Effect of propionate on mRNA expression of key genes for gluconeogenesis in liver of dairy cattle.

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.

Linking our understanding of mammary gland metabolism to amino acid nutrition.

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.

Acute metabolic decompensation due to influenza in a mouse model of ornithine transcarbamylase deficiency.

The urea cycle functions to incorporate ammonia, generated by normal metabolism, into urea. Urea cycle disorders (UCDs) 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 individuals 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 with 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 wild-type (WT) and spf-ash mice displayed similar cytokine profiles and lung viral titers in response to PR8 influenza infection. During infection, spf-ash mice displayed an increase in liver transaminases, suggesting a hepatic sensitivity to the inflammatory response and an altered hepatic immune response. Despite having no visible pathological changes by histology, WT and spf-ash mice had reduced CPS1 and OTC enzyme activities, and, unlike WT, spf-ash mice 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 UCD. 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 could be adapted to explore acute decompensation in other types of inborn errors of metabolism.

Quantification of Transcriptome Responses of the Rumen Epithelium to Butyrate Infusion using RNA-seq Technology.

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 (ɛ = 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.

Characterization of the longissimus lumborum transcriptome response to adding propionate to the diet of growing Angus beef steers.

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 <5%) were analyzed using Gene Ontology (GO) analysis (GOseq) to select terms where enrichment had occurred. Significant GO terms included regulation of cholesterol transport, regulation of sterol transport, and cellular modified amino acid metabolic process. Furthermore, the top four identified gene networks included lipid metabolism, small molecule biochemistry, carbohydrate metabolism, and molecular transport-related categories. Notably, changes in lipid metabolism specific genes reflect both increased oxidative and lipid synthetic capacities. Metabolism-related gene changes are reflective of expected enhancements in lean tissue accretion patterns exhibited in steers where high ruminal propionate relative to other short chain fatty acids is observed. Propionate feeding induced increased N retention in rapidly growing Angus cattle, and the observed alterations in LL tissue lipid metabolism-related gene networks are consistent with enhanced cell formation and function (protein synthesis, and lipogenic vs. lipolytic activities).

Muscle transcriptomic analyses in Angus cattle with divergent tenderness.

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.

High rates of mammary tissue protein turnover in lactating goats are energetically costly.

The high energetic demands and metabolism of amino acids (AA) within the lactating mammary gland have been ascribed to the requirements for milk component synthesis and tissue maintenance. Our objective in this work was to assess rates of protein synthesis from several AA so that the energetic costs of tissue maintenance could be better reflected. Lactating goats (n = 4) were given staggered infusions of 5 labeled forms of phenylalanine (Phe) initiated at 30, 12, 9, 6, and 3 h before goats were killed. [5-(13)CH(3)] Methionine (Met), [1-(13)C] leucine, and [1-(13)C] valine were also infused for 30 h, during which time, the glands were milked hourly and arteriovenous flux measurements were performed the last 6 h. A dynamic, compartmental model capable of simulating fluxes of AA through extracellular and intracellular free, slow and fast turnover tissue-bound, and milk protein pools was developed and fitted to the observed data. The udder removed 81% of the Phe present in plasma using 31% for milk protein synthesis and releasing 66% back into plasma. Transamination accounted for 40% of Phe flux in the mammary and transmethylation accounted for a portion of mammary Met flux. Mammary tissue protein synthesis was >300% the value of milk protein synthesis with fractional protein synthesis rates >130%/d. Assuming 4 mol of ATP/mol of peptide bond formed, we estimate that approximately 50% of ATP generated by the lactating mammary glands is used for synthesis of tissue (nonmilk) protein.

Glutamate is the major anaplerotic substrate in the tricarboxylic acid cycle of isolated rumen epithelial and duodenal mucosal cells from beef cattle.

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 <3% (P < 0.05). Although leucine and valine were catabolized to their respective keto-acids, these were not further metabolized to TCA cycle intermediates. Glucose, glutamine, leucine, and valine catabolism by ruminant gastrointestinal tract cells has been previously demonstrated, but in this study, their catabolism via the TCA cycle was limited. Further, although glutamate's contribution to TCA cycle fluxes was considerable, it was apparent that other substrates available in the media also contributed to the maintenance of TCA fluxes. Lastly, the results suggest that diet composition alters glucose, glutamate, and leucine catabolism by the TCA cycle of REC and DMC.

