Research Project: Molecular, Cellular, and Regulatory Aspects of Nutrition During Development

Location: Children's Nutrition Research Center

2019 Annual Report

Objectives
The goal of this research is to identify strategies to optimize the nutrition and health of infants and their development. CNRC researchers will: 1) investigate the impact of perinatal nutrition in the model system of premature piglets on the prevention of parenteral nutrition-associated liver disease (PNALD) and necrotizing enterocolitis (NEC); 2) define the role of liver receptor homolog-1 (LRH-1) as a factor in hepatic lipotropic responses, including the influence of methionine/choline deficient diets (MCD); 3) characterize the effect of the loss of hepatic LRH-1 in the lipotropic response to methyl pool supplementation in the standard mouse model of diet induced obesity; 4) determine the influence of leucine supplementation in stimulating protein synthesis, enhancing lean growth, and reducing protein degradation in healthy neonatal piglets and during catabolic conditions such as sepsis; 5) determine whether the deficit in lean deposition incurred with continuous as compared to intermittent bolus feeding during the neonatal period can be prevented by leucine supplementation or recuperated by initiation of intermittent bolus feeding; 6) determine whether citrulline plasma concentration is an early indicator for gut immaturity and gut dysfunction in a piglet model of prematurity; 7) determine if arginine and citrulline supplementation are able to reduce the incidence of necrotizing enterocolitis; 8) identify the cellular signaling networks that modify leptin-signal transducer and activator of transcription 3 (STAT3) signaling and potentially contribute to leptin resistance; 9) determine, using genetically engineered mouse models, the role of a cellular leptin signaling modifier in high fat diet-induced leptin resistance and subsequent alterations in energy and glucose homeostasis, and adiposity; 10) study the mechanism of circadian dysfunction-induced leptin resistance and the role of leptin resistance in obesity development; 11) determine the contributions of alpha Beta and yoT cells to inflammation in skeletal muscle; 12) determine the mechanisms leading to early anti-inflammatory macrophage polarization in mesenteric adipose tissue and the peritoneal cavity of C57BL/6J mice; 13) define how tissue healing is dysregulated in Western-style diet-induced obesity; 14) determine if Matrix Metalloproteinase 12 influences the development of insulin resistance and tissue inflammation in the context of high fat, Western-type diet-induced obesity; 15) determine if Matrix Metalloproteinase 12 influences white adipose tissue extracellular matrix remodeling under conditions of Western-type diet feeding.

Approach
The research will be accomplished using a variety of models and scientific tools to simulate the human newborn and/or child. Researchers will perform tissue transcriptomic profiling to identify novel genes, gene networks and metabolic pathways that are differentially affected by two lipid emulsions. We will also quantify expression of targeted genes involved in hepatic bile acid metabolism. In mouse models, physiologic studies will be performed that will include analysis of serum and hepatic levels of trigylcerides, free fatty acids, total cholesterol and total phosphatidylcholine. Using porcine models, we will perform protein synthesis experiments in leucine-infused endotoxemic pigs. Additionally we will use porcine models to determine if arginine and citrulline supplementation reduces the incidence of necrotizing enterocolitis. CNRC researchers will also employ an ex vivo model to determine if STAT3 signaling acts as a signaling hub for distinct signaling pathways mediating cellular leptin resistance. Complicated studies will be performed to study the mechanism of reciprocal interactions between the central circadian clock and hypothalamic arcuate nucleus in maintaining homeostasis of leptin signaling. Researchers will also study the role of circadian dysfunction of sympathetic nervous system (SNS) signaling in the development of leptin resistance and diet-induced obesity. And finally scientists will use a murine model (that develops chronic inflammation similar to that observed in obese humans) of diet-induced obesity and will use short- and long-term feeding techniques for the localization and phenotypic characterization of lymphocytes in skeletal muscle, and techniques for depletion of lymphocyte subsets.

Progress Report
To review the progress made during the year, please refer to the following projects: 3092-51000-065-10S (Project #1), 3092-51000-065-20S (Project #2), and 3092-51000-065-30S (Project #3).

