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High-yielding dairy cows in early lactation often encounter difficulties in meeting the energy requirements essential for maintaining milk production. This is primarily attributed to insufficient dry matter intake, which consequently leads to sustained lipolysis of adipose tissue. Fatty acids released by lipolysis can disrupt metabolic homeostasis. Autophagy, an adaptive response to intracellular environmental changes, is considered a crucial mechanism for regulating lipid metabolism and maintaining a proper cellular energy status. Despite its close relationship with aberrant lipid metabolism and cytolipotoxicity in animal models of metabolic disorders, the precise function of diacylglycerol o-acyltransferase 1 (DGAT1) in bovine adipose tissue during periods of negative energy balance is not fully understood, particularly regarding its involvement in lipolysis and autophagy. The objective of the present study was to assess the effect of DGAT1 on both lipolysis and autophagy in bovine adipose tissue and isolated adipocytes. 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 BHB, which were 3.19 mM (interquartile range = 0.20) and 0.50 mM (interquartile range = 0.06), respectively. Protein abundance of DGAT1 and phosphorylation levels of unc-51-like kinase 1 (ULK1), were greater in adipose tissue from cows with ketosis, whereas phosphorylation levels of phosphoinositide 3-kinase (PI3K), protein kinase B (AKT), and mammalian target of rapamycin (mTOR) were lower. Furthermore, when adipocytes isolated from the harvested adipose tissue of 15 healthy cows were transfected with DGAT1 overexpression adenovirus or DGAT1 small interfering RNA followed by exposure to epinephrine (EPI), it led to greater ratios and protein abundance of phosphorylated hormone-sensitive triglyceride lipase (LIPE) to total LIPE and adipose triglyceride lipase (ATGL), while inhibiting the protein phosphorylation levels of ULK1, PI3K, AKT, and mTOR. Overexpression of DGAT1 in EPI-treated adipocytes reduced lipolysis and autophagy, whereas silencing DGAT1 further exacerbated EPI-induced lipolysis and autophagy. Taken together, these findings indicate that upregulation of DGAT1 may function as an adaptive response to suppress adipocytes lipolysis, highlighting the significance of maintaining metabolic homeostasis in dairy cows during periods of negative energy balance., (The Authors. Published by Elsevier Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

Excessive concentrations of free fatty acids (FFA) are the main factors causing immune dysfunction and inflammation in dairy cows with ketosis. Polarization of macrophages (the process of macrophages freely switching from one phenotype to another) into M1 or M2 phenotypes is an important event during inflammation induced by environmental stimuli. In non-ruminants, mammalian target of rapamycin (mTOR)-mediated autophagy (a major waste degradation process) regulates macrophage polarization. Thus, the objective was to unravel the role of mTOR-mediated autophagy on macrophage polarization in ketotic dairy cows. Four experiments were performed as follows: (1) In vitro differentiated monocyte-derived macrophages from healthy dairy cows or dairy cows with clinical ketosis (CK) were treated with 100 ng/mL lipopolysaccharide (LPS) and 100 ng/mL interferon-γ (IFN-γ) or 10 ng/mL interleukin-4 (IL4) and 10 ng/mL interleukin-10 (IL10) for 24 h; (2) Immortalized bovine macrophages were treated with 0, 0.3, 0.6, 1.2 mM FFA and LPS and IFN-γ or IL4 and IL10 for 24 h; (3) Macrophages were pretreated with 2 μM 4,6-dimorpholino-N-(4-nitrophenyl)-1,3,5-triazin-2-amine (MHY1485) for 30 min before treatment with LPS and IFN-γ or IL4 and IL10; (4) Macrophages were pretreated with 100 nM rapamycin (RAPA) for 2 h before treatment with LPS and IFN-γ or IL4 and IL10. Compared with healthy cows, cows with CK had a greater mean fluorescence intensity (MFI) of CD86 + , but lower MFI of CD206 + and lower number of autophagosomes and autolysosomes in macrophages. Exogenous FFA treatment upregulated protein abundance of inducible nitric oxide synthase (iNOS) and mean fluorescence intensity of CD86, whereas it downregulated the protein abundance of arginase 1 (ARG1) and mean fluorescence intensity of CD206. In addition, FFA increased the p-p65/p65 protein abundance and tumor necrosis factor α (TNFA), interleukin-1B (IL1B), and interleukin-6 (IL6) mRNA abundance, but decreased LC3-phosphatidylethanolamine conjugate (LC3-II) protein abundance and autophagosomes and autolysosomes number. Pretreatment with MHY1485 promoted macrophage M1 polarization and inhibited macrophage M2 polarization via decreased mTOR-mediated autophagy. Activation of mTOR-mediated autophagy by pretreatment with RAPA attenuated the upregulation of inflammation in M1 macrophages that was induced by FFA. These data revealed that high concentrations of FFA promote macrophage M1 polarization in ketotic dairy cows via impairing mTOR-mediated autophagy., (The Authors. Published by Elsevier Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

