Fatty acids promote M1 polarization of monocyte-derived macrophages in healthy or ketotic dairy cows and a bovine macrophage cell line via impairing mTOR-mediated autophagy.

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.

Role of diacylglycerol O-acyltransferase 1 (DGAT1) in lipolysis and autophagy of adipose tissue from ketotic dairy cows.

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.

Free Fatty Acids Induce Apoptosis of Mammary Epithelial Cells of Ketotic Dairy Cows via the Mito-ROS/NLRP3 Signaling Pathway.

Early lactation increases metabolic stress in ketotic dairy cows, leading to mitochondrial damage, apoptosis, and inflammatory response in mammary epithelial cells. The pyrin domain 3 (NLRP3) pathway involving the mitochondrial reactive oxygen species (Mito-ROS)-induced nucleotide-binding oligomerization domain-like receptor has been recognized as a key mechanism in this inflammatory response and cell apoptosis. This study aimed to elucidate the underlying regulatory mechanism of Mito-ROS-NLRP3 pathway-mediated mammary epithelial cell apoptosis in dairy cows with ketosis. Mitochondrial damage and cellular apoptotic program and NLRP3 inflammasome activation were observed in the mammary gland of ketotic cows. Similar damage was detected in MAC-T cells treated with exogenous fatty acids (FFAs). However, NLRP3 inhibitor MCC950 pretreatment or Mito-ROS scavenging by MitoTEMPO attenuated apoptosis in FFA-induced MAC-T cells by inhibiting the NLRP3 inflammasome pathway. These findings reveal that the Mito-ROS-NLRP3 pathway activation is a potent mechanism underlying mammary epithelial cell apoptosis in response to metabolic stress in ketotic dairy cows, which further contributes to reduced milk yield.

Mitochondrial Calcium Uniporter Regulator 1 (MCUR1) Relieves Mitochondrial Damage Induced by Lipopolysaccharide by Mediating Mitochondrial Ca2+ Homeostasis in Bovine Mammary Epithelial Cells.

The metabolic stress triggered by negative energy balance after calving induces mitochondrial damage of bovine mammary epithelial cells. Mitochondrial calcium uniporter regulator 1 (MCUR1) is a key protein-coding gene that mediates mitochondrial calcium ion (Ca2+) uptake and plays an important role in mediating homeostasis of mitochondria. The aim of the present study was to elucidate the effects of MCUR1-mediated Ca2+ homeostasis on mitochondria of bovine mammary epithelial cells in response to an inflammatory challenge with lipopolysaccharide (LPS). Exogenous LPS resulted in upregulation of the MCUR1 mRNA and protein abundance, mitochondrial Ca2+ content, and mitochondrial reactive oxygen species (Mito-ROS) content while decreasing mitochondrial membrane potential, causing mitochondrial damage, and increasing the rate of apoptosis. Ryanodine pretreatment attenuated the upregulation of the mitochondrial Ca2+ content and Mito-ROS content induced by LPS. Overexpression of MCUR1 increased the mitochondrial Ca2+ content and Mito-ROS content, while it decreased mitochondrial membrane potential, damaged mitochondria, and induced cell apoptosis. In addition, knockdown of MCUR1 by small interfering RNA attenuated LPS-induced mitochondrial dysfunction by inhibiting mitochondrial Ca2+ uptake. Our results revealed that exogenous LPS induces MCUR1-mediated mitochondrial Ca2+ overload in bovine mammary epithelial cells, which leads to mitochondrial injury. Thus, MCUR1-mediated Ca2+ homeostasis may be a potential therapeutic target against mitochondrial damage induced by metabolic challenges in bovine mammary epithelial cells.

Activation of PINK1-mediated mitophagy protects bovine mammary epithelial cells against lipopolysaccharide-induced mitochondrial and inflammatory damage in vitro.

