Increased supply of methionine during a heat-stress challenge in lactating holstein cows alters mammary tissue mTOR signaling and its response to lipopolysaccharide

Abstract

The first objective was to investigate the effects of feeding rumen-protected methionine (RPM) during a heat stress (HS) challenge on abundance and phosphorylation of mechanistic target of rapamycin (mTOR)-related signaling proteins in mammary gland. The second objective was to investigate how HS and RPM may modulate the response of mammary gland explants to lipopolysaccharide (LPS) stimulation. Thirty-two multiparous, lactating Holstein cows (184 ± 59 DIM) were randomly assigned to 1 of 2 environmental treatment groups, and 1 of 2 dietary treatments [TMR with RPM (Smartamine M; Adisseo Inc.; 0.105% DM as top dress) or TMR without RPM (CON)] in a crossover design. There were 2 periods with 2 phases per period. In phase 1 (9 d), all cows were in thermoneutral conditions (TN) and fed ad libitum. During phase 2 (9 d), group 1 (n = 16) was exposed to HS using electric heat blankets while group 2 (n = 16) remained in TN but were pair-fed to HS counterparts to control for DMI decreases associated with HS. After a washout period (14 d), the study was repeated (period 2). Environmental treatments were inverted in period 2 (sequence), while dietary treatments remained the same. Mammary tissue was harvested via biopsy at the end of both periods. Tissue was used for protein abundance analysis and also for incubation with 0 or 3 μg/mL of LPS for 2 h and subsequently used for mRNA abundance. Data were analyzed using PROC MIXED in SAS. Analysis of protein abundance data included the effects of diet, environment and their interaction, and period and sequence to account for the crossover design. The explant data model also included the effect of LPS and its interaction with environment and diet. Abundance of phosphorylated mTOR and ratio of phosphorylated eukaryotic translation elongation factor 2 (p-EEF2) to total EEF2 in non-challenged tissue was greater with RPM supplementation (P = 0.04 for both) and in both cases tended to be greater with HS (P = 0.08 for both). Regardless of RPM supplementation, incubation with LPS upregulated mRNA abundance of IL8, IL6, IL1B, CXCL2, TNF, NFKB1 and TLR2 (P < 0.05). An environment × LPS interaction was observed for NFKB1 (P = 0.03); abundance was greater in LPS-treated explants from non-HS compared with HS cows. Abundance of CXCL2, NFKB1, NOS2, NOS1, and SOD2 was lower with HS (P < 0.05). While LPS did not alter abundance of mRNA associated with the antioxidant transcription factor NFE2L2 signaling (P = 0.59), explants from HS cows had lower abundance of NFE2L2 (P < 0.001) and CUL3 (P = 0.04). Overall, RPM supplementation may alter mTOR activation. Additionally, while HS reduced explant immune and antioxidant responses, RPM did not attenuate the inflammatory response induced by LPS in vitro.

Increased dietary methionine, lysine and histidine supply modulated the heat stress-induced metabolic remodeling of dairy cows

“…In the current experiment, we found increased plasma insulin concentrations during heat stress which was reverted in HS+AA by the end of the experimental period along with the recovery of milk protein content and decrease of milk urea (Ruiz-Gonzaĺez et al, 2022a). Lastly, Met was also suggested to activate the mTOR pathway in the mammary gland, however, the exact mechanisms are not yet known (Coleman et al, 2022). Our results indicated that improved milk protein content in HS+AA was associated with the increased availability of EAA, however, the exact interactions between Lys, BCAA, Met and the mTOR pathway are yet to be fully elucidated.…”

“…Under heat-stress conditions, milk yield and oxidative status of dairy cows was improved by increased Met supply, rather than MP (Mesgaran et al, 2022). In vitro studies on hyperthermic mammary gland epithelial cells indicated that increased Met supply can lead to a higher synthesis of the antioxidants taurine and glutathione and stimulate protein synthesis through the direct activation of the mechanistic target of rapamycin (mTOR) pathway or indirectly by increased branched-chain AA (BCAA) availability (Dong et al, 2018;Lopreiato et al, 2020;Coleman et al, 2022). However, the improved productive performance of heat-stressed cows after dietary Met supplementation was not consistent across the few reported studies (Pate et al, 2020;Mesgaran et al, 2022) and the mechanisms and pathways remain to be understood (Lopreiato et al, 2020;Mesgaran et al, 2022).…”

Effect of Rumen-Protected Methionine on Metabolic Profile of Liver, Muscle and Blood Serum Samples of Growing German Simmental Bulls Fed Protein-Reduced Diets

“…As such, it plays a pivotal role in epigenetics via DNA methylation [ 20 ] and forms a key part in the one-carbon metabolism in its biologically active form of S-adenosyl-methionine. Via the trans-sulfuration pathway and the intermediate metabolite cysteine, it is involved in the synthesis of the antioxidants glutathione and taurine [ 21 ].…”

“…They found that due to maternal methionine supply neonatal calves experienced beneficial effects on their antioxidant status. The supplementation of RPMET to peripartal dairy cows also led to an improved antioxidant status [ 21 , 36 , 37 ]. Alfaro et al [ 38 ] conducted a nutrigenetics study in beef cattle.…”