Effect of insulin on mRNA expression of genes related to milk synthesis in primary bovine mammary epithelial cells cultured in vitro
The crucial role for mTOR in the regulation of milk protein synthesis in the bovine mammary gland has been reported, but the molecular events associated with regulation of milk fat synthesis remain unknown. This study was conducted to examine the potential role of insulin in the presence of two lactogenic hormones, hydrocortisone and prolactin, on milk protein and fat synthesis. Primary bovine mammary epithelial cells cultured in vitro were treated in three different ways (no hormones (NH, control), 50 ng/mL hydrocortisone and 200 ng/mL prolactin (FP), or 100 ng/mL insulin, 50 ng/mL hydrocortisone and 200 ng/mL prolactin (IFP)). Expression of 17 key genes involved in four pathways were detected by real-time PCR. Statistical significance was evaluated by unpaired t-test analysis with SAS 9.0 software. Significance was declared at P < 0.05. Results showed that IFP group significantly increased the mRNA level of β-casein (CSN2), κ-casein (CSN3), Acetyl-CoA carboxylase (ACACA), fatty acid synthase (FASN) and Sterol Regulatory Element Binding Protein1 (SREBP1) compared to NH and FP groups (P < 0.05). Relative to the other groups, IFP group significantly up-regulated the expression of signal transducers and activators of transcription 5B (STAT5B) and E74-like factor 5 (ELF5) in JAK-STAT5 pathway, as well as Phosphatidylinositol 3-kinase (PI3K), Protein Kinase B (AKT1) and Eukaryotic initiation factor 4E (EIF4E) in PI3K/Akt/mTOR signaling pathway (P < 0.05). But the IFP hormone combinations had no effect on TSC1, TSC2 or RHEB transcription in AMPK signal pathway (P＞0.05). The results demonstrated that insulin may stimulate milk protein synthesis by JAK-STAT5 and PI3K/Akt/mTOR signaling pathways, and stimulate milk fat synthesis by PI3K/Akt/mTOR and SREBP signaling pathways in bovine mammary epithelial cells cultured in vitro. This research indicated insulin has an important role in milk protein and fat synthesis as the other two lactogenic hormones, hydrocortisone and prolactin.
Keywords: insulin, bovine mammary epithelial cells, real-time PCR