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

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.     

Long-acting insulins alter milk composition and metabolism of lactating dairy cows

This study investigated the effect of 2 different types of long-acting insulin on milk production, milk composition, and metabolism in lactating dairy cows. Multiparous cows (n = 30) averaging 88 d in milk were assigned to one of 3 treatments in a completely randomized design. Treatments consisted of control (C), Humulin-N (H; Eli Lilly and Company, Indianapolis, IN), and insulin glargine (L). The H and L treatments were administered twice daily at 12-h intervals via subcutaneous injection for 10 d. Cows were milked twice daily, and milk composition was determined every other day. Mammary biopsies were conducted on d 11, and mammary proteins extracted from the biopsies were analyzed by Western blot for components of insulin and mammalian target of rapamycin signaling pathways. Treatment had no effect on dry matter intake or milk yield. Treatment with both forms of long-acting insulin increased milk protein content and tended to increase milk protein yield over the 10-d treatment period. Analysis of milk N fractions from samples collected on d 10 of treatment suggested that cows administered L tended to have higher yields of milk protein fractions than cows administered H. Milk fat content and yield tended to be increased for cows administered long-acting insulins. Lactose content and yields were decreased by treatment with long-acting insulins. Administration of long-acting insulins, particularly L, tended to shift milk fatty acid composition toward increased short- and medium-chain fatty acids and decreased long-chain fatty acids. Plasma concentrations of glucose and urea N were lower for cows administered long-acting insulins; interactions of treatment and sampling time were indicative of more pronounced effects of L than H on these metabolites. Concentrations of nonesterified fatty acids and insulin were increased in cows administered long-acting insulins. Decreased concentrations of urea N in both plasma and milk suggested more efficient use of N in cows administered long-acting insulins. Western blot analysis of mammary tissue collected by biopsy indicated that the ratios of phosphorylated protein kinase b (Akt) to total Akt and phosphorylated ribosomal protein S6 (rpS6) to total rpS6 were not affected by long-acting insulins. Modestly elevating insulin activity in lactating dairy cows using long-acting insulins altered milk composition and metabolism. Future research should explore mechanisms by which either insulin concentrations or insulin signaling pathways in the mammary gland can be altered to enhance milk fat and protein production.     

Isoleucine,leucine, methionine,and threonine effects on mammalian target of rapamycin signaling in mammary tissue

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 2⁴ 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 4 h. 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.     

Dried distillers grains plus solubles with corn silage or alfalfa hay as the primary forage source in dairy cow diets

Nine multiparous (250 ± 6 d in milk) and 3 primiparous (204 ± 6 d in milk) Holstein cows were utilized in a 3 × 3 Latin square design to evaluate the lactation performance of cows fed a diet containing dried distillers grains plus solubles (DDGS) with either corn silage or alfalfa hay as forage. Cows were fed total mixed diets containing corn silage (CS), 50% corn silage and 50% alfalfa hay (CSAH), or alfalfa hay (AH) as the forage source. All diets had a 50:50 forage-to-concentrate ratio, contained 15% DDGS, and were formulated to be equal in metabolizable protein. Dry matter intake increased when cows were fed CSAH (24.9 kg/d) compared with CS (21.9 kg/d) and AH (20.9 kg/d). Yields of milk (26.5, 28.4, 29.0 kg/d for CS, CSAH, and AH, respectively) increased linearly as proportions of alfalfa fed increased but 4% fat-corrected milk and energy-corrected milk were not affected by treatment. Feed efficiency (1.28, 1.23, and 1.45 kg of energy-corrected milk/kg of intake) improved when AH was fed compared with CS or CSAH. Milk fat concentration (3.67, 3.55, and 3.49%) decreased linearly when alfalfa replaced corn silage, but was observed only in primiparous cows, not multiparous cows. Milk protein concentration (3.32, 3.29, and 3.29%) was not affected by diet although yield (0.90, 0.96, and 0.98 kg/d) tended to increase linearly when alfalfa was added to the diet. This may have been due to an increase in essential amino acid (AA) availability and uptake by the mammary gland or to greater crude protein intake in cows fed AH. In addition, replacing corn silage with alfalfa increased the uptake of Lys by the mammary gland. Methionine was the first-limiting AA based on the transfer efficiency of AA in arterial plasma to milk protein. However, Lys was the first-limiting AA in CS and CSAH and Met was first limiting in AH for mammary gland extraction efficiency of AA from plasma. In conclusion, replacing corn silage with alfalfa hay in diets containing 15% DDGS increased milk yield and tended to increase milk protein yield linearly in cows during late lactation. Feeding alfalfa hay as the sole forage source improved feed efficiency compared with diets containing corn silage.     

