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ABSTRACT: Through its influence on the gut microbiota, the feeding of Saccharomyces cerevisiae fermentation products (SCFP) has been a successful strategy to enhance the health of dairy cows during periods of physiological stresses. Although production and metabolic outcomes from feeding SCFP are well-known, its combined impacts on the ruminal microbiota and metabolome during gut barrier challenges remain unclear. To address this gap in knowledge, multiparous Holstein cows (97.1 ± 7.6 DIM [SD]; n = 8/group) fed a control diet (CON) or CON plus 19 g/d SCFP for 9 wk were subjected to a feed restriction (FR) challenge for 5 d, during which they were fed 40% of their ad libitum intake from the 7 d before FR. The DNA extracted from ruminal fluid was subjected to PacBio full-length 16S rRNA gene sequencing, real-time PCR of 12 major ruminal bacteria, and metabolomics analysis of up to 189 metabolites via GC/MS. High-quality amplicon sequence analyses were performed with the TADA (Targeted Amplicon Diversity Analysis), MicrobiomeAnalyst, PICRUSt2, and STAMP software packages, and metabolomics data were analyzed via MetaboAnalyst 5.0. Ruminal fluid metabolites from the SCFP group exhibited a greater α-diversity Chao 1 (P = 0.03) and Shannon indices (P = 0.05), and the partial least squares discriminant analysis clearly discriminated metabolite profiles between dietary groups. The abundance of CPla_4_termite_group, Candidatus Saccharimonas, Oribacterium, and Pirellula genus in cows fed SCFP was greater. In the SCFP group, concentrations of ethanolamine, 2-amino-4,6-dihydroxypyrimidine, glyoxylic acid, serine, threonine, cytosine, stearic acid, and pyrrole-2-carboxylic acid were greater in ruminal fluid. Both Fretibacterium and Succinivibrio abundances were positively correlated with metabolites across various biological processes: gamma-aminobutyric acid, galactose, butane-2,3-diol, fructose, 5-amino pentanoic acid, β-aminoisobutyric acid, ornithine, malonic acid, 3-hydroxy-3-methylbutyric acid, hexanoic acid, heptanoic acid, cadaverine, glycolic acid, β-alanine, 2-hydroxybutyric acid, methyl alanine, and alanine. In the SCFP group, compared with CON, the mean proportion of 14 predicted pathways based on metabolomics data was greater, whereas 10 predicted pathways were lower. Integrating metabolites and upregulated predicted enzymes (NADP+-dependent glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, serine: glyoxylate aminotransferase, and d-glycerate 3-kinase) indicated that the pentose phosphate pathway and photorespiration pathway were most upregulated by SCFP. Overall, SCFP during FR led to alterations in ruminal microbiota composition and key metabolic pathways. Among those, we identified a shift from the tricarboxylic acid cycle to the glyoxylate cycle, and nitrogenous base production was enhanced.

ABSTRACT: Excessive free fatty acid (FFA) oxidation and related metabolism are the major cause of oxidative stress and liver injury in dairy cows during the early postpartum period. In nonruminants, activation of transcription factor EB (TFEB) can improve cell damage and reduce the overproduction of mitochondrial reactive oxygen species. As a downstream target of TFEB, peroxisome proliferator-activated receptor γ coactivator 1 α (PGC-1α, gene name PPARGC1A) is a critical regulator of oxidative metabolism. Nuciferine (Nuc), a major bioactive compound isolated from the lotus leaf, has been reported to possess hepatoprotective activity. Therefore, the objective of this study was to investigate whether Nuc could protect bovine hepatocytes from FFA-induced lipotoxicity and the underlying mechanisms. A mixture of FFA was diluted in RPMI-1640 basic medium containing 2% low fatty acid bovine serum albumin to treat hepatocytes. Bovine hepatocytes were isolated from newborn calves and treated with various concentrations of FFA mixture (0, 0.3, 0.6, or 1.2 mM) or Nuc (0, 25, 50, or 100 μM), as well as co-treated with 1.2 mM FFA and different concentrations of Nuc. For the experiments of gene silencing, bovine hepatocytes were transfected with small interfering RNA targeted against TFEB or PPARGC1A for 36 h followed by treatment with 1.2 mM FFA for 12 h in presence or absence of 100 μΜ Nuc. The results revealed that FFA treatment decreased protein abundance of nuclear TFEB, cytosolic TFEB, total (t)-TFEB, lysosome-associated membrane protein 1 (LAMP1) and PGC-1α and mRNA abundance of LAMP1, but increased phosphorylated (p)-TFEB. In addition, FFA treatment increased the content of malondialdehyde (MDA) and hydrogen peroxide (H2O2) and decreased the activities of catalase (CAT) and glutathione peroxidase (GSH-Px) in bovine hepatocytes. Moreover, FFA administration enhanced the activities of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and lactose dehydrogenase (LDH) in the medium of FFA-treated hepatocytes, but reduced the content of urea. In FFA-treated bovine hepatocytes, Nuc administration increased TFEB nuclear localization and the protein abundance of t-TFEB, LAMP1, and PGC-1α and mRNA abundance of LAMP1, decreased the contents of MDA and H2O2 and the protein abundance of p-TFEB, and enhanced the activities of CAT and GSH-Px in a dose-dependent manner. Consistently, Nuc administration reduced the activities of ALT, AST, and LDH and increased the content of urea in the medium of FFA-treated hepatocytes. Importantly, knockdown of TFEB reduced the protein abundance of p-TFEB, t-TFEB, LAMP1, and PGC-1α and mRNA abundance of LAMP1, and impeded the beneficial effects of Nuc on FFA-induced oxidative damage in bovine hepatocytes. In addition, PPARGC1A silencing did not alter Nuc-induced nuclear translocation of TFEB, increase of the protein abundance of t-TFEB, LAMP1, and PGC-1α and mRNA abundance of LAMP1, or decrease of the protein abundance of p-TFEB, whereas it partially reduced the beneficial effects of Nuc on FFA-caused oxidative injury. Taken together, Nuc exerts protective effects against FFA-induced oxidative damage in bovine hepatocytes through activation of the TFEB/PGC-1α signaling pathway.

ABSTRACT: Endometritis is one of the most common causes of infertility in dairy cows, and is histopathologically characterized by inflammation and damage of endometrial epithelium. Interferon-tau (IFN-τ) is a novel type I interferon secreted by ruminant trophoblast cells with low cytotoxicity even at high doses. Previous studies suggested that IFN-τ plays an important role in inflammation. However, the mechanisms whereby IFN-τ may modulate the inflammatory responses in the bovine endometrium are unknown. In the present study, primary bovine endometrial epithelial cells (BEEC) isolated from fresh and healthy uterine horns were used for in vitro studies. The integrity of BEEC was assessed by immunofluorescence staining for cytokeratin 18 (CK-18, a known epithelial marker). For the experiments, BEEC were stimulated with different concentrations of lipopolysaccharide (LPS; 0–20 µg/mL) for different times (0–24 h). Cell viability and apoptosis were assessed via CCK-8 and flow cytometry. In a preliminary study, we observed that compared with the control group without LPS, 10 µg/mL of LPS stimulation for 24 h induced apoptosis. In a subsequent study, 20 or 40 ng/mL of IFN-τ alleviated LPS-induced apoptosis. Relative to the LPS group, western blotting further revealed that IFN-τ inhibited the protein abundance of TLR4 and phosphorylated (p-) p65 (p-p65) and Bax/Bcl-2 ratio, suggesting that IFN-τ can protect BEEC against inflammatory injury. Furthermore, the protein abundance of p-phosphoinositide 3-kinase (p-PI3K), p-protein kinase B (p-AKT), p-glycogen synthase kinase-3β (p-GSK3β), β-catenin, and p-forkhead box O1 (p-FoxO1) was lower in the LPS group, whereas IFN-τ upregulated their abundance. The use of LY294002, a specific inhibitor of PI3K/AKT, attenuated the upregulation of p-PI3K, p-AKT p-GSK3β, β-catenin, and p-FoxO1 induced by IFN-τ, and also blocked the downregulation of TLR4, p-p65, and Bax/Bcl-2 ratio. This suggested that the inhibition of TLR4 signaling by IFN-τ was mediated by the PI3K/AKT pathway. Furthermore, compared with the LPS group, the β-catenin agonist SB216763 led to greater p-FoxO1 and lower p-p65 and cell apoptosis. In contrast, knockdown of β-catenin using small interfering RNA had the opposite effects. To explore the role of FoxO1 on the inhibition of TLR4 by IFN-τ, we employed LY294002 to inhibit the PI3K/AKT while FoxO1 was knocked down. Results revealed that the knockdown of FoxO1 blocked the upregulation of TLR4 and p-p65 induced by LY294002, and enhanced the inhibition of IFN-τ on TLR4, p-p65, and cell apoptosis. Overall, these findings confirmed that IFN-τ can protect endometrial epithelial cells against inflammatory injury via suppressing TLR4 activation through the regulation of the PI3K/AKT/β-catenin/FoxO1 axis. These represent new insights into the molecular mechanisms underlying the anti-inflammatory function of IFN-τ in BEEC, and also provide a theoretical basis for further studies on the in vivo application of IFN-τ to help prevent negative effects of endometritis.

