducted to identify the proteins associated with liver lipidosis, oxidative stress, and antioxidative capacity. The top twenty proteins, according to their variable importance value, were selected for metabolic pathway enrichment analysis. Eighteen enriched KEGG pathways were identified, including metabolism, the citrate cycle, propanoate metabolism, the peroxisome, and type II diabetes mellitus. Our study showed that RPG supplementation reduced insulin sensitivity but increased the liver triglyceride concentration and the oxidative stress in early postpartum cows. Liver proteins related to lipidosis, oxidative stress, and antioxidative capacity, were positively associated with the glutamine metabolism, citric acid cycle, peroxisome, and type II diabetes pathways, which may indicate an increased risk of liver metabolic disorders caused by RPG supplementation in early postpartum cows.

Major Nutritional Metabolic Alterations Influencing the Reproductive System of Postpartum Dairy Cows.

Early successful conception of postpartum dairy cows is crucial in determining the optimum reproductive efficiency and profitability in modern dairy farming. Due to the inherent high production potential of modern dairy cows, the extra stress burden of peri-parturient events, and associated endocrine and metabolic changes causes negative energy balance (NEBAL) in postpartum cows. The occurrence of NEBAL is associated with excessive fat mobilization in the form of non-esterified fatty acids (NEFAs). The phenomenon of NEFA mobilization furthers with occurrence of ketosis and fatty liver in postpartum dairy cows. High NEFAs and ketones are negatively associated with health and reproductive processes. An additional burden of hypocalcemia, ruminal acidosis, and high protein metabolism in postpartum cows presents further consequences for health and reproductive performance of postpartum dairy cows. This review intends to comprehend these major nutritional metabolic alterations, their mechanisms of influence on the reproduction process, and relevant mitigation strategies.

Perspectives on the Fate of Absorbed Nitrogen in Dairy Farms

The demand for milk rises dramatically due to the increased global population without damaging the standard of living. As a result, the scale of the dairy industry in the world is growing rapidly. Consequently, a concomitant problem that needs to be faced is the environmental impact of the by-products produced during the operation of the dairy farm. Among the substances produced in dairy farms, the most harmful pollutant to the environment is nitrogen (N). In addition, the N pollution from dairy farms is significant and N has a great danger to human health and society. This study provides a systematic review on how to alleviate N pollution from dairy cows during milk production and the removal of ammonia and nitrate from dairy wastewater via biochar adsorption. First, from a physiological perspective, the metabolic pathways of amino acids and ammonia in the rumen, portal-drained viscera, liver, and mammary gland of dairy cows are elaborated, as well as the measures to improve N utilization. In the second part, the progress of research on the removal of ammonia and nitrate by biochar adsorption and the involved mechanisms are summarized. Modified biochar has a significant improvement in nitrate removal due to weakening the repulsive effect of ions. Based on the review of this paper, it can provide relevant theoretical support for future studies on N pollution mitigation from dairy farms.

Protein-synthesizing, bile-forming, urea-forming and carbohydrate functions in cows with fatty degeneration of the liver

In dairy farms of Ukraine, where highly productive dairy cows are kept, liver lesions are often diagnosed in the postpartum period. Postmortem studies of the liver of cows that were forcibly slaughtered showed that in mostly animals were diagnosed with fatty degeneration of the liver. The main causes of fatty hepatosis were violations of the structure of rations, imbalance of feeding on essential nutrients and biologically active substances, low content of easily digestible carbohydrates and high protein content. The study was performed on cows aged 4–5 years with productivity for the previous lactation of 5.600–7.500 L of milk, in a winter-stall period of keeping, 2–3 weeks after calving. According to clinical and biochemical blood tests, two groups of cows were formed – 50 clinically healthy and 50 cows with fatty liver disease. In cows diagnosed with fatty liver degeneration, the disease was manifested by decreased productivity and fatness, loss of appetite, oppression, hypotony of the rumen, reticulum and omasum. In some cows, there was pain at the liver area, increasing boundaries of hepatic dullness, jaundice of the visible mucous membranes and sclera. The blood serum of all cows with fatty liver disease established a decrease in albumin content, indicating impaired protein synthesis function of the liver. In some cows, the content of total protein in the serum increased due to globulin fractions, mainly gamma globulins. The ratio between the content of albumins and globulins decreased, which indicates the development in the blood of sick animals dysproteinemia. The development of fatty infiltration of the liver caused an increase in the concentration of bile acids in the serum of all sick cows. This is due to reduced conjugation and excretion of cholates by affected hepatocytes from the bile capillaries. The formation, absorption, conjugation, and excretion of bilirubin in the bile is disturbed, which causes the accumulation of total and conjugated bilirubin in the serum of sick animals. The cholesterol content in the blood of cows decreased, caused a violation of the esterification of its esters by hepatocytes. The established changes in the content of bile acids, total and conjugated bilirubin, and cholesterol in the blood of sick cows indicate a violation of bile secretion, bile production, and cholestasis development. In some cows with fatty liver degeneration, urea formation function and carbohydrate function are impaired, leading to a decrease in blood urea content and glucose.