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., (The Authors. Published by Elsevier Inc. and Fass Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

Bovine mammary epithelial cells undergo an increase in metabolic rate, mitochondrial dysfunction, and oxidative stress after calving. Nuclear factor erythroid 2-related factor 2 (NFE2L2), a master regulator of cellular redox homeostasis, plays crucial roles in the regulation of mitochondrial function. The objective of this study was to investigate the role of NFE2L2 on mitochondrial function in bovine mammary epithelial cells under hyperlipidemic conditions. Three experiments were conducted as follows: (1) the immortalized bovine mammary epithelial cell line MAC-T was treated with various concentrations of free fatty acids (FFA; 0, 0.6, 1.2, or 2.4 mM) for 24 h to induce stress; (2) MAC-T cells were transfected with small interfering RNA targeting NFE2L2 (si-NFE2L2) and scrambled nontarget negative control (si-Control) for 48 h; and (3) MAC-T cells were pretreated with 10 μM sulforaphane (SFN), an activator of NFE2L2, for 24 h followed by treatment with 1.2 mM FFA for an additional 24 h. Results indicated that exogenous FFA challenge induced linear and quadratic increases in concentrations of mitochondrial reactive oxygen species (ROS). Compared with 0 mM FFA, mitochondrial membrane potential, mRNA abundance of oxidative phosphorylation complexes (CO I-V), protein abundance of nuclear respiratory factor 1 (NRF1), peroxisome proliferator-activated receptor γ coactivator 1 α (PGC-1α), mitochondrial transcription factor A (TFAM), and NFE2L2 along with the contents of ATP, mitochondrial DNA (mtDNA), and total mitochondria were greater in the MAC-T challenged with 0.6 mM FFA group, but lower in the 1.2 and 2.4 mM FFA cultures. Knockdown of NFE2L2 via small interfering RNA led to greater mitochondrial ROS content and lower mitochondrial membrane potential along with contents of ATP, mtDNA, and total mitochondria. The SFN pretreatment upregulated protein abundance of NFE2L2 and attenuated the downregulation of NFE2L2 induced by FFA. Pretreatment with SFN attenuated the downregulation induced by FFA of PGC-1α, NRF1, and TFAM protein abundance along with contents of mtDNA and total mitochondria. Furthermore, SFN pretreatment attenuated the upregulation of mitochondrial ROS content, the downregulation of mitochondrial membrane potential, and the decreases in ATP, mtDNA, and mitochondrial content induced by FFA. Overall, data indicated that FFA inhibit NFE2L2, resulting in mitochondrial dysfunction and ROS production in bovine mammary epithelial cells. Thus, NFE2L2 may be a promising therapeutic target against metabolic challenge-driven mitochondrial dysfunction and oxidative stress in bovine mammary epithelial cells., (Copyright © 2021 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Dairy cows with ketosis exhibit signs of pronounced adipose tissue lipolysis and systemic inflammation, both of which exacerbate this metabolic disorder. In nonruminants, CIDEC plays a pivotal role in the formation of large unilocular lipid droplets. The present study aimed to ascertain the role of CIDEC in the lipolytic and inflammatory response of white adipose tissue (WAT) in vivo and in vitro. Subcutaneous adipose tissue and blood samples were collected from 15 healthy cows (blood β-hydroxybutyrate concentration < 1.2 mM) and 15 cows with clinical ketosis (blood β-hydroxybutyrate concentration > 3.0 mM) that had a similar number of lactations (median = 3, range = 2-4) and days in milk (median = 6 d, range = 3-9). Adipocytes isolated from 5 healthy Holstein calves (1 d old, female, 30-40 kg) were used for in vitro studies. Isolated adipocytes were treated with 0, 0.1, 1, or 10 ng/mL TNF-α for 3 h, transfected with CIDEC small interfering RNA for 48 h, or transfected with CIDEC overexpression adenovirus for 48 h followed by treatment with TNF-α (0.1 ng/mL) for 3 h. Serum concentrations of fatty acids were greater, and dry matter intake, milk