Evidence for metabolism of creatine by the conceptus, placenta, and uterus for production of ATP during conceptus development in pigs.

In pigs, the majority of embryonic mortality occurs when free-floating conceptuses (embryos/fetuses and associated placental membranes) elongate and the uterine-placental interface undergoes folding and develops areolae. Both periods involve proliferation, migration, and changes in morphology of cells that require ATP. We hypothesize that insufficient ATP in conceptus and uterine tissues contributes to conceptus loss in pigs. Creatine is stored in cells as phosphocreatine (PCr) for ATP regeneration through the creatine (Cr)-creatine kinase (CK)-PCr pathway. However, the expression of components of this pathway in pigs has not been examined throughout gestation. Results of qPCR analyses indicated increases in AGAT, GAMT, CKM, CKB, and SLC6A8 mRNAs in elongating porcine conceptuses and immunofluorescence microscopy localized GAMT, CKM, and CKB proteins to the trophectoderm of elongating conceptuses, to the columnar chorionic epithelial cells at the bottom of chorioallantoic troughs, and to endometrial luminal epithelium (LE) at the tops of the endometrial ridges of uterine-placental folds on Days 40, 60, and 90 of gestation. GAMT protein is expressed in endometrial LE at the uterine-placental interface, but immunostaining is more intense in LE at the bottoms of the endometrial ridges. Results of this study indicate that key elements of the pathway for creatine metabolism are expressed in cells of the conceptus, placenta, and uterus for potential production of ATP during two timepoints in pregnancy with a high demand for energy; elongation of the conceptus for implantation and development of uterine-placental folding during placentation.

Metabolic pathways for glucose and fructose: I synthesis and metabolism of fructose by ovine conceptuses.

Fructose, the most abundant hexose sugar in fetal fluids and blood of sheep and other ungulates and cetaceans, is synthesized from glucose via the polyol pathway in trophectoderm and chorion. However, the cell-specific and temporal expression of enzymes for the synthesis and metabolism of fructose in sheep conceptuses (embryo and placental membranes) and placentomes has not been characterized. This study characterized key enzymes involved in fructose synthesis and metabolism by ovine conceptuses throughout pregnancy. Day 17 conceptuses expressed mRNAs for the polyol pathway (SORD and AKR1B1) and glucose and fructose metabolism (HK1, HK2, G6PD, OGT, and FBP), but not those required for gluconeogenesis (G6Pase or PCK). Ovine placentomes also expressed mRNAs for SORD, AKR1B1, HK1, and OGT. Fructose can be metabolized via the ketohexokinase (KHK) pathway and isoforms, KHK-A and KHK-C, were expressed in ovine conceptuses from Day 16 of pregnancy and placentomes during pregnancy in a cell specific manner: KHK-A protein was more abundant in trophectoderm and cotyledons of placentomes, while KHK-C protein was more abundant in endoderm of Day 16 conceptuses and chorionic epithelium in placentomes. Expression of KHK mRNAs in placentomes was greatest at Day 30 of pregnancy (P < 0.05), but not different among days later in gestation. These results provide novel insights into the synthesis and metabolism of fructose via the uninhibited KHK pathway in ovine conceptuses to generate ATP via the TCA cycle, as well as substrates for the pentose cycle, hexosamine biosynthesis pathway and one-carbon metabolism required for conceptus development throughout pregnancy.

Metabolic pathways of glucose and fructose: II spatiotemporal expression of genes involved in synthesis and transport of lactate in ovine conceptuses.

Lactate, an abundant molecule in fetal fluids and blood of mammalian species is often overlooked as a metabolic waste product generated during pregnancy. Most of the glucose and fructose consumed by ovine conceptuses is converted to lactate, but proteins involved in lactate metabolism and transport have not been investigated. This study characterized total lactate produced by ovine conceptuses throughout gestation, as well as expression of mRNAs and proteins involved in lactate metabolism. Lactate increased in abundance in the uterine lumen during the preimplantation period and was more abundant than pyruvate. The abundance of lactate in allantoic and amniotic fluids increased with advancing days of gestation and most abundant on Day 125 of pregnancy (P < 0.05). Lactate dehydrogenase (LDH) subunits A (converts pyruvate to lactate) and B (converts lactate to pyruvate) were expressed by conceptuses throughout gestation. Lactate is transported via monocarboxylic acid transporters SLC16A1 and SLC16A3, both of which were expressed by the conceptus throughout gestation. Additionally, the interplacentomal chorioallantois from Day 126 expressed SLC16A1 and SLC16A3 and transported lactate across the tissue. Hydrocarboxylic acid receptor 1 (HCAR1), a receptor for lactate, was localized to the uterine luminal and superficial glandular epithelia of pregnant ewes throughout gestation, and conceptus trophectoderm during the peri-implantation period of gestation. These results provide novel insights into the spatiotemporal profiles of enzymes, transporters, and receptor for lactate by ovine conceptuses throughout pregnancy.