Metabogenomic analysis to functionally annotate the regulatory role of long non-coding RNAs in the liver of cows with different nutrient partitioning phenotype

Long non-coding RNAs (lncRNAs) hold gene regulatory potential, but require substantial further functional annotation in livestock. Applying two metabogenomic approaches by combining transcriptomic and metabolomic analyses, we aimed to identify lncRNAs with potential regulatory function for divergent nutrient partitioning of lactating crossbred cows and to establish metabogenomic interaction networks comprising metabolites, genes and lncRNAs. Through correlation analysis of lncRNA expression with transcriptomic and metabolomic data, we unraveled lncRNAs that have a putative regulatory role in energy and lipid metabolism, the urea and tricarboxylic acid cycles, and gluconeogenesis. Especially FGF21, which correlated with a plentitude of differentially expressed genes, differentially abundant metabolites, as well as lncRNAs, suggested itself as a key metabolic regulator. Notably, lncRNAs in close physical proximity to coding-genes as well as lncRNAs with natural antisense transcripts appear to perform a fine-tuning function in gene expression involved in metabolic pathways associated with different nutrient partitioning phenotypes.

NEFAs Influence the Inflammatory and Insulin Signaling Pathways Through TLR4 in Primary Calf Hepatocytes in vitro.

Transition dairy cows are often in a state of negative energy balance because of decreased dry matter intake and increased energy requirements, initiating lipid mobilization and leading to high serum β-hydroxybutyrate (BHBA) and non-esterified fatty acid (NEFAs) levels, which can induce ketosis and fatty liver in dairy cows. Inflammation and insulin resistance are also common diseases in the perinatal period of dairy cows. What is the relationship between negative energy balance, insulin resistance and inflammation in dairy cows? To study the role of non-esterified fatty acids in the nuclear factor kappa beta (NF-κB) inflammatory and insulin signaling pathways through Toll-like receptor 4 (TLR4), we cultured primary calf hepatocytes and added different concentrations of NEFAs to assess the mRNA and protein levels of inflammatory and insulin signaling pathways. Our experiments indicated that NEFAs could activate the NF-κB inflammatory signaling pathway and influence insulin resistance through TLR4. However, an inhibitor of TLR4 alleviated the inhibitory effects of NEFAs on the insulin pathway. In conclusion, all of these results indicate that high-dose NEFAs (2.4 mM) can activate the TLR4/NF-κB inflammatory signaling pathway and reduce the sensitivity of the insulin pathway through the TLR4/PI3K/AKT metabolic axis.

NEFA Promotes Autophagosome Formation through Modulating PERK Signaling Pathway in Bovine Hepatocytes.

Simple SummaryNegative energy balance can lead to the mobilization of large amounts of body fat. A large amount of non-esterified fatty acids (NEFA) produced by lipolysis not only can be used for energy generation by β-oxidation in the liver but can also act as a potential regulator of lipid metabolism. The present study provides evidence that NEFA can activate hepatocyte autophagy through the protein kinase R-like endoplasmic reticulum kinase (PERK) signaling pathway. Autophagy has been reported to contribute to lipid metabolism through promoting the breakdown of intracellular lipids. These findings enable a better understanding of the redistribution and utilization of energy during the perinatal period of dairy cows.During the perinatal period, the abnormally high plasma non-esterified fatty acids (NEFA) concentration caused by the negative energy balance (NEB) can impose a significant metabolic stress on the liver of dairy cows. Endoplasmic reticulum (ER) stress is an important adaptive response that can serve to maintain cell homeostasis in the event of stress. The protein kinase R-like endoplasmic reticulum kinase (PERK) pathway is the most rapidly activated cascade when ER stress occurs in cells and has an important impact on the regulation of hepatic lipid metabolism and autophagy modulation. However, it is unknown whether NEFA can affect autophagy through modulating the PERK pathway, under NEB conditions. In this study, we provide evidence that NEFA treatment markedly increased lipid accumulation, the phosphorylation level of PERK and eukaryotic initiation factor 2α (eIF2α), and the expression of glucose-regulated protein 78 (Grp78), activating transcription factor 4 (ATF4), and C/EBP homologous protein (CHOP). More importantly, NEFA treatment can cause a substantial increase in the protein levels of autophagy-related gene 7 (ATG7), Beclin-1 (BECN1), sequestosome-1 (p62), and microtubule-associated protein 1 light chain 3 (LC3)-II, and in the number of autophagosomes in primary bovine hepatocytes. The addition of GSK2656157 (PERK phosphorylation inhibitor) can significantly inhibit the effect of NEFA on autophagy and can further increase lipid accumulation. Overall, our results indicate that NEFA could promote autophagy via the PERK pathway in bovine hepatocytes. These findings provide novel evidence about the potential role of the PERK signaling pathway in maintaining bovine hepatocyte homeostasis.