yield, and serum glucose concentrations lower in cows with clinical ketosis. Protein and mRNA abundance of CIDEC were lesser in subcutaneous WAT of clinically ketotic versus healthy cows. Furthermore, the ratio of phosphorylated hormone sensitive lipase (p-LIPE) to LIPE, phosphorylated RELA (p-RELA) to RELA, and protein abundance of PNPLA2 and phosphorylated inhibitor of κBα (p-NFKBIA) were greater in dairy cows with clinical ketosis. The mRNA abundance of proinflammatory cytokines TNFA and IL1B were greater, and the anti-inflammatory cytokine IL10 was lower in WAT of dairy cows with clinical ketosis. In calf adipocytes, exogenous TNF-α (0.1, 1, or 10 ng/mL) decreased protein and mRNA abundance of CIDEC. In addition, exogenous TNF-α or knockdown of CIDEC reduced the secretion of the anti-inflammatory cytokine IL-10, but increased the ratio of p-LIPE to LIPE, p-RELA to RELA, protein abundance of PNPLA2 and p-NFKBIA, glycerol content, and the secretion of IL-1β in calf adipocytes. Overexpression of CIDEC in TNFα-treated adipocytes attenuated lipolysis and activation of the NF-κB signaling pathway. Overall, these data suggest that inhibition of lipid droplet-associated protein CIDEC by TNF-α contributes to the pronounced lipolysis and inflammation of calf adipocytes, and CIDEC is a relevant target in clinically ketotic cows., (Copyright © 2021 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Adipose tissue concentration of reactive oxygen species (ROS) increases in dairy cows with ketosis, suggesting that the tissue experiences oxidative stress. Autophagy, an adaptive response to cellular stress, has been shown to promote survival and plays a critical role in antioxidant responses. Dysregulation of adenosine 5'-monophosphate-activated protein kinase (AMPK) is closely related to antioxidant responses and autophagy of adipocytes in animal models of metabolic disorders, but its role in bovine adipose tissue during periods of stress is unknown. We hypothesized that AMPK may play important roles in the regulation of oxidative stress in adipose tissue of ketotic cows. Specific objectives were to evaluate autophagy status and AMPK activity in adipose tissue of ketotic cows, and their link with oxidative stress in isolated bovine adipocytes. Selection of 15 healthy and 15 clinically ketotic Holstein cows at 17 (±4) d postpartum was performed after a thorough veterinary evaluation for clinical symptoms and also based on serum β-hydroxybutyrate concentrations before collection of subcutaneous adipose tissue samples. Primary cultures of bovine adipocytes isolated from the harvested adipose tissue were stimulated with varying concentrations of H 2 O 2 (0, 50, 100, 200, or 400 μM) for 2 h. In another experiment, adipocytes were cultured with the AMPK activator A769662 or adenovirus-containing small interfering RNA (ad-AMPKα-siRNA) for 3 or 48 h, respectively, followed by H 2 O 2 exposure (200 μM) for 2 h. Compared with healthy cows, clinical ketosis led to increased abundance of AMPK and nuclear factor erythroid-derived 2-like 2 (NFE2L2), but lower abundance of Kelch-like ECH-associated protein 1 (KEAP1) in adipose tissue. Abundance of the key proautophagy proteins Beclin1, sequestosome 1 (SQSTM1), autophagy-related gene 7 (ATG7), ATG5, and ratio of microtubule-associated protein light chain 3 (LC3) II to LC3I were greater in adipose tissue of ketotic cows. In bovine adipocytes, treatment with H 2 O 2 induced accumulation of ROS and malondialdehyde (MDA), whereas H 2 O 2 stimulation inhibited activities of the antioxidant enzymes glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD). Addition of AMPK activator A769662 increased antioxidant response via activating NFE2L2 and its downstream targets heme oxygenase 1 (HMOX1), superoxide dismutase 1 (SOD1), catalase (CAT), and glutathione-S-transferase (GST) to improve H 2 O 2 -induced oxidative stress in adipocytes. Simultaneously, activation of AMPK increased abundance of Beclin1, SQSTM1, ATG7, ATG5, and ratio of LC3II to LC3I. In contrast, inhibition of AMPK downregulated abundance of NFE2L2, HMOX1, SOD1, CAT, Beclin1, SQSTM1, ATG7, ATG5, and ratio of LC3II to LC3I, and further aggravated H 2 O 2 -induced oxidative stress. Overall, these data indicate that activation of AMPK, as an adaptive mechanism for acute metabolic regulation of adipose tissue homeostasis, can induce antioxidant responses and autophagy, and further reduce oxidative stress in bovine adipocytes., (The Authors. Published by Elsevier Inc. and Fass Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).)