Nanoparticle-mediated delivery of tetrahydrobiopterin restores endothelial function in diabetic rats.

Endothelial dysfunction, underlying the vascular complications of diabetes and other cardiovascular disorders, may result from uncoupling of endothelial nitric oxide synthase (eNOS) activity due to decreased levels of tetrahydrobiopterin (BH4), a critical co-factor for eNOS. Some clinical trials attempting to deliver exogenous BH4 as a potential therapeutic strategy in vascular disease states have failed due to oxidation of BH4 in the circulation. We sought to develop a means of protecting BH4 from oxidation while delivering it to dysfunctional endothelial cells. Polymeric and solid lipid nanoparticles (NPs) loaded with BH4 were delivered by injection or oral gavage, respectively, to streptozotocin-induced diabetic rats. BH4 was measured in coronary endothelial cells and endothelium-dependent vascular reactivity was assessed in vascular rings. Lymphatic uptake of orally delivered lipid NPs was verified by sampling mesenteric lymph. BH4-loaded polymeric NPs maintained nitric oxide production by cultured endothelial cells under conditions of oxidative stress. BH4-loaded NPs, delivered via injection or ingestion, increased coronary endothelial BH4 concentration and improved endothelium-dependent vasorelaxation in diabetic rats. Pharmacodynamics assessment indicated peak concentration of solid lipid NPs in the systemic bloodstream 6 hours after ingestion, with disappearance noted by 48 hours. These studies support the feasibility of utilizing NPs to deliver BH4 to dysfunctional endothelial cells to increase nitric oxide bioavailability. BH4-loaded NPs could provide an innovative tool to restore redox balance in blood vessels and modulate eNOS-mediated vascular function to reverse or retard vascular disease in diabetes.

Dietary protein and amino acid intakes for mitigating sarcopenia in humans.

Adult humans generally experience a 0.5-1%/year loss in whole-body skeletal muscle mass and a reduction of muscle strength by 1.5-5%/year beginning at the age of 50 years. This results in sarcopenia (aging-related progressive losses of skeletal muscle mass and strength) that affects 10-16% of adults aged ≥ 60 years worldwide. Concentrations of some amino acids (AAs) such as branched-chain AAs, arginine, glutamine, glycine, and serine are reduced in the plasma of older than young adults likely due to insufficient protein intake, reduced protein digestibility, and increased AA catabolism by the portal-drained viscera. Acute, short-term, or long-term administration of some of these AAs or a mixture of proteinogenic AAs can enhance blood flow to skeletal muscle, activate the mechanistic target of rapamycin cell signaling pathway for the initiation of muscle protein synthesis, and modulate the metabolic activity of the muscle. In addition, some AA metabolites such as taurine, ß-alanine, carnosine, and creatine have similar physiological effects on improving muscle mass and function in older adults. Long-term adequate intakes of protein and the AA metabolites can aid in mitigating sarcopenia in elderly adults. Appropriate combinations of animal- and plant-sourced foods are most desirable to maintain proper dietary AA balance.

Recent Advances in the Nutrition and Metabolism of Dogs and Cats.

