Hexokinase 1 and 2 mediates glucose utilization to regulate the synthesis of kappa casein via ribosome protein subunit 6 kinase 1 in bovine mammary epithelial cells

Glucose plays a vital part in milk protein synthesis through the mTOR signaling pathway in bovine mammary epithelial cells (BMEC). The objectives of this study were to determine how glucose affects hexokinase (HK) activity in BMEC and investigate the regulatory effect of HK in kappa casein (CSN3) synthesis via the mechanistic target of rapamycin complex 1 (mTORC1) signaling pathway in BMEC. For this, HK1 and HK2 were knocked out in BMEC using the CRISPR/Cas9 system. The gene and protein expression, glucose uptake, and cell proliferation were measured. We found that glucose uptake, cell proliferation, CSN3 gene expression levels, and expression of HK1 and HK2 increased with increasing glucose concentrations. Notably, glucose uptake was significantly reduced in HK2 knockout (HK2KO) BMEC treated with 17.5 mM glucose. Moreover, under the same glucose treatment conditions, the proliferative ability and abundance of CSN3 were significantly diminished in both HK1 knockout (HK1KO) and HK2KO BMEC compared with that in wild-type BEMC. We further observed that the phosphorylation levels of ribosome protein subunit 6 kinase 1 (S6K1) were reduced in HK1KO and HK2KO BMEC following treatment with 17.5 mM glucose. As expected, the levels of glucose-6-phosphate and the mRNA expression levels of glycolysis-related genes were decreased in both HK1KO and HK2KO BMEC following glucose treatment. These results indicated that the knockout of HK1 and HK2 inhibited cell proliferation and CNS3 expression in BMEC under glucose treatment, which may be associated with the inactivation of the S6K1 and inhibition of glycolysis.