Increased metabolic stress during early lactation results in damage of mitochondria and inflammatory responses in bovine mammary epithelial cells, both of which could be aggravated by inhibition of mitophagy. PTEN-induced putative kinase 1 (PINK1)-mediated mitophagy is essential in the removal of damaged mitochondria and the regulation of inflammatory responses. The aim of the present study was to elucidate the role of PINK1-mediated mitophagy on mitochondrial damage and inflammatory responses in bovine mammary epithelial cells challenged with lipopolysaccharide (LPS). Exogenous LPS activated mitophagy and led to lower protein abundance of oxidative phosphorylation (OXPHOS) complexes (COI-V) and lower oxygen consumption rate (OCR) along with increased mitochondrial reactive oxygen species (Mito-ROS) content. These effects were also associated with increased protein abundance of Nucleotide-binding oligomerization domain-like receptor containing pyrin domain 3 (NLRP3) in a time-dependent manner. Pretreatment with 3-Methyladenine (3-MA) or knockdown of PINK1 aggravated the downregulation of COI-V protein abundance, the increase in Mito-ROS content, and the protein abundance of NLRP3, Cleaved-Caspase-1 and IL-1ß induced by LPS. Overexpression of PINK1 activated mitophagy and alleviated LPS-induced NLRP3 inflammasome activation by reducing Mito-ROS production. Overall, the data suggested that PINK1-mediated mitophagy is a crucial anti-inflammatory mechanism that removes damaged mitochondria in bovine mammary epithelial cells experiencing an increased inflammatory load.

ß-hydroxybutyrate impairs monocyte function via the ROS-NLR family pyrin domain-containing three inflammasome (NLRP3) pathway in ketotic cows.

Cows with ketosis display severe metabolic stress and immune dysfunction which renders them more susceptible to infections. Monocytes, one of the major subtypes of white blood cells, play an important role in innate immune defense against infections. Thus, the aim of this study was to investigate alterations in immune function, reactive oxygen species (ROS) production and activity of the NLR family pyrin domain containing 3 (NLRP3) inflammasome pathway in monocytes (CD14+) of cows with clinical ketosis (CK). Twelve healthy multiparous Holstein cows [blood ß-hydroxybutyrate (BHB) concentration < 1.2 mM] and 12 cows with CK (BHB > 3.0 mM) at 3 to 14 days in milk were used for blood sample collection. To determine effects of BHB on phagocytosis, ROS and protein abundance of the NLRP3 inflammasome pathway in vitro, monocytes isolated from healthy cows were treated with 3.0 mM BHB for 0, 6, 12 or 24 h. Dry matter intake (22.7 vs. 19.0 kg) was lower in cows with CK. Serum concentrations of fatty acids (0.30 vs. 0.88 mM) and BHB (0.52 vs. 3.78 mM) were greater in cows with CK, whereas concentration of glucose was lower (4.09 vs. 2.23 mM). The adhesion, migration and phagocytosis of monocytes were lower in cows with CK, but apoptosis and ROS content were greater. Protein abundance of NLRP3, cysteinyl aspartate specific proteinase 1 (caspase 1) and interleukin-1B p17 (IL1B p17) were greater in monocytes of cows with CK, while abundance of NADPH oxidase isoform 2 (NOX2) was lower. Compared with 0 h BHB, ROS content and apoptosis were greater in the monocytes challenged for 6, 12 or 24 h BHB. Compared with 0 h BHB, protein abundance of NLRP3, caspase 1, IL1B p17 and concentration of IL1B in medium were greater in the monocytes challenged for 6, 12 or 24 h BHB. However, compared with 0 h BHB, protein abundance of NOX2 and phagocytosis of monocytes were lower in the monocytes challenged for 6, 12 or 24 h BHB. Overall, the data suggested that exogenous BHB activated the ROS-NLRP3 pathway, which might be partly responsible for immune dysfunction of dairy cows with CK.

Effects of diacylglycerol O-acyltransferase 1 (DGAT1) on endoplasmic reticulum stress and inflammatory responses in adipose tissue of ketotic dairy cows.

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 ß-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.

Inhibiting nuclear factor erythroid 2 related factor 2-mediated autophagy in bovine mammary epithelial cells induces oxidative stress in response to exogenous fatty acids.

BACKGROUND: In early lactation, bovine mammary epithelial cells undergo serious metabolic challenges and oxidative stress both of which could be alleviated by activation of autophagy. Nuclear factor erythroid 2 related factor 2 (NFE2L2), a master regulator of cellular redox homeostasis, plays an important role in the regulation of autophagy and oxidative stress. Thus, the objective of this study was to investigate the role of NFE2L2-mediated autophagy on oxidative stress of bovine mammary epithelial cells in response to exogenous free fatty acids (FFA). RESULTS: Exogenous FFA induced linear and quadratic decreases in activities of glutathione peroxidase (GSH-Px), catalase (CAT), and superoxide dismutase (SOD), and increases in the contents of reactive oxygen species (ROS) and malondialdehyde (MDA). Protein abundance of LC3-phosphatidylethanolamine conjugate (LC3-II) and the number of autophagosomes and autolysosomes decreased in a dose-dependent manner, while protein abundance of p62 increased in cells challenged with FFA. Activation of autophagy via pre-treatment with Rap attenuated the FFA-induced ROS accumulation. Importantly, FFA inhibited protein abundance of NFE2L2 and the translocation of NFE2L2 into the nucleus. Knockdown of NFE2L2 by siRNA decreased protein abundance of LC3-II, while it increased protein abundance of p62. Furthermore, sulforaphane (SFN) pre-treatment attenuated the FFA-induced oxidative stress by activating NFE2L2-mediated autophagy. CONCLUSIONS: The data suggested that NFE2L2-mediated autophagy is an important antioxidant mechanism in bovine mammary epithelial cells experiencing increased FFA loads.