Domestic dogs (facultative carnivores) and cats (obligate carnivores) have been human companions for at least 12,000 and 9000 years, respectively. These animal species have a relatively short digestive tract but a large stomach volume and share many common features of physiological processes, intestinal microbes, and nutrient metabolism. The taste buds of the canine and feline tongues can distinguish sour, umami, bitter, and salty substances. Dogs, but not cats, possess sweet receptors. α-Amylase activity is either absent or very low in canine and feline saliva, and is present at low or substantial levels in the pancreatic secretions of cats or dogs, respectively. Thus, unlike cats, dogs have adapted to high-starch rations while also consuming animal-sourced foods. At metabolic levels, both dogs and cats synthesize de novo vitamin C and many amino acids (AAs, such as Ala, Asn, Asp, Glu, Gln, Gly, Pro, and Ser) but have a very limited ability to form vitamin D3. Compared with dogs, cats have higher requirements for AAs, some B-complex vitamins, and choline; greater rates of gluconeogenesis; a higher capacity to tolerate AA imbalances and antagonism; a more limited ability to synthesize arginine and taurine from glutamine/proline and cysteine, respectively; and a very limited ability to generate polyunsaturated fatty acids (PUFAs) from respective substrates. Unlike dogs, cats cannot convert either ß-carotene into vitamin A or tryptophan into niacin. Dogs can thrive on one large meal daily and select high-fat over low-fat diets, whereas cats eat more frequently during light and dark periods and select high-protein over low-protein diets. There are increasing concerns over the health of skin, hair, bone, and joints (specialized connective tissues containing large amounts of collagen and/or keratin); sarcopenia (age-related losses of skeletal-muscle mass and function); and cognitive function in dogs and cats. Sufficient intakes of proteinogenic AAs and taurine along with vitamins, minerals, and PUFAs are crucial for the normal structures of the skin, hair, bone, and joints, while mitigating sarcopenia and cognitive dysfunction. Although pet owners may have different perceptions about the feeding and management practice of their dogs and cats, the health and well-being of the companion animals critically depend on safe, balanced, and nutritive foods. The new knowledge covered in this volume of Adv Exp Med Biol is essential to guide the formulation of pet foods to improve the growth, development, brain function, reproduction, lactation, and health of the companion animals.

Roles of Nutrients in the Brain Development, Cognitive Function, and Mood of Dogs and Cats.

The brain is the central commander of all physical activities and the expression of emotions in animals. Its development and cognitive health critically depend on the neural network that consists of neurons, glial cells (namely, non-neuronal cells), and neurotransmitters (communicators between neurons). The latter include proteinogenic amino acids (e.g., L-glutamate, L-aspartate, and glycine) and their metabolites [e.g., γ-aminobutyrate, D-aspartate, D-serine, nitric oxide, carbon monoxide, hydrogen sulfide, and monoamines (e.g., dopamine, norepinephrine, epinephrine, and serotonin)]. In addition, some non-neurotransmitter metabolites of amino acids, such as taurine, creatine, and carnosine, also play important roles in brain development, cognitive health, behavior, and mood of dogs and cats. Much evidence shows that cats require dietary ω3 (α-linolenic acid, eicosapentaenoic acid, and docosahexaenoic acid) and ω6 (linoleic acid and arachidonic acid) polyunsaturated fatty acids for the development of the central nervous system. As an essential component of membranes of neurons and glial cells, cholesterol is also crucial for cognitive development and function. In addition, vitamins and minerals are required for the metabolism of AAs, lipids, and glucose in the nervous system, and also act as antioxidants. Thus, inadequate nutrition will lead to mood disorders. Some amino acids (e.g., arginine, glycine, methionine, serine, taurine, tryptophan, and tyrosine) can help to alleviate behavioral and mood disorders (e.g., depression, anxiety and aggression). As abundant providers of all these functional amino acids and lipids, animal-sourced foods (e.g., liver, intestinal mucosa, and meat) play important roles in brain development, cognitive function, and mood of dogs and cats. This may explain, in part, why dogs and cats prefer to eat visceral organs of their prey. Adequate provision of nutrients in all phases of the life cycle (pregnancy, lactation, postnatal growth, and adulthood) is essential for optimizing neurological health, while preventing cognitive dysfunction and abnormal behavior.

Characteristics of Nutrition and Metabolism in Dogs and Cats.

