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Dairy cows experiencing heat stress (HS) during the precalving portion of the transition period give birth to smaller calves and produce less milk and milk protein. Supplementation of rumen-protected methionine (RPM) has been shown to modulate protein, energy, and placenta metabolism, making it a potential candidate to ameliorate HS effects. We investigated the effects of supplementing RPM to transition cows under HS induced by electric heat blanket (EHB) on cow-calf performance. Six weeks before expected calving, 53 Holstein cows were housed in a tiestall barn and fed a control diet (CON, 2.2% Met of MP) or a CON diet supplemented with SmartamineM (MET, 2.6% Met of MP, Adisseo Inc., France). Four weeks precalving, all MET and half CON cows were fitted with an EHB. The other half of the CON cows were considered thermoneutral (TN), resulting in 3 treatments: CONTN (n = 19), CONHS (n = 17), and METHS (n = 17). Respiratory rate (RR), skin temperature (ST), and rectal temperature (RT) were measured thrice weekly and core body temperatures recorded biweekly. Postcalving BW and BCS were recorded weekly, and DMI was calculated and averaged weekly. Milk yield was recorded daily and milk components were analyzed every third DIM. Biweekly AA and weekly nonesterified fatty acids (NEFA), BHB, insulin, and glucose were measured from plasma. Calf birth weight and 24 h growth, thermoregulation, and hematology profile were measured and apparent efficiency of absorption (AEA) of immunoglobulins was calculated. Data were analyzed using the MIXED procedure of SAS with 2 preplanned orthogonal contrasts: CONTN versus the average of CONHS and METHS (C1) and CONHS versus METHS (C2). Relative to TN, EHB cows had increased RT during the postcalving weeks and increased RR and ST during the entire transition period. Body weight, BCS, DMI, and milk yield were not affected by the EHB or RPM. However, protein percentage and SNF were lower in CONHS, relative to METHS cows. At calving, METHS dams had higher glucose concentrations, relative to CONHS, and during the postcalving weeks, the EHB cows had lower NEFA concentrations than TN cows. Calf birthweight and AEA were reduced by HS, whereas RR was increased by HS. Calf withers height tended to be shorter and RT were lower in CONHS, compared with METHS heifers. Overall, RPM supplementation to transition cows reverts the negative effect of HS on blood glucose concentration at calving and milk protein percentage in the dams and increases wither height while decreasing RT in the calf., (The Authors. Published by Elsevier Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

The periparturient period is characterized by the increased demand for calcium (Ca) in dairy cows. This has resulted in the use of several different prepartal nutritional strategies to prevent hypocalcemia postpartum. The objective of our study was to determine the effects of feeding synthetic zeolite A (XZ), a diet with negative dietary cation-anion difference (-DCAD), or a positive-DCAD diet (CON) during the close-up period on peripartal mineral dynamics and hormones involved in calcium metabolism. To this end, 121 multiparous Holstein cows, blocked by lactation number and expected due date, were enrolled at 254 d of gestation and randomly assigned to 1 of 3 prepartum diets: CON (+190 mEq/kg; n = 40), -DCAD (-65 mEq/kg; n = 41), or a diet supplemented with sodium aluminum silicate (XZ; +278 mEq/kg, fed at 3.3% DM, targeting 500 g/d; n = 40; Protekta Inc.). Blood, urine, and saliva samples were collected from enrollment until parturition, with data analyzed and presented beginning 14 d before parturition (d -14) until parturition (d 0), and on d 1, 2, 3, 6, 9, 12, 15, 18, 21, 35, and 49 postpartum, to assess mineral and hormone dynamics. Total fecal collections were performed in a subset of 8 cows per treatment group to assess fecal mineral loss. Data were analyzed as a randomized complete block design in SAS. Cows fed XZ and -DCAD had higher blood Ca concentrations compared with CON-fed cows, with XZ-fed cows exhibiting the highest blood Ca concentrations pre- and postpartum. Cows fed XZ had decreased blood and salivary phosphorus (P), increased fecal water-extractable phosphate, and the highest blood calcium concentrations pre- and postpartum. Parathyroid hormone was unaffected by diet but was increased at parturition in all treatments. Serotonin concentrations were increased in -DCAD and XZ cows compared with CON during the prepartum period. Our data indicate that the XZ group's improvement in blood Ca concentrations pre- and postpartum is most likely regulated by a dietary P restriction. Taken together, these data suggest that XZ and -DCAD diets improve postpartum calcium metabolism; however, they appear to work through different mechanisms., (The Authors. Published by Elsevier Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

In dairy cows, the lactating mammary glands synthesize serotonin, which acts in an autocrine-paracrine manner in the glands and is secreted into the periphery. Serotonin signaling during lactation modulates nutrient metabolism in peripheral tissues such as adipose and liver. We hypothesized that the elevation of circulating serotonin during lactation would increase nutrient partitioning to the mammary glands, thereby promoting milk production. Our objective was to elevate circulating serotonin via intravenous infusion of the serotonin precursor 5-hydroxytryptophan (5-HTP) to determine its effects on mammary supply and extraction efficiency of AA, and milk components production. Twenty-two multiparous mid-lactation Holstein cows were intravenously infused with 5-HTP (1 mg/kg body weight) or saline, in a crossover design with two 21-d periods. Treatments were infused via jugular catheters for 1 h/d, on d 1 to 3, 8 to 10, and 15 to 17 of each period, to maintain consistent elevation of peripheral serotonin throughout the period. Milk and blood samples were collected in the last 96 h of each period. Whole-blood serotonin concentration was elevated above saline control for 96 h after the last 5-HTP infusion. Dry matter intake was decreased for cows receiving 5-HTP, and on average they lost body weight over the 21-d period, in contrast to saline cows who gained body weight. Milk production and milk protein yield were lower in cows receiving 5-HTP during the 3 infusion days, but both recovered to saline cow yields in the days after. Although milk fat yield exhibited a day-by-treatment interaction, no significant difference occurred on any given day. Milk urea nitrogen concentration was lower in 5-HTP cows on the days following the end of infusions, but not different from saline cows on infusion days. Meanwhile, plasma urea nitrogen was not affected by 5-HTP infusion. Circulating concentrations of AA were overall transiently decreased by 5-HTP, with concentrations mostly returning to baseline within 7 h after the end of 5-HTP infusion. Mammary extraction efficiency of AA was unaffected by 5-HTP infusion. Overall, both lactation performance and circulating AA were transiently reduced in cows infused with 5-HTP, despite sustained elevation of circulating serotonin concentration., (The Authors. Published by Elsevier Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