Regulation of protein synthesis in mammary glands of lactating dairy cows by starch and amino acids

The objective of this study was to evaluate local molecular adaptations proposed to regulate protein synthesis in the mammary glands. It was hypothesized that AA and energy-yielding substrates independently regulate AA metabolism and protein synthesis in mammary glands by a combination of systemic and local mechanisms. Six primiparous mid-lactation Holstein cows with ruminal cannulas were randomly assigned to 4 treatment sequences in a replicated incomplete 4 × 4 Latin square design experiment. Treatments were abomasal infusions of casein and starch in a 2 × 2 factorial arrangement. All animals received the same basal diet (17.6% crude protein and 6.61 MJ of net energy for lactation/kg of DM) throughout the study. Cows were restricted to 70% of ad libitum intake and abomasally infused for 36 h with water, casein (0.86 kg/d), starch (2 kg/d), or a combination (2 kg/d starch + 0.86 kg/d casein) using peristaltic pumps. Milk yields and composition were assessed throughout the study. Arterial and venous plasma samples were collected every 20 min during the last 8 h of infusion to assess mammary uptake. Mammary biopsy samples were collected at the end of each infusion and assessed for the phosphorylation state of selected intracellular signaling molecules that regulate protein synthesis. Animals infused with casein had increased arterial concentrations of AA, increased mammary extraction of AA from plasma, either no change or a trend for reduced mammary AA clearance rates, and no change in milk protein yield. Animals infused with starch had increased milk and milk protein yields, increased mammary plasma flow, reduced arterial concentrations of AA, and increased mammary clearance rates and net uptake of some AA. Infusions of starch increased plasma concentrations of glucose, insulin, and insulin-like growth factor-I. Starch infusions increased phosphorylation of ribosomal protein S6 and endothelial nitric oxide synthase, consistent with changes in milk protein yields and plasma flow, respectively. Phosphorylation of the mammalian target of rapamycin was increased in response to starch only when casein was also infused. Thus, cell signaling molecules involved in the regulation of protein synthesis differentially responded to these nutritional stimuli. The hypothesized independent effects of casein and starch on animal metabolism and cell signaling were not observed, presumably because of the lack of a milk protein response to infused casein.     

Milk synthetic response of the bovine mammary gland to an increase in the local concentration of amino acids and acetate

Rates of secretion of components into milk are a function of precursor concentrations and parameters that describe expression of the milk synthetic enzymes and their sensitivity to precursor concentrations. To establish the enzymatic sensitivities of milk fat yield and mammary acetate utilization to circulating acetate concentration, lactating cows were infused for 10 h with 0 or 40 g of acetate/h in an external iliac artery supplying one udder half. In addition, to investigate the possibility that energy supply influences the milk protein response to an elevated amino acid (AA) concentration, 2 different AA profiles were infused with and without acetate. Six cows, fed a total mixed ration of 21% crude protein ad libitum, were infused with AA at 0 g/h, 30 g/h in the profile of rumen microbes, or 30 g/h in the profile of milk proteins, in a 3 × 2 factorial arrangement with the 2 acetate treatments of 0 and 40 g/h, all in a 6 × 6 Latin square. Amino acid infusion caused a 60% increase, on average, in plasma concentration of AA entering the infused udder half. From the microbial AA profile, 49% of infused AA were taken up by the udder half, 42% of which occurred during the first pass. From the milk AA profile, 44% of infused AA were taken up by the udder half, 50% of which occurred during the first pass. There was an 8% increase in yield of milk protein with AA infusion, representing 7% capture, but no effect of the infused profile. Acetate infusion caused a decrease in the yields of milk protein and lactose when AA were infused, but not when AA were absent. Milk fat yields were not affected, although acetate concentrations in plasma entering the infused udder half increased by 123% and mammary uptakes increased by 128%. Mammary uptakes of long-chain fatty acids and β-hydroxybutyrate were not affected by acetate infusion, whereas glucose uptakes tended to increase. It was suggested that excess acetate may have been sequestered in adipose tissue in the udder. Yields of both protein and fat in milk showed a low sensitivity to the concentration of their precursors in circulation. It was concluded that the Km in Michaelis-Menten-type equations describing milk synthesis should be assigned a low value, and that the Vmax is regulated to bring about changes in milk yield and composition.     