Accomplishments
1. Intermittent bolus feeding promotes greater growth in newborns. More than 50,000 preterm infants born each year in the U.S. who cannot eat normally may be fed using a tube inserted into the stomach. These infants may be fed continuously or receive intermittent bolus feedings that are similar to that of a typical breakfast, lunch, dinner type feeding. Researchers in Houston, Texas, aimed to determine which feeding pattern, continuous or intermittent, promotes better growth and body composition in neonatal piglets as model for that of a human infant. These studies showed that intermittent bolus feeding promotes greater body weight gain than continuous feeding. Linear growth, lean mass, and vital organ growth were greater in pigs that were intermittent bolus fed compared to those continuously fed. These studies provide direct evidence that the intermittent bolus feeding is more advantageous than continuous feeding in improving lean body mass and growth of vital organs in neonates. These findings can be used by neonatologists and pediatric nurses to justify new recommendations that encourage intermittent bolus feeding of premature infants to promote better growth and earlier discharge from the hospital.

2. The citrulline generation test as a measurement of gut function. Many hospitalized premature infants have immature or diseased intestines, however, there is no effective and simple test to assess the health of the intestine in these babies. The small intestine produces the amino acid citrulline and measuring the citrulline concentration in blood samples has been proposed as a marker of intestinal health. Scientists in Houston, Texas, used neonatal pigs, as a model of human infants, to show that infusion of a large single injection of a tiny protein containing the amino acid, alanyl-glutamine, leads to increased blood citrulline and may reflect improved intestinal health. The amino acid glutamine is key for this test because it is used by the intestine to synthesize citrulline. This work shows proof of concept that the Citrulline Generation Test can become a useful tool to evaluate intestinal health in hospitalized premature infants, especially those that develop intestinal disease such as necrotizing enterocolitis.

3. Discovering new phospholipid hormones. Annually, more than 150,000 people under the age of 20 are diagnosed with type 2 diabetes. Researchers in Houston, Texas, have previously found that a unique type of fat, phosphatidylcholine (PC), can activate a cell signaling protein called LRH-1. LRH-1 is present in the liver and when activated can promote better control of blood sugar in diabetic children. Our lab used modern tools to study mice with defects in specific genes that make PC in the liver and discovered that blocking one of the two major genes involved in PC synthesis decreased LRH-1 activity. This finding gives researchers important insights into the identity of the natural PC form made in the liver that activate LRH-1. This research is important because these natural PC forms may be used as supplements to activate LRH-1 for the treatment of metabolic diseases linked to obesity, particularly type 2 diabetes.

4. The role of sympathetic circadian dysfunction in obesity and cancer. Obesity is closely associated with hypertension in humans, which, in addition to uncontrolled fat gain, is also characterized by over-activation of the sympathetic nervous system and increases the risk of chronic kidney and cardiovascular diseases. The sympathetic nervous system is important in the body's fight or flight response and helps to maintain homeostasis in the body. Researchers in Houston, Texas, found that a disruption in the circadian rhythm (our internal biological awake/sleep clock) elevates sympathetic activity. Blocking circadian disruption induced sympathetic over-activation by treatment with a drug beta-blocker) can reduce diet-induced obesity and completely inhibit obesity-related liver cancer. Beta-blockers have been safely used in treating various hypertension and cardiovascular disorders in humans since 1960s. These findings suggest that beta-blockers also have important therapeutic potentials in the prevention and treatment of obesity and obesity-related liver cancer in humans.

5. Discovery of a molecular pathway in the brain that impacts obesity. The brain is a critical site for the control of body weight and an understanding of how the brain reacts to excess nutrition and mediates obesity remains incomplete. To gain a better understanding of the underlying neural mechanisms of obesity, researchers in Houston, Texas, have developed a new laboratory tool which will help identify a critical molecular pathway that potentially promotes obesity. Using cellular imaging techniques enabled the scientists to visualize gene expression of a molecule that promotes obesity and identify a specific neural pathway involved in the cellular process that may drive obesity. Neural levels of the gene were successfully altered by manipulating the activity of the signaling pathway. These findings will provide a clue regarding the mechanisms that determine body weight and may lead to a novel approach to control body weight by manipulating this particular pathway.

