Stanniocalcin 2 Regulates Autophagy and Ferroptosis in Mammary Epithelial Cells of Dairy Cows Through the Mechanistic Target of Rapamycin Complex 1 Pathway.

BACKGROUND: Stanniocalcin 2 (STC2), a glycoprotein hormone, is extensively expressed in various organs and tissues, particularly in the mammary gland. STC2 plays a crucial role in enabling cells to adapt to stress conditions and avert apoptosis. The efficiency of milk production is closely linked to both the quantity and quality of mammary cells. Yet, there remains a dearth of research on the impact of STC2 on mammary cells' activity in dairy cows. OBJECTIVES: The objective of this study was to investigate the effects of STC2 on the viability of mammary epithelial cells in dairy cows and to elucidate the underlying mechanisms. METHODS: First, the Gene Expression Profiling and Interactive Analysis database was employed to perform survival analysis on STC2 expression in relation to prognosis using The Cancer Genome Atlas and GETx data. Subsequently, the basic physical and chemical properties, gene expression, and potential signaling pathways involved in the growth of dairy cow mammary epithelial cells were determined using STC2 knockdown. RESULTS: STC2 knockdown significantly suppressed autophagy in mammary epithelial cells of dairy cows. Moreover, STC2 knockdown upregulated glutathione peroxidase 4 protein expression, elicited an elevation in lipid ROS concentrations, and inhibited the mechanistic target of rapamycin complex 1 (mTORC1) signaling pathway, consequently repressing downstream genes involved in lipid synthesis regulated by mTORC1 and ultimately inducing ferroptosis. CONCLUSIONS: The findings of our study suggest that STC2 suppresses autophagy and ferroptosis through the activation of mTORC1. Mechanically, STC2 exerts an inhibitory effect on ferroptosis by activating antioxidative stress-related proteins, such as glutathione peroxidase 4, to suppress lipid ROS production and stimulating the mTORC1 signaling pathway to enhance the expression of genes associated with lipid synthesis.

Role of the p38/AKT Pathway in the Promotion of Cell Proliferation by Serum Heat Inactivation.

Serum is a common biomaterial in cell culture that provides nutrients and essential growth factors for cell growth. Serum heat inactivation is a common treatment method whose main purpose is to remove complement factors and viruses. As serum contains many heat-labile factors, heat inactivation may affect cell proliferation, migration, differentiation, and other functions. However, the specific mechanism of its effect on cell function has not been studied. Thus, we investigate the exact effects of heat-inactivated FBS on the viability of various cells and explore the possible molecular mechanisms. We treated HCT116, HT-29, and HepG2 cell lines with heat-inactivated (56 °C for 30 min) medium, DMEM, or fetal bovine serum (FBS) for different times (0, 10, 15, 30, 60, or 90 min); we found that heat-inactivated FBS significantly promoted the viability of these cells, whereas DMEM did not have this effect. Moreover, heat-inactivated FBS stimulated cells to produce a small amount of ROS and activated intracellular signaling pathways, mainly the p38/AKT signaling pathway. These results indicate that heat-inactivated FBS may regulate the p38/AKT signaling pathway by promoting the production of appropriate amounts of ROS, thereby regulating cell viability.

Resveratrol Promotes Gluconeogenesis by Inhibiting SESN2-mTORC2-AKT Pathway in Calf Hepatocytes.

BACKGROUND: The glucose requirement of dairy cows is mainly met by increasing the rate of hepatic gluconeogenesis. However, due to negative energy balance, the liver of periparturient cows is under oxidative stress induced by lipid over-mobilization, and hepatic gluconeogenesis is reduced. Studies have demonstrated that resveratrol, which is widely known for its antioxidant properties, can alter hepatic gluconeogenesis. However, it is not clear whether resveratrol could regulate hepatic gluconeogenesis by its antioxidant properties. OBJECTIVES: This study aims to investigate the precise effect of resveratrol in hepatic gluconeogenesis, the role of resveratrol on hydrogen peroxide (H2O2)-induced oxidative stress in hepatocytes and the potential mechanism using primary hepatocytes. METHODS: Primary hepatocytes were isolated from 5 healthy Holstein calves (1 d old, 30 to 40 kg, fasted) and treated with different concentrations of resveratrol (0, 5, 10, 25, or 50 µM) combined with or without H2O2 (0, 100, or 200 µM) induction for 12 h. RESULTS: Resveratrol enhanced the expression of gluconeogenic genes of calf hepatocytes in a dose-dependent manner (P < 0.05). Conversely, H2O2 suppressed the expression of gluconeogenic genes and induced oxidative stress (P < 0.05), which was improved by resveratrol in calf hepatocytes (P < 0.001). Furthermore, the mechanistic target of rapamycin complex 2 (mTORC2)-AKT pathway was found to negatively regulate gluconeogenesis. An AKT inhibitor was used to assess the role of the mTORC2-AKT pathway in the effects of resveratrol. The results showed resveratrol promoted hepatic gluconeogenesis by inhibiting the mTORC2-AKT pathway. Moreover, sestrin 2 (SESN2) upregulated the activity of mTORC2. We further found that resveratrol decreased SESN2 levels (P < 0.001). CONCLUSIONS: This study indicated that resveratrol enhances the gluconeogenic capacity of calf hepatocytes by improving H2O2-induced oxidative stress and modulating the activity of the SESN2-mTORC2-AKT pathway, implying that resveratrol may be a promising target for ameliorating liver oxidative stress in transition cows.

Promotion of intestinal epithelial cell apoptosis by enterotoxigenic Escherichia coli via PKA-mediated inhibition of mTORC1 activation.

Enterotoxigenic Escherichia coli (ETEC) is the main causative pathogen of diarrhea. It causes acute watery diarrhea that leads to rapid dehydration and prostration within hours. ETEC is still an important cause of neonatal and post-weaning diarrhea in pigs. However, the mechanism underlying ETEC-induced diarrhea is not yet clear. In this study, we investigated these mechanisms and found that the mTORC1 pathway plays a role in the host response to ETEC F4 infection. Specifically, we found that ETEC F4 treatment significantly repressed mTORC1 activity as well as cell proliferation, promoted apoptosis and regulated the expression of diarrhea-related genes via the promotion of PKA-mediated phosphorylation of SIN1, which plays a critical role in the assembly of mTORC2. These findings indicate that PKA is a checkpoint for ETEC-induced diarrhea. In terms of potential therapeutic strategies, we found that ZnSO4 dramatically rescued ETEC F4-induced the inhibition of mTORC1 activity and cell viability and the induction of apoptosis and alterations in the expression of diarrhea-related genes. Thus, the present findings demonstrate that ETEC F4 influences mTORC1 activation by inhibiting the assembly of mTORC2 through PKA-mediated phosphorylation of SIN1. Further, supplementation with ZnSO4 is an effective strategy for blocking the effect of ETEC F4 on mTORC1 activation, and it may have potential clinical applications in the treatment of ETEC F4-induced diarrhea.