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We aimed to quantify the rate of gluconeogenesis (GNG), non-essential amino-acid (NEAA) synthesis, and substrate partitioning to the Krebs cycle in embryonic (e) day e14 and e19 chicken embryos. An in ovo continuous tracer infusion approach was employed to test the hypotheses that GNG and NEAA synthesis in developing chicken embryo increases from e14 to e19. [13C6]Glucose or [13C3]glycerol was continuously infused (8 h) into the chorio-allantoic compartment of eggs on e14 and e19. Glucose entry rate, Cori cycling, and GNG were higher (P

Breast cancer is the second most common cancer among women in the US. The active form of vitamin D, 1,25-dihydroxyvitamin D (1,25(OH)2D), is proposed to inhibit cellular processes and to prevent breast cancer. The current studies investigated the effect of 1,25(OH)2D on glutamine metabolism during cancer progression employing Harvey-ras oncogene transformed MCF10A human breast epithelial cells (MCF10A-ras). Treatment with 1,25(OH)2D significantly reduced intracellular glutamine and glutamate levels measured by nuclear magnetic resonance (NMR) by 23 ± 2% each. Further, 1,25(OH)2D treatment reduced glutamine and glutamate flux, determined by [U-13C5] glutamine tracer kinetics, into the TCA cycle by 31 ± 0.2% and 17 ± 0.4%, respectively. The relative levels of mRNA and protein abundance of the major glutamine transporter, solute linked carrier family 1 member A5 (SLC1A5), was significantly decreased by 1,25(OH)2D treatment in both MCF10A-ras cells and MCF10A which overexpress ErbB2 (HER-2/neu). Consistent with these results, glutamine uptake was reduced by 1,25(OH)2D treatment and the impact was eliminated with the SLC1A5 inhibitor L-γ-Glutamyl-p-nitroanilide (GPNA). A consensus sequence to the vitamin D responsive element (VDRE) was identified in silico in the SLC1A5 gene promoter, and site-directed mutagenesis analyses with reporter gene studies demonstrate a functional negative VDRE in the promoter of the SLC1A5 gene. siRNA-SLC1A5 transfection in MCF10A-ras cells significantly reduced SLC1A5 mRNA expression as well as decreased viable cell number similar to 1,25(OH)2D treatment. SLC1A5 knockdown also induced an increase in apoptotic cells in MCF10A-ras cells. These results suggest 1,25(OH)2D alters glutamine metabolism in MCF10A-ras cells by inhibiting glutamine uptake and utilization, in part through down-regulation of SLC1A5 transcript abundance. Thus, 1,25(OH)2D down-regulation of the glutamine transporter, SLC1A5, may facilitate vitamin D prevention of breast cancer.

Glutamine is the preferred AA used by polymorphonuclear leukocytes (PMN) during the inflammatory response. However, the effect of other AA on bovine PMN response during inflammation and how this is altered by stage of lactation has not been fully elucidated. The objective of this study was to determine the effect of additional AA supplementation (pool of AA excluding Gln) on AA profiles, gene expression, and inflammatory function of PMN from dairy cows in early and mid lactation in vitro. We used 18 Holstein cows for this study. Polymorphonuclear leukocytes were isolated. Working solutions of AA (0 or 4 mM) and LPS (0 or 50μg/mL) were added to cell populations suspended in RPMI and incubated for 2h at 37°C. We used a subset of samples for gene and protein expression. Concentrations of AA in medium were determined using gas chromatography-mass spectrometry with norleucine as an internal standard. Apparent AA and glucose utilization were calculated by subtracting the concentration after from that of before incubation. Data were analyzed as a randomized block design. Challenge with LPS increased the expression of proinflammatory genes and AA supplementation decreased both the expression of some proinflammatory genes and the media concentrations of tumor necrosis factor-α. Neither stage of lactation, LPS challenge, nor AA supplementation altered the chemotactic or phagocytic abilities of PMN in vitro. Polymorphonuclear leukocytes supplemented with AA had greater concentrations and apparent utilization of most of the supplemented AA, whereas the unsupplemented group had greater apparent utilization of glucose. Alanine was not provided in the media but was present in spent media, and Ile, Gly, and Pro were greater in spent media than in media before incubation indicating synthesis of these AA. Regarding expression of genes involved in nutrient metabolism, the expression of G6PD, coding for the enzyme glucose 6-phosphate dehydrogenase, was increased and that of PDHA1, coding for the enzyme pyruvate dehydrogenase α 1, tended to increase with AA supplementation. Due to the lower concentration of tumor necrosis factor-α in media coupled with a downregulation of several proinflammatory genes, we concluded that AA, rather than Gln, alter the inflammatory response of bovine blood PMN. Independent from Gln, blood PMN from cows in early lactation may use certain AA as their primary carbon source for energy than cows in later lactation. Evaluating cows during the early postpartum period will provide additional information on the effect of stage of lactation and nutrient supplementation on PMN function.

Elevated needs for glucose in lactating dairy cows are met through a combination of increased capacity for gluconeogenesis and increased supply of gluconeogenic precursors, primarily propionate. This study evaluated the effects of propionate on mRNA expression of cytosolic phosphoenolpyruvate carboxykinase (PCK1), mitochondrial phosphoenolpyruvate carboxykinase (PCK2), pyruvate carboxylase (PC), and glucose-6-phosphatase (G6PC), key gluconeogenic enzymes, and capacity for glucose synthesis in liver of dairy cattle. In experiment 1, six multiparous mid-lactation Holstein cows were used in a replicated 3×3 Latin square design consisting of a 6-d acclimation or washout phase followed by 8h of postruminal infusion of either propionate (1.68mol), glucose (0.84mol), or an equal volume (10mL/min) of water. In experiment 2, twelve male Holstein calves [39±4 kg initial body weight (BW)] were blocked by birth date and assigned to receive, at 7d of age, either propionate [2mmol·h(-1)·(BW(0.75))(-1)], acetate [3.5mmol·h(-1)·(BW(.75))(-1)], or an equal volume (4mL/min) of saline. In both experiments, blood samples were collected at 0, 2, 4, 6, and 8h relative to the start of infusion and liver biopsy samples were collected at the end of the infusion for mRNA analysis. Liver explants from experiment 1 were used to measure tricarboxylic acid cycle flux and gluconeogenesis using (13)C mass isotopomer distribution analysis from (13)C3 propionate. Dry matter intake and milk yield were not altered by infusions in cows. Serum insulin concentration in cows receiving propionate was elevated than cows receiving water, but was not different from cows receiving glucose. Hepatic expression of PCK1 and G6PC mRNA and glucose production in cows receiving propionate were not different from cows receiving water, but tended to be higher compared with cows receiving glucose. Hepatic expression of PCK2 and PC mRNA was not altered by propionate infusion in cows. Blood glucose, insulin, and glucagon in calves receiving propionate were not different than controls. Calves receiving propionate had increased PCK1 mRNA, tended to have increased G6PC mRNA, and had similar PC mRNA compared with saline controls. These data indicate a tendency for in vivo effects of propionate to alter hepatic gene expression in mid-lactation cows and neonatal calves, which are consistent with a feed-forward effect of propionate to regulate its own metabolism toward gluconeogenesis through changes in hepatic PCK1 mRNA.