During the transition period, dairy cows are often exposed to negative energy balance (NEB), leading to lipid mobilization from adipose tissue into nonesterified fatty acids (NEFA), a common indicator of heightened illness risk. This study aimed to use blood near-infrared (NIR) spectra data to classify NEB into high or low categories, based on early-lactation cow NEFA thresholds. We collected a total of 186 plasma samples from 100 Holstein cows. The samples were categorized into critical thresholds, based on previous literature, of ≥0.60 and ≥0.70 mEq/L for identifying high NEB. Spectral data were preprocessed before the development of the predictive modes, which included the implementation of multiplicative scatter correction, standard normal variate (SNV), and first and second derivatives. The classification was performed using partial least square discriminant analyses (PLS-DA), and predictive performance was assessed using leave-one-out cross-validation. Predictive quality for each class was evaluated through specificity, precision, sensitivity, and F 1 score. The study showed promising results, with the SNV technique achieving higher F 1 scores. The model found 72.7% specificity, 78.9% precision, 80.8% sensitivity, and 79.8% F 1 score to classify animals with NEFA levels of ≥0.60 mEq/L, and 82.1% specificity, 78.7% precision, 80.8% sensitivity, and 79.7% F 1 score to classify animals with NEFA levels ≥0.70 mEq/L. These results indicate that NIR spectroscopy could serve as a tool for detecting cows under severe NEB, also showing potential for broader application across the entire transition period, as the spectral signal carried relevant information regarding cow metabolism. Furthermore, the combination of predictors derived from plasma spectra and other cow-level information can lead to more accurate disease alerts, given their relationship with the NEB., (© 2023.)

Energy partitioning in lactating cows affects milk production, feed efficiency, and body reserves, with the latter having health implications for the transition into the following lactation. One molecule likely involved in the regulation of energy partitioning is serotonin. The objective of this experiment was to explore how increasing circulating serotonin, by intravenous infusion of the serotonin precursor 5-hydroxytryptophan (5-HTP), affects metabolic responses to a glucose challenge in midlactation cows as a means to manipulate energy partitioning. We intravenously infused Holstein cows with 5-HTP (1 mg/kg bodyweight dissolved in saline, n = 11) or saline alone as control (n = 9) over 1 h/day for 3 days. Cows were fasted overnight on day 2. On day 3, fasted cows were given an intravenous bolus of glucose (0.092 g/kg bodyweight). Blood samples were collected for the following 120 min for metabolic and hormonal analysis. Infusion of 5-HTP elevated circulating concentrations of serotonin and free fatty acids, reduced the concentration of insulin and amino acids, and did not affect the concentration of glucose and glucagon before the glucose challenge. Surrogate insulin sensitivity indices indicated improved insulin sensitivity in 5-HTP cows, but due to the unique metabolism of lactating ruminants, these index changes may instead reflect effects in insulin-independent glucose disposal, like milk synthesis. Challenging 5-HTP-treated cows with a glucose bolus reduced the insulin spike and blunted the decrease in free fatty acids, compared to saline cows, without changing glucose dynamics. Overall, these results suggest that serotonin stimulates insulin-independent glucose disposal, requiring less insulin to maintain normoglycemia.

The aim of this experiment was to test whether insulin potentiates the effects of two abomasally infused amino acids (AA), leucine and methionine (LM), on mammary extraction efficiency of energetic and nitrogenous nutrients. Six lactating Holstein cows (155 ± 9 DIM) were ruminally-cannulated and had the right carotid artery subcutaneously transposed. Cows were fed a 20% metabolizable protein-restricted diet and abomasally infused with water (8 L/d) or AA (Met 26 g/d, Leu 70 g/d) for 8 h/d, for 7 days. On the last day of each period, cows were intravenously infused with saline (0.9% NaCl, 110 mL/h) or subjected to 8 h hyperinsulinemic clamp (IC) alongside abomasal infusions. For IC, insulin was infused at 1 µg/kg/h. Normoglycemia was maintained by varying glucose (50% w/v in water) infusion rate based on coccygeal vein glucose concentration. Carotid arterial and subcutaneous abdominal (mammary) vein blood samples were collected at 0, 1, 2, 4, and 6 h from the start of infusions. Milk weights and samples for baseline measurements of production were taken on day 5 PM, day 6 AM and PM, and day 7 AM of the experimental period. A final milk weight and sample was taken immediately after abomasal and intravenous infusions on day 7 PM for assessing the interaction between insulin and the infused AA. The experiment had an incompletely replicated Latin square design with a 2 × 2 factorial arrangement of treatments (abomasal and intravenous infusion). Baseline milk production when cows were only receiving abomasal infusions was largely unaffected by LM, but milk protein yield tended to be decreased. On day 7, LM tended to positively increase milk fat and de novo fatty acid content, and IC tended to decrease milk protein content. Both milk urea nitrogen and plasma urea nitrogen were decreased by IC. Circulating AA concentrations in plasma were decreased by both LM and IC, but mammary extraction efficiency was affected by neither. Infusion of LM had no effect on any energy metabolite analyzed. Circulating non-esterified fatty acid concentration was decreased by IC, with no effect on mammary extraction efficiency. Mammary extraction efficiency of both acetate and β-hydroxybutyrate were decreased by IC. Overall, while both circulating concentrations of energy metabolites and amino acids were decreased in response to treatments, this was not due to improved mammary extraction efficiency., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Poor maternal nutrition during gestation can negatively affect offspring growth, development, and health pre- and post-natally. Overfeeding during gestation or maternal obesity (MO) results in altered metabolism and imbalanced endocrine hormones in animals and humans which will have long-lasting and detrimental effects on offspring growth and health. In this study, we examined the effects of overnutrition during gestation on autophagy associated pathways in offspring heart muscles at two gestational and one early postnatal time point ( n = 5 for treated and untreated male and female heart respectively at each time point). Two-way ANOVA was used to analyze the interaction between treatment and sex at each time point. Our results revealed significant interactions of maternal diet by developmental stages for offspring autophagy signaling. Overfeeding did not affect the autophagy signaling at mid-gestation day 90 (GD90) in both male and female offspring while the inflammatory cytokines were increased in GD90 MO male offsrping; however, overfeeding during gestation significantly increased autophagy signaling, but not inflammation level at a later developmental stage (GD135 and day 1 after birth) in both males and females. We also identified a sexual dimorphic response in which female progeny were more profoundly influenced by maternal diet than male progeny regardless of developmental stages. We also determined the cortisol concentrations in male and female hearts at three developmental stages. We did not observe cortisol changes between males and females or between overfeeding and control groups. Our exploratory studies imply that MO alters autophagy associated pathways in both male and female at later developmental stages with more profound effects in female. This finding need be confirmed with larger sample numbers in the future. Our results suggest that targeting on autophagy pathway could be a strategy for correction of adverse effects in offspring of over-fed ewes., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Liu, Ding, Halderson, Arriola Apelo, Jones, Pillai, Hoffman, Reed, Govoni, Zinn and Guo.)