Effects of Pasture Type on Metabolism, Liver and Kidney Function, Antioxidant Status, and Plant Secondary Compounds in Plasma of Grazing, Jersey Dairy Cattle During Mid-lactation

Some pasture species are rich in phytochemicals, able to improve milk yield and quality and to reduce the environmental impacts of livestock farming. The phytochemicals interact with the different gene networks within the animal, such as nuclear factor erythroid 2-related factor 2 (NRF2), but their overall impact on animal health remains to be fully understood. The objective of this study was to identify the effects of pasture Legumes and non-leguminous Forbs containing high bioactive compounds on metabolism and activity of the liver, antioxidant response, kidney function, and inflammation of dairy cows using a large array of blood parameters associated with metabolism and the innate immune system. For this purpose, 26 parameters and the concentration of certain bioactive compounds were assessed in blood plasma, collected from the Jersey cows grazing either Grass, Legume, or Forb-based pastures. In addition, serum collected from all the cows was utilized to detect the changes in NRF2 activation in bovine mammary alveolar cells (MACT) and hepatocytes. Compared with Grass, the cows that grazed both Forb and Legume pastures had lower β-hydroxybutyric acid (BHB) and creatinine and larger vitamin E and the ferric reducing ability of the plasma, supporting an improved antioxidative status for these animals. Compared with both Grass and Legume, the cows that graze Forb pasture had lower urea and urea to creatinine ratio, and lower creatinine, indicating a better kidney function. The cows grazing Legume pasture had greater hematocrit, bilirubin, cholesterol, albumin, β-carotene, retinol, and thiol groups but lower ceruloplasmin, paraoxonase, and myeloperoxidase (MPO) than those grazed Grass and Forb pastures, indicating a positive effect of Legume pasture on the liver, oxidative stress, and red blood cells. The plasma of cows in the various pastures was enriched with various isoflavonoids, especially the cows grazed on Forb and Legume pastures, which likely contributed to improving the antioxidative status of those cows. However, this effect was likely not due to the higher activation of NRF2. Overall, these results indicate that Forb and Legume pastures rich in secondary metabolites do not strongly affect the metabolism but can improve the status of the liver and the kidney and improve the efficiency of N utilization and antioxidant response, compared with the Grass pasture.

Transcriptome analysis reveals disruption of circadian rhythms in late gestation dairy cows may increase risk for fatty liver and reduced mammary remodeling.

Circadian disruption increased insulin resistance and decreased mammary development in late gestation, nonlactating (dry) cows. The objective was to measure the effect of circadian disruption on transcriptomes of the liver and mammary gland. At 35 days before expected calving (BEC), multiparous dry cows were assigned to either control (CON) or phase-shifted treatments (PS). CON was exposed to 16-h light and 8-h dark. PS was exposed to 16-h light to 8-h dark, but phase of the light-dark cycle was shifted 6 h every 3 days. On day 21 BEC, liver and mammary were biopsied. RNA was isolated (n = 6 CON, n = 6 PS per tissue), and libraries were prepared and sequenced using paired-end reads. Reads mapping to bovine genome averaged 27 ± 2 million and aligned to 14,222 protein-coding genes in liver and 15,480 in mammary analysis. In the liver, 834 genes, and in the mammary gland, 862 genes were different (nominal P < 0.05) between PS and CON. In the liver, genes upregulated in PS functioned in cholesterol biosynthesis, endoplasmic reticulum stress, wound healing, and inflammation. Genes downregulated in liver function in cholesterol efflux. In the mammary gland, genes upregulated functioned in mRNA processing and transcription and downregulated genes encoded extracellular matrix proteins and proteases, cathepsins and lysosomal proteases, lipid transporters, and regulated oxidative phosphorylation. Increased cholesterol synthesis and decreased efflux suggest that circadian disruption potentially increases the risk of fatty liver in cows. Decreased remodeling and lipid transport in mammary may decrease milk production capacity during lactation.

PSIV-10 Effects of chronic molybdenum exposure in drinking water or feed on molybdenum and copper status and production performance of gestating and lactating beef cattle consuming a low-quality forage diet

Abstract The objective of this experiment was to evaluate the effects of Mo source (feed or water) on performance and mineral status of cows and calves fed a forage-based diet for two years (DM basis: 6.6% CP; 0.14% S, 6.7 mg Cu/kg, 2.4 mg Mo/kg). Fifty-four cow-calf pairs were stratified by cow age, BW, and liver Cu and Mo status, and randomly assigned to one of six treatments. Treatments consisted of: 1) Negative control (NC; no supplemental Mo or Cu); 2) Positive control [PC: NC + Cu (3 mg Cu/kg diet DM from CuSO4·5H2O)]; 3) NC + 500 µg Mo/L from MoNa2O4·2H2O in drinking water (Mo 500-water); 4) NC + 1000 µg Mo/L of MoNa2O4·2H2O in drinking water (Mo 1000-water); 5) Mo 1000-water + 3 mg Cu/kg diet DM from CuSO4·5H2O (Mo 1000-water+Cu); and 6) NC + 3 mg Mo/kg diet DM from MoNa2O4·2H2O (3.0 Mo-diet). Cattle were allowed ad-libitum access to feed, water, and a protein supplement throughout the experiment. During the winter months animals were housed in three replicate pens per treatment and during the summer months animals were housed in separate pastures by treatment where cow and calf feed and water intake could be measured separately. Data were analyzed as a completely randomized block design. Throughout the experiment no signs of molybdenosis were observed. There was a treatment x time (P &amp;lt; 0.01) interaction for cow liver Cu concentrations. Cows not receiving supplemental Cu had lower (P &amp;lt; 0.01) liver Cu concentrations than cows receiving supplemental Cu. Cow and calf performance, calf mineral status, cow liver Mo, and plasma and milk Mo and Cu concentrations were similar across treatments. These data indicate that Mo supplemented in water or feed at concentrations used in this experiment had minimal impact on Cu status and overall animal performance.

