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.

Dietary glycine supplementation enhances syntheses of creatine and glutathione by tissues of hybrid striped bass (Morone saxatilis ♀ × Morone chrysops ♂) fed soybean meal-based diets.

BACKGROUND: We recently reported that supplementing glycine to soybean meal-based diets is necessary for the optimum growth of 5- to 40-g (Phase-I) and 110- to 240-g (Phase-II) hybrid striped bass (HSB), as well as their intestinal health. Although glycine serves as an essential substrate for syntheses of creatine and glutathione (GSH) in mammals (e.g., pigs), little is known about these metabolic pathways or their nutritional regulation in fish. This study tested the hypothesis that glycine supplementation enhances the activities of creatine- and GSH-forming enzymes as well as creatine and GSH availabilities in tissues of hybrid striped bass (HSB; Morone saxatilis♀ × Morone chrysops♂). METHODS: Phase-I and Phase-II HSB were fed a soybean meal-based diet supplemented with 0%, 1%, or 2% glycine for 8 weeks. At the end of the 56-d feeding, tissues (liver, intestine, skeletal muscle, kidneys, and pancreas) were collected for biochemical analyses. RESULTS: In contrast to terrestrial mammals and birds, creatine synthesis occurred primarily in skeletal muscle from all HSB. The liver was most active in GSH synthesis among the HSB tissues studied. In Phase-I HSB, supplementation with 1% or 2% glycine increased (P < 0.05) concentrations of intramuscular creatine (15%-19%) and hepatic GSH (8%-11%), while reducing (P < 0.05) hepatic GSH sulfide (GSSG)/GSH ratios by 14%-15%, compared with the 0-glycine group; there were no differences (P > 0.05) in these variables between the 1% and 2% glycine groups. In Phase-II HSB, supplementation with 1% and 2% glycine increased (P < 0.05) concentrations of creatine and GSH in the muscle (15%-27%) and liver (11%-20%) in a dose-dependent manner, with reduced ratios of hepatic GSSG/GSH in the 1% or 2% glycine group. In all HSB, supplementation with 1% and 2% glycine dose-dependently increased (P < 0.05) activities of intramuscular arginine:glycine amidinotransferase (22%-41%) and hepatic γ-glutamylcysteine synthetase (17%-37%), with elevated activities of intramuscular guanidinoacetate methyltransferase and hepatic GSH synthetase and GSH reductase in the 1% or 2% glycine group. Glycine supplementation also increased (P < 0.05) concentrations of creatine and activities of its synthetic enzymes in tail kidneys and pancreas, and concentrations of GSH and activities of its synthetic enzymes in the proximal intestine. CONCLUSIONS: Skeletal muscle and liver are the major organs for creatine and GSH syntheses in HSB, respectively. Dietary glycine intake regulates creatine and GSH syntheses by both Phase-I and Phase-II HSB in a tissue-specific manner. Based on the metabolic data, glycine is a conditionally essential amino acid for the growing fish.

Dietary glycine supplementation activates mTOR signaling pathway in tissues of pigs with intrauterine growth restriction.