6. Reversibility of the effects of a high fat diet (HFD) on the cornea. When a high fat diet (HFD) is consumed, there are significant changes in the cornea of the eye that are of concern; such as an altered surface, tear production, and the size of specific glands in the eye lids. To determine if the diet effects on the cornea are reversible, researchers in Houston, Texas, conducted an experiment where mice were fed a HFD for 10 weeks and then switched to a normal diet for 5 weeks. The 5 weeks on the normal diet restored the normal nerve density in the cornea and the normal sensitivity to touch, but the increased inflammation was still present to a small extent. This indicated that the diet induced inflammation takes longer to recover than the nerve recovery. These results provide evidence that the changes in the nerves of the cornea induced by the HFD can be corrected by switching to a normal diet, and that the diet-induced inflammation may be difficult to correct by simply reverting to a normal diet.

Review Publications
Mohammad, M.A., Didelija, I.C., Stoll, B., Marini, J.C. 2018. Citrulline generation test: What does it measure? Journal of Parenteral and Enteral Nutrition. https://doi.org/10.1002/jpen.1462.
Plat, J., Baumgartner, S., Vanmierlo, T., Lutjohann, D., Calkins, K.L., Burrin, D.G., Guthrie, G., Thijs, C., Te Velde, A.A., Vreudenhil, A.C.E., Sverdlov, R., Garssen, J., Wouters, K., Trautwein, E.A., Wolfs, T.G., van Gorp, C., Mulder, M.T., Riksen, N.P., Groen, A.K., Mensink, R.P. 2019. Plant-based sterols and stanols in health & disease: Consequences of human development in a plant-based environment? Progress in Lipid Research. 74:87-102. https://doi.org/10.1016/j.plipres.2019.02.003.
Lin, S., Yang, X., Yuan, P., Yang, J., Wang, P., Zhong, H., Zhang, X., Che, L., Feng, B., Li, J., Zhuo, Y., Lin, Y., Xu, S., Wu, D.E., Burrin, D.G., Fang, Z. 2019. Undernutrition shapes the gut microbiota and bile acid profile in association with altered gut-liver FXR signaling in weaning pigs. Progress in Lipid Research. 67(13):3691-3701. https://doi.org/10.1016/j.plipres.2019.02.003.
Weischendorff, S., Kielsen, K., Nederby, M., Svendsen, L., Burrin, D.G., Heilmann, C., Ifversen, M., Sengelov, H., Molgaard, C., Muller, K. 2019. Reduced plasma amino acid levels during allogeneic haematopoietic stem cell transplantation are associated with systemic inflammation and treatment-related complications. Biology of Blood and Marrow Transplantation. https://doi.org/10.1016/j.bbmt.2019.03.018.
Lin, S., Stoll, B., Robinson, J., Pastor, J.J., Marini, J.C., Ipharraguerre, I.R., Hartmann, B., Holst, J.J., Cruz, S., Lau, P., Olutoye, O., Fang, Z., Burrin, D.G. 2019. Differential action of TGR5 agonists on GLP-2 secretion and promotion of intestinal adaptation in piglet short bowel model. American Journal of Physiology. https://doi.org/10.1152/ajpgi.00360.2018.
Welch-Jernigan, R.J., Abrahamse, E., Stoll, B., Smith, O., Wierenga, P.A., Van De Heijning, B., Renes, I., Burrin, D.G. 2018. Postprandial amino acid kinetics of milk protein mixtures are affected by composition, but not denaturation, in neonatal piglets. Current Developments in Nutrition. 3(4):1-11. https://doi.org/10.1093/cdn/nzy102.
