AMP-activated protein kinase controls lipid and lactose synthesis in bovine mammary epithelial cells.

Abstract

The synthesis of milk components in bovine mammary epithelial cells (BMEC) requires an adequate supply of energy. The AMP-activated protein kinase (AMPK) is a cellular energy gauge that controls anabolic and catabolic processes to maintain a balance between energy supply and demand. The objectives of this study were to assess the role of AMPK on de novo lipid and lactose synthesis, as well as its regulation by glucose and acetate availability in BMEC. We isolated primary BMEC from the mammary tissue of 3 lactating Holstein cows and differentiated them with lactogenic hormones for 4 d. We measured protein abundance, site-specific phosphorylation, and proteolytic processing by immunoblotting. We quantified the expression of genes involved in lipid and lactose synthesis using real-time quantitative PCR. We measured de novo lipid and lactose synthesis by incorporation of radioactive substrates. We analyzed data by ANOVA using a randomized complete block design with PROC MIXED in SAS. To assess the effect of AMPK activation on milk component synthesis, we treated BMEC with 100 μM A-769662 (A76; an allosteric activator of AMPK) or vehicle control for 16 h. Consistent with activation of AMPK, A76 increased phosphorylation of its downstream targets ACC Ser79 and TSC2 Ser1387 by 144% and 26%, respectively. Activation of AMPK decreased lipid synthesis by 19%. This effect was accompanied by increased expression of FABP3. Activation of AMPK reduced the proportion of mature SREBP-1c. In addition, AMPK activation reduced lactose synthesis by 24% and lowered the expression of SLC2A1, the gene encoding GLUT1. To assess the regulation of AMPK by energy substrate availability, we incubated BMEC in a control medium containing 4 mM D-glucose and 1 mM sodium acetate, or medium lacking glucose or acetate, for 4 h. Compared with the control medium, deprivation of glucose or acetate promoted AMPKα phosphorylation at Thr172 by 84% or 58%, respectively. Activation of AMPK was significantly increased in BMEC only when the medium was devoid of glucose for at least 4 h. We concluded that activation of AMPK inhibits de novo lipid and lactose synthesis in BMEC. Further studies are needed to assess the physiological relevance of AMPK activation for milk composition in vivo and to identify the mechanisms mediating its effects on milk component synthesis.