Domestic dogs and cats have evolved differentially in some aspects of nutrition, metabolism, chemical sensing, and feeding behavior. The dogs have adapted to omnivorous diets containing taurine-abundant meat and starch-rich plant ingredients. By contrast, domestic cats must consume animal-sourced foods for survival, growth, and development. Both dogs and cats synthesize vitamin C and many amino acids (AAs, such as alanine, asparagine, aspartate, glutamate, glutamine, glycine, proline, and serine), but have a limited ability to form de novo arginine and vitamin D3. Compared with dogs, cats have greater endogenous nitrogen losses and higher dietary requirements for AAs (particularly arginine, taurine, and tyrosine), B-complex vitamins (niacin, thiamin, folate, and biotin), and choline; exhibit greater rates of gluconeogenesis; are less sensitive to AA imbalances and antagonism; are more capable of concentrating urine through renal reabsorption of water; and cannot tolerate high levels of dietary starch due to limited pancreatic α-amylase activity. In addition, dogs can form sufficient taurine from cysteine (for most breeds); arachidonic acid from linoleic acid; eicosapentaenoic acid and docosahexaenoic acid from α-linolenic acid; all-trans-retinol from ß-carotene; and niacin from tryptophan. These synthetic pathways, however, are either absent or limited in all cats due to (a) no or low activities of key enzymes (including pyrroline-5-carboxylate synthase, cysteine dioxygenase, ∆6-desaturase, ß-carotene dioxygenase, and quinolinate phosphoribosyltransferase) and (b) diversion of intermediates to other metabolic pathways. Dogs can thrive on one large meal daily, select high-fat over low-fat diets, and consume sweet substances. By contrast, cats eat more frequently during light and dark periods, select high-protein over low-protein diets, refuse dry food, enjoy a consistent diet, and cannot taste sweetness. This knowledge guides the feeding and care of dogs and cats, as well as the manufacturing of their foods. As abundant sources of essential nutrients, animal-derived foodstuffs play important roles in optimizing the growth, development, and health of the companion animals.

Analysis of Gizzerosine in Foodstuffs by HPLC Involving Pre-column Derivatization with o-Phthaldialdehyde.

Gizzerosine [2-amino-9-(4-imidazolyl)-7-azanonanoic acid] is a toxic amino acid formed from histamine and lysine at high temperatures, and may be present in foodstuffs (e.g., fishmeal and meat-bone meal) for animals including cats and dogs. Here we developed a simple, rapid, sensitive, specific, and automated method for the analysis of gizzerosine in foodstuffs by high-performance liquid chromatography (HPLC) involving pre-column derivatization with o-phthaldialdehyde (OPA) in the presence of N-acetylcysteine (instead of the usual 2-mercaptoethanol or ethanethiol reagent). OPA reacted immediately (within 1 min) with gizzerosine in an autosampler at room temperatures (e.g., 20-25 °C), and their derivative was directly injected into the HPLC column. The highly fluorescent gizzerosine-OPA derivative was well separated from the OPA derivatives of all natural amino acids known to be present in physiological fluids (e.g., plasma), proteins and foodstuffs, and was detected at an excitation wavelength of 340 nm and an emission wavelength of 450 nm. The total time for chromatographic separation (including column regeneration) was 20 min per sample rather than 40 min and longer in previous HPLC methods. The detection limit for gizzerosine was at least 6 pmol/ml in an assay solution (HPLC vial) or at least 0.09 pmol per injection into the HPLC column. The analysis of gizzerosine was linear between 1 and 100 pmol per injection. When gizzerosine was extracted from foodstuffs, its detection limit was at least 875 pmol/g foodstuff or at least 0.21 mg/kg foodstuff. Our routine HPLC technique does not require any cleanup of samples or the OPA derivatization products (including the OPA-gizzerosine adduct), and is applicable for the analysis of gizzerosine in both foodstuffs and animal tissues.

Functions and Metabolism of Amino Acids in the Hair and Skin of Dogs and Cats.