Nuclear factor erythroid 2-related factor 2 protects bovine mammary epithelial cells against free fatty acid-induced mitochondrial dysfunction in vitro.

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.

Optimization of γ-Aminobutyric Acid (GABA) Accumulation in Germinating Adzuki Beans (Vigna angularis) by Vacuum Treatment and Monosodium Glutamate, and the Molecular Mechanisms.

This study aimed to investigate the optimal hypoxic and monosodium glutamate (MSG) stress conditions for the enrichment of γ-Aminobutyric acid (GABA) in germinating adzuki beans and to reveal the potential underlying molecular mechanisms of GABA accumulation. Using single-factor experiments and response surface model, we investigated the effects of germination time, germination temperature, vacuum time, and MSG concentration on GABA contents, and further explored the activity and gene expression of glutamate decarboxylase (GAD) and polyamine oxidase (PAO) critical rate restriction enzymes during GABA synthesis. The optimal soaking temperature, soaking time, and pH conditions were 35°C, 16 h, and 5, respectively. Furthermore, the optimal germination conditions for optimal GABA enrichment were 48 h, 1.99 mg/ml MSG concentration, germination temperature of 31.49°C, and vacuum time of 15.83 h. Under such conditions, the predicted GABA concentration was 443.57 ± 7.18 mg/100 g, with no significant difference between the predicted and experimental data. The vacuum + MSG (FZM) treatment has a maximum contribution rate of GABA to 38.29%, which significantly increase GABA content, and the increase was associated with increased GAD and PAO activity. In addition, MSG in combination with vacuum treatment could significantly induce VaGAD4 and VaGAD6 genes in 2 days germination of adzuki beans. According to the results of the present study, vacuum + MSG treatment is an effective approach to enhancing GABA accumulation in germinating adzuki beans, which could be employed in enhancing the functional quality of germinating adzuki beans.

Transcription factor EB (TFEB)-mediated autophagy protects bovine mammary epithelial cells against H2O2-induced oxidative damage in vitro.

BACKGROUND: Bovine mammary epithelial cells after calving undergo serious metabolic challenges and oxidative stress both of which could compromise autophagy. Transcription factor EB (TFEB)-mediated autophagy is an important cytoprotective mechanism against oxidative stress. However, effects of TFEB-mediated autophagy on the oxidative stress of bovine mammary epithelial cells remain unknown. Therefore, the main aim of the study was to investigate the role of TFEB-mediated autophagy in bovine mammary epithelial cells experiencing oxidative stress. RESULTS: H2O2 challenge of the bovine mammary epithelial cell MAC-T increased protein abundance of LC3-II, increased number of autophagosomes and autolysosomes while decreased protein abundance of p62. Inhibition of autophagy via bafilomycin A1 aggravated H2O2-induced reactive oxygen species (ROS) accumulation and apoptosis in MAC-T cells. Furthermore, H2O2 treatment triggered the translocation of TFEB into the nucleus. Knockdown of TFEB by siRNA reversed the effect of H2O2 on protein abundance of LC3-II and p62 as well as the number of autophagosomes and autolysosomes. Overexpression of TFEB activated autophagy and attenuated H2O2-induced ROS accumulation. Furthermore, TFEB overexpression attenuated H2O2-induced apoptosis by downregulating the caspase apoptotic pathway. CONCLUSIONS: Our results indicate that activation of TFEB mediated autophagy alleviates H2O2-induced oxidative damage by reducing ROS accumulation and inhibiting caspase-dependent apoptosis.

Adenosine 5'-monophosphate-activated protein kinase ameliorates bovine adipocyte oxidative stress by inducing antioxidant responses and autophagy.

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 ß-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 H2O2 (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 H2O2 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 H2O2 induced accumulation of ROS and malondialdehyde (MDA), whereas H2O2 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 H2O2-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 H2O2-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.

All-trans retinoic acid controls differentiation, proliferation, and lipolysis in isolated subcutaneous adipocytes from peripartal Holstein cows.