The mechanistic target of rapamycin (mTOR) cell signaling pathway serves as the central mechanism for the regulation of tissue protein synthesis and growth. We recently reported that supplementing 1% glycine to corn- and soybean meal-based diets enhanced growth performance between weaning and market weights in pigs with intrauterine growth restriction (IUGR). Results of recent studies have revealed an important role for glycine in activating mTOR and protein synthesis in C2C12 muscle cells. Therefore, the present study tested the hypothesis that dietary glycine supplementation enhanced the mTOR cell signaling pathway in skeletal muscle and other tissues of IUGR pigs. At weaning (21 d of age), IUGR pigs and litter mates with normal birth weights (NBW) were assigned randomly to one of two groups: supplementation with either 1% glycine or 1.19% L-alanine (isonitrogenous control) to a corn- and soybean meal-based diet. Tissues were obtained from the pigs within 1 wk after the feeding trial ended at 188 d of age to determine the abundances of total and phosphorylated forms of mTOR and its two major downstream proteins: eukaryotic initiation factor 4E-binding protein-1 (4EBP1) and ribosomal protein S6 kinase-1 (p70S6K). Results showed that IUGR decreased (P < 0.05) the abundances of both total and phosphorylated mTOR, 4EBP1, and p70S6K in the gastrocnemius muscle and jejunum. In the longissimus lumborum muscle of IUGR pigs, the abundances of total mTOR did not differ (P > 0.05) but those for phosphorylated mTOR and both total and phosphorylated 4EBP1 and p70S6K were down-regulated (P < 0.05), when compared to NBW pigs. These adverse effects of IUGR in the gastrocnemius muscle, longissimus lumborum muscle, and jejunum were prevented (P < 0.05) by dietary glycine supplementation. Interestingly, the abundances of total or phosphorylated mTOR, 4EBP1, and p70S6K in liver were not affected (P > 0.05) by IUGR or glycine supplementation. Collectively, our findings indicate that IUGR impaired the mTOR cell signaling pathway in tissues of pigs and that adequate glycine intake was crucial for maintaining active mTOR-dependent protein synthesis for the growth and development of skeletal muscle.

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.

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.

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.

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.

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.

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.

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.

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.

Dietary glycine supplementation enhances glutathione availability in tissues of pigs with intrauterine growth restriction.

This study tested the hypothesis that dietary supplementation with glycine enhances the synthesis and concentrations of glutathione (GSH, a major antioxidant) in tissues of pigs with intrauterine growth restriction (IUGR). At weaning (21 d of age), IUGR pigs and litter mates with normal birth weights (NBW) were assigned randomly to one of two groups, representing supplementation with 1% glycine or 1.19% l-alanine (isonitrogenous control) to a corn- and soybean meal-based diet. Blood and other tissues were obtained from the pigs within 1 wk after the feeding trial ended at 188 d of age to determine GSH, oxidized GSH (GSSG), and activities of GSH-metabolic enzymes. Results indicated that concentrations of GSH + GSSG or GSH in plasma, liver, and jejunum (P < 0.001) and concentrations of GSH in longissimus lumborum and gastrocnemius muscles (P < 0.05) were lower in IUGR pigs than in NBW pigs. In contrast, IUGR increased GSSG/GSH ratios (an indicator of oxidative stress) in plasma (P < 0.001), jejunum (P < 0.001), both muscles (P < 0.05), and pancreas (P = 0.001), while decreasing activities of γ-glutamylcysteine synthetase and GSH synthetase in liver (P < 0.001) and jejunum (P < 0.01); and GSH reductase in jejunum (P < 0.01), longissimus lumborum muscle (P < 0.01), gastrocnemius muscle (P < 0.05), and pancreas (P < 0.01). In addition, IUGR pigs had greater (P < 0.001) concentrations of thiobarbituric acid reactive substances (TBARS; an indicator of lipid peroxidation) in plasma, jejunum, muscles, and pancreas than NBW pigs. Compared with isonitrogenous controls, dietary glycine supplementation increased concentrations of GSH plus GSSG and GSH in plasma (P < 0.01), liver (P < 0.001), jejunum (P < 0.001), longissimus lumborum muscle (P = 0.001), and gastrocnemius muscle (P < 0.05); activities of GSH-synthetic enzymes in liver (P < 0.01) and jejunum (P < 0.05), while reducing GSSG/GSH ratios in plasma (P < 0.001), jejunum (P < 0.001), longissimus lumborum muscle (P < 0.001), gastrocnemius muscle (P = 0.01), pancreas (P < 0.05), and kidneys (P < 0.01). Concentrations of GSH plus GSSG, GSH, and GSSG/GSH ratios in kidneys were not affected (P > 0.05) by IUGR. Furthermore, glycine supplementation reduced (P < 0.001) TBARS concentrations in plasma, jejunum, muscles, and pancreas. Collectively, IUGR reduced GSH availability and induced oxidative stress in pig tissues, and these abnormalities were prevented by dietary glycine supplementation in a tissue-specific manner.