The hair and skin of domestic cats or dogs account for 2% and 12-24% of their body weight, respectively, depending on breed and age. These connective tissues contain protein as the major constituent and provide the first line of defense against external pathogens and toxins. Maintenance of the skin and hair in smooth and elastic states requires special nutritional support, particularly an adequate provision of amino acids (AAs). Keratin (rich in cysteine, serine and glycine) is the major protein both in the epidermis of the skin and in the hair. Filaggrin [rich in some AAs (e.g., serine, glutamate, glutamine, glycine, arginine, and histidine)] is another physiologically important protein in the epidermis of the skin. Collagen and elastin (rich in glycine and proline plus 4-hydroxyproline) are the predominant proteins in the dermis and hypodermis of the skin. Taurine and 4-hydroxyproline are abundant free AAs in the skin of dogs and cats, and 4-hydroxyproline is also an abundant free AA in their hair. The epidermis of the skin synthesizes melanin (the pigment in the skin and hair) from tyrosine and produces trans-urocanate from histidine. Qualitative requirements for proteinogenic AAs are similar between cats and dogs but not identical. Both animal species require the same AAs to nourish the hair and skin but the amounts differ. Other factors (e.g., breeds, coat color, and age) may affect the requirements of cats or dogs for nutrients. The development of a healthy coat, especially a black coat, as well as healthy skin critically depends on AAs [particularly arginine, glycine, histidine, proline, 4-hydroxyproline, and serine, sulfur AAs (methionine, cysteine, and taurine), phenylalanine, and tyrosine] and creatine. Although there are a myriad of studies on AA nutrition in cats and dogs, there is still much to learn about how each AA affects the growth, development and maintenance of the hair and skin. Animal-sourced foodstuffs (e.g., feather meal and poultry by-product meal) are excellent sources of the AAs that are crucial to maintain the normal structure and health of the skin and hair in dogs and cats.

Characteristics of the Digestive Tract of Dogs and Cats.

As for other mammals, the digestive system of dogs (facultative carnivores) and cats (obligate carnivores) includes the mouth, teeth, tongue, pharynx, esophagus, stomach, small intestine, large intestine, and accessory digestive organs (salivary glands, pancreas, liver, and gallbladder). These carnivores have a relatively shorter digestive tract but longer canine teeth, a tighter digitation of molars, and a greater stomach volume than omnivorous mammals such as humans and pigs. Both dogs and cats have no detectable or a very low activity of salivary α-amylase but dogs, unlike cats, possess a relatively high activity of pancreatic α-amylase. Thus, cats select low-starch foods but dogs can consume high-starch diets. In contrast to many mammals, the vitamin B12 (cobalamin)-binding intrinsic factor for the digestion and absorption of vitamin B12 is produced in: (a) dogs primarily by pancreatic ductal cells and to a lesser extent the gastric mucosa; and (b) cats exclusively by the pancreatic tissue. Amino acids (glutamate, glutamine, and aspartate) are the main metabolic fuels in enterocytes of the foregut. The primary function of the small intestine is to digest and absorb dietary nutrients, and its secondary function is to regulate the entry of dietary nutrients into the blood circulation, separate the external from the internal milieu, and perform immune surveillance. The major function of the large intestine is to ferment undigested food (particularly fiber and protein) and to absorb water, short-chain fatty acids (serving as major metabolic fuels for epithelial cells of the large intestine), as well as vitamins. The fermentation products, water, sloughed cells, digestive secretions, and microbes form feces and then pass into the rectum for excretion via the anal canal. The microflora influences colonic absorption and cell metabolism, as well as feces quality. The digestive tract is essential for the health, survival, growth, and development of dogs and cats.

Functions and Metabolism of Amino Acids in Bones and Joints of Cats and Dogs.

The bone is a large and complex organ (12-15% of body weight) consisting of specialized connective tissues (bone matrix and bone marrow), whereas joints are composed of cartilage, tendons, ligaments, synovial joint capsules and membranes, and a synovial joint cavity filled with synovial fluid. Maintaining healthy bones and joints is a dynamic and complex process, as bone deposition (formation of new bone materials) and resorption (breakdown of the bone matrix to release calcium and phosphorus) are the continuous processes to determine bone balance. Bones are required for locomotion, protection of internal organs, and have endocrine functions to maintain mineral homeostasis. Joints are responsible for resisting mechanical stress/trauma, aiding in locomotion, and supporting the overall musculoskeletal system. Amino acids have multiple regulatory, compositional, metabolic, and functional roles in maintaining the health of bones and joints. Their disorders are prevalent in mammals and significantly reduce the quality of life. These abnormalities in companion animals, specifically cats and dogs, commonly lead to elective euthanasia due to the poor quality of life. Multiple disorders of bones and joints result from genetic predisposition and are heritable, but other factors such as nutrition, growth rate, trauma, and physical activity affect how the disorder manifests. Treatments for cats and dogs are primarily to slow the progression of these disorders and assist in pain management. Therapeutic supplements such as Cosequin and formulated diets rich in amino acids are used commonly as treatments for companion animals to reduce pain and slow the progression of those diseases. Also, amino acids (e.g., taurine, arginine, glycine, proline, and 4-hydroxyproline), and glucosamine reduce inflammation and pain in animals with bone and joint disorders. Gaining insight into how amino acids function in maintaining bone and joint health can aid in developing preventative diets and therapeutic supplementations of amino acids to improve the quality of life in companion animals.

