Effects of AMP-activated protein kinase (AMPK) signaling and essential amino acids on mammalian target of rapamycin (mTOR) signaling and protein synthesis rates in mammary cells

Abstract

Regulation of mammary protein synthesis potentially changes the relationships between AA supply and milk protein output represented in current nutrient requirement models. Glucose and AA regulate muscle protein synthesis via cellular signaling pathways involving mammalian target of rapamycin (mTOR) and AMP-activated protein kinase (AMPK). The objective of this study was to investigate the effects of essential AA (EAA) and acetate or glucose on mTOR and AMPK signaling pathways and milk protein synthesis rates. A bovine mammary epithelial cell line, MAC-T, was subjected to different media containing 0 or 3.5 mmol/L EAA concentrations with 0 or 5 mmol/L acetate or 0 or 17.5 mmol/L glucose in 2 separate 2 × 2 factorial studies. In a separate set of experiments, lactogenic bovine mammary tissue slices were subjected to the same treatments except that the low EAA treatment contained a low level of EAA (0.18 mmol/L). Supplementation of EAA enhanced phosphorylation of mTOR (Ser2448) and eukaryotic initiation factor 4E binding protein 1 (4EBP1, Thr37/46), and reduced phosphorylation of eukaryotic elongation factor 2 (eEF2, Thr56) in MAC-T cells. Concentration of ATP and phosphorylation of AMPK increased and decreased, respectively, in the presence of EAA in MAC-T cells. Acetate, EAA, or glucose numerically reduced AMPK phosphorylation by about 16% in mammary tissue slices. Provision of EAA increased phosphorylation of mTOR and 4EBP1, intracellular total EAA concentration, and casein synthesis rates in mammary tissue slices, irrespective of the presence of acetate or glucose in the medium. Phosphorylation of mTOR had a marginally negative association with AMPK phosphorylation, which was positively related to eEF2 phosphorylation. Casein synthesis rates were positively and more strongly linked to mTOR phosphorylation than the negative link between eEF2 phosphorylation and casein synthesis rates. A 100% increase in mTOR phosphorylation was associated with an increase in the casein synthesis rate of 0.74%·h(-1), whereas a 100% increase in eEF2 phosphorylation was related to a decline in the casein synthesis rate of 0.33%·h(-1). Although AMPK phosphorylation was responsive to cellular energy status and had a negative effect on mTOR-mediated signals in bovine mammary epithelial cells, its effect on milk protein synthesis rates appeared to be marginal compared with the mTOR-mediated regulation of milk protein synthesis by EAA.

Mammary blood flow and metabolic activity are linked by a feedback mechanism involving nitric oxide synthesis

“…Acetate might have been a candidate to test the existence of feedback regulation because it is oxidized heavily in the mammary epithelium, but we have been unable to detect an effect of short-term, arterial acetate infusion on mammary ATP balance (Maas et al, 1995;Purdie et al, 2008). Similarly, glucose supplementation of mammary epithelial cells in vitro increased intracellular ATP concentrations, whereas acetate supplementation did not (Appuhamy et al, 2014).…”

Nutrient Requirements of Zebu and Crossbred Cattle - BR-CORTE

“…Menezes et al (2016) showed that reducing CP levels in diets of finishing cattle is possible without affecting animal performance besides to reduce N excretion to environment. Moreover, unbalanced diets represent economic losses, once protein is considered the most expensive nutrient in beef cattle diets (Appuhamy et al, 2014;Russel et al, 1992). Additionally, toxic effects related to N excess also lead to fertility impairment (Rhoads et al, 2006).…”

High Dietary Selenium Intake Alters Lipid Metabolism and Protein Synthesis in Liver and Muscle of Pigs

“…However, to the best of our knowledge, there was no information on effects of dietary selenium, in particular high selenium intake, on body protein metabolism, although protein synthesis was reported to negatively regulate insulin sensitivity (20,21) and a high protein diet inhibited the development of type 2 diabetes (22). Mammalian target of rapamycin (mTOR), ribosomal protein S6 kinase (P70), ribosomal protein S6 (S6), factor 4E binding protein 1 (4EBP1), and eukaryotic translation initiation factor 4E (EIF4E) are key factors involved in the protein synthesis pathway (23)(24)(25)(26)(27). Furthermore, AMP-activated protein kinase (AMPK) plays a key role in regulating energy metabolism (28) and showed a negative association with selenium intake in rats (29).…”