Feeding and postruminal infusion of propionate is known to increase N retention in ruminants. Our aim was to determine the role of rumen propionate on urea N recycling and gluconeogenesis in growing sheep. In Exp. 1, wether sheep ( = 6; 32.5 ± 3.57 kg BW) fitted with a rumen cannula were fed to 1.8 × ME requirement a concentrate-type ration (172 g CP/kg DM and 10.4 MJ ME/kg DM) and continuously infused into the rumen with isoenergetic (10% of dietary ME intake) solutions of either sodium acetate (control) or sodium propionate for 9-d periods in a crossover design. In Exp. 2, a different group of wether sheep ( = 5; 33.6 ± 3.70 kg BW) fitted with a rumen cannula were fed, on an isonitrogenous basis, either a control (151 g CP/kg DM and 8.4 MJ ME/kg DM) or sodium propionate-supplemented (139 g CP/kg DM and 8.9 MJ ME/kg DM) diet at 2-h intervals. [N] urea was continuously infused intravenously for the last 5 d of each period, and total urine was collected by vacuum and feces were collected by a harness bag. Over the last 12 h, [C]glucose was continuously infused intravenously and hourly blood samples were collected during the last 5 h. Propionate treatments increased ( < 0.001) the proportion of rumen propionate in both experiments. In Exp. 1, N retention was not affected by propionate infusion as compared with isoenergetic acetate. There was no effect on urea entry (synthesis) rate (UER) in Exp. 1; however, sodium propionate infusion tended ( < 0.1) to increase urinary urea elimination (UUE). In Exp. 2, feeding propionate increased ( < 0.01) N retention by 0.8 g N/d. In addition, UER was reduced by approximately 2 g urea N/d, leading to a reduction ( < 0.05) in UUE (7.0 vs. 6.2 g urea N/d). Between the 2 experiments, the proportion of UER recycled to the gut was greater with the forage-type diet in Exp. 2 (approximately 60%) compared with the concentrate-type diet in Exp. 1 (approximately 40%), although urea N fluxes across the gut remained unchanged in both experiments. In Exp. 1, glucose entry and gluconeogenesis were greater ( < 0.05) and plasma glucose tended ( < 0.1) to be greater with sodium propionate infusion than with sodium acetate infusion, but there was no difference in Cori cycling. In Exp. 2, glucose entry, gluconeogenesis, Cori cycling, and plasma glucose increased ( < 0.05) with dietary propionate. Our studies indicate that propionate inclusion in feed, but not continuous infusion in to the rumen, improves N utilization in growing sheep. The propionate effect is likely mediated by providing additional precursors for gluconeogenesis.

Butyrate, a major rumen VFA, has been indirectly linked to enhancement of urea recycling on the basis of increased expression of urea transporter in the rumen epithelia of steers fed a rumen butyrate-enhancing diet. Two studies were conducted to quantify the effect of elevated rumen butyrate concentrations on N balance, urea kinetics and rumen epithelial proliferation. Wether sheep (n= 4), fitted with a rumen cannula, were fed a pelleted ration (∼165 g CP/kg DM, 10.3 MJ ME/kg DM) at 1.8 × ME requirement. In Exp. 1, sheep were infused intraruminally with either an electrolyte buffer solution (Con-Buf) or butyrate dissolved in the buffer solution (But-Buf) during 8-d periods in a balanced crossover design. In Exp. 2, sheep were infused intraruminally with either sodium acetate (Na-Ac) or sodium butyrate (Na-But) for 9 d. All solutions were adjusted to pH 6.8 and 8.0 in Exp. 1 and 2, respectively, and VFA were infused at 10% of ME intake. [15N2] urea was continuously infused intravenously for the last 5 d of each period, and total urine and feces were collected. In Exp. 1, 2H5-phenylalanine was continuously infused intravenously over the last 12 h, after which a biopsy from the rumen papillae was taken for measurement of fractional protein synthesis rate (FSR). Butyrate infusion treatments increased (P = 0.1 in Exp. 1; P < 0.05 in Exp. 2) the proportion of rumen butyrate, and acetate infusion increased (P < 0.05) rumen acetate. All animals were in positive N balance (4.2 g N/d in Exp. 1; 7.0 g N/d in Exp. 2), but no difference in N retention was observed between treatments. In Exp. 2, urea entry (synthesis) rate was reduced ( < 0.05) by Na-But compared with the Na-Ac control. In Exp. 1, although But-Buf infusion increased the FSR of rumen papillae (35.3% ± 1.08%/d vs. 28.7% ± 1.08%/d; P < 0.05), urea kinetics were not altered by But-Buf compared with Con-Buf. These studies are the first to directly assess the role of butyrate in urea recycling and its effects on rumen papillae protein turnover in growing lambs. Under the feeding conditions used and the rate of continuous butyrate infusion into the rumen in the present studies, butyrate does not affect overall N retention in growing sheep. However, butyrate may play a role in the redistribution of urea N fluxes in the overall scheme of N metabolism.

The Met precursor 2-hydroxy-4-(methylthio) butanoic acid (HMB) is expected to be more extensively degraded in the rumen than its isopropyl ester (HMBi). A control and 3 isomolar treatments-0.097% dl-methionine, 0.11% HMBi (HMBi), and 0.055% HMBi plus 0.048% Met (Met + HMBi)-were dosed every 8h simultaneously with 3-times-daily feeding into continuous cultures. Starting on d 9, for 6 consecutive doses, both [1-(13)C]-l-Met and [methyl-(2)H3]-l-Met replaced part of the unlabeled dl-Met, [(13)C5]-dl-HMBi replaced a portion of the unlabeled dl-HMBi, and [1-(13)C]-l-Met plus [(13)C5]-dl-HMBi replaced a portion of the respective unlabeled doses for the Met + HMBi treatment. After the sixth dose (d 11), unlabeled Met or HMBi provided 100% of the doses to follow elimination kinetics of the labels in HMBi, free Met, and bacterial Met compartments. The free [1-(13)C]-l-Met recycled more and was recovered in bacterial Met to a lesser extent than was the free [methyl-(2)H3]-l-Met recycling and that was recovered in bacterial Met. Increasing HMBi inclusion (0, 50, and 100% substitution of the exogenously dosed Met on a molar equivalent basis) tended to increase HMBi escape from 54.7 to 71.3% for the 50 and 100% HMBi treatments, respectively. Despite HMBi substituting for and decreasing the dosage of Met, increasing HMBi increased accumulation of free Met in fermenter fluid. The HMBi (after de-esterification of the isopropyl group) presumably produces Met through the intermediate α-ketomethylthyiobutyrate with an aminotransferase that also has high affinity for branched-chain AA. We provide evidence that the HMBi-derived Met is likely released from bacterial cells and accumulates rather than being degraded, potentially as a result of lagging d-stereoisomer metabolism. More research is needed to evaluate racemization and metabolism of stereoisomers of HMBi, Met, and other AA in ruminal microbes.

Amino acids (AA) are not only building blocks of protein but are also key regulators of metabolic pathways in animals. Understanding the fate of AA is crucial to optimize utilization of AA for milk protein synthesis and, therefore, to reduce inefficiencies of nutrient utilization during lactation. By understanding the functional role of AA metabolism in mammary tissue, we can uncover pathways and molecular targets to improve AA utilization by mothers and neonates during the lactation period. The major objective of this article is to highlight recent advances in mammary AA transport, metabolism and utilization. Such knowledge will aid in refining dietary requirements of AA for lactating mammals, including women, sows and cows.

Since gut microbiota elicit host responses that impact human health and affect the nutritional value of animal products, the modulation of gut flora with a combination of natural prebiotics and probiotics, known as synbiotic, is an area of significant research interest. In this study, we evaluated the role of peanut fractions on the growth of Lactobacillus casei and Campylobacter jejuni, a probiotic and pathogenic bacteria, respectively. Moreover, in a co-culture system, we investigated whether there are synergistic effects between the bacterium in the presence of the peanut fraction. We observed that water-soluble (5 g/L) and alcalase-treated peanut fraction significantly stimulated L. casei growth by more than 1.5 logs at 24 h. However, the peanut fraction had no direct effect on C. jejuni growth. Analysis of the fatty acids profile suggested that the higher percentage of oleic acid present in peanuts might be responsible for stimulation of L. casei growth. The initial pH of medium ranging from 6 to 8 did not alter the growth of C. jejuni. In a co-culture system in the presence of the water soluble peanut fraction, the growth of C. jejuni was inhibited in a time dependent manner, reaching the highest level of inhibition (~ 2 logs) at 72 h, while no growth difference was observed in absence of peanut fractions or even in presence of alcalase-treated peanut fraction. In addition, L. casei competitively reduced the colonization of C. jejuni on human intestinal epithelial (INT407) cells. Our data suggest that peanut fraction induced the growth of L. casei, which in turn led to a reduction in the growth of C. jejuni as well as its colonization. Therefore, we hypothesize that regular consumption of peanuts may positively impact on the composition of human gut microbiota and decrease the burden of gastrointestinal infections by enteric bacterial pathogens.