Regulation of synthesis of polyamines by progesterone, estradiol, and their receptors in uteri of cyclic ewes†.

Progesterone (P4), estradiol (E2), and expression of their receptors (PGR and ESR1, respectively) by cells of the uterus regulate reproductive performance of mammals through effects on secretion and transport of nutrients into the uterine lumen. This study investigated the effect of changes in P4, E2, PGR, and ESR1 on expression of enzymes for the synthesis and secretion of polyamines. Suffolk ewes (n = 13) were synchronized to estrus (Day 0) and then, on either Day 1 (early metestrus), Day 9 (early diestrus), or Day 14 (late diestrus) of the estrous cycle, maternal blood samples were collected, and ewes were euthanized before obtaining uterine samples and uterine flushings. Endometrial expression of MAT2B and SMS mRNAs increased in late diestrus (P < 0.05). Expression of ODC1 and SMOX mRNAs decreased from early metestrus to early diestrus, and expression of ASL mRNA was lower in late diestrus than in early metestrus (P < 0.05). Immunoreactive PAOX, SAT1, and SMS proteins were localized to uterine luminal, superficial glandular, and glandular epithelia, stromal cells, myometrium, and blood vessels. Concentrations of spermidine and spermine in maternal plasma decreased from early metestrus to early diestrus and decreased further in late diestrus (P < 0.05). The abundances of spermidine and spermine in uterine flushings were less in late diestrus than early metestrus (P < 0.05). These results indicate that synthesis and secretion of polyamines are affected by P4 and E2, as well as the expression of PGR and ESR1 in the endometria of cyclic ewes.

Creatine metabolism at the uterine-placental interface throughout gestation in sheep†.

The placenta requires high levels of adenosine triphosphate to maintain a metabolically active state throughout gestation. The creatine-creatine kinase-phosphocreatine system is known to buffer adenosine triphosphate levels; however, the role(s) creatine-creatine kinase-phosphocreatine system plays in uterine and placental metabolism throughout gestation is poorly understood. In this study, Suffolk ewes were ovariohysterectomized on Days 30, 50, 70, 90, 110 and 125 of gestation (n = 3-5 ewes/per day, except n = 2 on Day 50) and uterine and placental tissues subjected to analyses to measure metabolites, mRNAs, and proteins related to the creatine-creatine kinase-phosphocreatine system. Day of gestation affected concentrations and total amounts of guanidinoacetate and creatine in maternal plasma, amniotic fluid and allantoic fluid (P < 0.05). Expression of mRNAs for arginine:glycine amidinotransferase, guanidinoacetate methyltransferase, creatine kinase B, and solute carrier 16A12 in endometria and for arginine:glycine amidinotransferase and creatine kinase B in placentomes changed significantly across days of gestation (P < 0.05). The arginine:glycine amidinotransferase protein was more abundant in uterine luminal epithelium on Days 90 and 125 compared to Days 30 and 50 (P < 0.01). The chorionic epithelium of placentomes expressed guanidinoacetate methyltransferase and solute carrier 6A13 throughout gestation. Creatine transporter (solute carrier 6A8) was expressed by the uterine luminal epithelium and trophectoderm of placentomes throughout gestation. Creatine kinase (creatine kinase B and CKMT1) proteins were localized primarily to the uterine luminal epithelium and to the placental chorionic epithelium of placentomes throughout gestation. Collectively, these results demonstrate cell-specific and temporal regulation of components of the creatine-creatine kinase-phosphocreatine system that likely influence energy homeostasis for fetal-placental development.

Metabolic pathways utilized by the porcine conceptus, uterus, and placenta.