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.

Aloin protects mice from diet-induced non-alcoholic steatohepatitis via activation of Nrf2/HO-1 signaling.

Aloin, a naturally occurring anthraquinone glycoside derived from the Aloe species, has antioxidant and anti-inflammatory activities, but its role in non-alcoholic steatohepatitis (NASH) remains unknown. This study was designed to investigate the anti-inflammatory, antioxidant, and anti-apoptotic effects of aloin and the underlying mechanisms during NASH. Wild-type or nuclear erythroid 2-related factor 2 (Nrf2) knock-out (KO) mice were fed a choline-deficient, l-amino acid-defined, high-fat (CDAAH) diet and treated with aloin (10, 20 or 40 mg per kg bw per day) by gavage for twelve weeks. Liver and blood samples were collected to evaluate liver function, protein abundance, and histopathological status. Supplementing aloin at 20 mg kg-1 was optimal for mitigating liver damage during NASH, as evidenced by reduced alanine transaminase and aspartate aminotransferase activity in serum. Supplementation with aloin significantly reduced serum concentration or liver protein abundance of malondialdehyde, tumor necrosis factor alpha, Interleukin (IL)-1ß and IL-6. Aloin treatment enhanced hepatic superoxide dismutase activity, glutathione and serum IL-10 levels in mice with NASH. Furthermore, supplementation with aloin inhibited hepatocyte apoptosis caused by Bcl-2 up-regulation and cleaved caspase-3 and Bax down-regulation. Mechanistically, by using Nrf2 KO mice, the protective effects of aloin were associated with enhanced antioxidant, anti-inflammatory and anti-apoptotic activity, all of which were mediated by Nrf2/heme oxygenase-1 (HO-1) signaling activation. Data suggested that aloin activates the Nrf2/HO-1 pathway and has protective potential against liver injury during NASH. Therefore, aloin supplementation might contribute to the prevention and treatment of NASH via activation of the Nrf2/HO-1 pathway.

Oxidative stress, NF-κB signaling, NLRP3 inflammasome, and caspase apoptotic pathways are activated in mammary gland of ketotic Holstein cows.

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 ß-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 IKKß 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.

Cardamonin Reduces Acetaminophen-Induced Acute Liver Injury in Mice via Activating Autophagy and NFE2L2 Signaling.

Cardamonin (CD), a naturally occurring chalcone derived from the Alpinia species, has been shown to exert antioxidant and anti-inflammatory activity, but its role in the prevention of acetaminophen- (APAP-) induced hepatotoxicity remains elusive. The objective of this study was to determine the protective effects of CD against APAP-induced acute liver injury (ALI) and the underlying mechanisms. Wild-type or transcription factor nuclear factor erythroid 2-related factor 2- (NFE2L2-) deficient mice were treated with CD (50 or 100 mg/kg, i.p.) or vehicle for 24 h. Subsequently, these mice were challenged with APAP (400 mg/kg, i.p.) for 6 h. Liver and blood samples were collected to evaluate liver injury and protein abundance. Treatment with CD significantly reduced APAP-induced hepatotoxicity. Furthermore, CD effectively reduced APAP-induced inflammation by inhibiting high mobility group box 1 (HMGB1), toll-like receptor 4 (TLR4), and NOD-like receptor protein 3 (NLRP3) signaling. In addition, CD induced activation of sequestosome 1 (p62) and NFE2L2 signaling and facilitated autophagy. By applying autophagy inhibitor 3-methyladenine (3-MA; 20 mg/kg, i.p.), further mechanistic exploration revealed that NFE2L2 deficiency promoted autophagic activity induced by CD treatment, which was conducive to the hepatoprotective effect of CD against APAP-induced hepatoxicity in NFE2L2-/- mice. Overall, data suggest that CD has hepatoprotective effect against APAP-induced ALI, which might contribute to the activation of NFE2L2 and autophagy.

Sodium butyrate reduces bovine mammary epithelial cell inflammatory responses induced by exogenous lipopolysaccharide, by inactivating NF-κB signaling.

Exogenous molecules derived from catabolic states (e.g., fatty acids, ß-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.

High levels of fatty acids inhibit ß-casein synthesis through suppression of the JAK2/STAT5 and mTOR signaling pathways in mammary epithelial cells of cows with clinical ketosis.