Reducing hepatic endoplasmic reticulum stress ameliorates the impairment in insulin signaling induced by high levels of β-hydroxybutyrate in bovine hepatocytes

Ketotic dairy cows exhibit a state of negative energy balance (NEB) characterized by elevated circulating levels of β-hydroxybutyrate (BHB) and fatty acids. Impaired hepatic insulin signaling in dairy cows occurs frequently during the transition into lactation, but its role on liver function during this period is not well known. In nonruminants, endoplasmic reticulum (ER) stress is a causal factor contributing to impaired insulin signaling in the liver. Thus, the aim of this study was to investigate the status of hepatic insulin and ER stress signaling and whether ER stress contributes to impaired insulin signaling in dairy cows with ketosis. Healthy (control cows, n = 10, BHB ≤0.6 mM) and ketotic (ketotic cows, n = 10, BHB ≥1.2 mM) cows at 3 to 10 d in milk were selected for liver biopsy and blood sampling before feeding. In vitro experiments were conducted with isolated hepatocytes from 5 healthy calves (1 d old, fasted female, 30-40 kg of body weight). Treatments included BHB (0, 0.9, 1.8, 3.6 mM), tauroursodeoxycholic acid (TUDCA, a canonical inhibitor of ER stress), and different incubation times (0.5, 1, 2, 3, 5, 7, 9, or 12 h). Ketotic cows had lower daily milk yield (median: 29.50 vs. 23.00 kg), higher plasma nonesterified fatty acid (NEFA) (median: 0.33 vs. 1.17 mM), BHB (median: 0.43 vs. 3.22 mM), aspartate aminotransferase (median: 70.58 vs. 155.70 U/L), alanine aminotransferase (median: 18.31 vs. 37.90 U/L), lower plasma glucose (median: 4.32 vs. 2.37 mg/dL), and revised quantitative insulin sensitivity check index (median: 0.39 vs. 0.37) compared with healthy cows. Increased abundance of phosphorylated insulin receptor substrate-1 (IRS1) and decreased abundance of phosphorylated protein kinase B (AKT) and glycogen synthase kinase-3β (GSK3β) in ketotic cows indicated a state of insulin resistance. In addition, abundance of phosphorylated protein kinase RNA-like ER kinase (PERK) and inositol requiring protein-1α (IRE1α), and cleavage of activating transcription factor-6 (ATF6) were greater in the liver of ketotic cows. In vitro, at the early stages of incubation, treatment with BHB upregulated abundance of phosphorylated of IRE1α, PERK, and the cleavage of ATF6, as well as several unfolded protein response genes [X-box-binding protein-1 (XBP1), 78 kDa glucose-regulated protein (GRP78), and C/EBP homologous protein (CHOP)]. Furthermore, in response to increasing doses of BHB, the phosphorylation level of PERK, IRE1α, and the cleavage of ATF6, and the abundance of XBP1, GRP78, and CHOP increased. In addition, BHB treatment increased phosphorylation of IRS1 and decreased phosphorylation of AKT and GSK3β, and upregulated abundance of gluconeogenic genes (phosphoenolpyruvate carboxykinase and glucose-6-phosphatase). Importantly, these changes were reversed by inhibiting ER stress with TUDCA treatment. Overall, the present study indicated that reversing ER stress during ketosis might help alleviate hepatic insulin resistance. Targeting ER stress may represent a potential therapeutic target for controlling the negative aspects of ketosis on liver function.

Proteome analysis identified proteins associated with mitochondrial function and inflammation activation crucially regulating the pathogenesis of fatty liver disease

BackgroundFatty liver disease prevalently occurs in commercial postpartum dairies, resulting in a worldwide high culling rate because of their subsequent limitations of production and reproduction performance.ResultsFatty liver-specific proteome and acetylome analysis revealed that energy metabolism suppression closely associated with mitochondrial dysfunction and inflammation activation were shown to be remarkable biological processes underlying the development of fatty liver disease, furthermore, acetylation modification of proteins could be one of the main means to modulate these processes. Twenty pivotal genetic factors/genes that differentially expressing and being acetylation modified in liver were identified and proposed to regulate the pathogenesis of fatty liver dairies. These proteins were confirmed to be differentially expressing in individual liver tissue, eight of which being validated via immunohistochemistry assay.ConclusionsThis study provided a comprehensive proteome and acetylome profile of fatty liver of dairy cows, and revealed potential important biological processes and essential regulators in the pathogenesis of fatty liver disease. Expectantly, understanding the molecular mechanisms of the pathogenesis of fatty liver disease in dairies, as an animal model of non-alcoholic fatty liver disease (NAFLD) in human beings, which is a clinico-pathologically defined process associated with metabolic syndrome, could inspire and facilitate the development of efficacious therapeutic drugs on NAFLD.

Sirtuin 1 is involved in oleic acid-induced calf hepatocyte steatosis via alterations in lipid metabolism-related proteins.