Dry period heat stress impairs subsequent milk production, but its impact on milk protein content and yield is inconsistent. We hypothesize that dairy cow exposure to dry period heat stress will reduce milk protein synthesis in the next lactation, potentially through modified amino acid (AA) transport and compromised mTOR signaling in the mammary gland. Cows were enrolled into heat-stressed (dry-HT, n = 12) or cooled (dry-CL, n = 12) treatments for a 46-day dry period then cooled after calving. Milk yield and composition and dry matter intake were recorded, and milk, blood, and mammary tissue samples were collected at 14, 42, and 84 days in milk (DIM) to determine free AA concentrations, milk protein fractions, and mammary AA transporter and mTOR pathway gene and protein expression. Dry matter intake did not significantly differ between treatments pre- or postpartum. Compared with dry-CL cows, milk yield was decreased (32.3 vs. 37.7 ± 1.6 kg/day) and milk protein yield and content were reduced in dry-HT cows by 0.18 kg/day and 0.1%. Further, dry-HT cows had higher plasma concentrations of glutamic acid, phenylalanine, and taurine. Gene expression of key AA transporters was upregulated at 14 and 42 DIM in dry-HT cows. Despite minor changes in mTOR pathway gene expression, the protein 4E-BP1 was upregulated in dry-HT cows at 42 DIM whereas Akt and p70 S6K1 were downregulated. These results indicate major mammary metabolic adaptations during lactation after prior exposure to dry period heat stress.

The objectives of this experiment were to evaluate and compare underivatized (UND) and precolumn derivatized (DER) methods for quantification of bovine plasma AA by isotope dilution ratio via liquid chromatography-electrospray ionization (ESI)-single quadrupole mass spectrometry. Linearity of the mass-to-charge ratio signal and area signal sensitivity of 12 C were evaluated for each AA with 5-point standard curves (range: 1.1-500 µ M ). Plasma from lactating dairy cows was isolated by centrifugation and deproteinized using 1 N perchloric acid with a final concentration of 0.5 N . Deproteinized plasma was filtered and injected into a 50 × 2-mm column (Imtakt) or extracted, derivatized, and injected into a 250 × 3-mm column (EZ:faast, Phenomenex) and analyzed via liquid chromatography-ESI-single quadrupole mass spectrometry. Coefficients of variation and recovery rates were evaluated using 4 replicates of pooled plasma samples spiked with each AA at concentrations of 10, 20, and 50 µ M . In addition, a subset of 24 plasma samples was used to directly compare methods using linear regression, correlation coefficient (r), concordance correlation coefficient (CCC), and Bland-Altman plot test. Both methods showed linearity within the dynamic range analyzed for all essential AA (coefficient of determination, R 2 ≥ 0.995) and most other AA, although the UND samples had poor linearity (R 2 ≤ 0.990) or peak resolution problems for Asp, Gly, Tyr, and Ser. Moreover, area signal sensitivity for  12 C AA was greater for DER samples than for UND samples [range: 2.2× (Pro) to 309.5× (Ala)]. Both methods had recovery rates ranging from 85.7 to 119.8.0%, and none differed from 100% except Gln [20 µ M (85.7%) and 50 µ M (87.6%)] and Val [50 µ M (119.8%)] using the UND method. The UND method had a coefficient of variation ranging from 0.9% (Val) to 7.8% (His), whereas for the DER method the range was 2.2% (Glu) to 8.8% (Asp). The highest correlation coefficient (>0.90) and CCC (>0.90) were observed for Arg, Ile, Leu, Met, Thr, Trp, Val, and Gln, with the Bland-Altman plot test showing minimal mean bias for these AA. Lowest values were observed for His (r = 0.46; CCC = 0.45), Lys (r = 0.76; CCC = 0.75), Ala (r = 0.83; CCC = 0.73), and Glu (r = 0.65; CCC = 0.42). The UND method showed linearity, precision, and accurate recovery rates for most AA, with most essential AA having comparable values between the 2 methods. However, the DER method had greater  12 C AA area signal sensitivity, linearity, and recovery rates., (© 2021.)

