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Methionine and arginine alter antioxidant and mechanistic target of rapamycin (mTOR) protein abundance in mammary explants stimulated with hydrogen peroxide.

D. N. Coleman

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06-23-2020

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Abstract:

T67
Methionine and arginine alter antioxidant and mechanistic target of rapamycin (mTOR) protein abundance in mammary explants stimulated with hydrogen peroxide.
D. N. Coleman*1, P. Totakul1,2, N. Onjea-uea1,3, Y. Liang1, F. F. Cardoso1, F. C. Cardoso1, J. J. Loor1. 1Department of Animal Sciences, University of Illinois Urbana, IL, 2Department of Animal Science, Faculty of Agriculture, Khon Kaen University Khon Kaen, Thailand, 3Institute of Agricultural Technology, Suranaree University of Technology Nakhon Ratchasima, Thailand.

The objective was to investigate effects of enhanced Met and Arg supply alone or in combination on protein abundance of mechanistic target of rapamycin (mTOR) and antioxidant signaling pathways in mammary gland explants during stimulation with hydrogen peroxide (HP). Mammary tissue from 4 Holstein cows (parity 4 � 1.4, DIM 248 � 38 before slaughter; mean � SD) was incubated in duplicate for 4, 8, 12 or 24h with 1 of the following media: ideal profile of essential AA as the control (IPAA; Lys:Met 2.9:1, Lys:Arg 2:1), increased Met (incMet; Lys:Met 2.5:1), increased Arg (incArg; Lys:Arg 1:1), or incMet plus incArg (Lys:Met 2.5:1 Lys:Arg 1:1) with or without exogenous HP (600 μM). Total protein was extracted to measure protein abundance using Western blot analysis. Data were analyzed as a 2 � 2 � 2 factorial using the MIXED procedure of SAS 9.4. Data from the IPAA treatment were analyzed over time to investigate the effect of incubation time. No differences were detected in the abundance of glutathione peroxidase 1 (GPX1), mTOR, phosphorylated (p) mTOR, AKT or p-AKT (P > 0.10) over time in IPAA treatments, indicating that mammary explants may be cultured for up to 24 h. After 8 h of incubation a tendency for an interaction between Met and HP was observed for GPX1 at 8 h (P = 0.09); stimulation with HP led to greater abundance of GPX1 compared with incubation without hydrogen peroxide, Met or Met and HP. However, there was no difference in the abundance of GPX1 between tissue treated with Met alone or in combination with HP. The abundance of p-mTOR was also greater in explants incubated with HP (P = 0.01). This increase in p-mTOR was associated with a tendency for an increase in the ratio of p-mTOR:total mTOR in explants incubated with HP (P = 0.09). The protein abundance of mTOR also tended to be increased by Arg (P = 0.09). Overall, preliminary evaluation of proteins indicates that supplying Met to mammary explants may help to maintain homeostasis in antioxidant metabolism during stimulation with HP. Further, supplying Arg to mammary explants may lead to increased mTOR signaling.

Keywords: amino acids, oxidative stress.