Effects of jugular infusions of isoleucine,leucine, methionine,threonine, and other amino acids on insulin and glucagon concentrations,mammalian target of rapamycin (mTOR) signaling,and lactational performance in goats

The supply and profile of absorbed AA may affect milk protein synthesis through hormonal changes and mammalian target of rapamycin (mTOR) signaling pathways; and Ile, Leu, Met, and Thr (ILMT) are the 4 AA that have been reported to have the greatest effect on mammary mTOR signaling. The extent to which ILMT and the other remaining AA (RAA) differ in their effects on milk protein synthesis needs to be systematically investigated. In this study, 5 lactating goats, averaging 120 ± 10 d in milk, fitted with jugular vein and carotid artery catheters, were fasted for 24 h, followed by intravenous infusions of a mixture containing AA and glucose for 8 h in a 5 × 5 Latin square design. The AA mixtures were formulated according to the profile of casein. The amounts of AA infused were calculated based on supplies of AA when metabolizable protein (MP) was at requirement (MR). Treatments were an infusate containing glucose without AA (NTAA); an infusate containing 3 × the MR of Ile, Leu, Met and Thr (3F0R); and infusates containing 3F0R plus 1, 2, or 3 × MR of RAA (3F1R, 3F2R, and 3F3R, respectively) according to amounts provided when fed to meet MP requirements for maintenance and lactation for each goat. Milk, arterial blood, and mammary tissue samples were collected immediately after halting the infusion. Relative to NTAA, supplementation of ILMT tended to increase milk protein production and plasma glucose concentrations, and increased milk and lactose production, but had no effects on production or content of milk fat. Graded supplementation of RAA tended to quadratically affect production of milk and lactose. Arterial glucose and glucagon concentrations decreased linearly, and plasma insulin concentrations decreased quadratically with increased RAA. Mammary p70-S6K1 phosphorylation was decreased by addition of ILMT compared with NTAA but increased linearly with increased RAA infusion. Furthermore, EIF4EBP1 gene expression was much lower for 3F-treated goats than for the NTAA treatment. Both MTOR and RPS6KB1 gene expressions were decreased quadratically with increased RAA supply. These results suggested that short-term milk protein yield tended to be increased by elevated ILMT availability, and this trend was not explained by variations in mammary mTOR signaling or pancreatic hormone secretions, whereas graded increase of RAA in combination with ILMT appeared to regulate the efficiency of conversion of glucose to lactose in a manner not involving milk protein production.     

Changes in production and mammary metabolism of dairy cows in response to essential and nonessential amino acid infusions

This study was undertaken to increase our understanding of the need of the mammary gland for the different types of AA and how the mammary gland alters its metabolism in response to a variable AA supply. Eight lactating Holstein cows (61 ± 4 DIM) were used in a replicated 4 × 4 Latin square balanced for residual effects with 14-d periods. The diet was formulated to supply 100% of the net energy requirement and 72% of the metabolizable protein requirement. The 4 treatments were 1) abomasal infusions of water, 2) essential AA at 359 g/d, 3) nonessential AA at 356 g/d, and 4) essential AA at 359 g/d + nonessential AA at 356 g/d (total of 715 g/d). The infusates had the same AA profile as casein with the exception that Met was increased to maintain a 3:1 ratio of digestible Lys to Met and because of solubility limitations all the Tyr was replaced by Phe and part of the Glu was replaced by Gln. Milk yield and milk protein yield were increased by the essential AA treatments compared with the other treatments. Mammary uptake of β-hydroxybutyrate plus lactate tended to increase with the essential AA treatments, whereas glucose mammary uptake tended to be higher with the nonessential AA treatments. With the essential AA treatments, the mammary uptake:milk protein output ratio for the group 1 AA (His, Met, Phe, Trp, and Tyr) did not differ from 1 but tended to increase; the ratio for the group 2 AA (Ile, Leu, Lys, and Val) did increase, significantly exceeding unity when essential AA were infused. These results indicate that the mammary gland alters differently its uptake of both AA and energy-yielding nutrients in response to the amount and profile of AA presented to it and that even under situations of protein deficiency nonessential AA supplementation does not enhance milk and milk protein synthesis.     