ABSTRACT: Bovine mastitis is the most common and costly disease affecting dairy cattle throughout the world. Enterococcus faecalis is one of the environmental origin mastitis-causing pathogens. The treatment of bovine mastitis is primarily based on antibiotics. Due to the negative impact of developing antibiotic resistance and adverse effects on soil and water environments, the trend toward use of nonantibiotic treatments is increasing. Phages may represent a promising alternative treatment strategy. However, it is unknown whether phages have therapeutic effects on E. faecalis-induced mastitis. Thus, the objective of this study was to investigate the degree of protection conferred by a phage during murine mastitis caused by multidrug-resistant E. faecalis. Enterococcus faecalis was isolated from the milk of dairy cows with mastitis, and a phage was isolated using the E. faecalis isolates as hosts. The bactericidal ability of the phage against E. faecalis and the ability to prevent biofilm formation were determined in vitro. The therapeutic potential of the phage on murine mastitis was evaluated in vivo. We isolated 14 strains of E. faecalis from the milk of cows with mastitis, all of which exhibited multidrug resistance, and most (10/14) could form strong biofilms. Subsequently, a new phage (EF-N13) was isolated using the multidrug-resistant E. faecalis N13 (isolated from mastitic milk) as the host. The phage EF-N13 belongs to the family Myoviridae, which has short latent periods (5 min) and high bursts (284 pfu/cell). The genome of EF-N13 lacked bacterial virulence-, antibiotic resistance-, and lysogenesis-related genes. Furthermore, bacterial loading in the raw milk medium was significantly reduced by EF-N13 and was unaffected by potential IgG antibodies. In fact, EF-N13 could effectively prevent the formation of biofilm by multidrug-resistant E. faecalis. All of these characteristics suggest that EF-N13 has potential as mastitis therapy. In vivo, 1 × 105 cfu/gland of multidrug-resistant E. faecalis N13 resulted in mastitis development within 24 h. A single dose of phage EF-N13 (1 × 104, 1 × 105, or 1 × 106 pfu/gland) could significantly decrease bacterial counts in the mammary gland at 24 h postinfection. Histopathological observations demonstrated that treatment with phage EF-N13 effectively alleviated mammary gland inflammation and damage. This effect was confirmed by the lower levels of proinflammatory cytokines IL-6, IL-1β, and tumor necrosis factor-α in the mammary gland treated with phage EF-N13 compared with those treated with phosphate-buffered saline. Overall, the data underscored the potential of phage EF-N13 as an alternative therapy for bovine mastitis caused by multidrug-resistant E. faecalis.

ABSTRACT: Hypocalcemia in dairy cows is associated with a decrease of neutrophil adhesion and phagocytosis, an effect driven partly by changes in the expression of store-operated Ca2+ entry (SOCE)-related molecules. It is well established in nonruminants that neutrophils obtain the energy required for immune function through glycolysis. Whether glycolysis plays a role in the acquisition of energy by neutrophils during hypocalcemia in dairy cows is unknown. To address this relationship, we performed a cohort study and then a clinical trial. Neutrophils were isolated at 2 d postcalving from lactating Holstein dairy cows (average 2.83 ± 0.42 lactations, n = 6) diagnosed as clinically healthy (CON) or with plasma concentrations of Ca2+

ABSTRACT: During the transition period in dairy cows, high circulating concentrations of nonesterified fatty acids (NEFA) increase hepatic lipid deposits and are considered a major pathological factor for liver damage. We investigated whether AdipoRon, a synthetic small-molecule agonist of adiponectin receptors 1 and 2 shown to prevent liver lipid accumulation in nonruminants, could alleviate NEFA-induced lipid accumulation and mitochondrial dysfunction. Bovine hepatocytes were isolated from 5 healthy Holstein female newborn calves (1 d of age, 30–40 kg, fasting), and independently isolated hepatocytes from at least 3 different calves were used for each subsequent experiment. The composition and concentration of NEFA used in this study were selected according to hematological criteria of dairy cows with fatty liver or ketosis. First, hepatocytes were cultured with various concentrations of NEFA (0, 0.6, 1.2, or 2.4 mM) for 12 h. In a second experiment, hepatocytes were treated with AdipoRon at different concentrations (0, 5, 25, or 50 μM for 12 h) and times (25 μM for 0, 6, 12, or 24 h) with or without NEFA (1.2 mM) treatment. In the last experiment, hepatocytes were treated with AdipoRon (25 μM), NEFA (1.2 mM), or both for 12 h after treatment with or without the autophagy inhibitor chloroquine. Hepatocytes treated with NEFA had increased protein abundance of sterol regulatory element-binding protein 1c (SREBP-1c) and mRNA abundance of acetyl-CoA carboxylase 1 (ACACA), and decreased protein abundance of peroxisome proliferator-activated receptor α (PPARA), proliferator-activated receptor gamma coactivator-1 α (PGC-1α), mitofusin 2 (MFN2), cytochrome c oxidase subunit IV (COX IV), and mRNA abundance of carnitine palmitoyltransferase 1A (CPT1A), along with lower ATP concentrations. AdipoRon treatment reversed these effects, suggesting this compound had a positive effect on lipid metabolism and mitochondrial dysfunction during the NEFA challenge. In addition, upregulated expression of microtubule-associated protein 1 light chain 3-II (LC3-II, encoded by MAP1LC3) and downregulated expression of sequestosome-1 (SQSTM1, also called p62) indicated that AdipoRon enhanced autophagic activity in hepatocytes. The fact that chloroquine impeded the beneficial effects of AdipoRon on lipid accumulation and mitochondrial dysfunction suggested a direct role for autophagy during NEFA challenge. Our results suggest that autophagy is an important cellular mechanism to prevent NEFA-induced lipid accumulation and mitochondrial dysfunction in bovine hepatocytes, which is consistent with other studies. Overall, AdipoRon may represent a promising therapeutic agent to maintain hepatic lipid homeostasis and mitochondrial function in dairy cows during the transition period.

ABSTRACT: Fatty liver is a major metabolic disorder of high-producing dairy cows during the transition period. In nonruminants, it is well established that insulin-induced gene 1 (INSIG1) plays a crucial role in regulating hepatic lipogenesis by controlling the anchoring of sterol regulatory element-binding protein 1 (SREBP-1) on the endoplasmic reticulum along with SREBP cleavage-activating protein (SCAP). Whether the INSIG1-SCAP-SREBP-1c transport axis is affected in cows experiencing fatty liver is unknown. Thus, the aim of this study was to investigate the potential role of INSIG1-SCAP-SREBP-1c axis in the progression of fatty liver in dairy cows. For in vivo experiments, 24 dairy cows at the start of their fourth lactation (median; range 3–5) and 8 d in milk (median; range 4–12 d) were selected into a healthy group [n = 12; triglyceride (TG) content 10%) according to their hepatic TG content. Blood samples were collected for detecting serum concentrations of free fatty acids, β-hydroxybutyrate, and glucose. Compared with healthy cows, cows with severe fatty liver had higher serum concentrations of β-hydroxybutyrate and free fatty acids and lower concentration of glucose. Liver biopsies were used to detect the status of INSIG1-SCAP-SREBP-1c axis, and the mRNA expression of SREBP-1c-target lipogenic genes acetyl-CoA carboxylase α (ACACA), fatty acid synthase (FASN), and diacylglycerol acyltransferase 1 (DGAT1). Cows with severe fatty liver had lower protein expression of INSIG1 in the hepatocyte endoplasmic reticulum fraction, greater protein expression of SCAP and precursor SREBP-1c in the hepatocyte Golgi fraction, and greater protein expression of mature SREBP-1c in the hepatocyte nuclear fraction. In addition, the mRNA expression of SREBP-1c-target lipogenic genes ACACA, FASN, and DGAT1 was greater in the liver of dairy cows with severe fatty liver. In vitro experiments were conducted on hepatocytes isolated from 5 healthy 1-d-old female Holstein calves, and hepatocytes from each calf were run independently. First, hepatocytes were treated with 0, 200, or 400 μM palmitic acid (PA) for 12 h. Exogenous PA treatment decreased INSIG1 protein abundance, enhanced the endoplasmic reticulum to Golgi export of SCAP–precursor SREBP-1c complex and the nuclear translocation of mature SREBP-1c, all of which was associated with increased transcriptional activation of lipogenic genes and TG synthesis. Second, hepatocytes were transfected with INSIG1-overexpressing adenovirus for 48 h and treated with 400 μM PA 12 h before the end of transfection. Overexpressing INSIG1 inhibited PA-induced SREBP-1c processing, upregulation of lipogenic genes, and TG synthesis in hepatocytes. Overall, the present in vivo and in vitro results indicated that the low abundance of INSIG1 contributed to SREBP-1c processing and hepatic steatosis in dairy cows. Thus, the INSIG1-SCAP-SREBP-1c axis may be a novel target for treatment of fatty liver in dairy cows.

