Impaired autophagy aggravates oxidative stress in mammary gland of dairy cows with clinical ketosis

Abstract

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

Nuciferine Ameliorates Nonesterified Fatty Acid-Induced Bovine Mammary Epithelial Cell Lipid Accumulation, Apoptosis, and Impaired Migration via Activating LKB1/AMPK Signaling Pathway

“…A recent study delineated an adaptive mechanism by which metabolic stress can be countered by activating autophagy in bMECs in response to a low concentration of fatty acids (0.6 mM). 64 Given that AMPK activity can lead to induction of autophagy, the increased AMPK activity in bMECs induced by 0.6 mM NEFA in the present study thus may contribute to this adaptive mechanism. Moreover, such an adaptive mechanism also likely operates in vivo as indicated by elevated AMPK activity and autophagy in the mammary gland tissue of dairy cows with hyperketonemia and mild increases in serum fatty acids.…”

“…Meanwhile, this was accompanied by a significant increase in TG content and SREBP-1c protein abundance. A recent study delineated an adaptive mechanism by which metabolic stress can be countered by activating autophagy in bMECs in response to a low concentration of fatty acids (0.6 mM) . Given that AMPK activity can lead to induction of autophagy, the increased AMPK activity in bMECs induced by 0.6 mM NEFA in the present study thus may contribute to this adaptive mechanism.…”

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

“…The blood concentration of βhydroxybutyric acid (BHB) was measured to confirm SCK and CK. According to the blood BHB concentrations and clinical symptoms, 6 10 CK (blood BHB concentrations >3.0 mmol/L), 10 SCK (blood BHB concentrations, 1.2−3.0 mmol/L), and 10 CON (blood BHB concentrations <0.6 mmol/L) cows with similar body weight were enrolled. The physiological parameters of the cows are presented in Table S1.…”

“…2,5 Excess FFA accumulation overwhelms the hepatic processing capacity, inducing β-oxidation, which increases ketone levels and causes ketosis in dairy cows. 6,7 During ketosis, the supraphysiological circulating concentration of FFA causes mammary gland injury in dairy cows, as there is a high demand for mammary glands to produce milk. 2,8 Thus, ketosis has been associated with reduced milk yield during early lactation, causing economic losses.…”

“…Ketosis is a common metabolic disease that occurs in dairy cows during early lactation and is associated with several other periparturient diseases and reduced milk yield. , Ketosis susceptibility has been associated with an imbalance between the increased nutrient demands of lactation and a decrease in dry matter intake (DMI) during the postpartum transition period, , which elevates fat mobilization and increases fatty acid (FFA) concentration in the blood. , Excess FFA accumulation overwhelms the hepatic processing capacity, inducing β-oxidation, which increases ketone levels and causes ketosis in dairy cows. , During ketosis, the supraphysiological circulating concentration of FFA causes mammary gland injury in dairy cows, as there is a high demand for mammary glands to produce milk. , Thus, ketosis has been associated with reduced milk yield during early lactation, causing economic losses …”