Sirtuin 1 (SIRT1), an NAD-dependent protein deacetylase, plays a central role in the control of lipid metabolism in nonruminants. However, the role of SIRT1 in hepatic lipid metabolism in dairy cows with fatty liver is not well known. Thus, we used isolated primary bovine hepatocytes to determine the role of SIRT1 in protecting cells against oleic acid (OA)-induced steatosis. Recombinant adenoviruses to overexpress (AD-GFP-SIRT1-E) or knockdown (AD-GFP-SIRT1-N) SIRT1 were used for transduction of hepatocytes. Calf hepatocytes isolated from five female calves (1 d old, 30 to 40 kg) were used to determine both time required and the lowest dose of OA that could induce triacylglycerol (TAG) accumulation. Analyses indicated that 0.25 mM OA for 24 h was suitable to induce TAG accumulation. In addition, OA not only led to an increase in TAG, but also upregulated mRNA and protein abundance of sterol regulatory element-binding transcription factor 1 (SREBF1) and downregulated SIRT1 and peroxisome proliferator-activated receptor-gamma coactivator 1 alpha (PPARGC1A). Thus, these in vitro conditions were deemed optimal for subsequent experiments. Calf hepatocytes were cultured and incubated with OA (0.25 mM) for 24 h, followed by adenoviral AD-GFP-SIRT1-E or AD-GFP-SIRT1-N transduction for 48 h. Overexpression of SIRT1 led to greater protein and mRNA abundance of SIRT1 along with fatty acid oxidation-related genes including PPARGC1A, peroxisome proliferator-activated receptor alpha (PPARA), retinoid X receptor α (RXRA), and ratio of phospho-acetyl-CoA carboxylase alpha (p-ACACA)/total acetyl-CoA carboxylase alpha (ACACA). In contrast, it resulted in lower protein and mRNA abundance of genes related to lipid synthesis including SREBF1, fatty acid synthase (FASN), apolipoprotein E (APOE), and low-density lipoprotein receptor (LDLR). The concentration of TAG decreased due to SIRT1 overexpression. In contrast, silencing SIRT1 led to lower protein and mRNA abundance of SIRT1, PPARGC1A, PPARA, RXRA, and greater protein and mRNA abundance of SREBF1, FASN, APOE, and LDLR. Further, those responses were accompanied by greater content of cellular TAG and total cholesterol (TC). Overall, data from these in vitro studies indicated that SIRT1 is involved in the regulation of lipid metabolism in calf hepatocytes subjected to an increase in the supply of OA. Thus, it is possible that alterations in SIRT1 abundance and activity in vivo contribute to development of fatty liver in dairy cows.

Ufbp1, a Key Player of Ufm1 Conjugation System, Protects Against Ketosis-Induced Liver Injury via Suppressing Smad3 Activation.

The dairy cattle suffer from severe liver dysfunction during the pathogenesis of ketosis. The Ufm1 conjugation system is crucial for liver development and homeostasis. Ufm1 binding protein (Ufbp1) is a putative Ufm1 target and an integral component, but its role in ketosis-induced liver injury is unclear so far. The purpose of this study is to explore the key role of Ufbp1 in liver fibrosis caused by ketosis in vivo and in vitro. Liver tissues were collected from ketotic cows and Ufbp1 conditional knockout (CKO) mice in vivo. However, Ufbp1–/– mouse embryonic fibroblast cells and Hela cells were used for in vitro validation. Subsequently, various assays were performed to reveal the underlying molecular mechanisms of the Ufbp1 protective effect. In this study, hepatic fibrosis, endoplasmic reticulum (ER) stress, and apoptosis were reported in the liver of ketotic cows, fibrotic markers (alpha-smooth muscle actin, Collagen1) and ER stress markers (glucose-regulated protein 78, CEBP homologous protein) were upregulated remarkably, and the apoptosis-related genes (Bcl2, Bax) were in line with expectations. Interestingly, Ufbp1 expression was almost disappeared, and Smad2/Smad3 protein was largely phosphorylated in the liver of ketotic cows, but Ufbp1 deletion caused Smad3 phosphorylation apparently, rather than Smad2, and elevated ER stress was observed in the CKO mice model. At the cellular level, Ufbp1 deficiency led to serious fibrotic and ER stress response, Smad3 was activated by phosphorylation significantly and then was translocated into the nucleus, whereas p-Smad2 was largely unaffected in embryonic fibroblast cells. Ufbp1 overexpression obviously suppressed Smad3 phosphorylation in Hela cells. Ufbp1 was found to be in full combination with Smad3 using endogenous immunoprecipitation. Taken together, our findings suggest that downregulation or ablation of Ufbp1 leads to Smad3 activation, elevated ER stress, and hepatocyte apoptosis, which in turn causes liver fibrosis. Ufbp1 plays a protective role in ketosis-induced liver injury.

Effects of starvation-induced negative energy balance on endoplasmic reticulum stress in the liver of cows.