Different models of lactation offer conflicting evidence as to whether insulin signaling is required for AA to stimulate mechanistic target of rapamycin complex 1 (mTORC1) activity. We hypothesized that insulin potentiates essential AA stimulation of mTORC1 activity in the MAC-T mammary epithelial cell line. Here, our objective was to assess mTORC1 signaling activity in response to insulin and individual or grouped essential AA. Insulin and essential AA concentrations in the treatment medium ranged from normo- to supraphysiological, with insulin at 0, 1, 10, or 100 nmol/L and essential AA at approximately 0, 0.01, 0.05, 0.1, 1, or 3× reference plasma levels. Effects and interaction of insulin and total essential AA were tested in a 3 × 5 factorial design (n = 3 replicates/treatment); insulin and the individual AA Leu, Met, Ile, and Arg were likewise tested in 3 × 4 factorials (n = 4). As the remaining individual AA His, Lys, Phe, Thr, Trp, and Val were expected to not affect mTORC1, these were tested only at the highest insulin level, 100 nmol/L (n = 4). For all of these, linear and quadratic effects of total and individual AA were evaluated. Essential AA were subsequently grouped by their positive (Leu, Met, Ile, Arg, and Thr; TOR-AA) or absent-to-negative effects (His, Lys, Phe, Trp, and Val; NTOR-AA), and tested for interaction in a 2 × 2 factorial design (n = 4), with each AA at its respective 1× plasma level, and insulin held at 100 nmol/L. All experiments consisted of 1 h treatment incubation, followed by Western blotting of cell lysates to measure phosphorylation and abundance of the mTORC1 pathway proteins Akt (Ser473); ribosomal protein S6 kinase p70 (S6K1, Thr389); eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1, Ser65); and ribosomal protein S6 (S6, Ser240/244). The Akt phosphorylation was overall increased by insulin, with a possible negative interaction with both total essential AA and the individual AA Leu. Total essential AA also increased S6K1 and 4E-BP1 phosphorylation in an insulin-dependent manner. The individual AA Leu, Met, Ile, and Arg increased S6K1 phosphorylation in an insulin-dependent manner. Similarly, Met and Arg increased 4E-BP1 phosphorylation in an insulin-dependent manner. Histidine, Lys, Trp, and Val did not affect S6K1 phosphorylation. However, S6K1 phosphorylation was linearly increased by Thr and quadratically decreased by Phe. Relative to the phosphorylation of S6K1 when cells were incubated with no essential AA, the NTOR-AA group had no effect, whereas the TOR-AA increased phosphorylation to the same degree observed with all 10 essential AA. Overall, we have found that insulin is required for essential AA to stimulate mTORC1 activity in MAC-T cells. In addition, the AA responsible for the bulk of mTORC1 activation in MAC-T are limited to Leu, Met, Ile, Arg, and Thr., (Copyright © 2020 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

For dairy production systems, nitrogen is an expensive nutrient and potentially harmful waste product. With three quarters of fed nitrogen ending up in the manure, significant research efforts have focused on understanding and mitigating lactating dairy cows' nitrogen losses. Recent changes proposed to the Nutrient Requirement System for Dairy Cattle in the US include variable efficiencies of absorbed essential AA for milk protein production. This first separation from a purely substrate-based system, standing on the old limiting AA theory, recognizes the ability of the cow to alter the metabolism of AA. In this review we summarize a compelling amount of evidence suggesting that AA requirements for milk protein synthesis are based on a demand-driven system. Milk protein synthesis is governed at mammary level by a set of transduction pathways, including the mechanistic target of rapamycin complex 1 (mTORC1), the integrated stress response (ISR), and the unfolded protein response (UPR). In tight coordination, these pathways not only control the rate of milk protein synthesis, setting the demand for AA, but also manipulate cellular AA transport and even blood flow to the mammary glands, securing the supply of those needed nutrients. These transduction pathways, specifically mTORC1, sense specific AA, as well as other physiological signals, including insulin, the canonical indicator of energy status. Insulin plays a key role on mTORC1 signaling, controlling its activation, once AA have determined mTORC1 localization to the lysosomal membrane. Based on this molecular model, AA and insulin signals need to be tightly coordinated to maximize milk protein synthesis rate. The evidence in lactating dairy cows supports this model, in which insulin and glucogenic energy potentiate the effect of AA on milk protein synthesis. Incorporating the effect of specific signaling AA and the differential role of energy sources on utilization of absorbed AA for milk protein synthesis seems like the evident following step in nutrient requirement systems to further improve N efficiency in lactating dairy cow rations.

Inhibition of mTOR (mechanistic Target Of Rapamycin) signaling by rapamycin promotes healthspan and longevity more strongly in females than males, perhaps because inhibition of hepatic mTORC2 (mTOR Complex 2) specifically reduces the lifespan of males. Here, we demonstrate using gonadectomy that the sex-specific impact of reduced hepatic mTORC2 is not reversed by depletion of sex hormones. Intriguingly, we find that ovariectomy uncouples lifespan from metabolic health, with ovariectomized females having improved survival despite paradoxically having increased adiposity and decreased control of blood glucose levels. Further, ovariectomy unexpectedly promotes midlife survival of female mice lacking hepatic mTORC2, significantly increasing the survival of those mice that do not develop cancer. In addition to identifying a sex hormone-dependent role for hepatic mTORC2 in female longevity, our results demonstrate that metabolic health is not inextricably linked to lifespan in mammals, and highlight the importance of evaluating healthspan in mammalian longevity studies., Competing Interests: SA, AL, JB, EM, MM, JT, CP, EB, NR, MA No competing interests declared, DL DWL has received funding from, and is a scientific advisory board member of, Aeovian Pharmaceuticals, which seeks to develop novel, selective mTOR inhibitors for the treatment of various diseases.