Mammary gland metabolite utilization in response to exogenous glucose or long-chain fatty acids at low and high metabolizable protein levels

We investigated mammary gland metabolism in lactating dairy cattle in response to energy from glucogenic (glucose; GG) or lipogenic (palm olein; LG) substrates at low (LMP) and high (HMP) metabolizable protein levels. According to a 6 × 6 Latin square design, 6 rumen-fistulated second-lactation Holstein-Friesian dairy cows (97 ± 13 d in milk) were abomasally infused with saline (LMP-C); isoenergetic infusions (digestible energy basis) of 1,319 g/d glucose (LMP-GG), 676 g/d palm olein (LMP-LG), or 844 g/d essential AA (EAA; HMP-C); or isoenergetic infusions of 1,319 g/d glucose + 844 g/d EAA (HMP-GG) or 676 g/d palm olein + 844 g/d EAA (HMP-LG). Each experimental period consisted of 5 d of continuous infusion followed by 2 d of rest. A total mixed ration (42% corn silage, 31% grass silage, and 27% concentrate on a dry matter basis) formulated to meet 100 and 83% of net energy and metabolizable protein requirements, respectively, was fed at 90% of ad libitum intake by individual cow. Arterial and venous blood samples were collected on d 5 of each period. Infusing GG or LG at the HMP level did not affect milk yield or composition differently than at the LMP level. Neither GG nor LG infusion stimulated milk protein or lactose yield, but fat yield tended to decrease with GG and tended to increase with LG. Infusion of GG increased arterial plasma concentrations of glucose and insulin and decreased concentrations of β-hydroxybutyrate (BHB), nonesterified fatty acids, long-chain fatty acids (LCFA), total AA, EAA, and group 2 AA. Infusion of LG increased arterial triacylglycerides (TAG) and LCFA but did not affect EAA concentrations. Compared with the LMP level, the HMP level increased arterial concentrations of BHB, urea, and all EAA groups and decreased the concentration of total non-EAA. Mammary plasma flow increased with GG and was not affected by LG or protein level. Uptake and clearance of total EAA and group 2 AA were affected or tended to be affected by GG × AA interactions, with their uptakes being lower and their clearances higher with GG, but only at the LMP level. Infusion of LG did not affect uptake or clearance of any AA group. The HMP level increased uptake and decreased clearance of all EAA groups and decreased non-EAA uptake. Infusion of GG tended to increase mammary glucose uptake, and tended to decrease BHB uptake only at the LMP level. Infusion of LG increased mammary uptake of TAG and LCFA and increased or tended to increase clearance of TAG and LCFA. We suspect GG increased mammary plasma flow to maintain intramammary energy and AA balance and stimulated lipogenesis in adipose, accounting for depressed arterial BHB and group 2 AA concentrations. Mammary glucose uptake did not cover estimated requirements for lactose and fat synthesis at the HMP level, except during HMP-GG infusion. Results of this study illustrate flexibility in mammary metabolite utilization when absorptive supply of glucogenic, lipogenic, and aminogenic substrate is increased.     

Deletion of arginine from an abomasal infusion of amino acids does not decrease milk protein yield in Holstein cows

This study was undertaken to determine if a limited supply of Arg would alter milk and milk protein yields, as well as mammary uptake of AA and energetic substrates. Six lactating Holstein cows (199 ± 5 d in milk) were used in a replicated 3 × 3 Latin square balanced for residual effects with 14-d periods. The diet was formulated to supply 100% of the National Research Council net energy requirement and 72% of the metabolizable protein requirement. The treatments were randomly distributed as abomasal infusions of (1) water (CTL), (2) a mixture of essential AA (EAA) excluding Arg (ARG−), or (3) a mixture of EAA including Arg (ARG+). The profile of EAA in the infusates was the same as that found in casein with the exception that methionine was increased to maintain a 3:1 ratio of digestible lysine:methionine (total dietary + infusion). Milk protein yield was increased by the ARG+ compared with the CTL treatment and deletion of Arg in the infusate (ARG−) did not impair this response. Deletion of Arg from the EAA mixture decreased the mammary uptake of Arg relative to that of the CTL treatment, and although the uptake:output ratio decreased from 2.52 (ARG+) to 2.12 (ARG−), it was still largely in excess of Arg secretion in milk protein. Otherwise, Arg deletion did not affect any of the measured parameters (no significant difference between ARG− and ARG+) except Arg and urea arterial concentrations. In support of the increased yields of milk protein and lactose, mammary uptake of the group 2 AA (Ile, Leu, Lys, and Val) increased and the uptake:output ratio tended to increase from 1.04 to 1.23. The mammary uptake:milk protein output ratio was not different from 1 and not different among treatments for the group 1 AA (His, Met, Phe + Tyr, Trp). Mammary uptake of energetic substrates did not vary across treatments, although milk lactose yield increased with the ARG+ treatment relative to CTL. These results indicate that deletion of Arg has minimal effects on milk and milk component yields when the remaining EAA are supplied in sufficient amounts despite decreased mammary Arg uptake and that group 2 AA seem to be involved in the mammary gland to support the lactose yield response to EAA infusion.     