Preadipocyte proliferation and differentiation are critical for normal adipose tissue development, including achieving a mature phenotype, characterized by its ability to accumulate triacylglycerol and release fatty acids. In nonruminants, it is well known that all-trans retinoic acid (ATRA), the most-active form of vitamin A, helps regulate proliferation, differentiation, and apoptosis in several types of cells including adipocytes. The purpose of this study was to evaluate the effect of ATRA on proliferation, apoptosis, differentiation, and lipolysis of primary bovine adipocytes isolated from subcutaneous adipose tissue of 5 healthy Holstein cows at 17 (±4 standard deviations) d postpartum. Cells were stimulated with increasing concentrations of ATRA (0.2, 2, and 20 nM) at the preconfluent (2 d) and postconfluent (8 d) preadipocyte stage or at the mature adipocyte stage (2 d). All concentrations of ATRA inhibited preconfluent preadipocyte proliferation with decreased proportion of S-phase cells and reduced protein abundance of cyclins (CCND1, CCND2, CCND3, CCNE1) and cyclin-dependent kinases (CDK2, CDK4, CDK6). Compared with vehicle, ATRA treatment induced apoptosis in preconfluent preadipocytes. Additionally, ATRA (0.2, 2, and 20 nM) supplementation also inhibited differentiation of postconfluent preadipocytes through downregulation of protein abundance of PPARγ and C/EBPα. After induction of differentiation, basal lipolysis in mature adipocytes increased upon treatment with all concentrations of ATRA. However, data on phosphorylated hormone-sensitive lipase or PLIN1 indicated that ATRA had no effect on epinephrine-stimulated lipolysis in mature adipocytes. Overall, these results demonstrate that ATRA might inhibit lipid accumulation by suppressing preadipocyte proliferation and differentiation, subsequently leading to apoptosis in postconfluent preadipocytes and promoting basal lipolysis in mature adipocytes. Overall, these in vitro responses provide some insights into the potential for nutritional management to modulate adipose tissue lipolysis, particularly in overconditioned cows during the dry period, which are more susceptible to suffer metabolic disorders due to excessive fat mobilization postpartum., (The Authors. Published by Elsevier Inc. and Fass Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).)

Ketosis is a serious metabolic disorder characterized by systemic and hepatic oxidative stress, inflammation, and apoptosis, as well as reduced milk yield. Because of the paucity of data on mammary responses during ketosis, the aim of this study was to evaluate alterations in oxidative stress, NF-κB signaling, NLRP3 inflammasome, and caspase apoptotic pathways in mammary gland of dairy cows with ketosis. Blood, mammary gland tissue, and milk samples were collected from healthy cows [Control, blood concentration of β-hydroxybutyrate (BHB) <0.6 mM, n = 10] and cows with subclinical ketosis (SCK, blood concentration of BHB >1.2 mM and <3 mM, n = 10) or clinical ketosis (CK, blood concentration of BHB >3 mM, n = 10) at median 8 d in milk (range = 6-12). Compared with Control, serum concentration of glucose was lower (3.91 vs. 2.86 or 2.12 mM) in cows with SCK or CK, whereas concentrations of fatty acids (0.25 vs. 0.57 or 1.09 mM) and BHB (0.42 vs. 1.81 or 3.85 mM) were greater. Compared with Control, the percentage of milk fat was greater in cows with SCK or CK. In contrast, the percentage of milk protein was lower in cows with SCK or CK. We detected no differences in milk lactose content across groups. Compared with Control, activities of glutathione peroxidase, superoxide dismutase, and catalase were lower in mammary gland tissue of cows with SCK or CK. In contrast, concentrations of hydrogen peroxide and malondialdehyde were greater in cows with SCK or CK. Compared with Control, mRNA abundances of TNFA, IL6, and IL1B were greater in mammary tissues of cows with SCK or CK. In addition, activity of IKKβ and the ratio of phosphorylated inhibitor of κBα to IκBα, and of phosphorylated NF-κB p65 to NF-κB p65, were also greater in mammary tissues of cows with SCK or CK. Subclinical or clinical ketosis also led to greater activity of caspase 1 and protein abundance of caspase 1, NLRP3, Bax, caspase 3, and caspase 9. In contrast, abundance of the antiapoptotic protein was lower in SCK or CK cows. The data indicate that the mammary gland of SKC or CK cows undergoes severe oxidative stress, inflammation, and cell death., (The Authors. Published by Elsevier Inc. and Fass Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).)