ABSTRACT: Cholesterol in the circulation is partly driven by changes in feed intake, but aspects of cholesterol metabolism during development of fatty liver are not well known. The objective of this study was to investigate mechanisms of cholesterol metabolism in calf hepatocytes challenged with high concentrations of fatty acids (FA). To address mechanistic insights regarding cholesterol metabolism, liver samples were collected from healthy control dairy cows (n = 6; 7–13 d in milk) and cows with fatty liver (n = 6; 7–11 d in milk). In vitro, hepatocytes isolated from 3 healthy female calves (1 d old) were challenged with or without a mix of 1.2 mM FA to induce metabolic stress. In addition, hepatocytes were processed with 10 µmol/L of the cholesterol synthesis inhibitor simvastatin or 6 µmol/L of the cholesterol intracellular transport inhibitor U18666A with or without the 1.2 mM FA mix. To evaluate the role of cholesterol addition, hepatocytes were treated with 0.147 mg/mL methyl-β-cyclodextrin (MβCD + FA) or 0.147 mg/mL MβCD with or without 10 and 100 µmol/L cholesterol before incubation with FA (CHO10 + FA and CHO100 + FA). In vivo data from liver biopsies were analyzed by 2-tailed unpaired Student's t-test. Data from in vitro calf hepatocytes were analyzed by one-way ANOVA. Compared with healthy cows, blood plasma total cholesterol and plasma low-density lipoprotein cholesterol content in cows with fatty liver was markedly lower, whereas the hepatic total cholesterol content did not differ. In contrast, compared with healthy controls, the triacylglycerol content in the liver and the content of FA, β-hydroxybutyrate, and aspartate aminotransferase in the plasma of cows with fatty liver were greater. The results revealed that both fatty liver in vivo and challenge of calf hepatocytes with 1.2 mM FA in vitro led to greater mRNA and protein abundance of sterol regulatory element binding transcription factor 1 (SREBF1) and fatty acid synthase (FASN). In contrast, mRNA and protein abundance of sterol regulatory element binding transcription factor 2 (SREBF2), acyl coenzyme A-cholesterol acyltransferase, and ATP-binding cassette subfamily A member 1 (ABCA1) were lower. Compared with the FA group, the cholesterol synthesis inhibitor simvastatin led to greater protein abundance of microsomal triglyceride transfer protein and mRNA abundance of SREBF2, 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), ACAT2, and lower ABCA1 and FASN protein abundance. In contrast, compared with the FA group, the cholesterol intracellular transport inhibitor U18666A + FA led to greater total cholesterol concentration and greater protein and mRNA abundance of FASN. Compared with the MβCD + FA group, the addition of 10 µmol/L cholesterol led to greater concentration of cholesteryl ester and excretion of apolipoprotein B100, and greater protein and mRNA abundance of ABCA1 and microsomal triglyceride transfer protein, and lower concentration of malondialdehyde. Overall, a reduction in cholesterol synthesis promoted FA metabolism in hepatocytes likely to relieve the oxidative stress caused by the high FA load. The data suggest that maintenance of normal cholesterol synthesis promotes very low-density lipoprotein excretion and can reduce lipid accumulation and oxidative stress in dairy cows that experience fatty liver.

ABSTRACT: The extent to which a nutrition-related disorder such as ketosis alters the ruminal microbiota or whether microbiota composition is related to ketosis and potential associations with host metabolism is unknown. We aimed to evaluate variations occurring in the ruminal microbiota of ketotic and nonketotic cows in the early postpartum period, and how those changes may affect the risk of developing the disease. Data on milk yield, dry matter intake (DMI), body condition score, and blood β-hydroxybutyrate (BHB) concentrations at 21 d postpartum were used to select 27 cows, which were assigned (n = 9 per group) to a clinical ketotic (CK, 4.10 ± 0.72 mmol BHB/L, DMI 11.61 ± 0.49 kg/d, ruminal pH 7.55 ± 0.07), subclinical ketotic (SK, 1.36 ± 0.12 mmol BHB/L, DMI 15.24 ± 0.34 kg/d, ruminal pH 7.58 ± 0.08), or control (NK, 0.88 ± 0.14 mmol BHB/L, DMI 16.74 ± 0.67/d, ruminal pH 7.61 ± 0.03) group. Cows averaged 3.6 ± 0.5 lactations and a body condition score of 3.11 ± 0.34 at the time of sampling. After blood serum collection for metabolomics analysis (1H nuclear magnetic resonance spectra), 150 mL of ruminal digesta was collected from each cow using an esophageal tube, paired-end (2 × 300 bp) sequencing of isolated DNA from ruminal digesta was performed via Illumina MiSeq, and sequencing data were analyzed using QIIME2 (v 2020.6) to measure the ruminal microbiota composition and relative abundance. Spearman correlation coefficients were used to evaluate relationships between relative abundance of bacterial genera and concentrations of serum metabolites. There were more than 200 genera, with approximately 30 being significant between NK and CK cows. Succinivibrionaceae UCG 1 taxa decreased in CK compared with NK cows. Christensenellaceae (Spearman correlation coefficient = 0.6), Ruminococcaceae (Spearman correlation coefficient = 0.6), Lachnospiraceae (Spearman correlation coefficient = 0.5), and Prevotellaceae (Spearman correlation coefficient = 0.6) genera were more abundant in the CK group and were highly positively correlated with plasma BHB. Metagenomic analysis indicated a high abundance of predicted functions related to metabolism (37.7%), genetic information processing (33.4%), and Brite hierarchies (16.3%) in the CK group. The 2 most important metabolic pathways for butyrate and propionate production were enriched in CK cows, suggesting increased production of acetyl coenzyme A and butyrate and decreased production of propionate. Overall, the combined data suggested that microbial populations may be related to ketosis by affecting short-chain fatty acid metabolism and BHB accumulation even in cows with adequate feed intake in the early postpartum period.

ABSTRACT: Fatty liver (i.e., hepatic lipidosis) is a prevalent metabolic disorder in dairy cows during the transition period, characterized by excess hepatic accumulation of triglyceride (TG), tissue dysfunction, and cell death. Detailed pathological changes, particularly hepatic fibrosis, during fatty liver remain to be determined. Liver fibrosis occurs as a consequence of liver damage, resulting from the excessive accumulation of extracellular matrix, which distorts the architecture of the normal liver, compromising its normal synthetic and metabolic functions. Thus, we aimed to investigate liver fibrosis status and its potential causal factors including oxidative stress, hepatocyte apoptosis, and production of inflammatory cytokines in the liver of cows with fatty liver. Forty-five dairy cows (parity, 3–5) were selected, and liver biopsy and blood were collected on the second week postpartum (days in milk, 10–14 d). On the basis of the degree of lipid accumulation in liver, selected cows were categorized into normal (n = 25; TG

