Location:2010 Annual Report
1a. Objectives (from AD-416)
1. Establish benchmark transcriptome profiles induced by uterine stress during gestation that are predictive of altered physiological mechanisms in key organs critical to the piglet immune and metabolic stress responses. 2. Identify critical gene(s), gene products, and their mechanism of action in the stress response of piglets associated with morbidity, growth rate, and body composition. 2.A. Identify key secretory proteins that are regulated by stress in the preweaning pig. 2.B. Identify key secretory proteins produced by adipose tissue that are regulated by stress in the preweaning pig. 3. Develop comprehensive in vitro models to analyze the mechanistic role of select, important developmental or metabolic factors in mediating the organism’s response to stresses via functional genomic approaches. 3.A. Determine the physiological mechanisms of key stress-regulatory proteins that regulate nutrient partitioning in the liver and adipose tissue. 3.B. Characterize the role of trophectoderm-derived estrogen as a modulator of “programmed” set points that determines the development potential of the peri-implantation embryo. 4. Define the repertoire of biomarkers that may be predictive of neonatal growth potential by using models of induced metabolic stress.
1b. Approach (from AD-416)
High preweaning mortality or impaired stress-related growth of live-born piglets continue to be major problems that negatively impact commercial swine production. Piglets exhibiting decreased vitality are at greater risk of morbidity or death and a decreased growth rate extending the farrow-to-market time, which results in increased producer costs. Considering embryo development as a continuum, it is plausible that abnormal piglet development and loss through adulthood is a consequence of aberrant embryonic/uterine development. This research will identify physiological mechanisms modulating piglet stress responses to pinpoint targets for interventions to improve “at risk” piglet survival. The research addresses three elemental issues 1) elucidation of the relationship between developmental perturbations and etiology of abnormal postnatal stress responses, 2) paucity of predictive screening tools for “at risk” neonates, and 3) lack of interventions to ameliorate postnatal development of “at risk” piglets. The impact of the uterine milieu on alterations of key physiological mechanisms that modulate stress response in metabolic or immune organs will be evaluated by comparative transcriptomic analysis between induced intrauterine growth retardation (IUGR), i.e. runting, and control concepti. To identify postnatal stress responses that are disrupted and persist ex utero as a consequence of in utero growth retardation or parturition complications and detect compensatory mechanisms, the gene expression of key metabolic and immune tissues from growth retarded piglets, (induced IUGR or spontaneous IUGR) and piglets exhibiting decreased vitality will be assessed by in-depth proteomic or transcriptomic analyses throughout the preweaning period. Functional analyses utilizing in vitro model systems and technologies, such as RNAi, will evaluate the mechanistic role of specific stress-related factors/pathways that are identified in metabolically important tissues. The relevance of putative stress-related factors/pathways will be assessed in vivo, employing distinct models of induced metabolic stress. The knowledge acquired will enable 1) the discovery of new biomarkers indicative of metabolic or immune stress response, 2) the identification physiological mechanisms/factors that can be targeted to develop new improved interventions that decrease mortality and days to market of “at risk” piglets and 3) the establishment of public “systems biology” database for specific gestational and environmental stresses.
3. Progress Report
Surgery and collection of control and runt tissues from our surgically-induced runting animal model were completed for fetal time points. An extra fetal time point was added owing to a new international collaboration - a comparative study to determine similar/dissimilar mechanisms in distinct runting models. To initiate longitudinal genomic studies of reprogramming in metabolic organs, mRNA was isolated; microarray analyses are underway and will be completed by September. A study of the regulation of endothelial nitric oxide synthase (ENOS) was begun; we hypothesize ENOS may help control a compensatory nutritional mechanism in the runt. Glucose can modulate ENOS expression via the insulin (INS) pathway so the mRNA level of key factors in the INS pathway was determined in normal and runt day 37 and 50 placenta. Differences were not found. To begin examining the biological role of ENOS, we optimized protein detection methods. The ENOS protein was decreased in control placenta at gestational day 50 compared to day 37, like its mRNA; however unlike the mRNA, the protein level was the same in runt and control at day 50. Aside from blood vessels, ENOS protein was expressed in placental trophoblast cells in contact with the endometrium. The presence of ENOS in these cells suggests it may have a role in mechanisms other than blood vessel regulation. An in vitro model was developed to study cytokine regulation of protein metabolism via factors, like nuclear transcription factor nuclear factor kappa B (NFKB), in hepatocytes isolated from young growing piglets. To determine the activation of NFKB in cultured neonatal hepatocytes, distinct antibodies were used to detect and localize metabolized NFKB component proteins impacted by the stress–inducing cytokine, tumor necrosis factor alpha (TNF). Short-term responses to TNF were followed at the protein level while several select marker genes were evaluated at the mRNA level after a 24 hr exposure in the presence of the metabolic regulatory hormones, insulin, glucagon, growth hormone or triiodothyronine. Future studies will examine if the piglet’s growth rate can influence the magnitude and complexity of these inflammatory responses. Experiments were conducted to examine the response of neonatal pig adipose tissue to specific cytokines, TNF and interleukin 6 (IL6), that can respond to stress in adult animals. Subcutaneous adipose tissue was obtained from 21 day old piglets and incubated with various concentrations of TNF or IL6. After a 24 hour of incubation, the mRNA expression of select genes was determined. Of the two cytokines, TNF was the more potent inducer of gene expression in adipose tissue. Both cytokines altered lipoprotein lipase (LPL) expression, a key gene for fatty acid uptake by adipose tissue. However, TNF inhibited while IL6 stimulated LPL expression. The mRNA expression of adipose-derived cytokines, monocyte chemotactic protein and interleukin 15, which mediate the insulin response and ensuing fat accumulation in adipose tissue, were also induced by TNF. Future studies will explore how the neonatal adipose tissue specifically responds to the stress-elicited, adipose-derived cytokines.