Dairy cows with fatty liver exhibit hepatic lipid accumulation and disturbances in fatty acid oxidation and lipid transport. Phosphatase and tensin homolog (PTEN), a lipid phosphatase, regulates intrahepatic fatty acid oxidation and lipid transport in mice. Whether PTEN play a role in fatty acid oxidation and very low density lipoprotein (VLDL) assembly in calf hepatocytes are unknown. Hepatocytes isolated from 3 healthy female Holstein calves (1 d old, 30-40 kg) were infected with empty adenovirus with green fluorescent protein for 48 h (Ad-GFP group) or infected with PTEN knockdown adenovirus for 48 h (Ad-shPTEN group), or cultured in RPMI-1640 without Ad-shPTEN or Ad-GFP (control group). Compared with the Ad-GFP group, PTEN knockdown decreased mRNA and protein abundance and the activity of fatty acid oxidation-related molecules, including acyl-coA synthetase long-chain 1, carnitine palmitoyltransferase 1, carnitine palmitoyltransferase 2, and 3-hydroxy acyl-coA dehydrogenase. Furthermore, PTEN knockdown decreased mRNA and protein abundance of VLDL assembly-related molecules, including apolipoprotein B100, apolipoprotein E, microsomal triglyceride transfer protein, and low density lipoprotein receptor. Importantly, PTEN knockdown promoted triglyceride accumulation in hepatocytes and reduced the VLDL content in culture medium. A subsequent study was conducted on the following 4 groups: cells infected with Ad-GFP for 48 h and then treated with 2% BSA for another 24 h (Ad-GFP + BSA); cells infected with Ad-GFP for 48 h and then treated with 1.2 mM free fatty acids (FFA) and 2% BSA for another 24 h (Ad-GFP + 1.2 mM FFA); cells infected with Ad-shPTEN for 48 h and then treated with 2% BSA for another 24 h (Ad-shPTEN + BSA); cells infected with Ad-shPTEN for 48 h and then treated with 1.2 mM FFA and 2% BSA for another 24 h (Ad-shPTEN + 1.2 mM FFA). Compared with Ad-GFP + BSA, the abundances of PTEN and of fatty acid oxidation- and VLDL assembly-related proteins were lower in the Ad-GFP + 1.2 mM FFA group. Importantly, PTEN knockdown heightened the increase in triglyceride accumulation of hepatocytes and the decrease in VLDL content in culture medium induced by FFA. Overall, these in vitro data indicate that FFA inhibits PTEN expression, leading to triglyceride accumulation and the inhibition of VLDL assembly in calf hepatocytes. These findings suggest that PTEN may be a potential therapeutic target for FFA-induced hepatic steatosis in dairy cows., (Copyright © 2020 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Exogenous molecules derived from catabolic states (e.g., fatty acids, β-hydroxybutyrate) during periods of stress such as the periparturient period or pathogen challenges [e.g., lipopolysaccharide (LPS)] can trigger an inflammatory response in tissues such as the liver and the mammary gland. Butyrate is one of the major short-chain fatty acids produced in the rumen, and work with non-ruminants has demonstrated that it can alter inflammatory processes. The primary objective of this study was to explore the preventive effect of sodium butyrate (SB) on LPS-induced inflammation in bovine mammary epithelial cells along with underlying molecular mechanisms. Immortalized bovine mammary epithelial cells (MAC-T) were treated with SB (0.1, 0.25, 0.5, 1, 2, or 5 mM) or with the histone deacetylase inhibitor trichostatin A (TSA; 6.25, 12.5, 25, or 50 nM) for 18 h, followed by a challenge with 1 µg/mL LPS for an additional 6 h. Pretreatment with SB prevented increase in apoptosis of LPS-challenged MAC-T cells in a dose-dependent manner. The LPS treatment upregulated mRNA abundance of tumor necrosis factor α (TNFA), interleukin-6 (IL6), and interleukin-1B (IL1B), whereas inhibition of histone deacetylase with TSA dampened this effect. More importantly, SB had clear dose-dependent effects on the inflammatory response by preventing upregulation of TNFA, IL6, and IL1B. Furthermore, pretreatment with TSA or SB attenuated the downregulation of histone H3 acetylation protein abundance induced by LPS. The greater ratio of p-IκB α/IκB α and p-p65/p65 protein abundance and the increase in nuclear localization of NF-κB p65 protein in response to LPS were attenuated by pretreatment with SB. Overall, the data indicated that exogenous SB alleviates mammary cell pro-inflammatory responses partly through post-translational mechanisms that diminish NF-κB signaling. Thus, the cytoprotective effect of SB against an inflammatory challenge might represent a preventive tool to help the mammary gland against pathogens such as those causing mastitis., (The Authors. Published by Elsevier Inc. and Fass Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).)