Intestinal protein supply alters amino acid, but not glucose, metabolism by the sheep gastrointestinal tract.

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 <100% of the additional supplies of leucine, valine, and isoleucine (0.6-0.66, P < 0.05) from casein infusion, whereas net absorption by the MDV accounted for 100% of the additional essential AA supply. Glucose absorption (negative) and utilization of arterial glucose supply by the GIT remained unchanged. There was a positive linear (P < 0.05) relation between transfer of plasma urea to the GIT and arterial urea concentration (MDV, P < 0.05, r = 0.90; PDV, P < 0.05, r = 0.93). The ruminant GIT appears to metabolize increasing amounts of the branched-chain AAs and certain nonessential AAs when the intestinal supply of protein is increased.

Influence of flavomycin on microbial numbers, microbial metabolism and gut tissue protein turnover in the digestive tract of sheep.

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 (n 5) 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 immediately post 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 (3.5 v. 1.2 x 10(9) per 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 amino acid need for milk synthesis is defined by the maximal uptake of plasma amino acids by porcine mammary glands.

To define dietary indispensable amino acid (IAA) needs for milk synthesis by the mammary glands (MG), 16 lactating sows were fed 1 of 4 isocaloric diets varying in protein concentrations (from 78 to 235 g/kg) with an ideal amino acid (AA) pattern. On d 9, 13, 17, and 21 of lactation, blood samples were obtained simultaneously from a carotid artery and the main mammary vein every 30 min over 6 h. A quadratic regression model of the log mammary arteriovenous difference (AVD) of plasma IAA (y) against daily intake of dietary IAA (X) was established. First, the reverse log intercept, defined as the mammary AVD at zero dietary AA supply, was used to quantify the contribution of endogenous IAA. The quantification was validated by body N balance coupled with AA composition analysis. Then, the estimated vertex (y(max), X(i)) was used in 2 aspects: 1) The maximal mammary uptake of plasma IAA, quantified by multiplying the maximal mammary AVD and plasma flow rate, was considered the physiological IAA need for milk synthesis. 2) Corresponding to the y(max), dietary IAA intake (X(i)) would represent the total dietary IAA requirement, i.e., the sum of maintenance need and milk synthesis need after adjustment for body weight loss. Thus, dietary IAA needs for milk synthesis were derived. Moreover, the estimate of lysine need for milk synthesis in this study was identical to an estimate obtained from multiple regression analysis of feeding trial data. We conclude that dietary IAA needs for milk synthesis can be quantified by the maximal uptakes of plasma IAA by porcine MG.

Amino acid availability affects amino acid flux and protein metabolism in the porcine mammary gland.

A kinetic model was used to examine transmembrane flux kinetics of lysine, methionine and valine across the porcine mammary gland (MG) under dietary amino acid (AA) limiting, adequate and excess conditions. Lactating sows (3 per treatment) were offered three diets: lysine-deficient [LD, 4.9 lysine and 9.9 valine (g/kg diet)], adequate (Control, 9.7 and 10.2) and valine-excess (VE, 9.8 and 13.4). On d 18 of lactation, 2-(15)N-lysine, 5-methyl-(2)H(3)-methionine and 1-(13)C-valine were infused into a jugular vein for 20.5 h. Milk and arterial and mammary venous blood samples were collected at 2- and 1-h intervals, respectively. Compared with Control, milk yield and litter growth rate decreased (P < 0.05) in sows fed the LD diet. Model estimates of mammary protein synthesis (PS), breakdown (PB) and net PS decreased (P < 0.05) in sows fed the LD diet. Net uptake of lysine decreased (P < 0.05) in sows fed the LD diet as a result of decreases in inward and outward transport of lysine. Inward transport of methionine tended to be reduced (P < 0.10) in sows fed the LD diet, resulting in a decrease in net methionine uptake. In sows fed the VE diet, PB was reduced (P < 0.05) and PS unchanged compared with Control. Outward transport of valine and net lysine uptake were reduced (P < 0.05), but net valine uptake was unchanged in sows fed the VE diet compared with Control. In conclusion, the kinetic model provided estimates of PS that were similar to empirical measurements of milk protein output and mammary protein accretion. Transport of lysine and methionine by the porcine MG is closely linked to regulation of mammary PS. Lysine availability has little effect on the transmembrane flux of valine.