Beef is a source of high quality protein for consumers. Beef tenderness significantly influences meat palatability, consumer expectation and industry profitability. To further elucidate the factors affecting beef tenderness, functional proteomics and bioinformatics interactome analyses were performed on Longissimus thoracis (LT) tissues from Angus cattle. Proteins were analyzed by SDS-PAGE and linearly regressed against tenderness measured by the Warner-Bratzler shear forces (WBSF). Seventeen electrophoresed bands appeared to be correlated with beef tenderness. Two of the bands with the most significant correlations were selected and submitted to LCMS/MS for analysis. Eight proteins, β-enolase (ENOB), L-lactate dehydrogenase A chain (LDHA), glyceraldehyde-3-phosphate dehydrogenase (G3P), carbonic anhydrase 3 (CAH3), phosphoglycerate mutase 2 (PGAM2), peroxiredoxin-6 (PRDX6), myosin heavy chain 1 (MYH1) and myosin heavy chain 7 (MYH7), were identified from these two bands. Interactions with these eight protein markers were constructed using Cytoscape and further functional annotation analysis indicated that these proteins of the interactome were involved in metabolic pathways, such as carbohydrate metabolic and catabolic processes, apoptosis, peptidase activity and responses to stress. The results of this study provide a novel insight into the functional interaction of proteins and beef tenderness.

Summary The urea cycle functions to incorporate ammonia, generated by normal metabolism, into urea. Urea cycle disorders (UCD) are caused by loss of function in any of the enzymes responsible for ureagenesis, and are characterized by life-threatening episodes of acute metabolic decompensation with hyperammonemia (HA). A prospective analysis of interim HA events in a cohort of patients with ornithine transcarbamylase (OTC) deficiency, the most common UCD, revealed that intercurrent infection was the most common precipitant of acute HA and was associated with markers of increased morbidity when compared to other precipitants. To further understand these clinical observations, we developed a model system of metabolic decompensation with HA triggered by viral infection (PR8 influenza) using spf-ash mice, a model of OTC deficiency. Both WT and spf-ash displayed similar cytokine profiles and lung viral titers in response to PR8 influenza infection. During infection, spf-ash displayed an increase in liver transaminases suggesting a hepatic sensitivity to the inflammatory response and an altered hepatic immune response. Despite having no visible pathologic changes by histology, WT and spf-ash had reduced CPS1 and OTC enzyme activities, and unlike WT, spf-ash failed to increase ureagenesis. Depression of urea cycle function was seen in liver amino acid analysis with reductions seen in aspartate, ornithine and arginine during infection. In conclusion, we developed a model system of acute metabolic decompensation due to infection in a mouse model of a urea cycle disorder. In addition, we have identified metabolic perturbations during infection in the spf-ash mice, including a reduction of urea cycle intermediates. This model of acute metabolic decompensation with HA due to infection in UCD serves as a platform for exploring biochemical perturbations and the efficacy of treatments, and may be adapted to explore acute decompensation in other types of inborn errors of metabolism.

Metabolic reprogramming that alters the utilization of glucose including the "Warburg effect" is critical in the development of a tumorigenic phenotype. However, the effects of the Harvey-ras (H-ras) oncogene on cellular energy metabolism during mammary carcinogenesis are not known. The purpose of this study was to determine the effect of H-ras transformation on glucose metabolism using the untransformed MCF10A and H-ras oncogene transfected (MCF10A-ras) human breast epithelial cells, a model for early breast cancer progression. We measured the metabolite fluxes at the cell membrane by a selective micro-biosensor, [(13)C6 ]glucose flux by (13)C-mass isotopomer distribution analysis of media metabolites, intracellular metabolite levels by NMR, and gene expression of glucose metabolism enzymes by quantitative PCR. Results from these studies indicated that MCF10A-ras cells exhibited enhanced glycolytic activity and lactate production, decreased glucose flux through the tricarboxylic acid (TCA) cycle, as well as an increase in the utilization of glucose in the pentose phosphate pathway (PPP). These results provide evidence for a role of H-ras oncogene in the metabolic reprogramming of MCF10A cells during early mammary carcinogenesis.

Short-chain fatty acids (SCFAs), such as butyrate, produced by gut microorganisms, play a critical role in energy metabolism and physiology of ruminants as well as in human health. In this study, the temporal effect of elevated butyrate concentrations on the transcriptome of the rumen epithelium was quantified via serial biopsy sampling using RNA-seq technology. The mean number of genes transcribed in the rumen epithelial transcriptome was 17,323.63 ± 277.20 (±SD; N = 24) while the core transcriptome consisted of 15,025 genes. Collectively, 80 genes were identified as being significantly impacted by butyrate infusion across all time points sampled. Maximal transcriptional effect of butyrate on the rumen epithelium was observed at the 72-h infusion when the abundance of 58 genes was altered. The initial reaction of the rumen epithelium to elevated exogenous butyrate may represent a stress response as Gene Ontology (GO) terms identified were predominantly related to responses to bacteria and biotic stimuli. An algorithm for the reconstruction of accurate cellular networks (ARACNE) inferred regulatory gene networks with 113,738 direct interactions in the butyrate-epithelium interactome using a combined cutoff of an error tolerance (ε = 0.10) and a stringent P-value threshold of mutual information (5.0 × 10–11). Several regulatory networks were controlled by transcription factors, such as CREBBP and TTF2, which were regulated by butyrate. Our findings provide insight into the regulation of butyrate transport and metabolism in the rumen epithelium, which will guide our future efforts in exploiting potential beneficial effect of butyrate in animal well-being and human health.

The objective was to determine the contributions of glucose to glycogen synthesis and glycerol to glycogen, glucose, and nonessential AA (NEAA) synthesis on embryonic day (e) 14/15 and e19/20. Chicken embryos from small (56.6 ± 0.88 g) and large eggs (71.7 ± 1.09 g) were repeatedly dosed with either [(13)C(3)]glycerol (14 mg/d for 4 d) or [(13)C(6)]glucose (15 mg/d for 3 d) into the chorio-allantoic fluid before blood and tissue collection. (13)C-Mass isotopomer enrichments in blood glucose, liver, and muscle glycogen, and blood and tissue NEAA were analyzed by mass spectrometry. Glucose metabolism did not differ between small- and large-egg embryos. Although glucose entry was 60% less for e20 compared with e15 embryos, e20 embryos conserved glucose more efficiently as a result of 2- to 3-fold greater (P < 0.001) rates of glucose carbon recycling. Importantly, the direct contribution of glucose to liver glycogen synthesis was minimal on e15, and on e20 direct incorporation of glucose into liver glycogen was only 17%. By comparison, [(13)C(3)]glycerol dosing led to the appearance of [M + 1], [M + 2], and [M + 3] isotopomers in blood glucose and in liver and muscle glycogen on e14 and e19. Here, the (13)C-isotopomer enrichments in blood glucose were ~2-fold greater (P < 0.05) in small- than in large-egg embryos on e14 and e19. Furthermore, [(13)C(3)]glycerol dosing led to substantial labeling of [M + 1], [M + 2], and [M + 3] isotopomers of alanine, aspartate, and glutamate in blood and in tissues where (13)C enrichments were greater (P < 0.05) in liver of small-egg embryos. In summary, this study provides unequivocal evidence that glycerol is a precursor for glucose and NEAA synthesis. Furthermore, glycerol, but not egg-derived glucose, is a major substrate for synthesis of liver and muscle glycogen and is an important anaplerotic substrate for the tricarboxylic acid cycle of embryos during later development.

Beef tenderness contributes significantly to variation of beef palatability, and is largely influenced by various genetic and environmental factors. To identify candidate genes and pathways related to beef tenderness, we analyzed the longissimus dorsi (LD) muscle of Angus cattle that had different degrees of tenderness, measured by Warner-Bratzler shear force (WBSF). Microarray and RT-PCR analyses identified 53 genes that were differentially expressed in LD samples categorized as either tough or tender, including myosin, heavy chain 3 skeletal muscle embryonic (MYH3), myosin heavy chain 8 skeletal muscle perinatal (MYH8), guanylate binding protein 5 (GBP5), fatty acid binding protein 4 (FABP4), Stearoyl-coenzyme A desaturase (SCD), Fatty acid synthase (FASN), ubiquitin-like with PHD and ring finger domains 1 (UHRF1). Most of these genes are involved in lipid metabolism and skeletal muscle contraction. Employing Gene ontology (GO) and Ingenuity Pathway Analysis (IPA), several GO terms and pathways were found to be related to hydrolase, peptidase and GTPase activity, lipid metabolism, small molecule biochemistry, molecular transport, and tissue development. Overall, this analysis provides insight into the metabolic relationships between muscle biology and beef quality.