Inflammation is critical in the progression from benign hepatic lipidosis to pathological hepatic steatosis. The objective of this study was to examine the potential role of the outer mitochondrial membrane protein mitofusin 2 (MFN2) in the etiology of hepatic steatosis in dairy cows during early lactation. Using a nested case-control design, we compared blood and liver samples from 10 healthy cows and 10 age-matched cows with moderate fatty liver. Cows with moderate fatty liver had high liver triacylglycerols, elevated plasma concentrations of free fatty acids (FFA) and β-hydroxybutyrate, and low concentrations of glucose. Cows with moderate fatty liver had overactivated inflammatory pathways in the liver, as indicated by increased abundance of phosphorylated nuclear factor κB (NF-κB) p65, NLR family pyrin domain containing 3 (NLRP3) and caspase-1 inflammasome protein, and elevated plasma concentrations and hepatic mRNA abundance of their molecular targets IL-1β, IL-6, and tumor necrosis factor α (TNF-α). In the cell culture model, we were able to replicate our findings in cows with moderate fatty liver: 1.2 mM exogenous FFA decreased the abundance of MFN2 and upregulated phosphorylation levels of the inhibitor of NF-κB (IκB) α and NF-κB p65, the IκB kinase β activity, and the abundance of NLRP3, caspase-1, IL-1β, IL-6, and TNF-α. Whereas MFN2 knockdown potentiated the FFA-induced activation of these inflammatory pathways, overexpression of MFN2 attenuated the detrimental effect of excess exogenous FFA by improving mitochondrial function and decreasing the release of reactive oxygen species, suggesting that MFN2 may be a potential therapeutic target for FFA-induced hepatic inflammation in dairy cows during early lactation., (Copyright © 2020 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Hyperketonemia is a metabolic disease in dairy cows, associated with negative nutrition balance (NNB) induced by low dry matter intake (DMI) and increased nutrient requirements. Hyperketonemia could induce metabolic stress, which might indirectly affect mammary tissue. Autophagy is a highly conserved physiological process that results in the turnover of intracellular material, and is involved in maintaining cellular homeostasis under the challenge of metabolic stress induced by NNB. The aim of this study was to investigate the autophagy status and autophagy-related pathways AMP-activated kinase α (AMPKα) and mechanistic target of rapamycin (mTOR) in the mammary glands of dairy cows with hyperketonemia. Cows with hyperketonemia [CWH, n = 10, blood β-hydroxybutyrate (BHB) concentration 1.2 to 3.0 mmol/L] and cows without hyperketonemia (CWOH, n = 10, BHB < 1.2 mmol/L) from 3 to 12 DIM were randomly selected from the herd. The mammary tissue and blood samples were collected from these cows between 0630 and 0800 h, before feeding, at 3 to 12 d in milk. Serum concentrations of glucose, BHB, and fatty acids were determined using an autoanalyzer with commercial kits between 0630 and 0800 h, before feeding. Concentrations of fatty acids, BHB (median and interquartile range: CWH, 2.44 and 1.3, 2.82 mM; CWOH, 0.49 and 0.41, 0.57 mM), and milk fat were greater in CWH. The DMI, glucose concentration, milk production, and milk protein levels were lower in CWH. The mRNA abundance of autophagosome formation-related gene, beclin 1 (BECN1), phosphatidylinositol 3-kinase catalytic subunit type 3 (PIK3C3), autophagy-related gene (ATG) 5, ATG7, ATG12, microtubule-associated protein 1 light chain 3 (MAP1LC3, also called LC3) and sequestosome-1 (SQSTM1, also called p62) were greater in the mammary glands of CWH. The protein abundance of LC3-II and phosphorylation level of Unc-51-like kinase 1 (ULK1) were greater in CWH, but the total ubiquitinated proteins and protein abundance of p62 were lower. Transmission electron microscopy showed an increased number of autophagosomes in the mammary glands of CWH. Furthermore, the phosphorylation of AMPKα was greater, but the phosphorylation of mTOR was lower in the mammary glands of CWH. These results indicate that activity of mTOR pathways and autophagy activity, and upregulation of AMPKα, may be response mechanisms to mitigate metabolic stress induced by hyperketonemia in the mammary glands of dairy cows., (Copyright © 2020 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