ABSTRACT: Excessive inflammation in bovine mammary endothelial cells (BMEC) due to mastitis leads to disease progression and eventual culling of cattle. Sirtuin 3 (SIRT3), a mitochondrial deacetylase, downregulates pro-inflammatory cytokines in BMEC exposed to high concentrations of nonesterified fatty acids by blunting nuclear factor-κB (NFκB) signaling. In nonruminants, SIRT3 is under the control of PGC1α, a transcriptional cofactor. Specific aims were to study (1) the effect of SIRT3 on inflammatory responses of lipopolysaccharide (LPS)-challenged bovine mammary epithelial cells (bovine mammary alveolar cells-T, MAC-T) models, and (2) the role of PGC1α in the attenuation of NFκB signaling via SIRT3. To address these objectives, first, MAC-T cells were incubated in triplicate with 0, 50, 100, 150, or 200 μg/mL LPS (derived from Escherichia coli O55:B5) for 12 h with or without a 2-h incubation of the NFκB inhibitor ammonium pyrrolidine dithiocarbamate (APDC, 10 μM). Second, SIRT3 was overexpressed using adenoviral expression (Ad-SIRT3) at different multiplicity of infection (MOI) for 6 h followed by a 12 h incubation with 150 μg/mL LPS. Third, cells were treated with the PGC1α agonist ZLN005 (10 μg/mL) for 24 h and then challenged with 150 μg/mL LPS for 12 h. Fourth, cells were initially treated with the PGC1α inhibitor SR-18292 (100 μM) for 6 h followed by a 6-h culture with or without 50 MOI Ad-SIRT3 and a challenge with 150 μg/mL LPS for 12 h. Data were analyzed using one-way ANOVA with subsequent Bonferroni correction. Linear and quadratic contrasts were used to determine dose-responses to LPS. There were linear and quadratic effects of LPS dosage on cell viability. Incubation with 150 and 200 μg/mL LPS for 12 h decreased cell viability to 78.6 and 34.9%, respectively. Compared with controls, expression of IL1B, IL6, and TNFA was upregulated by 5.2-, 5.9-, and 2.7-fold with 150 μg/mL LPS; concentrations of IL-1β, IL-6, and tumor necrosis factor-α (TNF-α) in cell medium also increased. Compared with the LPS group, LPS+APDC increased cell viability and reversed the upregulation of IL1B, IL6, and TNFA expression. However, mRNA and protein abundance of SIRT3 decreased linearly with increasing LPS dose. Ad-SIRT3 infection (50 MOI) reduced IL1B, IL6, and TNFA expression and also their concentrations in cell medium, and decreased pNFκB P65/NFκB P65 ratio and nuclear abundance of NFκB P65. The PGC1α agonist increased SIRT3 expression, whereas it decreased cytokine expression, pNFκB P65/NFκB P65 ratio, and prevented NFκB P65 nuclear translocation. Contrary to the agonist, the PGC1α inhibitor had opposite effects, and elevated the concentrations of IL-1β, IL-6, and TNF-α in cell medium. Overall, data suggested that SIRT3 activity could attenuate LPS-induced inflammatory responses in mammary cells via alterations in the PGC1α-NFκB pathway. As such, there may be potential benefits for targeting SIRT3 in vivo to help prevent or alleviate negative effects of mastitis.

ABSTRACT: Adipose tissue of ketotic dairy cows exhibits greater lipolytic rate and signs of inflammation, which further aggravate the metabolic disorder. In nonruminants, the endoplasmic reticulum (ER) is a key organelle coordinating metabolic adaptations and cellular functions; thus, disturbances known as ER stress lead to inflammation and contribute to metabolic disorders. Enhanced activity of diacylglycerol O-acyltransferase 1 (DGAT1) in murine adipocytes undergoing lipolysis alleviated ER stress and inflammation. The aim of the present study was to investigate the potential role of DGAT1 on ER stress and inflammatory response of bovine adipose tissue in vivo and in vitro. Adipose tissue and blood samples were collected from cows diagnosed as clinically ketotic (n = 15) or healthy (n = 15) following a veterinary evaluation based on clinical symptoms and serum concentrations of β-hydroxybutyrate, which were 4.05 (interquartile range = 0.46) and 0.52 mM (interquartile range = 0.14), respectively. Protein abundance of DGAT1 was greater in adipose tissue of ketotic cows. Among ER stress proteins measured, ratios of phosphorylated PKR-like ER kinase (p-PERK) to PERK and phosphorylated inositol-requiring enzyme 1 (p-IRE1) to IRE1, and protein abundance of cleaved ATF6 protein were greater in adipose tissue of ketotic cows. Furthermore, ratios of phosphorylated RELA subunit of NF-κB (p-RELA) to RELA and phosphorylated c-jun N-terminal kinase (p-JNK) to JNK were greater, whereas protein abundance of NF-κB inhibitor α (NFKBIA) was lower in adipose tissue of ketotic cows. In addition, mRNA abundance of proinflammatory cytokines including TNF and IL-6 was greater in adipose tissue of ketotic cows. To better address mechanistic aspects of these responses, primary bovine adipocytes isolated from the harvested adipose tissue of healthy cows were subjected to lipolysis-stimulating conditions via incubation with 1 μM epinephrine (EPI) for 2 h. In another experiment, adipocytes were cultured with DGAT1 overexpression adenovirus and DGAT1 small interfering RNA for 48 h, respectively, followed by EPI (1 μM) exposure for 2 h. Treatment with EPI led to greater ratios of p-PERK to PERK, p-IRE1 to IRE1, p-RELA to RELA, p-JNK to JNK, and cleaved ATF6 protein, whereas EPI stimulation inhibited protein abundance of NFKBIA. Furthermore, treatment with EPI upregulated the secretion of proinflammatory cytokines into culture medium, including TNF-α and IL-6. Overexpression of DGAT1 in EPI-treated adipocytes attenuated ER stress, the activation of NF-κB and JNK signaling pathways, and the secretion of inflammatory cytokines. In contrast, silencing DGAT1 further aggravated EPI-induced ER stress and inflammatory responses. Overall, these data indicated that activation of DGAT1 may act as an adaptive mechanism to dampen metabolic dysregulation in adipose tissue. As such, it contributes to relief from ER stress and inflammatory responses.

ABSTRACT: Peripartum dairy cows experience negative energy balance, characterized by high concentrations of blood free fatty acids (FFA) and immune dysfunction. Palmitic acid (PA), the most abundant saturated fatty acid in cow blood, is not only an energy precursor, but causes cellular dysfunction when in excess. Neutrophil extracellular traps (NET) are one of the arsenals of weapons neutrophils use to fight invading pathogens. However, given the marked increase in circulating PA during the peripartum period, it remains to be determined what effect (if any) PA has on NET release. Thus, the objective of this study was to evaluate the effect of PA on NET release and the underlying mechanism in vitro. Phorbol-12-myristate-13-acetate (PMA; 100 ng/mL, 3 h) was used to induce the release of NET in vitro. We isolated neutrophils from the peripheral blood of 5 healthy postpartum dairy cows with similar parity (median = 3, range = 2–4), milk yield (median = 27.84 kg/d per cow, range = 25.79–31.43 kg/d per cow), days in milk (median = 7 d, range = 4–10 d), and serum FFA 3.5 mM. Inhibition of double-stranded DNA (dsDNA) level, a marker of NET release, in response to PA was used to determine an optimal incubation time and concentration for in vitro experiments. Cells were maintained in RPMI-1640 basic medium without phenol red, treated with 600 μM PA for different times (4, 5, 6, and 7 h) in the presence or absence of PMA. There was a decrease for dsDNA level in the supernatant due to increased duration of PA treatment, with a peak response at 6 h. Thus, 6 h was selected as the challenge time. Then, cells were treated with different concentrations of PA (100, 200, 400, and 600 μM) for 6 h in the presence or absence of PMA. There was a decrease for dsDNA level in the supernatant due to increased dose of PA, with a peak response at 400 μM. Finally, 400 μM PA for 6 h was selected as the treatment for subsequent experiments. Protein abundance of citrullinated histone in the presence or absence of PMA was markedly lower in response to incubation with PA. Morphological observations by laser confocal microscopy and scanning electron microscopy showed that the ratio of NET-releasing cells decreased in response to incubation with PA. Autophagy is a potential key intermediate process in the regulation of NET by PA. To investigate the effect of PA on autophagy, we used chloroquine to block lysosomal degradation. Exogenous PA led to accumulation of sequestosome-1 and microtubule-associated protein 1 light chain 3-II, and no further accumulation in the presence of chloroquine, all of which suggested an impairment of autophagic flux. To verify the role of autophagy in NET, we used rapamycin to promote autophagic flux; 100 nM rapamycin attenuated the suppressive effect of PA on NET release indicated by greater dsDNA levels, accumulation of citrullinated histone, and ratio of NET-releasing neutrophils. Overall, these data demonstrate PA inhibits NET release by suppressing autophagic flux, which provides information for understanding the immune dysfunction in postpartum cows.

