Effect of hyperthermia on cell viability, amino acid transfer, and milk protein synthesis in bovine mammary epithelial cells

Abstract

The reduction of milk yield caused by heat stress in summer is the main condition restricting the economic benefits of dairy farms. To examine the impact of hyperthermia on bovine mammary epithelial (MAC-T) cells, we incubated the MAC-T cells at thermal-neutral (37°C, CON group) and hyperthermic (42°C, HS group) temperatures for 6 h. Subsequently, the cell viability and apoptotic rate of MAC-T cells, apoptosis-related genes expression, casein and amino acid transporter genes, and the expression of the apoptosis-related proteins were examined. Compared with the CON group, hyperthermia significantly decreased the cell viability (p < 0.05) and elevated the apoptotic rate (p < 0.05) of MAC-T cells. Moreover, the expression of heat shock protein (HSP)70, HSP90B1, Bcl-2-associated X protein (BAX), Caspase-9, and Caspase-3 genes was upregulated (p < 0.05). The expression of HSP70 and BAX (pro-apoptotic) proteins was upregulated (p < 0.05) while that of B-cell lymphoma (BCL)2 (antiapoptotic) protein was downregulated (p < 0.05) by hyperthermia. Decreased mRNA expression of mechanistic target of rapamycin (mTOR) signaling pathway-related genes, amino acid transporter genes (SLC7A5, SLC38A3, SLC38A2, and SLC38A9), and casein genes (CSNS1, CSN2, and CSN3) was found in the heat stress (HS) group (p < 0.05) in contrast with the CON group. These findings illustrated that hyperthermia promoted cell apoptosis and reduced the transport of amino acids into cells, which inhibited the milk proteins synthesis in MAC-T cells.

Keywords: Amino acid transport; Apoptosis; Heat stress; Hyperthermia; Milk protein synthesis.

Fig. 1.. Impacts of heat stress on the apoptosis and cell viability of MAC-T cells.

(A) The MAC-T cells viability was evaluated after being thermal treatment at 42°C for 6 h; (B) Populations of early and late apoptotic MAC-T cells cultured at 37°C for 6 h, as determined by flow cytometry; (C) Populations of early and late apoptotic MAC-T cells cultured at 42°C for 6 h, as determined by flow cytometry. (D) The early apoptotic (EA) and late apoptotic (LA) rates of MAC-T cells after being treated for 6 h. * p < 0.05, ** p < 0.01. HS, heat stress.

Fig. 2.. Impacts of heat stress on the expression of heat shock and apoptosis-related genes.

* p < 0.05, ** p < 0.01. HSP70, heat shock protein 70; HSP90B1, heat shock protein 90B1; BAX, Bcl-2-associated X protein; BCL2, B-cell lymphoma 2; HS, heat stress.

Fig. 3.. Impacts of heat stress on the expression of heat shock and apoptosis-related proteins.

* p < 0.05.HSP70, heat shock protein 70; BAX, Bcl-2-associated X protein; BCL2, B-cell lymphoma 2; HS, heat stress.

Fig. 4.. Impacts of heat stress on the expression of mTOR signaling pathway-related genes.

* p < 0.05. AKT1, serine/threonine kinase 1; mTOR, mechanistic target of rapamycin kinase; RPS6, ribosomal protein S6; RPS6KB1, ribosomal protein S6 kinase B1; HS, heat stress.

Fig. 5.. Impacts of heat stress on the expression of casein and amino acid transporter genes.

(A) The expression of casein genes; (B) The expression of amino acid transporter genes. * p < 0.05, ** p < 0.01. CSN1S1, casein alpha s1; CSN2, casein beta; CSN3, casein kappa; SLC7A5, solute carrier family 7 member 5; SLC38A2, solute carrier family 38 member 2; SLC38A3, solute carrier family 38 member 3; SLC38A9, solute carrier family 38 member 9; HS, heat stress.