Amino acids and glucose have been shown to regulate protein synthesis in the mammary gland through their effects on cellular signaling pathways. Acetate might also have an effect on protein synthesis via the AMP-activated kinase signaling pathway, because it is the main energy source for the mammary secretory cell. Thus, the objective of this experiment was to evaluate the effects of casein and energy-yielding nutrients (acetate and glucose), and their combination, on performance and mammary metabolism. Six multiparous Holstein cows, averaging 49 kg of milk/d, were used in a 6 × 6 Latin square design with 14-d periods. Cows were fed to 100% National Research Council requirements for metabolizable protein (MP) and energy (ME) for 9 d, after which they were feed-restricted for 5 d to 85% of their individual ad libitum intake and then abomasally infused with 1 of 6 treatments. Treatments were acetate (A), glucose (G), each at 5% of ad libitum ME intake, casein (C) at 15% of ad libitum MP intake, A + C, G + C, or a saline solution (negative control). Casein infused alone increased milk protein yield numerically, with 25% recovery of the infused casein in milk protein. Glucose infused alone increased milk and milk protein yield and promoted the highest efficiency of nitrogen utilization (37%), with an efficiency of MP use for milk protein of 58%. We discovered no effect of treatment on mammary plasma flow, and the increase in milk protein yield with glucose infusion was brought about by greater mammary AA clearance rate. Infusion of casein and glucose together further increased milk protein yield in an additive fashion, and 47% of the infused casein was recovered in milk protein. Acetate infused alone had no effect on milk protein yield but increased milk fat yield numerically, suggesting that the greater amount of acetate taken up by the mammary gland was used for milk fat synthesis. Infusion of acetate and casein together yielded responses similar to those of casein alone. In conclusion, glucose has a major effect on stimulating milk protein synthesis, and the mammary gland has the ability to increase its supply of nutrients to match its synthetic capacity., (Copyright © 2020 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Background: Understanding the mechanisms of N utilization for lactation can lead to improved requirement estimates and increased efficiency, which modern dairy diets currently fail to maximize. The mechanistic target of rapamycin complex 1 (mTORC1) is a central hub of translation regulation, processing extra- and intra-cellular signals of nutrient availability and physiological state, such as amino acids and energy. We hypothesized that dietary amino acids regulate lactation through mTORC1, such that inhibition of mTORC1 will lead to decreased lactation performance when amino acids are not limiting. Our objectives were to assess lactation performance in lactating mice undergoing dietary and pharmacologic interventions designed to alter mTORC1 activity., Methods: First lactation mice ( N  = 18; n  = 6/treatment) were fed an adequate protein diet (18% crude protein), or an isocaloric protein-restricted diet (9% crude protein) from the day after parturition until lactation day 13. A third group of mice was fed an adequate protein diet and treated with the mTORC1 inhibitor rapamycin (4 mg/kg every other day) intraperitoneally, with the first two groups treated with vehicle as control. Dams and pups were weighed daily, and feed intake was recorded every other day. Milk production was measured every other day beginning on lactation day 4 by the weigh-suckle-weigh method. Tissues were collected after fasting and refeeding., Results: Milk production and pup weight were similarly decreased by both protein restriction and rapamycin treatment, with final production at 50% of control ( P  = 0.008) and final pup weight at 85% of control ( P  < 0.001). Mammary phosphorylation of mTORC1's downstream targets were decreased by protein restriction and rapamycin treatment ( P  < 0.05), while very little effect was observed in the liver of rapamycin treated mice, and none by protein restriction., Conclusions: Overall, sufficient supply of dietary amino acids was unable to maintain lactation performance status in mice with pharmacologically reduced mammary mTORC1 activity, as evidenced by diminished pup growth and milk production, supporting the concept that mTORC1 activation rather than substrate supply is the primary route by which amino acids regulate synthesis of milk components., Competing Interests: Competing interestsSIAA is a consultant for Kemin Industries Inc., (© The Author(s) 2020.)

Objective: To describe a method for permanent transposition of the common carotid artery (CCA) in standing cattle., Study Design: Experimental study., Animals: Eight healthy, adult, lactating Holstein-Friesian cows., Methods: Cows were restrained with the head and neck extended by using halters, head catch, and squeeze chute. Surgery was performed under local anesthesia and intravenous sedation. The right CCA was approached through a skin incision dorsal and parallel to the jugular vein. The skin incision was extended through the brachiocephalicus and longus capitus muscles. When the vessel was present, ligation of accessory vessels of the CCA and internal jugular vein was performed to facilitate exposure. The artery was sharply dissected from the carotid sheath and elevated by using Penrose drains. The muscles were closed in two layers, leaving the artery in a subcutaneous position. The incision was protected with a tie-over bandage for 1 week. Sampling from the CCA was initiated approximately 6 weeks after surgery., Results: The CCA was successfully transposed and used for repeated arterial blood sampling in all eight cows. No cows had intraoperative complications or evidence of surgical site infection. One cow had a postoperative suture reaction at the site of a suture used for maintaining the tie-over bandage. All arteries remained patent for use in subsequent studies., Conclusion: Permanent translocation of the CCA was successful in all cows in this study and consistently allowed serial arterial blood sampling., Clinical Significance: Common carotid artery translocation is possible without general anesthesia in adult cattle and is useful in studies requiring serial sampling of arterial blood., (© 2020 The American College of Veterinary Surgeons.)

The mechanistic target of rapamycin (mTOR) is an evolutionarily conserved protein kinase that regulates growth and metabolism. mTOR is found in two protein complexes, mTORC1 and mTORC2, that have distinct components and substrates and are both inhibited by rapamycin, a macrolide drug that robustly extends lifespan in multiple species including worms and mice. Although the beneficial effect of rapamycin on longevity is generally attributed to reduced mTORC1 signaling, disruption of mTORC2 signaling can also influence the longevity of worms, either positively or negatively depending on the temperature and food source. Here, we show that loss of hypothalamic mTORC2 signaling in mice decreases activity level, increases the set point for adiposity, and renders the animals susceptible to diet-induced obesity. Hypothalamic mTORC2 signaling normally increases with age, and mice lacking this pathway display higher fat mass and impaired glucose homeostasis throughout life, become more frail with age, and have decreased overall survival. We conclude that hypothalamic mTORC2 is essential for the normal metabolic health, fitness, and lifespan of mice. Our results have implications for the use of mTORC2-inhibiting pharmaceuticals in the treatment of brain cancer and diseases of aging., (© 2019 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.)

Rapamycin, an inhibitor of mechanistic Target Of Rapamycin Complex 1 (mTORC1), extends lifespan and shows strong potential for the treatment of age-related diseases. However, rapamycin exerts metabolic and immunological side effects mediated by off-target inhibition of a second mTOR-containing complex, mTOR complex 2. Here, we report the identification of DL001, a FKBP12-dependent rapamycin analog 40x more selective for mTORC1 than rapamycin. DL001 inhibits mTORC1 in cell culture lines and in vivo in C57BL/6J mice, in which DL001 inhibits mTORC1 signaling without impairing glucose homeostasis and with substantially reduced or no side effects on lipid metabolism and the immune system. In cells, DL001 efficiently represses elevated mTORC1 activity and restores normal gene expression to cells lacking a functional tuberous sclerosis complex. Our results demonstrate that highly selective pharmacological inhibition of mTORC1 can be achieved in vivo, and that selective inhibition of mTORC1 significantly reduces the side effects associated with conventional rapalogs.