ABSTRACT: Sustained lipolysis and insulin resistance increase the risk of metabolic dysfunction in dairy cows during the transition period. Proinflammatory cytokines are key regulators of adipose tissue metabolism in nonruminants, but biological functions of these molecules in ruminants are not well known. Thus, the objective of this study was to investigate whether tumor necrosis factor-α (TNF-α) could affect insulin sensitivity and lipolysis in bovine adipocytes as well as the underlying mechanisms. Bovine adipocytes (obtained from the omental and mesenteric adipose depots) isolated from 5 Holstein female calves (1 d old) with similar body weight (median: 36.9 kg, range: 35.5–41.2 kg) were differentiated and used for (1) treatment with different concentrations of TNF-α (0, 0.1, 1, or 10 ng/mL) for 12 h; (2) pretreatment with 10 μM lipolytic agonist isoproterenol (ISO) for 3 h, followed by treatment with or without 10 ng/mL TNF-α for 12 h; and (3) pretreatment with the c-Jun N-terminal kinase (JNK) inhibitor SP600125 (20 μM for 2 h) and nuclear factor kappa B (NF-κB) inhibitor BAY 11–7082 (10 μM for 1 h) followed by treatment with or without 10 ng/mL TNF-α for 12 h. The TNF-α increased glycerol content in supernatant, decreased triglyceride content and insulin-stimulated phosphorylation of protein kinase B suggesting activation of lipolysis and impairment of insulin sensitivity. The TNF-α reduced cell viability, upregulated mRNA abundance of Caspase 3 (CASP3), an apoptosis marker, and increased activity of Caspase 3. In addition, increased phosphorylation of NF-κB and JNK, upregulation of mRNA abundance of interleukin-6 (IL-6), TNFA, and suppressor of cytokine signaling 3 (SOCS3) suggested that TNF-α activated NF-κB and JNK signaling pathways. Furthermore, ISO plus TNF-α-activated NF-κB and JNK signaling pathway to a greater extent than TNF-α alone. Combining TNF-α and ISO aggravated TNF-α-induced apoptosis, insulin insensitivity and lipolysis. In the absence of TNF-α, inhibition of NF-κB and JNK did not alter glycerol content in supernatant, triglyceride content or insulin-stimulated phosphorylation of protein kinase B. In the presence of TNF-α, inhibition of NF-κB and JNK alleviated TNF-α-induced apoptosis, insulin insensitivity and lipolysis. Overall, TNF-α impairs insulin sensitivity and induces lipolysis and apoptosis in bovine adipocytes, which may be partly mediated by activation of NF-κB and JNK. Thus, the data suggested that NF-κB and JNK are potential therapeutic targets for alleviating lipolysis dysregulation and insulin resistance in adipocytes.

ABSTRACT: Mitochondria are the main site of fatty acid oxidation and reactive oxygen species (ROS) formation. Damaged or dysfunctional mitochondria induce oxidative stress and increase the risk of lipid accumulation. During the process of mitophagy, PTEN induced kinase 1 (PINK1) accumulates on damaged mitochondria and recruits cytoplasmic Parkin to mitochondria. As an autophagy receptor protein, sequestosome-1 (p62) binds Parkin-ubiquitinated outer mitochondrial membrane proteins and microtubule-associated protein 1 light chain 3 (LC3) to facilitate degradation of damaged mitochondria. In nonruminants, clearance of dysfunctional mitochondria through the PINK1/Parkin-mediated mitophagy pathway contributes to reducing ROS production and maintaining metabolic homeostasis. Whether PINK1/Parkin-mediated mitophagy plays a similar role in dairy cow liver is not well known. Thus, the objective of this study was to investigate mitophagy status in dairy cows with fatty liver and its role in free fatty acid (FFA)-induced oxidative stress and lipid accumulation. Liver and blood samples were collected from healthy dairy cows (n = 10) and cows with fatty liver (n = 10) that had a similar number of lactations (median = 3, range = 2 to 4) and days in milk (median = 6 d, range = 3 to 9 d). Calf hepatocytes were isolated from 5 healthy newborn female Holstein calves (1 d of age, 30–40 kg). Hepatocytes were transfected with small interfering RNA targeted against PRKN for 48 h or transfected with PRKN overexpression plasmid for 36 h, followed by treatment with FFA (0.3 or 1.2 mM) for 12 h. Mitochondria were isolated from fresh liver tissue or calf hepatocytes. Serum concentrations of β-hydroxybutyrate were higher in dairy cows with fatty liver. Hepatic malondialdehyde (MDA) and hydrogen peroxide (H2O2) were greater in cows with fatty liver. The lower protein abundance of PINK1, Parkin, p62, and LC3-II in hepatic mitochondrial fraction of dairy cows with fatty liver indicated the mitophagy was impaired. In hepatocytes, knockdown of PRKN decreased protein abundance of p62 and LC3-II in the mitochondrial fraction, and increased contents of triacylglycerol (TG), MDA, and H2O2. In addition, protein abundances of PINK1, Parkin, p62, and LC3-II were lower in the mitochondrial fraction from hepatocytes treated with 1.2 mM FFA than the hepatocytes treated with 0.3 mM FFA, whereas the content of TG, MDA, and H2O2 increased. In 1.2 mM FFA-treated hepatocytes, PRKN overexpression increased protein abundance of p62 and LC3-II in the mitochondrial fraction and decreased contents of TG, MDA, and H2O2. Together, our data demonstrate that low abundance of mitophagy markers is associated with ROS overproduction in dairy cows with fatty liver and impaired mitophagy induced by a high concentration of FFA promotes ROS production and lipid accumulation in female calf hepatocytes.

ABSTRACT: Ketosis is a common metabolic disorder in peripartal dairy cows that is caused by excessive mobilization of fat and incomplete hepatic metabolism of fatty acids (FFA). Recent data in nonruminant models revealed that sortilin 1 (SORT1) is involved in a variety of lipid metabolism–related diseases. It plays important roles in the regulation of triglyceride (TAG) and total cholesterol (TC) levels. In this study, we first used liver biopsies from healthy cows (serum β-hydroxybutyrate concentration 3.0 mM) to assess alterations in cholesterol synthesis, transport, and excretion. Then, to assess mechanistic links between SORT1 and fatty acid–mediated cholesterol metabolism, hepatocytes isolated from 4 healthy female calves (1 d old, 35–45 kg) were challenged with or without a mixture of free fatty acids (FFA; 1.2 mM) to induce metabolic stress. Hepatocytes were then treated with empty adenovirus vectors (with green fluorescent protein; Ad-GFP) or with SORT1-overexpressing adenovirus (Ad-SORT1) for 6 h or with SORT1 inhibitor (SORT1i) for 2 h, followed by a challenge with (Ad-GFP+FFA, Ad-SORT1+FFA, or SORT1i+FFA) or without (Ad-GFP, Ad-SORT1, or SORT1i) 1.2 mM FFA mixture for 12 h. Data analysis of calf hepatocyte treatment comparisons were assessed by 2-way ANOVA, and multiplicity for each experiment was adjusted using the Bonferroni procedure. Expression levels of factors related to cholesterol synthesis, transport, and excretion in liver tissue of cows with ketosis was lower. Hepatocytes challenged with FFA had lower concentrations of TC and mRNA and protein abundances of sterol regulatory element-binding protein 2 (SREBF2), acetyl acyl coenzyme A-cholesterol acyltransferase 2 (ACAT2), ATP-binding cassette transporter A1 (ABCA1), ABC subfamily G member 5 (ABCG5), and ABC subfamily G member 8 (ABCG8). Compared with FFA challenge alone, SORT1i + FFA led to greater protein abundance of 3-hydroxy-3-methyl-glutaryl coenzyme A reductase (HMGCR), ACAT2, and ABCG5, and greater mRNA abundance of ABCG5. Compared with FFA challenge alone, SORT1 overexpression led to lower protein abundance of SREBF2. In contrast, protein abundance of ABCA1 was greater. Overall, our data suggested that exogenous FFA induced abnormal cholesterol metabolism in hepatocytes, whereas a high abundance of SORT1 affected cholesterol esterification and potentially influx into bile. Thus, downregulation of hepatic SORT1 might be a cholesterol-regulated protective mechanism in the presence of a marked increase in FFA.

