Rumen-protected choline and methionine during the periparturient period affect choline metabolites, amino acids, and hepatic expression of genes associated with one-carbon and lipid metabolism.

Feeding supplemental choline and Met during the periparturient period can have positive effects on cow performance; however, the mechanisms by which these nutrients affect performance and metabolism are unclear. The objective of this experiment was to determine if providing rumen-protected choline, rumen-protected Met, or both during the periparturient period modifies the choline metabolitic profile of plasma and milk, plasma AA, and hepatic mRNA expression of genes associated with choline, Met, and lipid metabolism. Cows (25 primiparous, 29 multiparous) were blocked by expected calving date and parity and randomly assigned to 1 of 4 treatments: control (no rumen-protected choline or rumen-protected Met); CHO (13 g/d choline ion); MET (9 g/d DL-methionine prepartum; 13.5 g/d DL-methionine, postpartum); or CHO + MET. Treatments were applied daily as a top dress from ∼21 d prepartum through 35 d in milk (DIM). On the day of treatment enrollment (d -19 ± 2 relative to calving), blood samples were collected for covariate measurements. At 7 and 14 DIM, samples of blood and milk were collected for analysis of choline metabolites, including 16 species of phosphatidylcholine (PC) and 4 species of lysophosphatidylcholine (LPC). Blood was also analyzed for AA concentrations. Liver samples collected from multiparous cows on the day of treatment enrollment and at 7 DIM were used for gene expression analysis. There was no consistent effect of CHO or MET on milk or plasma free choline, betaine, sphingomyelin, or glycerophosphocholine. However, CHO increased milk secretion of total LPC irrespective of MET for multiparous cows and in absence of MET for primiparous cows. Furthermore, CHO increased or tended to increase milk secretion of LPC 16:0, LPC 18:1, and LPC 18:0 for primi- and multiparous cows, although the response varied with MET supplementation. Feeding CHO also increased plasma concentrations of LPC 16:0 and LPC 18:1 in absence of MET for multiparous cows. Although milk secretion of total PC was unaffected, CHO and MET increased secretion of 6 and 5 individual PC species for multiparous cows, respectively. Plasma concentrations of total PC and individual PC species were unaffected by CHO or MET for multiparous cows, but MET reduced total PC and 11 PC species during wk 2 postpartum for primiparous cows. Feeding MET consistently increased plasma Met concentrations for both primi- and multiparous cows. Additionally, MET decreased plasma serine concentrations during wk 2 postpartum and increased plasma phenylalanine in absence of CHO for multiparous cows. In absence of MET, CHO tended to increase hepatic mRNA levels of betaine-homocysteine methyltransferase and phosphate cytidylyltransferase 1 choline, α, but tended to decrease expression of 3-hydroxy-3-methylglutaryl-coenzyme A synthase 2 and peroxisome proliferator activated receptor α irrespective of MET. Although shifts in the milk and plasma PC profile were subtle and inconsistent between primi- and multiparous cows, gene expression results suggest that supplemental choline plays a probable role in promoting the cytidine diphosphate-choline and betaine-homocysteine S-methyltransferase pathways. However, interactive effects suggest that this response depends on Met availability, which may explain the inconsistent results observed among studies when supplemental choline is fed.

Glycerol is a major substrate for glucose, glycogen, and nonessential amino acid synthesis in late-term chicken embryos.

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.

Uncoupling hypoxia signaling from oxygen sensing in the liver results in hypoketotic hypoglycemic death.

As the ultimate electron acceptor in oxidative phosphorylation, oxygen plays a critical role in metabolism. When oxygen levels drop, heterodimeric hypoxia-inducible factor (Hif) transcription factors become active and facilitate adaptation to hypoxia. Hif regulation by oxygen requires the protein von Hippel-Lindau (pVhl) and pVhl disruption results in constitutive Hif activation. The liver is a critical organ for metabolic homeostasis, and Vhl inactivation in hepatocytes results in a Hif-dependent shortening in life span. While albumin-Cre;Vhl(F/F) mice develop hepatic steatosis and impaired fatty acid oxidation, the variable penetrance and unpredictable life expectancy has made the cause of death elusive. Using a system in which Vhl is acutely disrupted and a combination of ex vivo liver perfusion studies and in vivo oxygen measurements, we demonstrate that Vhl is essential for mitochondrial respiration in vivo. Adenovirus-Cre mediated acute Vhl disruption in the liver caused death within days. Deprived of pVhl, livers accumulated tryglicerides and circulating ketone and glucose levels dropped. The phenotype was reminiscent of inborn defects in fatty acid oxidation and of fasted PPARα-deficient mice and while death was unaffected by pharmacologic PPARα activation, it was delayed by glucose administration. Ex vivo liver perfusion analyses and acylcarnitine profiles showed mitochondrial impairment and a profound inhibition of liver ketone and glucose production. By contrast, other mitochondrial functions, such as ureagenesis, were unaffected. Oxygen consumption studies revealed a marked suppression of mitochondrial respiration, which, as determined by magnetic resonance oximetry in live mice, was accompanied by a corresponding increase in liver pO(2). Importantly, simultaneous inactivation of Hif-1ß suppressed liver steatosis and rescued the mice from death. These data demonstrate that constitutive Hif activation in mice is sufficient to suppress mitochondrial respiration in vivo and that no other pathway exists in the liver that can allow oxygen utilization when Hif is active precluding thereby metabolic collapse.

Gluconeogenesis differs in developing chick embryos derived from small compared with typical size broiler breeder eggs.

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.

Salvage of blood urea nitrogen in sheep is highly dependent on plasma urea concentration and the efficiency of capture within the digestive tract.

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)N2]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.

Application of stable isotopes and mass isotopomer distribution analysis to the study of intermediary metabolism of nutrients.

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.