ObjectiveEndoplasmic reticulum (ER) stress engages the unfolded protein response (UPR) that serves as an important mechanism for modulating hepatic fatty acid oxidation and lipogenesis. Chronic fasting in mice induced the UPR activation to regulate lipid metabolism. However, there is no direct evidence of whether negative energy balance (NEB) induces ER stress in the liver of cows. This study aimed to elucidate the relationship between the NEB attributed to feed deprivation and ER stress in bovine hepatocytes.MethodsBlood samples and liver biopsy tissues were collected from 6 non-lactating cows before and after their starvation for 48 h. The blood non-esterified fatty acids (NEFA), β-hydroxybutyric acid (BHBA) and glucose level were analyzed. Real-time quantitative polymerase chain reaction and Western blotting were used to explore the regulation of genes associated with UPR and lipid metabolism.ResultsThe starvation increased the plasma BHBA and NEFA levels and decreased the glucose level. Additionally, the starvation caused significant increases in the mRNA expression level of spliced X-box binding protein 1 (XBP1s) and the protein level of phosphorylated inositol-requiring kinase 1 alpha (p-IRE1α; an upstream protein of XBP1) in the liver. The mRNA expression levels of peroxisome proliferator-activated receptor alpha and its target fatty acid oxidation- and ketogenesis-related genes were significantly upregulated by the starvation-mediated NEB. Furthermore, we found that the mRNA expression levels of lipogenic genes were not significantly changed after starvation.ConclusionThese findings suggest that in the initial stage of NEB in dairy cows, the liver coordinates an adaptive response by activating the IRE1 arm of the UPR to enhance ketogenesis, thereby avoiding a fatty liver status.

Longitudinal changes in fatty acid metabolism and in the mitochondrial protein import system in overconditioned and normal conditioned cows: A transcriptional study using microfluidic quantitative PCR

This study investigated the effect of body condition around calving on the hepatic mRNA expression of genes involved in fatty acid (FA) metabolism and mitochondrial protein import system of dairy cows during the transition period. Fifteen weeks before their anticipated calving date, 38 multiparous Holstein cows were selected based on their current and previous body condition scores (BCS) and allocated to either a high or a normal BCS group (19 cows each). They received different diets to reach targeted differences in BCS and backfat thickness (BFT) until dry-off. At dry-off, normal BCS (NBCS) cows had a BCS <3.5 and BFT <1.2 cm, and the high BCS (HBCS) cows had a BCS >3.75 and BFT >1.4 cm. The expression of targeted genes in the liver was assayed by reverse-transcription quantitative real-time PCR using microfluidics integrated fluidic circuit chips on a subset of 5 cows from each group. Liver biopsies were collected at d -49, +3, +21, and +84 relative to parturition. The mRNA abundance of 47 genes related to lipid metabolism including carnitine metabolism, FA uptake and transport, lipoprotein export, carnitine metabolism, mitochondrial and proximal FA oxidation, ketogenesis, AMP-activated protein kinase/mammalian target of rapamycin pathway, and mitochondrial protein import system was assessed in liver tissue. The mRNA abundances of FA binding protein (FABP)6 (in both groups), and FABP1 and solute carrier family 22 member 5 (SLC22A5) in HBCS were upregulated (>1.5-fold change, FC) in early lactation (at d +3 and +21 postpartum) compared with antepartum (d -49), indicating promoted FA uptake and intracellular transport in the liver due to the metabolic adaptations of elevated lipo-mobilization after parturition. The upregulation of SLC22A5 and SLC25A20 after parturition was more pronounced in HBCS than in NBCS cows, suggesting a need for increasing the capacity of FA uptake, and FA transport into the hepatocyte. The increased mRNA abundance of carnitine palmitoyltransferase 1A, after parturition and to a greater extent in HBCS (FC = 4.1) versus NBCS (FC = 2.1) indicates a physiological increase in the capacity of long-chain fatty acyl-CoA entry into the liver mitochondria compared with antepartum (ap; d -49 relative to calving). The greater hepatic mRNA abundance of genes encoding enzymes involved in mitochondrial FA oxidation in HBCS than in NBCS points to an increased rate of mitochondrial β-oxidation. The hepatic mRNA abundance of 3-hydroxy-3-methylglutaryl-CoA synthase 2 and 3-hydroxy-3-methylglutaryl-CoA were upregulated after parturition (d +21/d +3 pp) to a greater extent in HBCS than in NBCS cows, indicating that excess acetyl-CoA generated via β-oxidation was increasingly used for ketogenesis. We observed for the first time that the mRNA abundance of genes involved in the translocase of the inner membrane (TIM) complex (TIM22 and TIM23) in the hepatic mitochondrial protein import system were undergoing distinct changes during the transition from late pregnancy to early lactation in dairy cows. Even though sample size in this study was relatively small, the results support that overconditioning around calving may contribute to mitochondrial FA overload and greater ketogenesis at the level of transcription in the liver of early lactation cows.

Intracellular Ca2+ signaling and ORAI calcium release-activated calcium modulator 1 are associated with hepatic lipidosis in dairy cattle.