ABSTRACT: Subacute ruminal acidosis (SARA), a common digestive disease in dairy cows, is accompanied by systemic inflammation and high concentrations of histamine in blood. Histamine-induced neutrophil adhesion may play an important role in the systemic inflammation experienced by cows during SARA. Autophagy, an intracellular degradation system, regulates recycling of membrane-associated integrin and may be involved in histamine-induced adhesion of bovine neutrophils. In the present study, 20 multiparous mid-lactation cows (average body weight 486 ± 24 kg) fitted with ruminal fistula were assigned to a control group (n = 10) or a SARA group (n = 10). We induced SARA by feeding different combinations of wheat-barley pellets and chopped alfalfa hay for 8 wk; SARA was defined as a ruminal pH

ABSTRACT: Endoplasmic reticulum (ER) stress can be induced by various stimuli and triggers the unfolded protein response to activate intracellular signaling pathways that are mediated by 3 ER-resident sensors: inositol requiring protein-1α (IRE1α), PKR-like ER kinase (PERK), and activating transcription factor-6 (ATF6). In nonruminants, ER stress plays a critical role in hepatic insulin resistance. However, whether ER stress plays a role in nonesterified fatty acid (NEFA)-induced hepatic insulin resistance in dairy cows is still unknown. Experiments were conducted using primary bovine hepatocytes isolated from 5 healthy calves (body weight: 30–40 kg; 1 d old). First, hepatocytes were treated with NEFA (1.2 mM) for 0.5, 1, 2, 3, 5, 7, 9, or 12 h. Treatment with NEFA elevated abundance of phosphorylated IRE1α and PERK, and cleavage of ATF6, along with the ER stress-associated genes XBP1, ATF4, and DNAJC3, resulting in both linear and quadratic effects. Furthermore, ER Tracker red staining and transmission electron microscopy results indicated that ER was dilated and degranulated in response to NEFA treatment, suggesting that ER stress was induced by NEFA treatment in bovine hepatocytes. Second, to assess the effect of ER stress on NEFA-induced insulin resistance, hepatocytes were treated with different concentrations of NEFA (0, 0.6, 1.2, or 2.4 mM) for 5 h with or without tauroursodeoxycholic acid (TUDCA, a canonical inhibitor of ER stress). Here, NEFA induced insulin resistance by increasing the abundance of insulin receptor substrate-1 (IRS1) phosphorylation at the inhibitory residue Ser 307 (S307) and decreasing the abundance of phosphorylated protein kinase B (AKT) and glycogen synthase kinase-3β (GSK3β) in a dose-dependent manner. This was accompanied by upregulation of an abundance of gluconeogenic genes [phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6-Pase)]. These detrimental effects of NEFA on insulin signaling could be reversed with TUDCA treatment, indicating a mechanistic link between ER stress and NEFA-induced insulin resistance. In a third experiment, pGPU6/GFP/Neo vectors containing short hairpin RNA targeting IRE1α were used to silence IRE1α transcription, and GSK2656157 (PERK phosphorylation inhibitor) and 4-(2-aminoethyl) benzenesulfonyl fluoride (AEBSF; an inhibitor of ATF6) were used to block PERK and ATF6 branches, respectively. Notably, the silencing of the IRE1α branch improved NEFA-induced insulin resistance by decreasing phosphorylation of IRS1 (S307) and increasing phosphorylation of AKT and GSK3β, and reducing PEPCK and G6-Pase mRNA abundance, which was likely dependent on IRE1α kinase activity. Similarly, blockage of the PERK branch increased phosphorylation of AKT and GSK3β, and reduced PEPCK and G6-Pase mRNA abundance, but had no effect on phosphorylation of IRS1 (S307). However, results showed that inhibition of the ATF6 branch had no effects on phosphorylation of IRS1, AKT, and GSK3β, and instead found increasing PEPCK and G6-Pase mRNA abundance. Taken together, data in the present study found that impeding IRE1α and PERK signaling might aid in relieving hepatic insulin resistance. However, the more detailed mechanisms of how IRE1α and PERK signaling contribute to hepatic insulin resistance in dairy cows remain to be determined.

ABSTRACT: When ketosis occurs, supraphysiological levels of free fatty acids (FFA) can cause oxidative injury to the mammary gland and autophagy can regulate the cellular oxidative status. The aim of this study was to investigate the autophagy status of mammary tissue and its associations with oxidative stress in healthy and clinically ketotic dairy cows. Mammary tissue and blood samples were collected from healthy cows [n = 15, β-hydroxybutyrate (BHB) 3.0 mM) at 3 to 15 (average = 7) days in milk. For in vitro study, bovine mammary epithelial cells (BMEC) isolated from healthy cows were treated with 0, 0.3, 0.6, or 1.2 mM FFA for 24 h. Furthermore, BMEC were pretreated with 100 nM rapamycin, an autophagy activator, for 4 h or 50 mM 3-methyladenine (3-MA), an autophagy inhibitor, for 1 h, followed by treatment with or without FFA (1.2 mM) for another 24 h. Oxidation indicators and autophagy-related protein abundance were measured. Compared with healthy cows, serum concentrations of FFA, BHB, and malondialdehyde were greater in clinically ketotic cows, but milk production (kg/d), milk protein (kg/d), activities of superoxide dismutase, catalase, and glutathione peroxidase were lower. Abundances of mRNA and protein of autophagy-related gene 5 (ATG5) and 7 (ATG7) were lower, but sequestosome-1 (SQSTM1, also called p62) greater in mammary tissue of clinically ketotic cows. The mRNA abundance of microtubule-associated protein 1 light chain 3 (MAP1LC3, also called LC3) and protein abundance of LC3-II were lower in mammary tissue of clinically ketotic cows. In vitro, exogenous FFA increased the content of malondialdehyde and reactive oxygen species, but decreased the activities of superoxide dismutase, catalase, and plasma glutathione peroxidase. Compared with the 0 mM FFA group, abundance of ATG5, ATG7, LC3-II was greater, but p62 was lower in the 0.6 mM FFA-treated cells. Similarly, abundance of ATG5, ATG7, and LC3-II was lower, but p62 greater in the 1.2 mM FFA-treated cells relative to 0 mM FFA group. Culture with rapamycin alleviated oxidative stress induced by 1.2 mM FFA, whereas 3-MA aggravated it. Overall, results indicated that a low concentration (0.6 mM) of FFA can induce oxidative stress and activate autophagy in BMEC. At higher concentrations of FFA (1.2 mM), autophagy is impaired and oxidative stress is aggravated. Autophagy is a mechanism for BMEC to counteract FFA-induced stress. As such, it could serve as a potential target for further development of novel strategies against oxidative stress.

ABSTRACT: High circulating concentrations of fatty acids cause triacylglycerol (TAG) accumulation in hepatocytes of dairy cows, a common metabolic disorder after calving. Low secretion of apolipoprotein B (APOB) and very low density lipoprotein (VLDL) are thought to be the major factors for TAG accumulation in hepatocytes. Recent data in nonruminant models revealed that sortilin 1 (SORT1) is a key regulator of VLDL secretion in part due to its ability to bind APOB. Thus, SORT1 could play a role in the susceptibility of dairy cows to develop fatty liver. To gain mechanistic insights in vivo and in vitro, we performed experiments using liver biopsies or isolated primary hepatocytes. For the in vivo study, blood and liver samples were collected from healthy multiparous dairy cows (n = 6; 9.0 ± 2.1 d in milk) and cows with fatty liver (n = 6; 9.7 ± 2.2 d in milk). In vitro, hepatocytes isolated from 4 healthy female calves (1 d old, 42–51 kg) were challenged with (fatty acids) or without (control) a 1.2 mM mixture of fatty acids in an attempt to induce metabolic stress. Furthermore, hepatocytes were treated with empty adenovirus vectors (Ad-GFP) or SORT1 overexpressing adenovirus (Ad-SORT1) for 6 h, or SORT1 inhibitor for 2 h followed by a challenge with (Ad-GFP + fatty acids, Ad-SORT1 + fatty acids, or SORT1 inhibitor + fatty acids) or without (Ad-GFP, Ad-SORT1, or SORT1 inhibitor) the 1.2 mM mixture of fatty acids for 12 h. Data from liver biopsies were compared using a 2-tailed unpaired Student's t-test. Data from calf hepatocytes were analyzed by one-way ANOVA. Data revealed that both fatty liver and in vitro challenge with fatty acids were associated with greater concentrations of TAG and mRNA and protein abundance of SORT1, SREBF1, FASN, and ACACA. In contrast, mRNA and protein abundance of CPT1A and APOB, and mRNA abundance of MTTP were markedly lower. Compared with fatty acid challenge alone, SORT1 overexpression led to greater concentration of TAG and mRNA abundance of SREBF1, FASN, ACACA, DGAT1, and DGAT2, and protein abundance of SREBF1, FASN, and ACACA. In contrast, concentration of secreted VLDL-APOB and mRNA abundance of APOB and MTTP, and protein abundance of CPT1A, APOB, and MTTP were lower. Compared with fatty acid challenge alone, SORT1 inhibitor + fatty acids led to lower concentrations of TAG and mRNA abundance of SREBF1, FASN, and DGAT2, and protein abundance of FASN, ACACA, and DGAT1. Concentrations of secreted VLDL-APOB and mRNA abundance of CPT1A and protein abundance of CPT1A and APOB were greater. Overall, in vitro data suggested that greater SORT1 abundance induced by exogenous fatty acids caused a reduction in VLDL-APOB secretion and increased hepatocyte TAG synthesis. Such mechanism was also apparent in tissue from cows with fatty liver. Thus, targeted downregulation of hepatic SORT1 could represent a viable mechanism to unload lipid during conditions where the influx of fatty acids increases markedly.