The aim of this study was to determine whether subtle changes in the energy-to-N ratio of medium-concentrate diets alters hepatic export protein synthesis and the partition of protein metabolism in the whole body of growing rams. Rams (n = 6; 41.5 +/- 2.6 kg of BW) were fitted with catheters for measurement of Leu and Phe tracer kinetics across the portal drained-viscera (PDV) and liver. Rams were assigned to receive 3 dietary treatments according to a duplicated Latin square design. Animals received forage-concentrate-based diets that were balanced for ME and available N (CON), 20% imbalanced (reduced) in available N (LN), or 20% imbalanced in ME (LE). After 15 d on each experimental diet, [ring-(2)H(5)]Phe (4.3 micromolxkg(-1)xh(-1)) and [1-(13)C]Leu (8.6 micromolxkg(-1)xh(-1)) were continuously infused into the vena cava for 10 h and, over the last 7 h of infusion, matched sets of blood samples were taken. Daily BW gain was less (P < 0.05) for the LE (0.191 kgxd(-1)) diet compared with CON (0.265 kg/d) and LN (258 kgxd(-1)) diets. Compared with CON, whole body irreversible loss rate (ILR) of Leu and Phe was less (10 to 16%, P < 0.02) for LN and LE diets, which for Leu reflected its decreased (20 to 24%, P < 0.05) net PDV absorption. The decreased whole body ILR is due to a decreased PDV ILR in both diets with a relative contribution of the PDV to the whole body ILR decreased (P < 0.05) in the LN (27%) diet compared with the CON (36%) and LE (33%) diets. This decreased PDV ILR was associated with a decreased net Leu PDV uptake in LN and LE diets (-25 and -20%, respectively; P < 0.05). Conversely, the decreased whole body Phe ILR is explained by a decreased hepatic ILR (and contribution to the whole body ILR) and was associated with a decreased net hepatic uptake of Phe in LN (-25%) and LE (-20%) diets compared with CON (P = 0.03). The fractional and absolute synthesis rates of total proteins and albumin were decreased by 10% in LE animals (P < 0.05), whereas they were not affected by the LN diet. These results suggest a specific decreased utilization of Leu at the PDV due to a specific sparing mechanism in the LN diet. Conversely, a decreased Phe utilization occurred in the liver in both diets (due to a decreased export protein synthesis and a probable decreased oxidation in LE diet, whereas only oxidation is reduced in LN diet).

We hypothesized that, as the supply of preformed glucose diminishes during development, the embryo would transition to a greater rate of gluconeogenesis (GNG) and that GNG would be greater in embryos from small vs. typical size eggs. Gluconeogenesis by embryos from small (51.1 +/- 3.46 g) and typical size (65 +/- 4.35 g) broiler breeder eggs was measured by dosing [(13)C(6)]glucose (15 mgxegg(-1)) into the chorio-allantoic fluid for 3 consecutive days to achieve isotopic steady-state before blood collection on embryonic day (e) 12, e14, e16, and e18 (4 to 5 eggsxsize(-1)xd(-1)). The (13)C-Mass isotopomer enrichment of blood glucose was determined by gas chromatography-mass spectrometry. On e14, e16, and e18, but not on e12, embryos from small eggs weighed less (P < 0.05) than typical size eggs. For both sizes of eggs, blood glucose concentration, glucose entry rate (g.d(-1)), and Cori cycling and glucose (13)C-recycling (% of entry rate) increased (P < 0.05) with development. On e12 and e14, rates of glucose entry and Cori cycle flux were greater (P < 0.05) for embryos from small eggs. When standardized to BW (g.100 g of BW(-1)xd(-1)), glucose entry and Cori and non-Cori cycle fluxes were greater for embryos from small eggs. From e12 through e18, blood concentrations of gluconeogenic AA (threonine, glutamine, arginine, proline, isoleucine, and valine) were 25 to 48% less (P < 0.01) in embryos from small eggs. In conclusion, embryos from small eggs exhibit greater rates of GNG earlier in development compared with typical size eggs and, perhaps as a consequence, their reduced embryonic growth may result from diverting greater supplies of AA toward GNG.

The objective of this study was to test whether mammary AA metabolism and blood flow (BF) fluctuate in relation to changes in milk protein yield brought about by unilateral milking frequency. To investigate these relationships, the vascular systems of the mammary glands of 4 lactating goats were separated by ligation and flow probes were fitted around the external pudic artery of each gland. Goats were subjected to 3 consecutive periods (5 d each) during which 1) both glands were milked twice daily (2×; period 1); 2) glands were unilaterally milked, with gland A milked 8 times daily and gland B milked incompletely (30% of period 1 milk yield retained in the gland) once daily (8× vs. 1×; period 2); and 3) both glands milked twice daily (2×; period 3). On d 5 of periods 1 (2×) and 2 (8× vs. 1×), arteriovenous [1- 13 C]Leu kinetics were monitored for each gland. Milk protein yield was greater for gland A compared with gland B. After 2× milking resumed in period 3, yields for both glands returned to period 1 levels. Similarly, BF increased (19%) for gland A (8×), whereas BF was decreased (21%) for gland B (1×). For both glands, BF returned to previous levels when 2× milking resumed in period 3. Mammary net uptakes and oxidation of Leu were greater for the gland milked 8× compared with that milked 1×. Net uptakes from blood of Ile, Leu, Val, Lys, Pro, and Arg were greater (39–79%) for the 8× compared with the 1× gland. In conclusion, the excess removal and oxidation by the mammary gland of some amino acids appears an obligatory requirement to support increases or decreases in milk protein yield when milking frequency is altered.

The aim of this study was to test whether feeding of diets containing lower proportions of ruminally degradable protein (RDP) but with a constant proportion of ruminally undegradable protein (RUP) alters feed intake, milk production and yield, and the apparent efficiency of N utilization by mid-lactation dairy cows. During the covariate period (d 1 to 28), 40 mid-lactation cows (36 Holstein and 4 Jersey x Holstein cross-breds) were fed a common diet formulated to contain 11.3% of diet dry matter (DM) as RDP. During the treatment period (d 29 to 47), cows were randomly assigned to 1 of 4 diets formulated to contain 11.3, 10.1, 8.8, or 7.6% RDP, whereas ruminally undegradable protein remained constant at 7.1% of DM. All diets contained 47.5% forage and 52.5% concentrate on a DM basis. Dry matter intake was significantly reduced for the 7.6% RDP diet. The lowest RDP content was associated with a trend for reduced milk yield. Dietary RDP had no effect on body weight or milk fat, protein, and lactose contents. Milk protein yield was not affected by RDP level; however, milk fat yield decreased linearly as dietary RDP was reduced. Concentrations of plasma essential amino acids were unaffected, whereas milk urea-N concentrations decreased linearly as dietary RDP content was reduced. The apparent efficiency of N utilization for milk N production increased from 27.7% on the 11.3% RDP diet to 38.6% on the 7.6% RDP diet. The dietary RDP requirement of cows in this study was apparently met between 15.9 and 14.7% dietary crude protein. Milk production was not significantly affected by the 8.8% RDP (15.9% crude protein) diet even though the NRC (2001) model predicted that RDP supply was 87% of that required, suggesting the current NRC recommendations for RDP may be overestimated for mid-lactation dairy cows in this study.

The aims of this study were 1) to determine whether transfer of blood urea to the gastrointestinal tract (GIT) or the efficiency of capture of urea N within the GIT is more limiting for urea N salvage, and 2) to establish the relationship between plasma urea concentration and recycling of urea N to the GIT. We used an i.v. urea infusion model in sheep to elevate the urea entry rate and plasma concentrations, thus avoiding direct manipulation of the rumen environment that otherwise occurs when feeding additional N. Four growing sheep (28.1 ± 0.6 kg of BW) were fed a low-protein (6.8% CP, DM basis) diet and assigned to 4 rates of i.v. urea infusion (0, 3.8, 7.5, or 11.3 g of urea N/d; 10-d periods) in a balanced 4 x 4 Latin square design. Nitrogen retention (d 6 to 9), urea kinetics ([ 15 N 2 ]urea infusion over 80 h), and plasma AA were determined. Urea infusion increased apparent total tract digestibility of N (29.9 to 41.3%) and DM (47.5 to 58.9%), and N retention (1.45 to 5.46 g/d). The plasma urea N entry rate increased (5.1 to 21.8 g/d) with urea infusion, as did the amount of urea N entering the GIT (4.1 to 13.2 g/d). Urea N transfer to the GIT increased with plasma urea concentration, but the increases were smaller at greater concentrations of plasma urea. Anabolic use of urea N within the GIT also increased with urea infusion (1.43 to 2.98 g/d; P = 0.003), but anabolic use as a proportion of GIT entry was low and decreased (35 to 22%; P = 0.003) with urea infusions. Consequently, much (44 to 67%) of the urea N transferred to the GIT returned to the liver for resynthesis of urea (1.8 to 9.2 g/d; P < 0.05). The present results suggest that transfer of blood urea to the GIT is 1) highly related to blood urea concentration, and 2) less limiting for N retention than is the efficiency of capture of recycled urea N by microbes within the GIT.