Molina, T.L., Stoll, B., Mohammad, M., Mohila, C.A., Call, L., Cui, L., Guthrie, G., Kunichoff, D., Lin, S., Welch-Jernigan, R., Nielsen, J., Premkumar, M., Robinson, J., Smith, V., Teets, H., Obelitz-Ryom, K., Hagan, J., Cruz, S., Lau, P., Puyau, M., Fang, Z., Shypailo, R., Manjarin, R., Butte, N., Burrin, D.G., Olutoye, O., Thymann, T., Sangild, P. 2019. New generation lipid emulsions increase brain DHA and improve body composition, but not short-term neurodevelopment in parenterally-fed preterm piglets. Brain Behavior and Immunity. https://doi.org/10.1016/j.bbi.2019.04.031.
Wang, P., Zhong, H., Song, Y., Yuan, P., Li, Y., Lin, S., Zhang, X., Li, J., Che, L., Feng, B., Lin, Y., Xu, S., Zhuo, Y., Tian, G., Chen, D., We, D., Burrin, D.G., Fang, Z. 2019. Targeted metabolomics analysis of maternal-placental-fetal metabolism in pregnant swine reveals links in fetal bile acid homeostasis and sulfation capacity. American Journal of Physiology - Gastrointestinal and Liver Physiology. 317:G8-G16. https://doi.org/10.1152/ajpgi.00056.2019.
El-Kadi, S.W., Boutry, C., Suryawan, A., Gazzaneo, M.C., Orellana, R.A., Srivastava, N., Nguyen, H.V., Kimball, S.R., Fiorotto, M.L., Davis, T.A. 2018. Intermittent bolus feeding promotes greater lean growth than continuous feeding in a neonatal piglet model. American Journal of Clinical Nutrition. 108(4):830-841. https://doi.org/10.1093/ajcn/nqy133.
Wang, X., Yuan, Y., Didelija, I.C., Mohammad, M.A., Marini, J.C. 2018. Ex vivo enteroids recapitulate in vivo citrulline production in mice. Journal of Nutrition. 148(9):1415-1420. https://doi.org/10.1093/jn/nxy126.
Robinson, J.L., Smith, V.A., Stoll, B., Agarwal, U., Premkumar, M.H., Lau, P., Cruz, S.M., Manjarin, R., Olutoye, O., Burrin, D.G., Marini, J.C. 2018. Prematurity reduces citrulline-arginine-nitric oxide production and precedes the onset of necrotizing enterocolitis in piglets. American Journal of Physiology - Gastrointestinal and Liver Physiology. 5:9. https://doi.org/10.1152/ajpgi.00198.2018.
Cordonier, E.L., Liu, T., Saito, K., Chen, S.S., Xu, Y., Fukuda, M. 2019. Luciferase reporter mice for in vivo monitoring and ex vivo assessment of hypothalamic signaling of Socs3 expression. Journal of the Endocrine Society. 3(7):1246-1260. https://doi.org/10.1210/js.2019-00077.
Rudar, M., Fiorotto, M.L., Davis, T.A. 2018. Regulation of muscle growth in early postnatal life in a swine model. Annual Review of Animal Biosciences. 7:309-335. https://doi.org/10.1146/annurev-animal-020518-115130.
Fekry, B., Ribas-Latre, A., Baumgartner, C., Deans, J.R., Kwok, C., Patel, P., Fu, L., Berdeaux, R., Sun, K., Kolonin, M.G., Wang, S.H., Yoo, S., Sladek, F.M., Eckel-Mahan, K. 2018. Incompatibility of the circadian protein BMAL1 and HNF 4a in hepatocellular carcinoma. Nature Communications. https://doi.org/10.1038/s41467-018-06648-6.
Kaneko, K., Fu, Y., Lin, H.Y., Cordonier, E.L., Mo, Q., Gao, Y., Yan, T., Naylor, J., Howard, V., Saito, K., Xu, P., Chen, S.S., Xu, Y., Williams, K.W., Ravn, P., Fukuda, M. 2019. Gut-derived GIP activates central Rap1 to impair neural leptin sensitivity during overnutrition. Journal of Clinical Investigation. https://doi.org/10.1172/JCI126107.
Donelson, J., Wang, Q., Monroe, T.O., Jiang, X., Zhou, J., Yu, H., Mo, Q., Sun, Q., Marini, J.C., Wang, X., Nakata, P.A., Hirschi, K.D., Wang, J., Rodney, G.G., Wehrens, X.T., Cheng, N. 2019. Cardiac-specific ablation of glutaredoxin 3 leads to cardiac hypertrophy and heart failure. Physiological Reports. https://doi.org/10.14814/phy2.14071.