The membrane transporter AT-1/SLC33A1 translocates cytosolic acetyl-CoA into the lumen of the endoplasmic reticulum (ER), participating in quality control mechanisms within the secretory pathway. Mutations and duplication events in AT-1/SLC33A1 are highly pleiotropic and have been linked to diseases such as spastic paraplegia, developmental delay, autism spectrum disorder, intellectual disability, propensity to seizures, and dysmorphism. Despite these known associations, the biology of this key transporter is only beginning to be uncovered. Here, we show that systemic overexpression of AT-1 in the mouse leads to a segmental form of progeria with dysmorphism and metabolic alterations. The phenotype includes delayed growth, short lifespan, alopecia, skin lesions, rectal prolapse, osteoporosis, cardiomegaly, muscle atrophy, reduced fertility, and anemia. In terms of homeostasis, the AT-1 overexpressing mouse displays hypocholesterolemia, altered glycemia, and increased indices of systemic inflammation. Mechanistically, the phenotype is caused by a block in Atg9a-Fam134b-LC3β and Atg9a-Sec62-LC3β interactions, and defective reticulophagy, the autophagic recycling of the ER. Inhibition of ATase1/ATase2 acetyltransferase enzymes downstream of AT-1 restores reticulophagy and rescues the phenotype of the animals. These data suggest that inappropriately elevated acetyl-CoA flux into the ER directly induces defects in autophagy and recycling of subcellular structures and that this diversion of acetyl-CoA from cytosol to ER is causal in the progeria phenotype. Collectively, these data establish the cytosol-to-ER flux of acetyl-CoA as a novel event that dictates the pace of aging phenotypes and identify intracellular acetyl-CoA-dependent homeostatic mechanisms linked to metabolism and inflammation., (© 2018 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.)

Key Points: We recently found that feeding healthy mice a diet with reduced levels of branched-chain amino acids (BCAAs), which are associated with insulin resistance in both humans and rodents, modestly improves glucose tolerance and slows fat mass gain. In the present study, we show that a reduced BCAA diet promotes rapid fat mass loss without calorie restriction in obese mice. Selective reduction of dietary BCAAs also restores glucose tolerance and insulin sensitivity to obese mice, even as they continue to consume a high-fat, high-sugar diet. A low BCAA diet transiently induces FGF21 (fibroblast growth factor 21) and increases energy expenditure. We suggest that dietary protein quality (i.e. the precise macronutrient composition of dietary protein) may impact the effectiveness of weight loss diets., Abstract: Obesity and diabetes are increasing problems around the world, and although even moderate weight loss can improve metabolic health, reduced calorie diets are notoriously difficult to sustain. Branched-chain amino acids (BCAAs; leucine, isoleucine and valine) are elevated in the blood of obese, insulin-resistant humans and rodents. We recently demonstrated that specifically reducing dietary levels of BCAAs has beneficial effects on the metabolic health of young, growing mice, improving glucose tolerance and modestly slowing fat mass gain. In the present study, we examine the hypothesis that reducing dietary BCAAs will promote weight loss, reduce adiposity, and improve blood glucose control in diet-induced obese mice with pre-existing metabolic syndrome. We find that specifically reducing dietary BCAAs rapidly reverses diet-induced obesity and improves glucoregulatory control in diet-induced obese mice. Most dramatically, mice eating an otherwise unhealthy high-calorie, high-sugar Western diet with reduced levels of BCAAs lost weight and fat mass rapidly until regaining a normal weight. Importantly, this normalization of weight was mediated not by caloric restriction or increased activity, but by increased energy expenditure, and was accompanied by a transient induction of the energy balance regulating hormone FGF21 (fibroblast growth factor 21). Consumption of a Western diet reduced in BCAAs was also accompanied by a dramatic improvement in glucose tolerance and insulin resistance. Our results link dietary BCAAs with the regulation of metabolic health and energy balance in obese animals, and suggest that specifically reducing dietary BCAAs may represent a highly translatable option for the treatment of obesity and insulin resistance., (© 2017 The Authors. The Journal of Physiology © 2017 The Physiological Society.)

Recently, it has become apparent that dietary macronutrient composition has a profound impact on metabolism, health and even lifespan. Work from many laboratories now suggest that dietary protein quality - the precise amino acid composition of the diet, as well as possibly the source of dietary protein - may also be critical in regulating the impact of diet on health. Perhaps in part due to the naturally low methionine content of plants, vegan diets are associated with a decreased risk of diabetes and improved insulin sensitivity, but this association is confounded by the lower overall protein intake of vegans. Here, we test the effect of consuming isocaloric rodent diets with similar amino acid profiles derived from either plant protein or dairy protein. We find that male C57BL/6J mice consuming either diet have similar glycemic control, as assessed by glucose, insulin, and pyruvate tolerance tests, and have similar overall body composition. We conclude that short-term feeding of plant protein has no positive or negative effect on the metabolic health of young male C57BL/6J mice, and suggest that dietary interventions that alter either dietary protein levels or the levels of specific essential amino acids are more likely to improve metabolic health than alterations in dietary protein source.

In this issue of Molecular Cell, Moloughney et al. (2016) find that mTORC2 responds to falling levels of glucose and glutamine catabolites, promoting glutaminolysis and preserving the TCA cycle and hexosamine biosynthesis., (Copyright © 2016 Elsevier Inc. All rights reserved.)