Pigs have the highest rate of intrauterine growth restriction (IUGR) among livestock species. These pigs, which have low birth weights (<1.1 kg) and account for ~15% to 20% of newborn pigs, are often culled after birth because they have lower growth performance and feed efficiency due to multiple factors (including oxidative stress in tissues), when compared with litter mates with normal birth weights (NBW). Much evidence shows that glutathione, which is a tripeptide synthesized from glutamate, glycine, and cysteine via enzymes (biological catalysts, γ-glutamylcysteine synthetase, and glutathione synthetase), is a major low-molecular-weight antioxidant in animal cells. Based on the findings of our recent study that dietary glycine supplementation enhanced the growth performance of IUGR pigs from weaning to market weight, the current study tested the hypothesis that this nutritional strategy increased the synthesis and availability of glutathione in their tissues. Our results indicated that the key organs of the digestive system (the small intestine, liver, and pancreas) as well as both longissimus lumborum and gastrocnemius muscles of IUGR pigs had lower concentrations of glutathione as compared with NBW pigs, due to reductions in both the activities of glutathione-synthetic enzymes and the availability of glycine. Dietary supplementation with 1% glycine prevented these metabolic deficiencies in tissues of IUGR pigs. Our findings support the notion that IUGR pigs fed conventional corn- and soybean meal-based diets do not synthesize adequate glutathione and that dietary glycine supplementation plays an important role in enhancing the availability of glutathione and mitigating oxidative stress to improve health and growth in these compromised animals.

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.

Dietary glycine supplementation improves the growth performance of 110- to 240-g (phase II) hybrid striped bass (Morone saxatilis â™€× Morone chrysops ♂) fed soybean meal-based diets.

We recently reported that supplementing glycine to soybean meal (SBM)-based diets is necessary for optimum growth of 5- to 40-g (phase I) hybrid striped bass (HSB). The present study tested the hypothesis that supplementing glycine to SBM-based diets may enhance the growth of 110- to 240-g (phase II) HSB. HSB (the initial body weight of approximately 110 g) were fed an SBM (58%)-based diet supplemented with 0%, 1%, or 2% of glycine, with l-alanine serving as the isonitrogenous control. There were four tanks per dietary group, with four fish per tank. The fish were fed their respective diets to apparent satiation twice daily. The feed intake and body weight of fish were recorded daily and every 2 wk, respectively. At the end of the 56-d feeding trial, plasma and tissue samples were collected to determine amino acid concentrations and histological alterations, and tissues were used to measure the oxidation of l-glutamate, l-glutamine, l-aspartate, and glycine. Results showed that dietary supplementation with 1% and 2% glycine dose-dependently increased (P < 0.05) the concentration of glycine in the plasma of HSB by 48% and 99%, respectively. Compared with the 0%-glycine group, dietary supplementation with 1% glycine did not affect (P > 0.05) the feed intake of HSB but increased (P < 0.05) their final body weight, weight gain, and gain:feed ratio during the whole period by 13%, 29%, and 21%, respectively. Compared with the 1% glycine group, dietary supplementation with 2% glycine increased (P < 0.05) the feed intake, final body weight, and weight gain of HSB by 13%, 7%, and 14%, respectively. Compared with the 0%-glycine group, fish fed with the 1%-glycine and 2%-glycine diets had a greater (P < 0.05) villus height in the proximal intestine, when compared with the 0%-glycine group. Collectively, these results indicated that SBM-based diets did not provide sufficient glycine for phase II HSB (110 to 240 g) and that dietary glycine supplementation is essential for their optimum growth and intestinal structure.