Effects of glucagon infusions on protein and amino acid composition of milk from dairy cows

Changing the composition of milk proteins and AA affects the nutritional and physical properties of dairy products. Intravenous infusions of glucagon decreases milk protein production and concentration by promoting the use of gluconeogenic blood AA for hepatic glucose synthesis. Little is known about how the diversion of AA to gluconeogenesis affects the composition of milk proteins and AA. The objective was to quantify changes in composition of milk protein and AA in response to i.v. glucagon infusions. Three separate experiments were used: 1) 8 Holstein cows were fed ad libitum and infused with glucagon at 10 mg/d for 14 d, 2) 7 Holstein cows were feed restricted and infused with glucagon at 10 mg/d for 14 d, and 3) 4 Brown Swiss cows were infused with glucagon at 5 and 10 mg/d for 2 d each. Milk and milk component yields and milk protein and amino acid composition of samples, collected with blood samples at the first and last day of the glucagon infusion period, were compared with those collected 1 d before and after the glucagon infusion period. Glucagon infusions decreased milk protein production and concentration in each experiment by at least 0.2 ± 0.05 kg/d and 4 ± 0.4 g/L, respectively. The decrease was accompanied by changes in milk protein composition, the most consistent being an increase in κ-casein (1.68 ± 0.27%). Overall, glucagon infusions resulted in higher proportions of κ-casein and α_S2-casein (1.34 ± 0.51%) and smaller proportions of α_S1-casein (−3.83 ± 1.75%) and α-lactalbumin (−0.91 ± 0.32%). Glucagon had little impact on milk AA composition except an increase in glycine (0.26 ± 0.11%). The results suggest that milk protein synthesis is regulated by many factors including AA and glucose availability.     

Methionine supply during the periparturient period enhances insulin signaling,amino acid transporters,and mechanistic target of rapamycin pathway proteins in adipose tissue of Holstein cows

Enhanced postruminal supply of Met during the periparturient period increases dry matter intake and milk yield. In nonruminants, adipose tissue is responsive to AA supply, and can use AA as fuels or for protein synthesis regulated in part via insulin and mechanistic target of rapamycin (mTOR) signaling. Whether enhancing supply of Met has an effect on insulin and mTOR pathways in adipose tissue in peripartal cows is unknown. Multiparous Holstein cows were assigned from ?28 to 60 d relative to parturition to a basal diet (control; 1.47 Mcal/kg of dry matter and 15.3% crude protein prepartum; 1.67 Mcal/kg and 17.7% crude protein postpartum) or the control plus ethyl-cellulose rumen-protected Met (RPM). The RPM was fed individually at a rate of 0.09% of dry matter intake prepartum and 0.10% postpartum. Subcutaneous adipose tissue harvested at ?10, 10, and 30 d relative to parturition (days in milk) was used for quantitative PCR and Western blotting. A glucose tolerance test was performed at ?12 and 12 d in milk to evaluate insulin sensitivity. Area under the curve for glucose in the pre- and postpartum tended to be smaller in cows fed Met. Enhanced Met supply led to greater overall mRNA abundance of Gln (SLC38A1), Glu (SLC1A1), l-type AA (Met, Leu, Val, Phe; SLC3A2), small zwitterionic α-AA (SLC36A1), and neutral AA (SLC1A5) transporters. Abundance of AKT1, RPS6KB1, and EIF4EBP1 was also upregulated in response to Met. A diet × day interaction was observed for protein abundance of insulin receptor due to Met cows having lower values at 30 d postpartum compared with controls. The diet × day interaction was significant for hormone-sensitive lipase due to Met cows having greater abundance at 10 d postpartum compared with controls. Enhanced Met supply upregulated protein abundance of insulin-responsive proteins phosphorylated (p)-AKT, peroxisome proliferator-activated receptor gamma, and fatty acid synthase. Overall abundance of solute carrier family 2 member 4 tended to be greater in cows fed Met. A diet × day interaction was observed for mTOR protein abundance due to greater values for RPM cows at 30 d postpartum compared with controls. Enhanced RPM supply upregulated overall protein abundance of solute carrier family 1 member 3, p-mTOR, and ribosomal protein S6. Overall, data indicate that mTOR and insulin signaling pathways in adipose tissue adapt to the change in physiologic state during the periparturient period. Further studies should be done to clarify whether the activation of p-AKT or increased availability of AA leads to the activation of mTOR.     