To improve dietary protein use efficiency in lactating cows, mammary protein synthesis responses to AA, energy substrates, and hormones must be better understood. These entities exert their effects through stimulation of mRNA translation via control of initiation and elongation rates at the cellular level. A central protein kinase of this phenomenon is the mammalian target of rapamycin (mTOR), which transfers the nutritional and hormonal stimuli onto a series of proteins downstream through a cascade of phosphorylation reactions that ultimately affect protein synthesis. The objective of this work was to further develop an existing mechanistic model of mTOR phosphorylation responses to insulin and total essential AA to include the effects of specific essential AA and acetate mediated by signaling proteins including protein kinase B (Akt), adenosine monophosphate activated protein kinase (AMPK), and mTOR and to add a representation of milk protein synthesis. Data from 6 experiments in MAC-T cells and mammary tissue slices previously conducted in our laboratory were assembled and used to parameterize the dynamic system of differential equations representing Akt, AMPK, and mTOR in their phosphorylated and dephosphorylated states and the resulting regulation of milk protein synthesis. The model predicted phosphorylated Akt, mTOR, AMPK, and casein synthesis rates with root mean square prediction errors of 16.8, 28.4, 33.0, and 54.9%, respectively. All other dependent variables were free of mean and slope bias, indicating an adequate representation of the data. Whereas mTOR was not very sensitive to changes in insulin or acetate levels, it was highly sensitive to leucine and isoleucine, and this signal appeared to be effectively transduced to casein synthesis. Although prior work had observed a relationship with additional essential AA, and data supporting those conclusions were present in the data set, we were unable to derive significant relationships with any essential AA other than leucine and isoleucine. The signaling properties and dynamics of AMPK under nutrient depletion and sufficiency, the responses to additional essential AA, and the consequent effects on protein synthesis remain to be better understood., (Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Protein-restricted (PR), high-carbohydrate diets improve metabolic health in rodents, yet the precise dietary components that are responsible for these effects have not been identified. Furthermore, the applicability of these studies to humans is unclear. Here, we demonstrate in a randomized controlled trial that a moderate PR diet also improves markers of metabolic health in humans. Intriguingly, we find that feeding mice a diet specifically reduced in branched-chain amino acids (BCAAs) is sufficient to improve glucose tolerance and body composition equivalently to a PR diet via metabolically distinct pathways. Our results highlight a critical role for dietary quality at the level of amino acids in the maintenance of metabolic health and suggest that diets specifically reduced in BCAAs, or pharmacological interventions in this pathway, may offer a translatable way to achieve many of the metabolic benefits of a PR diet., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Rapamycin (sirolimus) is a macrolide immunosuppressant that inhibits the mechanistic target of rapamycin (mTOR) protein kinase and extends lifespan in model organisms including mice. Although rapamycin is an FDA-approved drug for select indications, a diverse set of negative side effects may preclude its wide-scale deployment as an antiaging therapy. mTOR forms two different protein complexes, mTORC1 and mTORC2; the former is acutely sensitive to rapamycin whereas the latter is only chronically sensitive to rapamycin in vivo. Over the past decade, it has become clear that although genetic and pharmacological inhibition of mTORC1 extends lifespan and delays aging, inhibition of mTORC2 has negative effects on mammalian health and longevity and is responsible for many of the negative side effects of rapamycin. In this review, we discuss recent advances in understanding the molecular and physiological effects of rapamycin treatment, and we discuss how the use of alternative rapamycin treatment regimens or rapamycin analogs has the potential to mitigate the deleterious side effects of rapamycin treatment by more specifically targeting mTORC1. Although the side effects of rapamycin are still of significant concern, rapid progress is being made in realizing the revolutionary potential of rapamycin-based therapies for the treatment of diseases of aging., (© The Author 2016. Published by Oxford University Press on behalf of The Gerontological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Inhibition of the mTOR (mechanistic target of rapamycin) signaling pathway by the FDA-approved drug rapamycin promotes life span in numerous model organisms and delays age-related disease in mice. However, the utilization of rapamycin as a therapy for age-related diseases will likely prove challenging due to the serious metabolic and immunological side effects of rapamycin in humans. We recently identified an intermittent rapamycin treatment regimen-2mg/kg administered every 5 days-with a reduced impact on glucose homeostasis and the immune system as compared with chronic treatment; however, the ability of this regimen to extend life span has not been determined. Here, we report for the first time that an intermittent rapamycin treatment regimen starting as late as 20 months of age can extend the life span of female C57BL/6J mice. Our work demonstrates that the anti-aging potential of rapamycin is separable from many of its negative side effects and suggests that carefully designed dosing regimens may permit the safer use of rapamycin and its analogs for the treatment of age-related diseases in humans., (© The Author 2016. Published by Oxford University Press on behalf of The Gerontological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Inhibition of the mechanistic target of rapamycin (mTOR) signaling pathway by the FDA-approved drug rapamycin has been shown to promote lifespan and delay age-related diseases in model organisms including mice. Unfortunately, rapamycin has potentially serious side effects in humans, including glucose intolerance and immunosuppression, which may preclude the long-term prophylactic use of rapamycin as a therapy for age-related diseases. While the beneficial effects of rapamycin are largely mediated by the inhibition of mTOR complex 1 (mTORC1), which is acutely sensitive to rapamycin, many of the negative side effects are mediated by the inhibition of a second mTOR-containing complex, mTORC2, which is much less sensitive to rapamycin. We hypothesized that different rapamycin dosing schedules or the use of FDA-approved rapamycin analogs with different pharmacokinetics might expand the therapeutic window of rapamycin by more specifically targeting mTORC1. Here, we identified an intermittent rapamycin dosing schedule with minimal effects on glucose tolerance, and we find that this schedule has a reduced impact on pyruvate tolerance, fasting glucose and insulin levels, beta cell function, and the immune system compared to daily rapamycin treatment. Further, we find that the FDA-approved rapamycin analogs everolimus and temsirolimus efficiently inhibit mTORC1 while having a reduced impact on glucose and pyruvate tolerance. Our results suggest that many of the negative side effects of rapamycin treatment can be mitigated through intermittent dosing or the use of rapamycin analogs., (© 2015 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.)

When fed to meet the metabolizable protein requirements of the National Research Council, dairy cows consume an excess of N, resulting in approximately 75% of dietary N being lost to the environment as urine and feces. Reductions in environmental N release could be attained through an improvement in N efficiency. The objective of this study was to determine if the predicted reduction in milk yield associated with feeding a low-protein diet to lactating dairy cows could be avoided by dietary supplementation with 1 or more ruminally protected (RP) AA. Fourteen multiparous and 10 primiparous Holstein cows, and 24 multiparous Holstein × Jersey crossbred cows were used in a Youden square design consisting of 8 treatments and 3 periods. The 8 dietary treatments were (1) a standard diet containing 17% crude protein [CP; positive control (PC)], (2) a 15% CP diet [negative control (NC)], (3) NC plus RP Met (+M), (4) NC plus RP Lys (+K), (5) NC plus RP Leu (+L), (6) NC plus RP Met and Lys (+MK), (7) NC plus RP Met and Leu (+ML), and (8) NC plus RP Met, Lys, and Leu (+MKL). Dry matter intake was not affected by treatment. Crude protein intake was lower for NC and RP AA treatments compared with the PC treatment. No detrimental effect was detected of the low-CP diet alone or in combination with AA supplementation on milk and fat yield. However, milk protein yield decreased for NC and +MKL diets, and lactose yield decreased for the +MKL compared with the PC diet. Milk urea N concentrations were lower for all diets, suggesting that greater N efficiency was achieved by feeding the low-protein diet. Minimal effects of treatments on arterial plasma essential AA concentrations were detected, with only Ile and Val being significantly lower in the NC than in the PC diet. Phosphorylation ratios of signaling proteins known to regulate mRNA translation were not affected by treatments. This study highlights the limitations of requirement models aggregated at the protein level and the use of fixed postabsorptive efficiency to calculate milk protein requirements. Milk protein synthesis regulation by signaling pathways in vivo is still poorly understood., (Copyright © 2014 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