ABSTRACT: Ketosis occurs most frequently in the peripartal period and is associated with liver injury and steatosis. Lysosomes serve as the terminal degradative station and contribute to liver homeostasis through their role in the digestion of dysfunctional organelles and lipid droplets. Transcription factor EB (TFEB) has been identified as a master regulator of lysosomal function. Thus, the objective of the present study was to investigate the status of lysosomal function and TFEB transcriptional activity and potential changes in abundance of upstream effectors of TFEB identified in nonruminants, including mechanistic target of rapamycin kinase complex 1 (mTORC1), protein kinase B (Akt), glycogen synthase kinase β (GSK3β), and extracellular signal-regulated kinase1/2 (ERK1/2), and to explore which factor induces the above changes. Liver and blood samples were collected from healthy cows (n = 10) and ketotic cows (n = 10) that had a similar number of lactations (median = 3, range = 2–4) and days in milk (median = 6 d, range = 3–9 d). Calf hepatocytes were isolated from Holstein calves and treated with 10 ng/mL growth hormone (GH), 3.0 mM β-hydroxybutyrate (BHB), 1.5 ng/mL interleukin-18 (IL-18), 0.15 ng/mL tumor necrosis factor-α (TNF-α), or 1.2 mM free fatty acid (FFA) for 12 h. Serum levels of FFA and activities of alanine aminotransferase and aspartate aminotransferase were greater in ketotic cows, whereas glucose was lower. Additionally, ketotic dairy cows exhibited higher serum concentrations of GH, IL-18, and TNF-α, and lower serum concentration of insulin. The lower protein abundance of lysosome-associated membrane protein 1 (LAMP1) and mRNA abundance of LAMP1 indicated that hepatic lysosomal mass was lower in ketotic cows. Furthermore, lower protein abundance of cathepsin D (CTSD) and mRNA abundance of CTSD and V0 domain of the vacuolar ATPase along with lower activity of β-N-acetylglucosaminidase indicated impairment in hepatic lysosomal function due to ketosis. The lower nuclear abundance, total protein, and mRNA abundance of TFEB and peroxisome proliferator-activated receptor γ coactivator 1 α along with greater phosphorylated (p)-TFEB in the liver of ketotic cows indicated an impairment of hepatic TFEB transcriptional activity. The protein abundances of phosphorylated mTOR (p-mTOR) and its downstream effectors ribosomal protein S6 kinase B (RPS6KB) and eukaryotic factor 4E-binding protein 1 (EIF4EBP1) were greater, whereas p-Akt, p-GSK3β, and p-ERK1/2 were lower in the liver of ketotic cows. Importantly, elevated phosphorylation of mTOR, RPS6KB, and EIF4EBP1 was observed in calf hepatocytes treated with GH, BHB, IL-18, TNF-α, and FFA. Moreover, BHB, TNF-α, and FFA, not GH and IL-18, reduced TFEB transcriptional activity and impaired lysosomal function in calf hepatocytes. Taken together, these data suggest that BHB, TNF-α, and FFA overactivate the hepatic mTORC1 signaling pathway during ketosis and further impaired TFEB transcriptional activity and lysosomal function, which may contribute to liver injury and steatosis.

ABSTRACT: Reduced feed intake during the transition period renders cows unable to meet their energy needs for maintenance and lactation, leading to a state of negative energy balance. Severe negative energy balance initiates fat mobilization and increases circulating levels of free fatty acids (FFA), which could induce hepatic mitochondrial dysfunction, oxidative stress, and apoptosis. Enhancing the hepatic supply of propionate (major gluconeogenic substrate) is a feasible preventive and therapeutic strategy to alleviate hepatic metabolic disorders during the transition period. Whether propionate supply affects pathways beyond gluconeogenesis during high FFA loads is not well known. Thus, the objective of this study was to investigate whether propionate supply could protect calf hepatocytes from FFA-induced mitochondrial dysfunction, oxidative stress, and apoptosis. Hepatocytes were isolated from 5 healthy calves (1 d old, female, 30–40 kg, fasting) and treated with various concentrations of propionate (0, 1, 2, and 4 mM propionate for 12 h) or for different times (2 mM propionate for 0, 3, 6, 12 and 24 h). Furthermore, hepatocytes were treated with propionate (2 mM), fatty acids (1.2 mM), or both for 12 h with or without 50 nM PGC-1α (peroxisome proliferator-activated receptor-gamma coactivator-1 alpha) small interfering RNA. Compared with the control group, protein abundance of PGC-1α was greater with 2 and 4 mM propionate treatment groups. Furthermore, protein abundance of TFAM (mitochondrial function marker mitochondrial transcription factor A) and VDAC1 (voltage-dependent anion channel 1) was greater with 1, 2, and 4 mM propionate, and COX4 (cyclooxygenase 4) was greater with 2 and 4 mM propionate groups. In addition, propionate supply led to an increase in protein abundance of PGC-1α, TFAM, VDAC1, and COX4 over time. Flow cytometry revealed that propionate treatment increased the number of mitochondria in hepatocytes compared with control group, but inhibition of PGC-1α abolished these beneficial effects. The lower protein abundance of PGC-1α, TFAM, COX4, and VDAC1 and activities of superoxide dismutase and glutathione peroxidase, along with greater production of reactive oxygen species, malondialdehyde, and apoptosis rate in response to treatment with high concentrations of FFA suggested an impairment of mitochondrial function and induction of oxidative stress and apoptosis. In contrast, propionate treatment hastened these negative effects. Knockdown of PGC-1α by small interfering RNA impeded the beneficial role of propionate on FFA-induced mitochondrial dysfunction, oxidative stress, and apoptosis. Overall, results demonstrated that propionate supply alleviates mitochondrial dysfunction, oxidative stress, and apoptosis in FFA-treated calf hepatocytes by upregulating PGC-1α. Together, the data suggest that PGC-1α may be a promising target for preventing or improving hepatic function during periods such as the transition into lactation where the FFA load on the liver increases.

ABSTRACT: Ketosis in dairy cows often occurs in the peripartal period and is accompanied by immune dysfunction. High concentrations of β-hydroxybutyrate (BHB) in peripheral blood during ketosis inhibits the release of neutrophil extracellular traps (NET) and contributes to immune dysfunction. However, the mechanisms whereby BHB affects NET release remains unclear. In this study, 5 healthy peripartal dairy cows (within 3 wk postpartum) with serum BHB concentrations 3.5 mM were used as blood donors. Blood samples were collected before feeding, and the isolated polymorphonuclear neutrophils were incubated with 3 mM BHB for different times. Inhibition of Cit-H3 (citrullinated histone 3) protein abundance, a marker of NET activation, in response to BHB was used to determine an optimal incubation time for in vitro experiments. Four hours was selected as the optimal duration of BHB treatment. Phorbol-12-myristate-13-acetate (PMA) was used to induce the release of NET in vitro. The BHB treatment with or without PMA treatment decreased protein abundance of Cit-H3 and PAD4 (arginine deiminase 4) and increased neutrophil elastase. Immunofluorescence and scanning electron microscope analyses revealed that BHB treatment inhibited PMA-induced NET release. The BHB treatment also decreased double strain DNA content in the supernatant, further confirming the inhibitory effect of BHB on NET release. Furthermore, BHB treatment decreased the level of intracellular reactive oxygen species (ROS), phosphorylation level of p47, and protein abundance of Rac2, suggesting that BHB-induced NET inhibition may have been caused by decreased NADPH oxidase-derived ROS. The phosphorylation level of phosphoinositide 3-kinase (PI3K), an important upstream regulator of NADPH oxidase, was attenuated by BHB treatment. To confirm the involvement of PI3K signaling pathway in BHB-induced NET inhibition, 740Y-P, a potent activator of PI3K signaling pathway, was used. Data indicated that 740Y-P relieved the inhibitory effects of BHB on ROS production and NADPH oxidase activation. Importantly, as revealed by immunofluorescence and scanning electron microscopy analyses, 740Y-P also dampened the inhibitory effect of BHB on NET release and the protein abundance of Cit-H3 and PAD4. Overall, the present study revealed that high concentration of BHB impairs NET release through inhibiting PI3K-mediated NADPH oxidase ROS production. These findings help partly explain the immune dysfunction in cows experiencing negative energy balance or ketosis in early lactation.