Effect of amino acid or casein supply on whole-body, splanchnic, and mammary glucose kinetics in lactating dairy cows

This study was conducted to establish how AA supplied in a free form or as protein (casein, CN) affect the whole-body rate of appearance (WB Ra) of glucose, splanchnic and mammary glucose kinetics, and milk lactose secretion in lactating dairy cows. Five Holstein cows fitted with a rumen cannula and permanent indwelling catheters in the abomasum, portal, hepatic, and mesenteric veins, and one mesenteric artery, were used in a Youden square with 4 periods of 14 d each. Cows were fed a hay-based diet providing 100 and 70% of their net energy and metabolizable protein (MP) requirements, respectively. Treatments consisted of abomasal infusions of water (70% of MP requirements: control, Con), free AA (95% of MP requirements: AA1; and 120% of MP requirements: AA2), or CN (95% of MP requirements: CN1). The free AA mixture had the same profile as CN. On d 14 of each period, [6,6-²H₂]glucose (25.8 mmol/h) was infused into a jugular vein, and blood samples (n = 8) were taken over 4 h from arterial, portal, hepatic, and mammary sources to measure glucose enrichment and concentration. Splanchnic and mammary plasma flows were determined by downstream dilution of para-aminohippurate and with the Fick principle, respectively. The last 6 milkings of each period were weighed and sampled to measure the yields of milk and components. The AA1 and CN1 treatments were not different for any of the measured parameters. Supplying AA linearly increased glucose WB Ra (AA2 vs. Con: +151 mmol/h) and liver net flux (+149 mmol/h). Utilization of glucose from the plasma compartment by the portal-drained viscera and liver and true portal absorption were not affected by AA supply. From these observations, we suggest that the increased WB Ra was due to increased net hepatic production. The AA from the infusion, in excess of that used to cover the increase in milk protein, were converted to glucose with an apparent efficiency close to 100% of maximum theoretical efficiency. Milk and lactose yields increased linearly with infusions of AA, by 14 and 16% with AA2 treatment, respectively. However, mammary glucose uptake was not significantly altered by AA infusions; this suggests that the mammary gland exerts active control on the uptake and utilization of glucose. For all treatments, the sum of true portal glucose absorption and true hepatic glucose production contributed more than 99% of WB Ra in the lactating cow; this would suggest that renal glucose synthesis makes only a small contribution to WB Ra under these conditions.     

Effects of dietary supplements of folic acid and vitamin B12 on metabolism of dairy cows in early lactation

The present experiment was undertaken to determine the effects of dietary supplements of folic acid and vitamin B₁₂ given from 3 wk before to 8 wk after calving on lactational performance and metabolism of 24 multiparous Holstein cows assigned to 6 blocks of 4 cows each according to their previous milk production. Supplementary folic acid at 0 or 2.6 g/d and vitamin B₁₂ at 0 or 0.5 g/d were used in a 2 × 2 factorial arrangement. Supplementary folic acid increased milk production from 38.0 ± 0.9 to 41.4 ± 1.0 kg/d and milk crude protein yield from 1.17 ± 0.02 to 1.25 ± 0.03 kg/d. It also increased plasma Gly, Ser, Thr, and total sulfur AA, decreased Asp, and tended to increase plasma Met. Supplementary B₁₂ decreased milk urea N, plasma Ile, and Leu and tended to decrease Val but increased homocysteine, Cys, and total sulfur AA. Liver concentration of phospholipids was higher in cows fed supplementary B₁₂. Plasma and liver concentrations of folates and B₁₂ were increased by their respective supplements, but the increase in plasma folates and plasma and liver B₁₂ was smaller for cows fed the 2 vitamins together. In cows fed folic acid supplements, supplementary B₁₂ increased plasma glucose and alanine, tended to decrease plasma biotin, and decreased K_m of the methylmalonyl-coenzyme A mutase in hepatic tissues following addition of deoxyadenosylcobalamin, whereas it had no effect when cows were not fed folic acid supplements. There was no treatment effect on plasma nonesterified fatty acids as well as specific activity and gene expression of Met synthase and methylmalonyl-coenzyme A mutase in the liver. Ingestion of folic acid supplements by cows fed no supplementary B₁₂ increased total lipid and triacylglycerols in liver, whereas these supplements had no effect in cows supplemented with B₁₂. The increases in milk and milk protein yields due to folic acid supplements did not seem to be dependent on the vitamin B₁₂ supply. However, when vitamin B₁₂ was given in combination with folic acid, utilization of the 2 vitamins seems to be increased, probably more so in extrahepatic tissues. Metabolic efficiency seems also to be improved as suggested by similar lactational performance and dry matter intake for cows fed supplementary folic acid but increased plasma glucose and decreased hepatic lipids in cows fed folic acid and vitamin B₁₂ together.     