Glycine is the simplest but the most abundant amino acid in the bodies of animals including fish and pigs. The content of glycine in plant-sourced feedstuffs (e.g., soybean meal) is generally low. Glycine can be synthesized de novo in all animals and, therefore, has traditionally been classified as a nutritionally nonessential amino acid for fish and mammals. However, a capacity for the synthesis of glycine does not necessarily mean its adequate formation by animals. Growing evidence shows that either neonatal pigs fed milk protein-based diets or postweaning pigs regardless of their birth weights do not synthesize sufficient glycine, and must ingest supplemental glycine (e.g., 1% in diets) for optimum growth performance. Similar results have been reported for 5- to 40-g (phase I) juvenile hybrid striped bass (HSB) fed and largemouth bass fed soybean meal-based diets. The present study tested the hypothesis that supplementing glycine to soybean meal-based diets may enhance the growth of 110- to 240-g (phase II) HSB. Results of the current investigation indicate that glycine is also inadequate for normal intestinal structure or maximum growth in phase II HSB fed soybean meal-based diets. Supplementing 1% or 2% glycine to these diets increased protein accretion, weight gain, and feed efficiency in HSB while improving their intestinal structure. These findings indicate an important role for a sufficient provision of dietary glycine in the optimal nutrition, health, and growth of finishing HSB, and have broad implications for developing low-fishmeal diets to enhance fish production and sustain animal agriculture (including aquaculture).

Dietary glycine supplementation enhances the growth performance of hybrid striped bass (Morone saxatilis â™€× Morone chrysops ♂) fed soybean meal-based diets.

This study was conducted to test the hypothesis that supplementing 1% and 2% glycine to soybean meal (SBM)-based diets can improve the growth performance of juvenile hybrid striped bass (HSB). The basal diets contained 15% fishmeal and 58% SBM (DM basis). Alanine was used as the isonitrogenous control in different diets. All diets contained 44% crude protein and 10% lipids (DM basis). There were four tanks (15 fish per tank) per dietary group, with the mean of the initial body weight (BW) of fish being 5.3 g. Fish were fed to apparent satiation twice daily, and their BW was recorded every 2 wk. The trial lasted for 8 wk. Results indicated that the BW, weight gain, protein efficiency ratio, and retention of dietary lipids in fish were enhanced (P < 0.05) by dietary supplementation with 1% or 2% glycine. In addition, dietary supplementation with glycine did not affect (P > 0.05) the feed intake of fish but increased (P < 0.05) the retention of dietary nitrogen, most amino acids, and phosphorus in the body, compared to the 0% glycine group. Dietary supplementation with 1% and 2% glycine dose-dependently augmented (P < 0.05) the villus height of the proximal intestine and reduced the submucosal thickness of the gut, while preventing submucosal and lamina propria hemorrhages. Compared with the 0% glycine group, dietary supplementation with 1% or 2% glycine decreased (P < 0.05) the proportion of skeletal-muscle fibers with diameters of 40 to 60 µm but increased (P < 0.05) the proportion of skeletal-muscle fibers with diameters of 80 to 100 µm and > 100 µm. Collectively, these findings indicate that glycine in SBM-based diets is inadequate for maximum growth of juvenile HSB and that dietary supplementation with 1% or 2% glycine is required to improve their weight gain and feed efficiency. Glycine is a conditionally essential amino acid for this fish.

Animal agriculture (including aquaculture) provides high-quality protein for improving human nutrition and health. The United States is the top producer of hybrid striped bass (HSB) in the world as both food and sport fish. Fishmeal has traditionally been used as the major protein feedstuff in HSB diets, but feeding fish with fishmeal is not sustainable in the industry. Over the past four decades, there have been extensive studies to replace fishmeal with plant-sourced feedstuffs (mainly soybean meal) in aquafeeds at variable success. It has now been recognized that the content of glycine (the most abundant amino acid in the animal body) in soybean meal is only about half of that in fishmeal. Results of this study indicate that glycine is inadequate for normal intestinal structure or maximum growth in HSB fed soybean meal-based diets. Supplementing 1% or 2% glycine to these diets increased protein accretion, skeletal-muscle hypertrophy, and weight gain in HSB, while improving their intestinal structure. These findings indicate an important role for a sufficient provision of dietary glycine in the optimal nutrition, health, and growth of HSB, and have broad implications for developing low-fishmeal diets to enhance fish production and sustain animal agriculture.