Conceptus elongation and early placentation involve growth and remodeling that requires proliferation and migration of cells. This demands conceptuses expend energy before establishment of a placenta connection and when they are dependent upon components of histotroph secreted or transported into the uterine lumen from the uterus. Glucose and fructose, as well as many amino acids (including arginine, aspartate, glutamine, glutamate, glycine, methionine, and serine), increase in the uterine lumen during the peri-implantation period. Glucose and fructose enter cells via their transporters, SLC2A, SLC2A3, and SLC2A8, and amino acids enter the cells via specific transporters that are expressed by the conceptus trophectoderm. However, porcine conceptuses develop rapidly through extensive cellular proliferation and migration as they elongate and attach to the uterine wall resulting in increased metabolic demands. Therefore, coordination of multiple metabolic biosynthetic pathways is an essential aspect of conceptus development. Oxidative metabolism primarily occurs through the tricarboxylic acid (TCA) cycle and the electron transport chain, but proliferating and migrating cells, like the trophectoderm of pigs, enhance aerobic glycolysis. The glycolytic intermediates from glucose can then be shunted into the pentose phosphate pathway and one-carbon metabolism for the de novo synthesis of nucleotides. A result of aerobic glycolysis is limited availability of pyruvate for maintaining the TCA cycle, and trophectoderm cells likely replenish TCA cycle metabolites primarily through glutaminolysis to convert glutamine into TCA cycle intermediates. The synthesis of ATP, nucleotides, amino acids, and fatty acids through these biosynthetic pathways is essential to support elongation, migration, hormone synthesis, implantation, and early placental development of conceptuses.

The ovine conceptus utilizes extracellular serine, glucose, and fructose to generate formate via the one carbon metabolism pathway.

Highly proliferative cells rely on one carbon (1C) metabolism for production of formate required for synthesis of purines and thymidine for nucleic acid synthesis. This study was to determine if extracellular serine and/or glucose and fructose contribute the production of formate in ovine conceptuses. Suffolk ewes (n = 8) were synchronized to estrus, bred to fertile rams, and conceptuses were collected on Day 17 of gestation. Conceptuses were either snap frozen in liquid nitrogen (n = 3) or placed in culture in medium (n = 5) containing either: 1) 4 mM D-glucose + 2 mM [U-13C]serine; 2) 6 mM glycine + 4 mM D-glucose + 2 mM [U-13C]serine; 3) 4 mM D-fructose + 2 mM [U-13C]serine; 4) 6 mM glycine + 4 mM D-fructose + 2 mM [U-13C]serine; 5) 4 mM D-glucose + 4 mM D-fructose + 2 mM [U-13C]serine; or 6) 6 mM glycine + 4 mM D-glucose + 4 mM D-fructose + 2 mM [U-13C]serine. After 2 h incubation, conceptuses in their respective culture medium were homogenized and the supernatant analyzed for 12C- and 13C-formate by gas chromatography and amino acids by high performance liquid chromatography. Ovine conceptuses produced both 13C- and 12C-formate, indicating that the [U-13C]serine, glucose, and fructose were utilized to generate formate, respectively. Greater amounts of 12C-formate than 13C-formate were produced, indicating that the ovine conceptus utilized more glucose and fructose than serine to produce formate. This study is the first to demonstrate that both 1C metabolism and serinogenesis are active metabolic pathways in ovine conceptuses during the peri-implantation period of pregnancy, and that hexose sugars are the preferred substrate for generating formate required for nucleotide synthesis for proliferating trophectoderm cells.

Plasma Biomarkers and Fractional Exhaled Nitric Oxide in the Diagnosis of Eosinophilic Esophagitis.