ABSTRACT: Ketosis in dairy cows, a common metabolic disorder during the peripartal period, is accompanied by systemic inflammation and high concentrations of blood β-hydroxybutyrate (BHB). Neutrophil apoptosis plays a key role in maintaining the balance of inflammation and functional capacity of circulating neutrophils in ketotic cows. The kinases ERK1/2 and AKT, as well as their downstream Bcl-2 family-mediated mitochondrial signaling, are important apoptosis-regulating pathways in neutrophils. The objective of our study was to investigate the effects of BHB on neutrophil apoptosis and the underlying regulatory mechanisms during ketosis. Neutrophils were isolated from 5 multiparous cows (within 3 wk postpartum) with serum BHB concentrations 3.5 mM. In a series of experiments, neutrophils were treated with increasing concentrations of BHB (0, 0.6, 2, and 3 mM for 10 h) and time (0, 2, 4, 6, 8, and 10 h with 2 mM). Subsequently, a 2 mM BHB dose was used to challenge neutrophils for 8 h. Apoptosis rate of neutrophils and protein abundance of cleaved caspase 3 were lower after BHB treatment. Treatment with BHB decreased protein and mRNA abundance of the pro-apoptotic genes Bax (BAX) and Bad (BAD), whereas it increased mitochondrial membrane potential (MMP) and protein and mRNA of the anti-apoptotic genes Bcl-xL (BCL2L1) and Mcl-1 (MCL1). This indicated that a mitochondrial pathway was involved in the inhibition of neutrophil apoptosis via BHB. In addition, both SCH772984 (an inhibitor of the ERK1/2 signaling pathway) and MK-2206 (an inhibitor of the AKT signaling pathway) alleviated the BHB-induced anti-apoptotic function of the Bcl-2 family and the inhibition of MMP. Overall, our data demonstrated that high concentrations of BHB inhibit apoptosis in bovine neutrophils by activating the ERK1/2 and AKT signaling pathways. These findings provide a theoretical basis for the understanding of systemic inflammation in ketotic cows.

ABSTRACT: Hypocalcemia in dairy cows is associated with decreased neutrophil phagocytosis, adhesion capacity, migration, and reactive oxygen species (ROS) production through alterations in ORAI calcium release-activated calcium modulator 1 (ORAI1). Neutrophils can resist the invasion of pathogenic microorganisms by releasing neutrophil extracellular traps (NET). However, the mechanisms controlling NET formation during hypocalcemia are unknown. To address the role of ORAI1 in NET formation, neutrophils were isolated at 2 d postcalving from lactating Holstein dairy cows (n = 10 per group) diagnosed as clinically healthy (control) or with plasma concentrations of Ca2+

ABSTRACT: Subclinical ketosis (SCK) in dairy cows, a common metabolic disorder during the peripartal period, is accompanied by systemic inflammation. Excessive release of azurophil granule (AG) contents during degranulation of polymorphonuclear neutrophils (PMN) could contribute to systemic inflammation in SCK cows. Although the increase in blood free fatty acids (FFA) in SCK cows may promote AG degranulation from PMN, the underlying mechanisms are unclear. Thirty multiparous cows (within 3 wk postpartum) with similar lactation numbers (median = 3, range = 2–4) and days in milk (median = 6, range = 3–15) were classified based on serum β-hydroxybutyrate (BHB) level as control (n = 15, BHB < 0.6 mM) or SCK (n = 15, 1.2 mM < BHB < 3.0 mM). Cows with SCK had greater levels of serum haptoglobin, serum amyloid A, IL-1β, IL-6, IL-8 and tumor necrosis factor-α. These proinflammatory factors had strong positive correlations with myeloperoxidase (MPO), a marker protein of PMN AG, whose content was greater in the serum of SCK cows. Both the number of AG and the protein abundance of MPO were lower in PMN isolated from SCK cows. Additionally, we found a greater ratio of blood CH138A+/CD63high cells and greater mean fluorescence intensity of CD63 on the PMN membrane, further confirming the greater degree of AG degranulation in cows with SCK. In vitro FFA dose response (0, 0.3, 0.6, 1.2, and 2.4 mM for 4 h) and time course (0, 0.5, 1, 2, and 4 h with 0.6 mM) experiments were performed on PMN isolated from control cows. The increase in MPO content in extracellular supernatant resulting from those experiments led to the selection of 0.6 mM FFA for 1 h duration as conditions for subsequent studies. After FFA treatment, release of intracellular MPO was increased along with increased levels of CD63 mean fluorescence intensity on the PMN membrane, confirming that FFA promoted degranulation of AG. In addition, FFA treatment increased reactive oxygen species (ROS) production by PMN, an effect that was attenuated by incubation with diphenyleneiodonium chloride (DPI), a NADPH oxidase-derived ROS inhibitor. The mitochondrial-derived ROS inhibitor carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP) did not affect ROS in response to FFA treatment. Treatment with FFA increased p47 phosphorylation and mRNA abundance of NCF1, NCF2, and CYBB in PMN. Furthermore, DPI, but not FCCP, dampened the degranulation of PMN AG induced by FFA in vitro. These data suggested that the degranulation of AG in PMN induced by FFA was mediated by NADPH oxidase-derived ROS. As verified ex vivo, PMN from SCK cows had greater levels of ROS, phosphorylation of p47, and mRNA abundance of NCF1, NCF2, and CYBB. Overall, the present study revealed that high blood concentrations of FFA in SCK cows induce the production of NADPH oxidase-derived ROS, thereby promoting degranulation of AG in PMN. The stimulatory effect of FFA on the release of AG content during degranulation, especially MPO, provides a new insight into the systemic inflammation experienced by peripartal cows with SCK.

ABSTRACT: Ketosis in dairy cows often occurs in the peripartal period and is accompanied by immune dysfunction. High concentrations of β-hydroxybutyrate (BHB) in peripheral blood during ketosis are closely related to the impairment of polymorphonuclear neutrophil (PMN) chemotaxis and contribute to immune dysfunction. The specific effect of BHB on PMN chemotaxis in dairy cows and the underlying molecular mechanisms are unclear. Here, 30 multiparous cows (within 3 wk postpartum) classified based on serum BHB as control (n = 15, BHB 3.0 mM) were used. Blood samples were collected before feeding, and the isolated PMN were treated with platelet-activating factor for 0.5 h to activate their migration. Scanning electron microscopy revealed a longer tail in PMN of ketotic cows. In addition, the phosphorylation and transcription levels of myosin light chain 2 (MLC2) increased in PMN of ketotic cows. Polymorphonuclear neutrophils from control dairy cows were incubated with 3.0 mM BHB for different times in vitro, and 6 h was selected as the proper duration of BHB stimulation according to its inhibition effect on PMN migration using an under-agarose PMN chemotaxis model. Similarly, BHB stimulation in vitro resulted in inhibition of migration distance and deviation of migration direction of PMN, as well as a longer tail in morphology in the scanning electron microscope data, suggesting that BHB-induced PMN migration inhibition may be mediated by impairing the trailing edge contraction. To confirm this hypothesis, sotrastaurin (Sotra)—a specific inhibitor of protein kinase C (PKC), which is the core regulator of cell contraction—was used with or without BHB treatment in vitro. Sotra was pretreated 0.5 h before BHB treatment. Accordingly, BHB treatment increased the phosphorylation level of PKC and MLC2, the protein abundance of RhoA and rho-kinase 1 (ROCK1), and the mRNA abundance of PRKCA, MYL2, RHOA, and ROCK1 in PMN. In contrast, these effects of BHB on PMN were dampened by Sotra. As demonstrated by immunofluorescence experiments in vitro, the BHB-induced inhibition of trailing edge contraction of PMN was relieved by Sotra. In addition, Sotra also dampened the effects of BHB on PMN migration in vitro. Furthermore, as verified by in vivo experiments, compared with the control cows, both abundance and activation of PKC signaling were enhanced in PMN of ketotic cows. Overall, the present study revealed that high concentrations of blood BHB impaired PMN migration distance through inhibition of the trailing edge contraction, mediated by enhancing the activation of PKC-MLC2 signaling. These findings help explain the dysfunctional immune state in ketotic cows and provide information on the pathogenesis of infectious diseases secondary to ketosis.