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.

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.

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.

Knockdown of phosphatase and tensin homolog (PTEN) inhibits fatty acid oxidation and reduces very low density lipoprotein assembly and secretion in calf hepatocytes.

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.

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.