OBJECTIVES: Eosinophilic esophagitis (EoE) is a chronic disease which requires endoscopy with biopsies for diagnosis and monitoring. We aimed to identify a panel of non-invasive markers that could help identify patients with active EoE. METHODS: In this prospective cohort study, we enrolled 128 children aged 5-18 years old, scheduled for endoscopy for suspected esophageal or peptic disease. On the day of the endoscopy, fractionated exhaled nitric oxide (FeNO) was measured; and blood was collected for peripheral absolute eosinophil count (AEC), plasma amino acids, and plasma polyamine analysis. Patients were grouped into controls (n = 91), EoE in remission (n = 16), or active EoE (n = 21), based on esophageal eosinophilia and history of EoE. RESULTS: AEC was not statistically significant different among the groups compared ( P = 0.056). Plasma amino acids: citrulline (CIT), ß-alanine (ß-ALA), and cysteine (CYS) were higher in active EoE compared to controls ( P < 0.05). The polyamine spermine was lower in active EoE versus controls ( P < 0.05). Receiver operator characteristic (ROC) curve to assess the predictive capability of a combined score made of FeNO, ß-ALA, CYS, and spermine had an area under curve (AUC) of 0.90 (95% CI: 0.80-0.96) in differentiating active EoE from controls and 0.87 (95% CI: 0.74-1.00) when differentiating active EoE from EoE in remission. CONCLUSION: A panel comprising FeNO, 2 plasma amino acids (ß-ALA, CYS) and the polyamine spermine can be used as a non-invasive tool to differentiate active EoE patients from controls.

Understanding placentation in ruminants: a review focusing on cows and sheep.

Mammals differ regarding their placentae, but in all species placental trophoblasts interact intimately with the uterine endometrium to mediate the transfer of nutrients from the mother to the embryo/fetus through the closely juxtaposed microcirculatory systems of the uterus and placenta. Placentation in ruminants is intermediate between the non-invasive type, as observed in the epitheliochorial placenta of pigs, and the invasive type, as observed in the haemochorial placentae of mice and humans. In ruminants, placental trophoblast cells invade uterine endometrial tissue, but invasion is believed to be limited to the endometrial luminal epithelium (LE). In the LE there are varying degrees of syncytialisation among species, with syncytialisation being more extensive in sheep than cows. The hallmarks of placentation in ruminants include: (1) an extended period in which conceptuses (embryos and associated placental membranes) elongate and must be supported by secretions (histotroph) from the uterus; (2) a cascade involving an array of adhesion molecules that includes integrin-mediated attachment of the conceptus trophoblast to the endometrial LE for implantation; (3) syncytialisation of the developing early placenta, a process for which there is currently limited understanding; and (4) development of placentomes that define the cotyledonary placentae of cows and sheep, and provide haemotrophic support of fetal development.

Nutrition and Gut Health: Recent Advances and Implications for Development of Functional Foods.

The small intestine is a highly differentiated and complex organ with many nutritional, physiological, and immunological functions [...].

Elongating porcine conceptuses can utilize glutaminolysis as an anaplerotic pathway to maintain the TCA cycle†.

During the peri-implantation period of pregnancy, the trophectoderm of pig conceptuses utilize glucose via multiple biosynthetic pathways to support elongation and implantation, resulting in limited availability of pyruvate for metabolism via the TCA cycle. Therefore, we hypothesized that porcine trophectoderm cells replenish tricarboxylic acid (TCA) cycle intermediates via a process known as anaplerosis and that trophectoderm cells convert glutamine to α-ketoglutarate, a TCA cycle intermediate, through glutaminolysis. Results demonstrate: (1) that expression of glutaminase (GLS) increases in trophectoderm and glutamine synthetase (GLUL) increases in extra-embryonic endoderm of conceptuses, suggesting that extra-embryonic endoderm synthesizes glutamine, and trophectoderm converts glutamine into glutamate; and (2) that expression of glutamate dehydrogenase 1 (GLUD1) decreases and expression of aminotransferases including PSAT1 increase in trophectoderm, suggesting that glutaminolysis occurs in the trophectoderm through the GLS-aminotransferase pathway during the peri-implantation period. We then incubated porcine conceptuses with 13C-glutamine in the presence or absence of glucose in the culture media and then monitored the movement of glutamine-derived carbons through metabolic intermediates within glutaminolysis and the TCA cycle. The 13C-labeled carbons were accumulated in glutamate, α-ketoglutarate, succinate, malate, citrate, and aspartate in both the presence and absence of glucose in the media, and the accumulation of 13C-labeled carbons significantly increased in the absence of glucose in the media. Collectively, our results indicate that during the peri-implantation period of pregnancy, the proliferating and migrating trophectoderm cells of elongating porcine conceptuses utilize glutamine via glutaminolysis as an alternate carbon source to maintain TCA cycle flux.