Dietary supplementation with L-leucine reduces nitric oxide synthesis by endothelial cells of rats.

This study tested the hypothesis that elevated L-leucine concentrations in plasma reduce nitric oxide (NO) synthesis by endothelial cells (ECs) and affect adiposity in obese rats. Beginning at four weeks of age, male Sprague-Dawley rats were fed a casein-based low-fat (LF) or high-fat (HF) diet for 15 weeks. Thereafter, rats in the LF and HF groups were assigned randomly into one of two subgroups (n = 8/subgroup) and received drinking water containing either 1.02% L-alanine (isonitrogenous control) or 1.5% L-leucine for 12 weeks. The energy expenditure of the rats was determined at weeks 0, 6, and 11 of the supplementation period. At the end of the study, an oral glucose tolerance test was performed on all the rats immediately before being euthanized for the collection of tissues. HF feeding reduced (P < 0.001) NO synthesis in ECs by 21% and whole-body insulin sensitivity by 19% but increased (P < 0.001) glutamine:fructose-6-phosphate transaminase (GFAT) activity in ECs by 42%. Oral administration of L-leucine decreased (P < 0.05) NO synthesis in ECs by 14%, increased (P < 0.05) GFAT activity in ECs by 35%, and reduced (P < 0.05) whole-body insulin sensitivity by 14% in rats fed the LF diet but had no effect (P > 0.05) on these variables in rats fed the HF diet. L-Leucine supplementation did not affect (P > 0.05) weight gain, tissue masses (including white adipose tissue, brown adipose tissue, and skeletal muscle), or antioxidative capacity (indicated by ratios of glutathione/glutathione disulfide) in LF- or HF-fed rats and did not worsen (P > 0.05) adiposity, whole-body insulin sensitivity, or metabolic profiles in the plasma of obese rats. These results indicate that high concentrations of L-leucine promote glucosamine synthesis and impair NO production by ECs, possibly contributing to an increased risk of cardiovascular disease in diet-induced obese rats.

Synthesis of glycine from 4-hydroxyproline in tissues of neonatal pigs with intrauterine growth restriction.

This study tested the hypothesis that the synthesis of glycine from 4-hydroxyproline (an abundant amino acid in milk and neonatal blood) was impaired in tissues of piglets with intrauterine growth restriction (IUGR), thereby contributing to a severe glycine deficiency in these compromised neonates. At 0, 7, 14, and 21 days of age, IUGR piglets were euthanized, and tissues (liver, small intestine, kidney, pancreas, stomach, skeletal muscle, and heart) were obtained for metabolic studies, as well as the determination of enzymatic activities, cell-specific localization, and expression of mRNAs for glycine-synthetic enzymes. The results indicated relatively low enzymatic activities for 4-hydroxyproline oxidase (OH-POX), proline oxidase, serine hydroxymethyltransferase, threonine dehydrogenase (TDH), alanine: glyoxylate transaminase, and 4-hydroxy-2-oxoglutarate aldolase in the kidneys and liver from 0- to 21-day-old IUGR pigs, in the pancreas of 7- to 21-day-old IUGR pigs, and in the small intestine and skeletal muscle (except TDH) of 21-day-old IUGR pigs. Accordingly, the rates of conversion of 4-hydroxyproline into glycine were relatively low in tissues of IUGR piglets. The expression of mRNAs for glycine-synthetic enzymes followed the patterns of enzymatic activities and was also low. Immunohistochemical analyses revealed the relatively low abundance of OH-POX protein in the liver, kidney, and small intestine of IUGR piglets, and the lack of OH-POX zonation in their livers. These novel results provide a metabolic basis to explain why the endogenous synthesis of glycine is insufficient for optimum growth of IUGR piglets and have important implications for improving the nutrition and health of other mammalian neonates including humans with IUGR.