Abomasal protein infusion in postpartum transition dairy cows: Effect on performance and mammary metabolism

The effect of increasing the postpartum metabolizable protein (MP) supply on performance and mammary metabolism was studied using 8 Holstein cows in a complete randomized design. At parturition, cows were assigned to abomasal infusion of water (CTRL) or casein (CAS). Arterial and epigastric venous blood samples were taken 14 d before expected parturition and at 4, 15, and 29 d in milk (DIM). To compensate previously estimated deficiency of essential AA and to avoid oversupply, casein protein infusion was graduated with 696 ± 1, 490 ± 9, and 212 ± 10 g/d at 4, 15 and 29 DIM, respectively. Dry matter intake was unaffected by CAS. Compared with CTRL, MP supply was greater at 4 DIM with CAS but did not differ by 29 DIM. Milk yield was greater with CAS (+7.2 ± 1.3 kg/d from 1 to 29 DIM). Milk protein yield was greater with CAS at 4 DIM and averaged 1,664 ± 39 g/d compared with 1,212 ± 86 g/d for CTRL, but did not differ at 29 DIM (1,383 ± 48 g/d). The ratio of MP total supply to requirement was numerically greater at 4 DIM for CAS compared with CTRL, indicating less postpartum protein deficiency. In contrast, a greater net energy deficiency tended to be induced with CAS, but the greater milk yield allowed a large part of mobilized fat to be secreted in milk. Arterial concentration of total essential AA increased sharply after parturition for CAS compared with slight decreases for CTRL. The patterns of arterial concentrations combined with arterial-mammary venous concentration differences indicated that Lys, Leu, and Tyr were the first-limiting AA at 4 DIM with CTRL. Mammary plasma flow was unaffected by treatment, indicating similar perfusion of mammary tissue. The greater milk yield with CAS was associated with greater mammary uptake of individual essential AA, tendencies to greater uptake of glucose, lactate, and β-hydroxybutyrate, whereas uptakes of volatile fatty acids were unaffected. Despite similar MP supply by 29 DIM, milk and lactose yields were greater with CAS indicating a persistent response to increased postpartum MP supply. In conclusion, the postpartum MP deficiency can have a substantial negative effect in dairy cows as the major outcome of increasing the postpartum MP supply was increased milk, milk protein, and lactose yield, as well as an enhanced MP balance. Potential positive effects for other body functions than milk synthesis are discussed. Future investigations are needed to delineate how to transfer the effect into practical feeding strategies.     

Lipopolysaccharide challenge of the mammary gland in bovine induced a transient glandular shift to anaerobic metabolism

Support of milk production in modern dairy cows demands a large proportion of its own metabolic resources, such as glucose, which might be required under stressful situations. The aim of the experiment was to test the hypothesis that acute immune stress shifts oxidative metabolism to glycolysis. Two mammary quarters in 6 Holstein cows were infused with lipopolysaccharide (LPS), whereas the 2 counter quarters served as controls to the treatment. An additional 6 cows were infused with saline and served as running controls. The LPS challenge induced dramatic transient increases in milk lactate (75-fold) and malate (11-fold) concentrations (both markers of glycolysis) at 24 h posttreatment. No significant changes in lactate and malate concentrations were recorded in control quarters and control animals, indicating that the effect of LPS was restricted to the treated gland. The LPS challenge induced a dramatic transient decrease in milk yield, and lactose and citrate (a marker of mitochondrial metabolism) secretion at 24 h posttreatment. The kinetics were inversely proportional to those of lactate and malate concentrations. Thus, our data suggest that LPS challenge induces acute conversion of epithelial cell metabolism from principally mitochondrial-oxidative to principally cytosolic (glycolytic), which allows the diversion of metabolic resources normally used to synthesize milk to support the immune system. An in vitro bacterial growth test showed that concentrations of lactate, malate, and lactose equivalent to those found in the in vivo experiment delayed and reduced the growth of a pathogenic Escherichia coli strain, suggesting that they play a role in diminution of bacterial multiplication in the mammary gland.     