Ketosis is a metabolic disease of dairy cows often characterized by high concentrations of ketone bodies and fatty acids, but low milk protein and milk production. The Janus kinase 2 (JAK2)-signal transducer and activator of transcription 5 (STAT5) and the mechanistic target of rapamycin (mTOR) signaling pathways are central for the regulation of milk protein synthesis. The effect of high levels of fatty acids on these pathways and ß-casein synthesis are unknown in dairy cows with clinical ketosis. Mammary gland tissue and blood samples were collected from healthy (n = 15) and clinically-ketotic (n = 15) cows. In addition, bovine mammary epithelial cells (BMEC) were treated with fatty acids, methionine (Met) or prolactin (PRL), respectively. In vivo, the serum concentration of fatty acids was greater (P > 0.05) and the percentage of milk protein (P > 0.05) was lower in cows with clinical ketosis. The JAK2-STAT5 and mTOR signaling pathways were inhibited and the abundance of ß-casein was lower in mammary tissue of cows with clinical ketosis (P > 0.05). In vitro, high levels of fatty acids inhibited the JAK2-STAT5 and mTOR signaling pathways (P > 0.05) and further decreased the ß-casein synthesis (P > 0.05) in BMEC. Methionine or PRL treatment, as positive regulators, activated the JAK2-STAT5 and mTOR signaling pathways to increase the ß-casein synthesis. Importantly, the high concentration of fatty acids attenuated the positive effect of Met or PRL on mTOR, JAK2-STAT5 pathways and the abundance of ß-casein (P > 0.05). Overall, these data indicate that the high concentrations of fatty acids that reach the mammary cells during clinical ketosis inhibit mTOR and JAK2-STAT5 signaling pathways, and further suppress ß-casein synthesis.

Nuclear Factor E2-Related Factor 2 Mediates Oxidative Stress-Induced Lipid Accumulation in Adipocytes by Increasing Adipogenesis and Decreasing Lipolysis.

Aims: Nuclear factor E2-related factor 2 (Nrf2) is a regulator of cellular oxidative stress and is also involved in lipid metabolism in adipocytes. However, it remains unknown as to whether Nrf2 is the link between oxidative stress and the induction of lipid accumulation in adipocytes. Results: Here, we show that oxidative stress is markedly increased in white adipose tissue from mice with high-fat diet-induced or genetically (ob/ob)-induced obesity and from human subjects with obesity. Notably, in response to oxidative stress, Nrf2 expression and activity were induced, further promoting lipid accumulation in adipocytes and exacerbating the development of obesity. In contrast, Nrf2 ablation alleviated oxidative stress-induced lipid accumulation. Mechanistically, oxidative stress promoted Nrf2 recruitment to the sterol regulatory element binding protein 1 promoter, inducing target gene transcription and subsequent lipogenesis. In addition, Nrf2 mediated oxidative stress-inhibited lipolysis in adipocytes via the protein kinase A pathway. Innovation and Conclusion: Our data provide a novel insight that Nrf2, as a critical signaling node, links oxidative stress to the induction of fat accumulation in adipocytes.

Lycopene alleviates H2O2-induced oxidative stress, inflammation and apoptosis in bovine mammary epithelial cells via the NFE2L2 signaling pathway.

During the transition into lactation, bovine mammary epithelial cells (bMECs) are likely subjected to altered redox balance due to the high metabolic rate associated with the onset of lactation. In non-ruminants, lycopene (LYC), a naturally occurring hydrocarbon carotenoid, has attracted considerable attention as a potential natural agent against oxidative stress. The aim of this study was to investigate whether LYC alleviates oxidative injury in bMECs induced by H2O2 and the underlying molecular mechanisms. The primary bMEC and bovine MEC line MAC-T cells were treated with H2O2 (500 µM) and/or LYC (0.5, 1 or 2 µM) for 24 h. The results showed that treatment with LYC decreased H2O2-induced accumulation of intracellular reactive oxygen species (ROS), inflammatory cytokine (TNF-α, IL-6, and IL-1ß) expression and the apoptosis rate. These effects were associated with the activation of the NFE2L2-antioxidant response element (ARE) pathway coupled with inactivation of the nuclear factor-κB (NF-κB) inflammatory and caspase/Bcl2 apoptotic pathways. The bMECs were transfected with NFE2L2 siRNA for 48 h and/or treated with H2O2 (500 µM) and/or LYC (2 µM) for another 24 h. The fact that transfection with NFE2L2 siRNA abrogated the protection of LYC against H2O2-induced accumulation of intracellular ROS, inflammatory cytokine expression and apoptosis suggested that this antioxidant transcription factor is essential for the protective mechanism induced by LYC. These results suggest that LYC might be a potent antioxidant in vivo that could be administered to ruminant animals during stressful periods such as the transition into lactation.