This study was intended to establish the extent which amino acids (AAs) and glucose are net metabolized by the gastrointestinal tract (GIT) of ruminant sheep when intestinal protein supply is varied. Wether sheep (n = 4, 33 +/- 2.0 kg) were fitted with catheters for measurement of net absorption by the mesenteric (MDV) and portal-drained (PDV) viscera and a catheter inserted into the duodenum for casein infusions. Sheep received a fixed amount of a basal diet that provided adequate metabolizable energy (10.9 MJ/d) but inadequate metabolizable protein (75 g/d) to support 300-g gain per day. Four levels of casein infusion [0 (water), 35, 70, and 105 g/d], each infused for 5.5 d, were assigned to sheep according to a 4 x 4 Latin square design. [methyl-(2)H(3)]leucine was infused (8 h) into the duodenum while [1-(13)C]leucine plus [6-(2)H(2)]glucose were infused (8 h) into a jugular vein. With the exception of glutamate and glutamine, net absorption of AAs increased linearly (P < 0.05, R(2) = 0.46-1.79 for MDV; P < 0.05, R(2) = 0.6-1.58 for PDV) with casein infusion rate. Net absorption by the PDV accounted for

Genomic investigations in animals have begun to reveal the metabolic and physiological functions of genes and protein products. However, a thorough understanding of the genomic roadmaps will require investigative approaches that yield qualitative and quantitative information on the activities, fluxes, and connectivity of pathways involved in nutrient use in farm animals; that is, the metabolic phenotype. Recently, the commercial availability of stable isotope (13C, 15N, 2H)-labeled compounds and highly accurate mass spectrometers has made it possible to probe the details of metabolic pathways involved in macronutrient use. For years, the biological sciences have exploited uniformly 13C-labeled substrates (e.g., glucose, amino acids, nucleic acids) and 13C-mass isotopomer distribution (MID) in their metabolic investigations, whereas their use in the animal sciences is very limited. When [U-13C] substrates are fed, infused, or added to cell incubations, the 13C-skeletons distribute throughout metabolic networks. 13C-Mass isotopomer distribution in intermediates and end products of the pathways provides a signature of the fluxes and activities of pathway enzymes traveled by the precursor molecule. This paper highlights aspects of animal nutrition and metabolism in which [U-13C] substrates and MID can be applied to investigations of amino acid, carbohydrate, and fat metabolism. We will focus on [U-13C] glucose as a tracer in chickens, fish, sheep, and cell cultures to investigate the interconnectivity of the pathways of macronutrient and nucleic acid metabolism, and provide demonstration of the central position of the Krebs cycle in preserving metabolic flexibility via anaplerotic and cataplerotic sequences. Exploitation of this approach in animal sciences offers endless opportunities to provide missing details of the biochemical networks of nutrient use that may prove to be strictly under genomic control.

The objective of this experiment was to determine the capability of ruminant gut tissues to detoxify ammonia-N using short-term incubations of isolated cells in vitro. Ruminal epithelial cells (REC) and duodenal mucosal cells (DMC) were isolated from growing Texel-Polypay ram lambs (n=4) fed a pelleted forage:concentrate-based diet. Immediately after isolation, primary cells were incubated for 60min with glucose (1m M ), glutamate (1m M ), [ 15 N]ammonium chloride (5, 10, 20, or 40m M ), and 1 of 4 combinations of substrates (1m M each) that could support urea synthesis [control, N -carbamoylglutamate (NCG); NCG + ornithine (ONCG); and ONCG + aspartate (AONCG)]. Treatments were arranged in a 4×4 factorial design. Incorporation of ammonia- 15 N into alanine, citrulline, arginine, and urea was determined by gas chromatography-mass spectrometry. For both cell types, ammonia-N transfer to alanine was lower when incubation medium contained NCG compared with control, whereas use of ammonia-N for net alanine synthesis increased quadratically with ammonia concentration regardless of substrate treatment. For REC, ammonia-N was not incorporated into citrulline, arginine, or urea, nor into arginine or urea by DMC. Ammonia-N use for net citrulline synthesis exhibited an inverse relationship with ammonia concentration, decreasing linearly as media ammonia concentration increased. Thus, ala-nine synthesis may be a significant metabolic pathway for ruminant gut tissues to detoxify ammonia-N when it is presented luminally at high concentrations as compared with detoxification by the ornithine-urea cycle. Furthermore, DMC do exhibit a metabolic capability to incorporate ammonia-N into citrulline, but low or absent activity of downstream enzymes of the ornithine-urea cycle appears to limit ammonia-N transfers to urea.

Flavomycin is an antibiotic that promotes growth in ruminant and non-ruminant livestock. The aim of this study was to determine the mechanism of action of flavomycin in sheep by measuring microbial numbers, microbial metabolism and gut tissue protein turnover at different sites in the digestive tract. Two weight-matched groups (n5) of male castrate lambs (30 kg) received 800 g grass cubes/d for 6 weeks, with one group receiving 20 mg/d flavomycin during the last 2 weeks. Samples of digesta and gut tissue segments were obtained immediatelypost mortem, 90 min after a flood-dose of [ring-D5]phenylalanine. Viable bacterial counts and volatile fatty acid concentrations were highest in ruminal digesta, followed by the colon and caecum, then the duodenum and ileum. The only effect of flavomycin was an increased bacterial count in the rumen (3·5v.1·2×109per g;P=0·04). Acetate was proportionally greater and propionate and butyrate were lower in the caecum and colon than the rumen. Flavomycin had no effect on volatile fatty acid proportions or ammonia concentrations. Bacteria growing on peptides as sole C source were not affected by flavomycin. Proteolytic, peptidolytic and amino acid deamination activities were similar in the rumen, caecum and colon; they tended to be lower in animals receiving flavomycin. Protein turnover in ruminal wall and duodenal tissues, measured by a flood-dose technique, decreased with flavomycin (P=0·075 and 0·027, respectively). Thus, flavomycin differs from ionophores in its mode of action. It may influence protein metabolism of both digesta and tissue throughout the ruminant digestive tract.

The objective of this study was to deter- mine whether the porcine mammary gland responds to increasing dietary CP concentration through changes in AA arteriovenous difference (a-v). Sixteen Landrace × Yorkshire lactating sows were provided ad libitum access to one of four isocaloric diets varying in CP con- centration (7.8, 13.0, 18.2, and 23.5 %; as-fed basis). Litters were adjusted to 11 pigs within 48 h of birth. Sows were fitted with catheters in the carotid artery and main mammary vein on d 4. On d 10, 14, 18, and 22 of lactation, arterial and venous blood samples were obtained every 30 min over 6 h. Milk yield was esti- mated on d 11 and 21 using the D2O dilution technique. Final litter sizes on d 21 were 10.3, 11, 9.5, and 11 piglets for sows fed the 7.8, 13.0, 18.2, and 23.5% CP diets, respectively. Piglet ADG tended (P = 0.088) to increase with increasing dietary CP concentration and were 186, 221, 220, and 202 g for sows fed the 7.8, 13.0, 18.2, and 23.5% CP diet, respectively. Daily total milk yield on d 21 (kg milk/d) tended (P = 0.099) to increase, and average milk yield per nursed piglet (kg of milk pig −1 d −1 ) increased (P < 0.05) with increasing CP con- centration and were, on a per-piglet basis, 0.95, 1.19