In current dairy production systems, an average of 25% of dietary N is captured in milk, with the remainder being excreted in urine and feces. About 60% of total N losses occur postabsorption. Splanchnic tissues extract a fixed proportion of total inflow of each essential AA (EAA). Those EAA removed by splanchnic tissues and not incorporated into protein are subjected to catabolism, with the resulting N converted to urea. Splanchnic affinity varies among individual EAA, from several fold lower than mammary glands' affinity for the branched-chain AA to similar or higher affinity for Phe, Met, His, and Arg. On average, 85% of absorbed EAA appear in peripheral circulation, indicating that first-pass removal is not the main source of loss. Essential AA in excess of the needs of the mammary glands return to general circulation. High splanchnic blood flow dictates that a large proportion of EAA that return to general circulation flow through splanchnic tissues. In association with this constant recycling, EAA are removed and catabolized by splanchnic tissues. This results in splanchnic catabolism equaling or surpassing the use of many EAA for milk protein synthesis. Recent studies have demonstrated that EAA, energy substrates, and hormones activate signaling pathways that in turn regulate local blood flow, tissue extraction of EAA, and rates of milk protein synthesis. These recent findings would allow manipulation of dairy diets to maximize mammary uptake of EAA and reduce catabolism by splanchnic tissues. Dairy cattle nutrient requirement systems consider EAA requirements in aggregate as metabolizable protein (MP) and assume a fixed efficiency of MP use for milk protein. Lysine and Met sufficiency is only considered after MP requirements have been met. By doing so, requirement systems limit the scope of diet manipulation to achieve improved gross N efficiency. Therefore, this review focuses on understanding the dynamics of EAA metabolism in mammary and splanchnic tissues that would lead to improved requirement prediction systems. Inclusion of variable individual EAA efficiencies derived from splanchnic and mammary responses to nutrient and hormonal signals should help reduce dietary protein levels. Supplementing reduced crude protein diets with individual EAA should increase gross N efficiency to more than 30%, reducing N excretion by the US dairy industry by 92,000 t annually., (Copyright © 2014 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Specific AA affect rates of milk protein synthesis in the mammary glands of lactating cows. The objective of this study was to quantify the rate of αS1-casein synthesis in response to Ile, Leu, Met, and Thr supplementation, and to test the single-limiting AA theory for milk protein synthesis by exploring interactions among these AA. Effects of Ile, Leu, Met, and Thr were studied in vitro with a composite design containing a central point repeated 4 times, with 2 axial points per AA and a complete 2(4) factorial. Other AA were at the concentration in Dulbecco's modified Eagle medium/F12 medium (DMEM). The experiment was replicated with mammary tissue from 5 lactating cows. Mammary tissue slices (0.12 ± 0.02 g) were incubated for 4h at 37°C in 5 mL of treatment medium containing (2)H5-Phe. Caseins were precipitated from cell homogenate supernatants. Enrichment with (2)H5-Phe of the N[34]LLRFFVAPFPE αS1 peptide was determined by matrix-assisted laser desorption/ionization-tandem time-of-flight (MALDI-TOF-TOF), which was used to determine enrichment of Phe in the transfer (t)RNA pool and αS1-casein fractional synthesis rates (CFSR). Data were analyzed with a polynomial mixed model containing linear, quadratic, and 2-factor interactions for Ile, Leu, Met, and Thr, and cow and residual as random factors. Interactions were not significant at P<0.1 and were removed from the model. Increasing concentrations of Ile, Leu, Met, and Thr simultaneously increased CFSR curvilinearly with a predicted maximum response of 4.32 ± 0.84%/h at 63% of DMEM concentrations. The maximum response to each of the 4 AA was at 71, 49, 60, and 32% of the concentration in DMEM, for Ile, Leu, Met, and Thr, respectively. These values correspond to 270, 120, 440, and 140% the plasma concentrations of Ile, Leu, Met, and Thr observed in lactating cows fed to meet National Research Council requirements, respectively. The CFSR estimated at those maxima were similar among AA (3.6 ± 0.6%/h). Individual AA effects on CFSR did not correlate with mammalian target of rapamycin (mTOR) signaling. Independent responses of CFSR to individual essential AA observed in this study contradict the single-limiting AA theory assumed in current requirement systems. The saturable responses in CFSR to these 4 AA also highlight the inadequacy of using a fixed postabsorptive AA efficiency approach for determining AA requirements for milk protein synthesis., (Copyright © 2014 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Improved representation of postabsorptive N metabolism in lactating dairy cows requires a better understanding of protein synthesis regulation in the mammary glands. This study aimed to determine the quantitative effects of Ile, Leu, Met, and Thr on the phosphorylation state of signaling proteins that regulate protein synthesis. The experiment used a composite design with a central point, 2 axial points per AA, and a complete 2(4) factorial. All of the other AA were provided at the concentrations in Dulbecco's modified Eagle's medium. The experiment was replicated with tissues from 5 lactating cows. Mammary tissue slices (0.12 ± 0.02 g) were incubated for 4h. Total and site-specific phosphorylated mammalian target of rapamycin (mTOR; Ser2448), eukaryotic elongation factor (eEF) 2 (Thr56), ribosomal protein S6 (Ser235/236), and eukaryotic initiation factor 2α (Ser51) were determined by western immunoblotting. Tissue concentrations of the 4 AA studied responded linearly to media supply. Addition of Ile, Leu, Met, or Thr had no effect on eukaryotic initiation factor 2α phosphorylation. Isoleucine and Thr positively affected mTOR phosphorylation. However, the 2 AA had an antagonistic relationship. Similarly, Ile linearly increased ribosomal protein S6 phosphorylation, and Thr inhibited the Ile effect. In addition, eEF2 phosphorylation was linearly decreased by Ile and Leu. Threonine curvilinearly decreased eEF2 phosphorylation, Ile and Leu negatively interacted on eEF2, and Thr tended to inhibit Leu effects on eEF2. This work demonstrated saturable responses and interactions between AA on activation of the mTOR pathway. Incorporation of these concepts into milk protein response models will help to improve milk and milk protein yield predictions and increase postabsorptive N efficiency and reduce N excretion by dairy cows., (Copyright © 2014 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)