High blood concentrations of nonesterified fatty acids (NEFA) and altered lipid metabolism are key characteristics of fatty liver in dairy cows. In nonruminants, the mitochondrial membrane protein mitofusin 2 (MFN2) plays important roles in regulating mitochondrial function and intrahepatic lipid metabolism. Whether MFN2 is associated with hepatic lipid metabolism in dairy cows with moderate fatty liver is unknown. Therefore, to investigate changes in MFN2 expression and lipid metabolic status in dairy cows with moderate fatty liver, blood and liver samples were collected from healthy dairy cows (n = 10) and cows with moderate fatty liver (n = 10). To determine the effects of MFN2 on lipid metabolism in vitro, hepatocytes isolated from healthy calves were used for small interfering RNA-mediated silencing of MFN2 or adenovirus-mediated overexpression of MFN2 for 48 h, or treated with 0, 0.6, 1.2, or 2.4 mM NEFA for 12 h. Milk production and plasma glucose concentrations in dairy cows with moderate fatty liver were lower, but concentrations of NEFA and β-hydroxybutyrate (BHB) were greater in dairy cows with moderate fatty liver. Dairy cows with moderate fatty liver displayed hepatic lipid accumulation and lower abundance of hepatic MFN2, peroxisome proliferator-activated receptor-α (PPARα), and carnitine palmitoyltransferase 1A (CPT1A). However, sterol regulatory element-binding protein 1c (SREBP-1c), acetyl CoA carboxylase 1 (ACACA), fatty acid synthase (FASN), and diacylglycerol acyltransferase 1 (DGAT1) were more abundant in the livers of dairy cows with moderate fatty liver. In vitro, exogenous NEFA treatment upregulated abundance of SREBP-1c, ACACA, FASN, and DGAT1, and downregulated the abundance of PPARα and CPT1A. These changes were associated with greater lipid accumulation in calf hepatocytes, and MFN2 silencing aggravated this effect. In contrast, overexpression of MFN2-ameliorated exogenous NEFA-induced lipid accumulation by downregulating the abundance of SREBP-1c, ACACA, FASN, and DGAT1, and upregulating the abundance of PPARα and CPT1A in calf hepatocytes. Overall, these data suggest that one cause for the negative effect of excessive NEFA on hepatic lipid accumulation is the inhibition of MFN2. As such, these mechanisms partly explain the development of hepatic steatosis in dairy cows., (Copyright © 2019 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Dairy cows with ketosis display excessive lipolysis in adipose tissue. Heat-shock protein B7 (HSPB7), a small heat-shock protein, plays important roles in mediating cytoprotective responses to oxidative stress in rodent adipose tissue. Accordingly, it is assumed that HSPB7 may also play important roles in the antioxidant response in adipose tissue of ketotic cows. Therefore, the aim of this study is to investigate (1) the redox state of adipose tissue in ketotic cows and (2) the role and mechanism of HSPB7 on the regulation of oxidative stress in adipocytes from preruminant calves. An in vivo study consisting of 15 healthy and 15 clinically ketotic cows was performed to harvest subcutaneous adipose tissue and blood samples. In addition, adipocytes isolated from calves were treated with different concentrations of H 2 O 2 (0, 12.5, 25, 50, 100, or 200 μM) for 2 h, transfected with adenovirus-mediated overexpression of HSPB7 for 48 h, or transfected with small interfering RNA of HSPB7 for 48 h followed by exposure to H 2 O 2 (200 μM) for 2 h. Serum concentrations of nonesterified fatty acids and β-hydroxybutyrate were greater in cows with clinical ketosis, whereas serum concentration of glucose was lower. Compared with healthy cows, the malondialdehyde content was greater but the activity of glutathione peroxidase and superoxide dismutase was lower in adipose tissue of clinically ketotic cows. The abundance of HSPB7 and nuclear factor, erythroid 2 like 2 (NFE2L2) was greater in adipose tissue of clinically ketotic cows. In vitro, H 2 O 2 treatment induced the overproduction of reactive oxygen species and malondialdehyde, and inhibited the activity of antioxidant enzymes glutathione peroxidase and superoxide dismutase in adipocytes from preruminant calves. The low concentration of H 2 O 2 (12.5, 25, and 50 μM) increased the abundance of HSPB7 and NFE2L2, but high concentrations of H 2 O 2 (100 or 200 μM) reduced the abundance of HSPB7 and NFE2L2. The overexpression of HSPB7 improved the H 2 O 2 -induced oxidative stress in adipocytes via increasing the abundance of NFE2L2 and its downstream target genes heme oxygenase-1 (HMOX1) and NADH quinone oxidoreductase 1 (NQO1). Knockdown of HSPB7 markedly inhibited the expression of NFE2L2, HMOX1, and NQO1 and further exacerbated H 2 O 2 -induced oxidative stress. Overall, these results indicate that activation of the HSPB7-NFE2L2 pathway increases cellular antioxidant capacity, thereby alleviating oxidative stress in bovine adipocytes., (Copyright © 2019 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