The objective of this study was to evaluate oxidative metabolism of glucose, glutamate, and glutamine by isolated ovine enterocytes in the presence of other metabolic fuels in vitro. A mixed mucosal primary cell culture containing enterocytes was isolated from crossbred wether sheep (n = 6) fed a mixed forage-concentrate diet and incubated for 90 min with 1 mM U- 14 C-glucose, -glutamate, or -glutamine and additional substrates (water as negative control, acetate, propionate, butyrate, glucose, glutamate, or glutamine) at concentrations of 0.1, 1.0, and 10.0 mM. Oxidation of labeled substrates to CO 2 and net production of lactate and pyruvate in incubation media were measured. Oxidation of glucose and glutamine to CO 2 was decreased (P < 0.05) by 5 to 40% in the presence of additional substrates except acetate. Our observation that glutamine oxidation can be decreased by the presence of additional substrates is contrary to observations in the literature using enterocytes from nonruminants, indicating that ruminant enterocytes might rely on glutamine to a lesser extent as an energy source. Net glucose utilization was decreased (P < 0.05) 16% by propionate (10 mM) compared with control but was not affected by the other additional substrates. Glutamate oxidation to CO 2 was decreased 28% (P < 0.05) in the presence of propionate (10 mM) or by 17 and 33% in the presence of glutamine (1.0 and 10 mM, respectively), but not by that of the other additional substrates. Acetate did not affect the oxidation of glucose, glutamate, and glutamine. Propionate decreased (P < 0.05) the oxidation of glucose and glutamate only at the highest concentration (10 mM), indicating that the sparing effects of propionate on substrate oxidation are affected by its concentration in the incubation media. These observations indicate that ruminant enterocytes possess metabolic flexibility for oxidative metabolism of glucose, glutamine, and glutamate depending on the type and concentration of available additional substrates.

The metabolic response of the liver during periods of inflammation is poorly understood. The objective of this study was to characterize the effects of nutrient supply and lipopolysaccharide (LPS) challenge on hepatic intermediate metabolism of early- and mid-lactation cows by employing gas chromatography-mass spectrometry with stable isotope tracer. Twelve multiparous Holstein-Friesian cows in early (n = 6; 12 ± 4.2 d in milk) and mid (n = 6; 115 ± 13.5 d in milk) lactation were used for this study. Liver biopsies were performed on all cows. Liver slices (40-60 mg) were incubated in a 37°C water bath for 2 h with either control (phosphate buffered saline), pyruvate (PYR; 1mM unlabeled pyruvate and 1mM [(13)C3]pyruvate), pyruvate + propionate (PYR+PRO; 1mM unlabeled pyruvate, 1mM [(13)C3]pyruvate, and 2mM sodium propionate), or pyruvate + AA (PYR+AA; 1mM unlabeled pyruvate, 1mM [(13)C3]pyruvate, and 2mM AA solution), and LPS (0.0 or 0.2 μg/mL) was added to flasks per treatment. Enrichment of isotopomers in metabolic equilibrium with Krebs cycle intermediates was assessed. Pyruvate fluxes and the enzymatic activity of pyruvate carboxylase (PC) versus pyruvate dehydrogenase (PDH) and phosphoenol pyruvate carboxykinase (PEPCK) were calculated. Media were analyzed for concentrations of tumor necrosis factor-α (TNF-α), glucose, and haptoglobin. Data were analyzed as randomized block (stage of lactation) design in a factorial arrangement of nutrient treatments by LPS dose. Challenge with LPS increased the mRNA abundance of TNF-α, haptoglobin, and serum amyloid A 2, and the concentration of TNF-α in media. Challenge with LPS increased mRNA abundance of PC but reduced the enrichment of (13)C1[M1] and (13)C2[M2]alanine and tended to reduce the enzymatic activity of PEPCK. Incubation with PYR+PRO and PYR+AA increased the flux of pyruvate to acetyl CoA. However, only PYR+PRO increased the enzymatic activity of PEPCK and PDH versus PC and decreased the mRNA abundance of PC. Cows in early lactation tended to receive a greater contribution of pyruvate to the oxaloacetate flux via the lower PDH versus PC activity and a higher mRNA abundance of PC than cows in mid lactation. Our results suggest that regardless of stage of lactation and nutrient supplement, hepatic gluconeogenesis was impaired during inflammation. Further research examining how various nutrients support liver function and improve the immunometabolic response of liver during inflammation is warranted.

In dairy rations, Met is often a limiting amino acid that is provided by rumen-undegradable protein and rumen-protected sources of Met. A Met precursor, 2-hydroxy-4-(methylthio) butanoic acid (HMB) has undergone considerable study for ruminal and postruminal metabolism, whereas its isopropyl ester (HMBi) has been evaluated primarily with respect to its supply of metabolizable Met rather than as a preformed source of Met for microbial metabolism. A control and 3 isomolar Met treatments-0.097% dl-Met, 0.048% dl-Met plus 0.055% HMBi (Met + HMBi treatment), and 0.11% HMBi-were pulse-dosed every 8h into continuous cultures simultaneously with feeding. Treatment had no effect on digestibilities of acid-detergent fiber or true organic matter. Digestibilities of neutral detergent fiber and hemicellulose were linearly decreased with increasing HMBi inclusion. Concentration of NH3-N tended to decrease linearly and quadratically, and NH3-N flow tended to decrease linearly, with increasing HMBi inclusion; in contrast, the proportion of bacterial N derived from NH3-N increased linearly. Peptide N increased linearly and tended to be affected quadratically (highest for the HMBi treatment). Acetate and propionate production both decreased with increasing HMBi, but acetate declined more such that acetate:propionate increased linearly. Isobutyrate production decreased, but isovalerate and valerate increased with increasing HMBi inclusion. Relative changes in population abundance were not detected by denaturing gradient gel electrophoresis. In the second study, which was done in batch culture, Met treatments consisted of control, 0.097% l-Met, 0.097% l-Met, 0.125% dl-HMBi, 0.098% dl-HMB, 0.250% dl-HMBi (2× HMBi), 0.049% dl-Met + 0.063% dl-HMBi (Met + HMBi), and 0.098% dl-HMB + 0.039% isopropanol. All of these Met treatments were unlabeled (i.e., at natural abundance of (13)C) but simultaneously dosed with equivalent dosages of [1-(13)C]-l-Met. All 8 treatments were inoculated with faunated or partially defaunated inocula. Protozoal abundance had minor effect on measurements. The unlabeled l-Met treatment had the lowest (13)C enrichment of Met in the microbial pellet followed by Met + HMBi and then d-Met or dl-HMB, which were lower than remaining treatments. The percentage of the [1-(13)C]-l-Met dose recovered in microbial Met was lowest for the l-Met treatment; intermediate for d-Met, dl-HMB (with or without isopropanol), and Met + HMBi treatments; and highest for HMBi, 2× HMBi, and control. Results suggest that racemization of d-Met lags behind l-Met. The similar conversions of the HMBi and 2× HMBi treatments compared with the control suggests a low degradation of HMBi to provide unlabeled Met to dilute the [1-(13)C]-l-Met dose for protein synthesis. The lack of treatment by time interaction suggests that these initial responses carried through during the 24h of incubation. The proportion of HMBi available to ruminal microbes can influence microbial metabolism, potentially through formation of l-Met.

Studies were designed to investigate the effect of 1,25 dihydroxyvitamin D (1,25D) on glutamine metabolism during cancer progression employing untransformed (10A) and ras oncogene transformed MCF10A human breast epithelial cells (ras). Glutamine restriction reduced cell number in both cell lines, and 1,25D ameliorated these reductions. Intracellular glutamine and glutamate levels, determined by NMR, were increased 25% in ras cells compared to 10A cells, and 1,25D reduced these levels by 23% in ras cells. Fractional glutamine and glutamate flux, utilizing U13C5-L-glutamine, into the TCA cycle was increased in ras cells by 21% and 18%, respectively, compared to 10A cells. 1,25D decreased glutamine flux into the TCA cycle by 18% and 31% in 10A and ras cells, respectively, and decreased glutamate flux into the TCA cycle by 13% and 17% in 10A and ras cells, respectively. The mRNA expression of the glutamine transporter, solute linked carrier family 1 member A5 (SLC1A5), was decreased by 34% in ras compared to ...