Fatty liver is a common metabolic disorder afflicting dairy cows during the periparturient period and is closely associated with endoplasmic reticulum (ER) stress. The onset of ER stress in humans and mice alters hepatic lipid metabolism, but it is unknown if such event contributes to fatty liver in dairy cows soon after parturition. ORAI calcium release-activated calcium modulator 1 (ORAI1) is a key component of the store-operated Ca2+ entry mechanism regulating cellular Ca2+ balance. The purpose of this study was to investigate the role of ORAI1 on hepatic lipidosis via ER stress in dairy cows. Liver tissue biopsies were collected from Holstein cows diagnosed as healthy (n = 6) or with hepatic lipidosis (n = 6). Protein and mRNA abundance of ER stress-related targets, lipogenic targets, or the transcription regulator SREBP1 and ORAI1 were greater in cows with lipidosis. In vitro, hepatocytes were isolated from four healthy female calves and used for culture with a 1.2 mM mixture of fatty acids (oleic, linoleic, palmitic, stearic, and palmitoleic acid) for various times (0, 3, 6, 9, or 12 h). As incubation time progressed, increases in concentration of Ca2+ and abundance of protein kinase RNA-like ER kinase (PERK), inositol-requiring protein 1α (IRE1α), and activating transcription factor-6 (ATF6) protein in response to exogenous fatty acids underscored a mechanistic link among Ca2+, fatty acids, and ER stress. In a subsequent study, hepatocytes were transfected with small interfering RNA (siORAI1) or the ORAI1 inhibitor BTP2 for 48 h or 2 h followed by a challenge with the 1.2 mM mixture of fatty acids for 6 h. Compared with control group, silencing or inhibition of ORAI1 led to decreased abundance of fatty acid synthesis (FASN, SREBP1, and ACACA) and ER stress-related proteins in bovine hepatocytes. Overall, data suggested that NEFA through ORAI1 regulate intracellular Ca2+ signaling, induce ER stress, and lead to lipidosis in isolated hepatocytes.

Aspects of transition cow metabolomics—Part II: Histomorphologic changes in the liver parenchyma throughout the transition period, in cows with different liver metabotypes and effects of a metaphylactic butaphosphan and cyanocobalamin treatment

The aims of this study were to evaluate histopathologic changes during the transition period, describe the histopathological features of the metabotypes identified in Part I (Schären et al., 2021b), and investigate effects of a metaphylactic treatment with butaphosphan and cyanocobalamin (BCC) on the liver parenchyma. Eighty German Holstein cows (mean 305-d production: 10,957 kg, range: 6,480-15,193 kg; mean lactation number: 3.9, range: 2-9) from a commercial dairy farm in Saxony, Germany, were enrolled in a randomized, prospective, triple-blinded study. Two groups received a treatment with BCC (5 or 10 mL/100 kg of body weight 10% butaphosphan and 0.005% cyanocobalamin, Catosal, Bayer Animal Health, n = 20 each) and one group a placebo treatment (NaCl 0.9%, n = 40). Liver biopsy specimens were collected 14 d antepartum (AP) and 7, 28, and 42 d postpartum (PP), routinely processed for histologic examination, and stained with hematoxylin and eosin, Sudan III, periodic acid-Schiff, and picrosirius red stains. The sections were assessed for fat and glycogen content and degenerative, inflammatory, fibrotic, and proliferative changes. The statistical analysis included the effects of the sampling day, the lactation number, the treatment, and the metabotype (A = medium, B = minor, C = large alterations in the liver metabolome profile between AP and PP status). There was mild to moderate fat infiltration in the liver of 37% of cows in the last 2 wk AP, and moderate to severe fat infiltration in 66% of cows in the first days PP. The degree of fat infiltration increased from 2 wk AP until the end of the first week PP, and then decreased until the end of the study period, at which time about 25% of cows had moderate to severe fatty infiltration. Lipidosis was positively correlated with the severity of liver cell degeneration, and negatively correlated with the degree of glycogen deposits. Complete glycogen depletion of hepatocytes was not observed in cows, even in the presence of severe hepatic lipidosis. Moderate to severe lymphocytic hepatitis was seen in 39% of cows throughout the study period, and cows with lactation numbers 5 or greater had perisinusoidal fibrosis more often than younger cows. Severe fibrosis and cirrhosis of the liver did not occur. Metabotype B animals exhibited a higher chance of fatty infiltration, lower glycogen storage, and perisinusoidal fibrosis and for this metabotype positive correlations were calculated between increased fat deposition in the liver and marked glycogen depletion, and increased degenerative, inflammatory, fibrotic, and proliferative changes of hepatic tissue. For the treatment with BCC, no significant effect was observed. In summary, during the transition period, the liver of dairy cows is characterized by fat accumulation and glycogen depletion and histologic signs of hepatitis and hepatocyte degeneration. These histomorphologic changes were accentuated in animals exhibiting little alterations in their liver metabolome profile across the transition period (metabotype B) and support the assumption of a decreased grass silage quality as a causative factor.

Aspects of transition cow metabolomics—Part III: Alterations in the metabolome of liver and blood throughout the transition period in cows with different liver metabotypes