1. Identification of a biological marker associated with impaired growth rate in piglets. Biological markers, predictive of growth rate, are needed to identify poor postnatal performance prior to weaning in normal birth weight pigs. This would allow the producer to administer earlier treatments that improve piglet growth, health, and well-being; this is important to maintain regular and consistent swine production that preserves the Global Food Security priority of the USDA. Researchers at ARS, Beltsville, MD collected blood samples from normal birth weight baby pigs within a day after birth and the growth rate of each piglet was followed until weaning. A relationship between the quantity of a predominant blood protein (alpha-1 acid glycoprotein) and the rate of postnatal growth was found; piglets with a lower concentration of the blood protein after birth had a faster growth rate. This research has pinpointed a protein that can be investigated further for its potential use as a readily available and simple diagnostic tool to identify slow growing normal weight vulnerable pigs immediately following birth.
2. Factors of the immune system regulate gene expression in the fat tissue of the neonatal piglet. Environmental (e.g. temperature) and biological (e.g. disease) stresses that impact the neonatal pig result in a mortality rate that is now approaching 20%. Distinct hormone-like factors called cytokines are produced by the immune system and can be released in response to stress; cytokines are good indicators of stress in swine. Fat tissue also produces cytokines (adipokines), which are thought to play major roles in obesity and associated diseases (metabolic syndrome and insulin resistant diabetes) in human; thus, an understanding of the regulation of adipokines in swine could also be important for their health and well-being. This is relevant to the USDA priority of Childhood Nutrition and Health. Researchers at ARS Beltsville, MD were the first to investigate the role of two stress-induced cytokines, tumor necrosis factor alpha (TNF) and interleukin 6 (IL6), in regulating adipokines and genes that control fat metabolism between birth and weaning, a time when fat tissue is one of the fastest growing tissues in the piglet. Both cytokines altered the levels of distinct adopkines; however IL6 enhanced genes involved in fat metabolism and TNF inhibited genes involved in fat metabolism. These data indicate that cytokines released during stress could alter fat metabolism in the piglet during a critical survival period.
3. Piglet growth rate alters genes in tissues important for normal metabolism. Prior to weaning, piglets of normal birth weight may exhibit different growth rates that can make some pigs underperform and become economically inefficient to the producer. Measureable biological markers have not been identified that predict which piglet will become an underperforming hog. To identify genes that may serve as early markers for divergence in growth rate, researchers at ARS, Beltsville, MD delineated the gene level for factors regulating the immune system, hormones, or metabolism in fat, skeletal muscle, and liver tissue obtained from fast or slow growing piglets within litters prior to weaning. No common gene was found to differ across all three tissues between fast and slow growing piglets; however within each distinct tissue several genes were found whose level correlated with fast or slow growth. This study has identified potential changes in genes that function in metabolism, hormone or immune processes that can be investigated further to determine if they have a role piglet growth.
4. Neonatal piglet growth rate affects body composition. During the preweaning period, a significant number of piglets born at normal weight exhibit poor growth and other signs of a failure to thrive. However, whether slower growth rate is accompanied by changes in the content of total body fat, lean (non-fat and non-bone tissue), and water, that is, body composition, in normal birth weight piglets is not known. Researchers at ARS, Beltsville, MD utilized a technology, Quantitative Magnetic Resonance, that can measure fat, lean, and water content in live piglets, to evaluate changes in body composition of 235 piglets from birth to weaning (postnatal day one to 23). Though measurements revealed a wide range in body composition and growth rates, body composition was more closely related to body weight, the measure of growth rate, than to postnatal age or birth weight. This research demonstrated that the body composition of a slower growing normal birth weight piglet is different than an age-matched faster growing piglet. It also suggested that factors other than birth weight have a greater influence on piglet growth rate and body composition during the preweaning period.
5. Significant Activities that Support Special Target Populations
Four month detail at the NIH Eunice Kennedy Shriver National Institute of Child Health and Human Development in the Center for Population Research, Reproductive Sciences Branch to write a Program Announcement (PA) for a joint NIH/USDA-NIFA funding initiative that was established to promote research of high priority to both biomedicine and agriculture that maintains relevance to the mission of both agencies. The funding opportunity is targeted towards under-funded agricultural and veterinarian researchers at land-grant based institutions to help support agriculture-focused research and stimulate the interest of new researchers in agricultural research. The saliency of the PA is that it promotes the use of agricultural species as refined animal models for specific developmental and pathological studies, two of which are relevant to this project, metabolism in adipose tissue as related to obesity and nutrition and, developmental origins of adult disease germane to fetal reprogramming of metabolic organs in intrauterine growth retarded offspring. The PA was completed, revised and approved by the USDA-NIFA, and is currently proceeding through formal NIH approval. This initiative is a significant step towards the promotion of interagency cooperation, recognition of the importance of maintaining/supporting agriculture-based research, and the acknowledgement of the interdependency of human and agricultural animal health.
Ramsay, T.G., Richards, M.P., Li, C.J., Caperna, T.J. 2010. IGF-I mediated inhibition of leptin receptor expression in porcine hepatocytes. Comparative Biochemsitry and Physiology. 155(1):43-48.