Mammary uptake, portal-drained visceral flux, and hepatic metabolism of free and peptide-bound amino acids in cows fed steam-flaked or dry-rolled sorghum grain diets

Our objectives were to measure net fluxes of free AA (FAA) and peptide-bound AA (PBAA) across portal-drained viscera, liver, splanchnic tissues, and mammary tissues, and milk AA output of lactating Holstein cows (n = 8, 86 ± 8 d in milk). Cows were fed an alfalfa-based total mixed ration containing 40% steam-flaked (SFS) or dry-rolled (DRS) sorghum grain. The total mixed rations were offered at 12-h intervals in a crossover design. Blood samples were obtained from indwelling catheters in portal, hepatic, and mammary veins and from mesenteric or costoabdominal arteries every 2 h from each cow and diet. Intake of dry matter was 17.9 and 18.6 kg/d of the SFS and DRS diets, respectively, but dropped to 16.3 kg/d for cows fed the SFS diet in the last 3 experimental days, sampling day included. Milk and milk crude protein yields (kg/12-h sampling) were 13.85 vs. 13.25 and 0.425 vs. 0.396 for cows fed SFS or DRS, respectively, and were not affected by the considerable drop in dry matter intake of cows fed the SFS diet during the last 3 experimental days. The portal-drained visceral flux of total essential FAA was 417 and 442 g/12 h (SEM 63) in cows fed SFS and DRS, respectively. However, the portal-drained visceral flux of 7 essential PBAA out of the 9 determined was numerically greater in cows fed the SFS diet, and total essential PBAA in that treatment was 77.4 ± 22.2 compared with 35.4 ± 50.2 g/12 h for cows fed the DRS diet. This phenomenon was again observed in a greater total splanchnic flux (FAA + PBAA) of 462 and 371 g/12 h in SFS- and DRS-fed cows, respectively. Mammary uptake of essential AA from both pools (free and peptide bound), and recovery of essential AA in milk, was again numerically higher in SFS-fed cows. In addition to FAA, quantifying the contribution of PBAA may improve our understanding of tissue use of AA substrates, and this may ultimately lead to improved diet formulations with respect to intestinal absorption and mammary uptake of AA.     

Effect of substitution of soybean meal by canola meal or distillers grains in dairy rations on amino acid and glucose availability

Canola meal (CM) or by-products of ethanol production (dried distillers grain, DDG) may offer an economical alternative to soybean meal (SBM) in North American dairy rations. These protein supplements can effectively replace SBM and, in 2 recent meta-analyses, CM had a positive effect on milk and milk protein yields compared with SBM. The objective of this study was to determine if the positive responses observed with inclusion of CM in dairy rations could be explained by an increased availability of His, Lys, Met, or glucose. Eight Holstein dairy cows were used in a replicated 4 × 4 Latin square with 14-d periods. Cows were fed isonitrogenous (17.2% crude protein) and isoenergetic (1.56 Mcal/kg of net energy of lactation) diets formulated to slightly exceed nutrient requirements. Diets contained 38% grass hay and 62% corn-based concentrate including SBM, CM, corn high-protein DDG (HPDDG), or wheat DDG plus solubles (WDDGS) as the single protein supplement. The effect of protein supplements on availability of His, Lys, Met, and glucose was estimated using variations in the whole-body (WB) flux of these nutrients, determined by isotopic dilution. As planned, dry matter intake and milk and milk protein yields were not affected by treatments and averaged 23.7, 31.4, and 1.14 kg/d, respectively. Lactose yield did not differ among diets although milk lactose content tended to be lower with CM and WDDGS diets than with SBM and HPDDG diets. Lysine availability was affected by treatments: the highest WB irreversible loss rate (ILR) was observed for the CM diet (371 g/d) and the lowest for HPDDG diet (290 g/d); values for SBM and WDDGS were intermediate (330 and 316 g/d, respectively). Availability of His and Met did not vary among diets and WB ILR averaged, respectively, 129 and 124 g/d; the CM diet, however, had numerically the highest His and Met ILR. Plasma concentrations of most of the essential AA were higher with the CM diet and lower with the HPDDG diet, the exception being Leu for which the concentration was highest for the HPDDG diet. Glucose WB rate of appearance was altered by diet, with the highest mean observed for SBM (3,036 g/d) and the lowest for CM (2,795 g/d); the 2 diets with the lowest WB glucose rate of appearance (CM and WDDGS) also had the lowest dietary starch concentration. Overall, this study suggested that positive responses in milk and milk protein yields observed with inclusion of CM in dairy rations could be linked to a greater supply of metabolizable protein, including some essential AA, especially His, Lys, and Met, as glucose availability was certainly not increased in cows fed the CM diet.