The liver plays a central role in the postpartum (PP) energy metabolism of the transition dairy cow; however, studies describing the liver metabolome during this period were lacking. The aim of the presented study was therefore to compare the alterations in the liver and blood metabolome of transition dairy cows. For this purpose, an on-farm trial with 80 German Holstein cows (mean lactation number: 3.9; range: 2-9) was performed, with thorough documentation of clinical traits and clinical chemistry, as well as production data. Liver biopsies and blood samples were collected at d 14 (mean: 12 d, range: 1-26 d) antepartum (AP), d 7 (7, 4-13) and 28 (28, 23-34; mean, earliest-latest) PP for targeted mass spectroscopy-based metabolomics analysis using the AbsoluteIDQ p180 kit (Biocrates Life Sciences). Statistical analysis was performed using multivariate (partial least squares discriminant analysis) as well as univariate methods (linear mixed model). Multivariate data analysis of the liver metabolome revealed 3 different metabotypes (A = medium, B = minor, C = large alterations in the liver metabolome profile between AP and PP). In metabotype C, an increase of almost all acylcarnitines, lysophosphatidylcholines (lysoPC), sphingomyelins, and some phosphatidylcholines (PC, mainly at 7 d PP) was observed after calving. In contrast to metabotypeC, the clinical data of the metabotype B animals indicated a higher PP lipomobilization and occurrence of transition cow diseases. The liver metabolome profile of these animals most likely mirrors a failure of adaptation to the PP state. This strong occurrence of metabotypes was much less pronounced in the blood metabolome. Additionally, differences in metabolic patterns were observed across the transition period when comparing liver and blood matrices (e.g., in different biogenic amines, acylcarnitines and sphingolipids). In summary, the blood samples at 7dPP showed lower acylcarnitines and PC, with minor alterations and a heterogeneous pattern in AA, biogenic amines, and sphingomyelins compared with 14 d AP. In contrast to 7 d PP, the blood samples at 28 PP revealed an increase in several AA, lysoPC, PC, and sphingomyelins in comparison to the AP state, irrespective of the metabotype. In the liver biopsies metabotype B differed from metabotype C animals ante partum by following metabolites: higher α aminoadipic acid, lower AA, serotonin, taurine, and symmetric dimethylarginine levels, lower or higher concentrations of certain acylcarnitines (higher: C2, C3, C5, C4:1; lower: C12:1, C14:1-OH, C16:2), and lower lysoPC (a C16:0, C18:0, C20:3, C20:4) and hexose levels. In blood samples, fewer differences were observed, with lower serotonin, acylcarnitine C16:2, lysoPC (a C16:0, C17:0, C18:0 and C18:1), PC aa C38:0, and PC ae C42:2. The results show that the use of only the blood metabolome to assess liver metabolism may be hampered by the fact that blood profiles are influenced by the metabolism of many organs, and metabolomics analysis from liver biopsies is a more suitable method to identify distinct metabotypes. Future studies should investigate the stability and reproducibility of the metabotype and phenotypes observed, and the possible predictive value of the metabolites already differing AP between metabotype B and C.

Aspects of transition cow metabolomics—Part I: Effects of a metaphylactic butaphosphan and cyanocobalamin treatment on the metabolome in liver, blood, and urine in cows with different liver metabotypes

Dairy cows in modern production systems are at risk to develop metabolic disorders during the transition period. Reasons for individual differences in susceptibility, as well as the underlying pathomechanisms, are still only partially understood. The development of metaphylactic treatment protocols is needed. In this context, an on-farm prospective 3-fold blinded randomized study involving 80 German Holstein cows was performed throughout 1 yr. The trial involved a thorough recording of the production and clinical traits, clinical chemistry, and liver biopsies and blood and urine sampling at d 14 (mean: 12 d, range: 1-26 d) antepartum (AP), and d 7 (7, 4-13) and 28 (28, 23-34) postpartum (PP) for metabolomics analyses. Two groups received a treatment with butaphosphan and cyanocobalamin (BCC) at either the dosage recommended by the manufacturer or the double dosage (5 or 10 mL/100 kg of body weight 10% butaphosphan and 0.005% cyanocobalamin (Catosal, Bayer Animal Health), n = 20 in each group, parity: 4.2 ± 2.0 and 3.4 ± 1.3, respectively (mean ± SD)] and one group a placebo treatment (NaCl 0.9%, n = 40, parity: 4.0 ± 1.9). The animals were treated at 6 time points (7, 6, and 5 d AP, and 1, 2, and 3 d PP) via intravenous injection. Mass spectroscopy-based targeted metabolomics analysis of blood plasma and liver samples were performed using the AbsoluteIDQ p180 kit (Biocrates Life Sciences), whereas the urine samples were analyzed by nuclear magnetic resonance spectroscopy. Statistical analysis was performed using multivariate [partial least squares discriminant analysis (PLS-DA)] and univariate methods (linear mixed model). Multivariate data analysis (PLS-DA plots) of the liver metabolome revealed 3 different metabotypes (A = medium, B = minor, C = large alterations in liver metabolome profile between AP and PP status). Metabotype B animals were characterized by higher PP lipomobilization (stronger PP body condition decrease and higher blood bilirubin, fatty acids, gamma-glutamyltransferase, and triglyceride levels) and a higher occurrence of transition cow diseases, compared with the animals in metabotype C. Analysis of the feeding data showed that the period of metabotype B animals (calving in a distinct time frame) was characterized by a decreased grass silage quality. The PP liver metabolome of the metabotype C animals was characterized by higher concentrations of AA, acylcarnitines, lysoPC and sphingomyelins compared with metabotype B. For the metaphylactic treatment with BCC a dose-dependent effect was confirmed, differing between the metabotypes. In all matrices and metabotypes at various time points significant treatment effects were observed, with different profiles in clinical chemistry and as well in metabolomics data. The most clear-cut treatment effect was observed in metabotype B in the liver at 7 d PP, characterized by an increase in several acylcarnitines and phosphatidylcholines, indicating a more efficient influx and oxidation of fatty acids in mitochondria and thereby an increase in energy supply and more efficient triglyceride export in the liver. The results from the liver metabolomics analysis support the application of an indication-based metaphylactic treatment with BCC.