Milk pricing schemes place economic importance on milk components. Most current nutrient requirement models do not predict milk component yields accurately. Deaggregation of energy and protein terms in those models may improve prediction accuracy. Descriptions of energy metabolism by the major postabsorptive tissues have progressed over the last 20 years. More recent efforts have been directed at representing amino acid metabolism. Mammary amino acid metabolism appears to be a function of amino acid supply and regulatory elements. Regulation of uptake and blood flow occurs and is represented in some models. Intracellular metabolism of amino acids and possibly energy are determinants of removal. Both the rate of amino acid oxidation and use for protein synthesis appear to be functions of intracellular concentrations. Experimental observations suggest that representation of protein synthesis as a linear function of the first-limiting amino acid is inadequate. A multi-substrate Michaelis–Menten equation form is more consistent with experimental observations and appears to yield better predictions as compared to the single-limiting model. Consideration of energy supply as a driver of milk protein synthesis also appears to be warranted. Additional knowledge of the substrate response surface for protein synthesis and how it is regulated is needed.

Whole-body and splanchnic metabolism of dietary amino acids derived from casein (CAS) or the corresponding crystalline L-amino acid mixture (AA) were compared. Male adult rats were adapted for 9 d to two isoenergetic, isonitrogenous diets (15 g/100 g protein, 5 g/100 g fat) containing either CAS or AA. On d 10, the rats were fed a single mixed meal (3 g dry mass) containing either intrinsically (13)C-labeled goat casein or the amino acid mixture containing [U-(13)C(6)] leucine and [alpha-(15)N] lysine. Rats were killed before and 1, 3, 5 and 7 h after meal ingestion and samples of plasma, stomach wall and contents, small intestine and liver were collected. (13)C and (15)N enrichments of free and protein-bound amino acids in plasma and tissues were analyzed by gas chromatography-combustion isotope ratio mass spectrometry. Urinary nitrogen excretion was higher (P < 0.05) and weight gain lower (P < 0.05) in rats given the AA diet, indicating a lower whole-body net protein synthesis. Free (13)C-leucine from the AA diet appeared in the intestinal mucosa free pool more rapidly (P < 0.05) than the CAS-(13)C-leucine, probably due to the faster transit through the stomach of the AA group. However, the incorporation of dietary leucine into plasma and liver proteins was higher in the CAS group 7 h after the meal (P < 0.05), whereas lysine incorporation into liver protein was higher in the AA group (P < 0.05). We conclude that whole-body protein homeostasis is better supported by dietary casein-bound than crystalline free amino acids, and that protein-bound leucine, but not lysine, is used more efficiently for liver protein synthesis than dietary free leucine.

Wallace, R. J., Newbold, C. J., Bequette, B. J., MacRae, J. C., Lobley, G. E. (2001). Increasing the flow of protein from ruminal fermentation - Review. Asian-Australian Journal of Animal Science, 14 (6), 885-893.

An arteriovenous kinetics technique was used to monitor mammary gland lysine and protein metabolism in goats (n = 4) at two stages of lactation (80 +/- 17 vs. 233 +/- 14 DIM) in response to an i.v. infusion of lysine (Lys) plus methionine (Met). At each stage of lactation [2-15N] and [1-13C; 6,6-2H2] Lys kinetics were performed on the last day of 5-d i.v. infusion of saline followed by Lys (370 mg/h) plus Met (84 mg/h, LM). Milk and protein yields and dry matter intake were higher in early than in late lactation, but LM infusion did not affect these variables. Regardless of stage of lactation, the absolute and fractional oxidation rates of Lys by the mammary gland increased in response to LM infusion. When corrected for Lys oxidation, net uptake of Lys by the gland was less than milk protein Lys secretion. However, correction for the contribution of peptides (15.8%) to Lys uptake brought net Lys uptake close into balance with milk Lys secretion. The present data suggests that when Lys is in excess of requirements, the mammary gland appears to dispose of the extra supply via the oxidative mechanism.

Dietary manipulation of milk fat content and/or fatty acid composition is becoming a feasible, and for certain niche–markets, attractive means of boosting the nutritive value and acceptability of milk and its secondary products. However it is not yet possible to indicate equivalent ways of manipulating milk protein content and/or composition. This paper will consider current knowledge on milk protein biosynthesis and the opportunities for nutritional manipulation. Recent infusion studies which have linked changes in mammary amino acid metabolism with changes in milk protein content will be examined in an attempt to elucidate key features of the metabolic regulation of the dairy cow which need to be addressed if the British consumer is to have the choice of higher protein (possibly designer protein) milk products. This review will (in part) utilise data from a joint project at the Rowett Institute and Reading University funded by a consortium comprising government (MAFF, BBSRC and SOAEFD) and agribusiness (MDC, Purina Mills and Hendrix).

An isotope dilution model for partitioning phenylalanine and tyrosine uptake by the mammary gland of the lactating dairy cow is constructed and solved in the steady state. The model contains four intracellular and four extracellular pools and conservation of mass principles are applied to generate the fundamental equations describing the behaviour of the system. The experimental measurements required for model solution are milk secretion and plasma flow rate across the gland in combination with phenylalanine and tyrosine concentrations and plateau isotopic enrichments in arterial and venous plasma and free and protein bound milk during a constant infusion of [1-(13)C]phenylalanine and [2,3,5,6-(2)H]tyrosine tracer. If assumptions are made, model solution enables determination of steady state flows for phenylalanine and tyrosine inflow to the gland, outflow from it and bypass, and flows representing the synthesis and degradation of constitutive protein and phenylalanine hydroxylation. The model is effective in providing information about the fates of phenylalanine and tyrosine in the mammary gland and could be used as part of a more complex system describing amino acid metabolism in the whole ruminant.

We hypothesized that 5′-adenosine monophosphate-activated protein kinase (AMPK), a ubiquitous energy sensor, plays a key role in coordinating nutrient use in developing broiler breeder embryos. To test this hypothesis, we measured AMPK activity in the liver of embryos developing in small versus large (typical) eggs. Small (53.2 ± 1.0 g) eggs from 26-wk-old and large (69.0 ± 1.9 g) eggs from 42-wk-old broiler breeder hens were acquired and measurements were made on embryonic day (e) 11, 14, 17, and 20, and on posthatch d 1. The initial weight of dry yolk to albumen was higher (2.3 vs. 1.7) in large versus small eggs, and embryo weight differed from e17 onward. The AMPK activity was higher in livers of embryos from large versus small eggs across all sampling days, and activity was highest on e14 for both sizes of eggs. We speculate that higher AMPK activity in livers of embryos from large eggs may facilitate increased metabolism of their larger nutrient supplies to support faster growth rate in ovo. Taken together, our results are the first to investigate the relationship between liver AMPK activity and egg size and nutrient composition in developing chicken embryos.

This study was designed to investigate the impact of 1,25 dihydroxyvitamin D (1,25(OH)2D) on glucose metabolism during early cancer progression. Untransformed and ras-oncogene transfected (ras) MCF10A human breast epithelial cells were employed to model early breast cancer progression. 1,25(OH)2D modified the response of the ras cells to glucose restriction, suggesting 1,25(OH)2D may reduce the ras cell glucose addiction noted in cancer cells. To understand the 1,25(OH)2D regulation of glucose metabolism, following four-day 1,25(OH)2D treatment, metabolite fluxes at the cell membrane were measured by a nanoprobe biosensor, [13C6]glucose flux by 13C-mass isotopomer distribution analysis of media metabolites, intracellular metabolite levels by NMR, and gene expression of related enzymes assessed. Treatment with 1,25(OH)2D reduced glycolysis as flux of glucose to 3-phosphoglycerate was reduced by 15% (P = 0.017) and 32% (P < 0.003) in MCF10A and ras cells respectively. In the ras cells, 1,25(OH)2D reduced lactate dehydrogenase activity by 15% (P < 0.05) with a concomitant 10% reduction in the flux of glucose to lactate (P = 0.006), and reduction in the level of intracellular lactate by 55% (P = 0.029). Treatment with 1,25(OH)2D reduced flux of glucose to acetyl-coA 24% (P = 0.002) and 41% (P < 0.001), and flux to oxaloacetate 34% (P = 0.003) and 33% (P = 0.027) in the MCF10A and ras cells, respectively, suggesting a reduction in tricarboxylic acid (TCA) cycle activity. The results suggest a novel mechanism involving the regulation of glucose metabolism by which 1,25(OH)2D may prevent breast cancer progression.

