Abstract #T189

# T189
Insulin potentiates the effect of individual amino acids on mechanistic target of rapamycin complex 1 (mTORC1) signaling in mammary epithelial cells.
J. Zhang*1,2, V. Pszczolkowski2,3, S. Arriola Apelo1,3, 1Department of Dairy Science, University of Wisconsin-Madison, Madison, WI, 2State Key Laboratory of Animal Nutrition, Beijing Engineering Technology Research Center of Raw Milk Quality and Safety Control, College of Animal Science and Technology, China Agricultural University, Beijing, China, 3Endocrinology and Reproductive Physiology Graduate Training Program, University of Wisconsin-Madison, Madison, WI.

The mechanistic target of rapamycin complex 1 (mTORC1) integrates endocrine and nutritional signals to modulates metabolic functions. The objective of this study was to investigate the role of insulin in essential AA regulation of mTORC1 signaling in mammary epithelial cells. Serum starved MAC-T cells were incubated for 1 h in media containing 5.6 mM glucose, 10 mM sodium acetate, and 1.25 mM non-essential AAs, and lacking EAAs. To confirm the effect of individual AAs on mTORC1 activity, cells were treated with 100 nM insulin and each essential AA at 0, 1×, or 3× the reference plasma level of total essential AA in a lactating dairy cow, considering the profile observed in milk proteins (i.e., for 1× 55 μM Arg, 55 μM His, 130 μM Ile, 210 μM Leu, 150 μM Lys, 50 μM Met, 90 μM Phe, 100 μM Thr, 20 μM Trp, 160 μM Val). The ratio of phosphorylated to total protein for the mTORC1 substrates and mRNA translation regulators S6 kinase 1 (P-S6K1 T389) and translation initiation factor 4E-binding protein 1 (P-4E-BP1 S65) were determined by Western blotting. Essential AAs that stimulated mTORC1 activity were studied in individual 4 by 3 factorial studies with each AA at 0, 0.5×, 1×, and 3× plasma concentration (as above), and insulin at 0, 10, or 100 nM . Data were analyzed with the lm function in R. Only the EAA Arg, Ile, Leu and Met significantly increased S6K1 and 4E-BP1 phosphorylation in a quadratic fashion (x and x2P < 0.05). For these 4 AAs, insulin significantly (P < 0.05) affected polynomial parameters of S6K1 and 4E-BP1 phosphorylation, decreasing with the decrease in insulin concentration. At the lowest concentration of insulin, linear and quadratic parameters for any of the 4 AA effects on S6K1 and 4E-BP1 were different than zero (P > 0.05). Our results challenge the conventional representation of the relationship between individual AAs and energy, at least for their effect on the regulatory network of milk protein translation (i.e., mTORC1 signaling). These results also entertain the idea of energy beyond calories, in which insulinemic and non-insulinemic energy sources could play contrasting roles in production outcomes.

Key Words: essential amino acid, insulin, mechanistic target of rapamycin complex 1 (mTORC1)