High blood concentrations of nonesterified fatty acids (NEFA) during ketosis represent a source of fatty acids for milk fat synthesis and explain the increase in milk fat content in ketotic cows. Cell death-inducing DFFA-like effector a (CIDEA) is a lipid droplet coat protein with important roles in the regulation of milk fat synthesis and secretion in mice. Whether ketosis alters the expression of CIDEA in mammary gland tissue and the extent to which it may contribute to regulation of milk fat synthesis and secretion are unknown. Mammary gland tissue and blood samples were collected from healthy (n = 15) and clinically ketotic (n = 15) cows. Mammary epithelial cells isolated from cows were infected with CIDEA overexpression adenovirus for 48 h, treated with 0, 0.3, 0.6, or 1.2 mM NEFA for 24 h, or infected with CIDEA-silencing adenovirus for 48 h and treated with 1.2 mM NEFA for 24 h. Serum concentrations of NEFA and β-hydroxybutyrate were greater in cows with clinical ketosis, and milk production and dry matter intake were lower in cows with clinical ketosis. However, compared with healthy cows, the content of milk fat of cows with clinical ketosis was greater. Compared with healthy cows, abundance of mRNA and protein of CIDEA, fatty acid synthase (FASN), acetyl-coA carboxylase 1 (ACACA), butyrophilin (BTN1A1), and xanthine dehydrogenase (XDH) was greater in mammary tissue of cows with clinical ketosis. Overexpression of CIDEA in cultured mammary epithelial cells increased the abundance of FASN, ACACA, XDH, and BTN1A1, and increased triacylglycerol (TAG) content in mammary epithelial cells. Exogenous NEFA increased the abundance of CIDEA, FASN, ACACA, XDH, and BTN1A1, and increased TAG content in mammary epithelial cells. Importantly, knockdown of CIDEA reversed the upregulation of FASN, ACACA, XDH, and BTN1A1 abundance and TAG content induced by NEFA treatment. Overall, these data suggest that high levels of NEFA stimulate the expression of CIDEA and enhance de novo fatty acid synthesis and milk fat secretion. As such, these mechanisms explain in part the elevation of milk fat content in dairy cows with clinical ketosis., (Copyright © 2019 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Fatty liver is a common metabolic disorder in dairy cows during the transition period. Perilipin 5 (PLIN5), a lipid droplet coat protein, plays important roles in the development of hepatic steatosis in mice and humans. Whether PLIN5 plays a role in the development of fatty liver in dairy cows is unknown. An in vivo study consisting of 10 healthy and 10 cows with fatty liver was performed to harvest liver tissue and blood samples. In addition, hepatocytes isolated from calves were infected with PLIN5 overexpression adenovirus for 48 h; treated with 0, 0.6, 1.2, or 2.4 mM nonesterified fatty acids (NEFA) for 24 h; or infected with PLIN5 silencing adenovirus for 48 h and then treated with 1.2 mM NEFA for 24 h. Serum concentrations of NEFA and β-hydroxybutyrate were greater in cows with fatty liver. Milk production and plasma glucose concentrations were lower in cows with fatty liver. The results revealed that PLIN5 is highly expressed in steatotic liver and localized to lipid droplets. The abundance of fatty acid and triacylglycerol (TAG) synthesis-related proteins including sterol regulatory element binding protein-1c, fatty acid synthase, acetyl-coA carboxylase 1, diacylglycerol acyltransferase 1, and diacylglycerol acyltransferase 2 was greater in the liver of cows with fatty liver. In contrast, the abundance of microsomal triglyceride transfer protein (MTP), apolipoprotein B100, and apolipoprotein E was lower in the liver of cows with fatty liver. Consequently, cows with fatty liver exhibited severe hepatic TAG accumulation and lower blood concentration of very low density lipoprotein apolipoprotein B (VLDL-ApoB). Overexpression of PLIN5 and exogenous NEFA in cultured hepatocytes increased the abundance of sterol regulatory element binding protein-1, fatty acid synthase, acetyl-coA carboxylase 1, diacylglycerol acyltransferase 1, and diacylglycerol acyltransferase 2 but decreased the abundance of microsomal triglyceride transfer protein, apolipoprotein B100, and apolipoprotein E, which promoted TAG synthesis and inhibited VLDL-ApoB assembly, inducing lipid accumulation. Importantly, knockdown of PLIN5 attenuated the upregulation of TAG synthesis and downregulation of VLDL-ApoB assembly induced by NEFA. Overall, these data suggest that NEFA activate PLIN5, leading to TAG accumulation and inhibition of VLDL assembly. As such, these mechanisms explain in part the development of hepatic steatosis in dairy cows., (Copyright © 2019 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)