INTRODUCTION Four Holstein-Friesian cows were used to investigate the effects of intravascular infusions of AA mixtures on milk constituents. Cows were in wk 11 to 28 of lactation and were fed a basal concentrate (142 g of CPkg of DM) and grass silage ( 149 g of CPkg of DM) in a. 60:40 ratio (percentage of DM). Cows were fed hourly, and feed intake was fixed at 95% of ad libitum intake for each experimental period. Each cow received a 4-d jugular saline infusion, followed by a 5-d jugular infusion of a mixture of AA. Two mixtures of AA were used in a crossover design. The first mixture contained both the essential AA and nonessential AA found in milk protein (total AA); this mixture was infused at 400 g of Md. The other mixture represented the essential AA fraction only and was infused at 208 gld. Infusion of total AA increased milk protein concentration from 32.4 to 35.0 gkg, and essential AA increased milk protein concentration from 32.5 to 36.9 gkg; milk protein yield increased by 87 g/d (total AA) and 143 gld (essential AA). Intravascular administration of AA specifically stimulated milk protein concentration, and the efficiency with which the AA were used was higher than had been previously reported when AA supply was increased either by dietary supplementation or by abomasal infusion.

The response in whole-body and splanchnic tissue mass and isotope amino acid transfers in both plasma and blood has been studied in sheep offered 800 g lucerne (Medicago sutiva) pellets/d. Amino acid mass transfers were quantified over a 4 h period,by arterio-venous procedures, across the portal-drained viscera (PDV) and liver on day 5 of an intravenous infusion of either vehicle or the methylated products, choline (0.5 g/d) plus creatine (10 g/d). Isotopic movements were monitored over the same period during a 10 h infusion of a mixture of U-13C-labelled amino acids obtained from hydrolysis of labelled algal cells. Sixteen amino acids were monitored by gas chromatography-mass spectrometry, with thirteen of these analysed within a single chromatographic analysis. Except for methionine, which is discussed in a previous paper, no significant effects of choline plus creatine infusion were observed on any of the variables reported. Whole-body protein irreversible-loss rates ranged from 158 to 245 g/d for the essential amino acids, based on the relative enrichments (dilution of the U-13C molecules by those unlabelled) of free amino acids in arterial plasma, and 206-519 g/d, when blood free amino acid relative enrichments were used for the calculations. Closer agreement was obtained between lysine, threonine, phenylalanine and the branched-chain amino acids. Plasma relative enrichments always exceeded those in blood (P < 0.001), possibly due to hydrolysis of peptides or degradation of protein within the erythrocyte or slow equilibration between plasma and the erythrocyte. Net absorbed amino acids across the PDV were carried predominantly in the plasma. Little evidence was obtained of any major and general involvement of the erythrocytes in the transport of free amino acids from the liver. Net isotope movements also supported these findings. Estimates of protein synthesis rates across the PDV tissues from [U-13C] leucine kinetics showed good agreement with previous values obtained with single-labelled leucine. Variable rates were obtained between the essential amino acids, probably due to different intracellular dilutions. Isotope dilution across the liver was small and could be attributed predominantly to uni-directional transfer from extracellular sources into the hepatocytes and this probably dominates the turnover of the intracellular hepatic amino acid pools.

Development of management paradigms that enhance the rate of gain and qualitative characteristics of beef carcass development has the potential to impact production and nutrient use efficiency but also mitigate losses to the environment. We used eight Black Angus beef steers (272.5 ± 17.6 kg initial body wt) fed a forage-based pelleted diet alone ( n = 4) or supplemented with sodium propionate included ( n = 4) for 42 days. High-quality RNA was extracted from the longissimus lumborum and subjected to transcriptome sequencing using RNA-seq technology. Trimmed reads were aligned to the bovine reference genome (Btau4.0, release 63) and uniquely mapped reads from control and propionate treatment groups were subject to further analysis using edgeR. Candidates were filtered to account for multiple testing and differentially expressed genes (153 at a false discovery rate of

Four lactating cows equipped with rumen and duodenal cannulas were fed a diet of grass silage and concentrates containing either 12.4 or 17.2% CP (DM basis) in a change-over design. Additional protein was supplied as white fish meal. Fish meal did not affect molar proportions of VFA in the rumen, but duodenal NAN supply was increased .69 g/g of N in supplementary feed. In Experiment 2, three lactating dairy cows that had been prepared with catheters across the mammary gland were fed the same diets using a switchback design. Blood samples were taken to determine changes in metabolite flux to the mammary gland. In both experiments, milk production and protein yield were non-significantly increased by addition of fish meal. Milk urea output was increased from 3.18 to 4.74 g/d by fish meal supplementation, reflecting increased arterial concentrations of urea. Concentrations of glucose, VFA, and BHBA in blood showed no substantial changes because of dietary supplementation of fish meal. Supply of essential AA increased 26% with fish meal supplementation, mammary uptake increased 34%, but milk protein output only increased 5%. The low efficiency of conversion of supplementary protein to milk protein appears to be related to the inability of the gland to utilize the additional AA.

International audience; We investigated the effect of relative changes in dietary nitrogen (N) and energy supply and the subsequent variations in net portal appearance (NPA) of nitrogenous and energy nutrients on the net amino acid (AA) uptake by the liver and net N supply to the peripheral tissues. Six lambs were catheterised across the splanchnic tissues and received, in a replicated Latin square, one of three dietary treatments. The diets were formulated to either match the requirements of N and energy (C), or supply only 0.8 of the N requirement (LN) or 0.8 of the energy requirement (LE). Net fluxes of AA and urea-N were measured across the portal-drained viscera, and estimation of arterial hepatic flow allowed the estimation of hepatic fluxes. Catheters were implanted into the portal and hepatic veins as well as in the abdominal aorta for the measurement of AA fluxes. Animals fed the LN diet showed more efficient N retention (0.59 of digested N) than did the C and LE diet (0.50 and 0.33, respectively; P < 0.001). The NPA of total AA-N for the LN diet was only 0.60 of the value measured for the control (C) diet (P < 0.01). Despite this, the total estimated AA-N net splanchnic fluxes were not significantly different across the three diets (3.3, 1.9 and 2.6 g total AA-N/day for C, LN and LE, respectively, P = 0.52). Thus, different metabolic regulations must have taken place across the liver between the three experimental diets. A combination of decreased net uptake of total AA-N by the liver of animals in the LN diet (0.61 of the C diet; P = 0.002) and reduced urinary urea-N production (0.52 of the C diet; P = 0.001) spared AA from catabolism in the LN diet relative to the other two diets. For the LE diet, the urinary urea-N output was 1.3 times the value of the C diet (P = 0.01). This may relate to an increased catabolism of AA by the muscle and/or, to a lesser extent, to an increased utilisation of AA for gluconeogenesis in the liver. These effects may explain the reduced whole body protein retention observed with the LE diet.

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 x 4 Latin square design experiment. Treatments were abomasal infusions of casein and starch in a 2 x 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.

In this study, we aimed to determine the contribution of substrates to tricarboxylic acid (TCA) cycle fluxes in rumen epithelial cells (REC) and duodenal mucosal cells (DMC) isolated from Angus bulls (n = 6) fed either a 75% forage (HF) or 75% concentrate (HC) diet. In separate incubations, [(13)C(6)]glucose, [(13)C(5)]glutamate, [(13)C(5)]glutamine, [(13)C(6)]leucine, or [(13)C(5)]valine were added in increasing concentrations to basal media containing SCFA and a complete mixture of amino acids. Lactate, pyruvate, and TCA cycle intermediates were analyzed by GC-MS followed by (13)C-mass isotopomer distribution analysis. Glucose metabolism accounted for 10-19% of lactate flux in REC from HF-fed bulls compared with 27-39% in REC from HC and in DMC from bulls fed both diets (P < 0.05). For both cell types, as concentration increased, an increasing proportion (3-63%) of alpha-ketoglutarate flux derived from glutamate, whereas glutamine contributed

Blood and milk concentrations of the acute phase protein lipopolysaccharide-binding protein (LBP) were evaluated in cows with naturally occurring mastitis. Blood and milk samples were collected from 101 clinically healthy dairy cows and 17 dairy cows diagnosed with clinical mastitis, and the LBP concentrations of the samples were measured by an ELISA. Concentrations of LBP were greater in the blood and milk of cows with clinical mastitis than in those with healthy quarters. Concentrations of LBP also differed between uninfected and subclinically infected quarters with low somatic cell count. Blood concentrations of LBP in cows with subclinical intramammary infections could not be differentiated from those of cows with all healthy quarters. Together, these data demonstrate that increased blood and milk concentrations of LBP can be detected in dairy cows with naturally acquired intramammary infections that cause clinical mastitis.