Source: VIRGINIA POLYTECHNIC INSTITUTE submitted to NRP
MECHANISMS OF NEUROPEPTIDE Y`S EFFECTS ON ADIPOSE TISSUE DEPOSITION IN CHICKENS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
Reporting Frequency
Annual
Accession No.
1005751
Grant No.
2015-67015-23359
Cumulative Award Amt.
$450,000.00
Proposal No.
2014-04996
Multistate No.
(N/A)
Project Start Date
May 1, 2015
Project End Date
Apr 30, 2019
Grant Year
2015
Program Code
[A1231]- Animal Health and Production and Animal Products: Improved Nutritional Performance, Growth, and Lactation of Animals
Recipient Organization
VIRGINIA POLYTECHNIC INSTITUTE
(N/A)
BLACKSBURG,VA 24061
Performing Department
Animal and Poultry Sciences
Non Technical Summary
The broiler chicken, which has been genetically selected for efficient feed utilization, is an economical and nutritious source of protein. A greater understanding of how fat (adipose) develops will facilitate strategies to affect the proportion of lean mass and fat and thus reduce the incidence of metabolic disorders in breeders. The Virginia Lines of chickens are the result of more than 55 generations of selection for low (LWS) or high (HWS) body weight at 56 days of age with a current 10-fold difference in body weight at selection age. The HWS chickens are compulsive feeders that accumulate excess fat, whereas the LWS chickens are relatively lean. Thus, they represent a model that can be used to study physiology and mechanisms that regulate fat deposition. Our preliminary studies showed that neuropeptide Y (NPY), a potent stimulator of hunger, also stimulates fat deposition, with differences in responsiveness between the LWS and HWS lines. We hypothesize that NPY signaling differs in the adipose tissue of LWS and HWS. We will determine if LWS and HWS adipose tissue respond differently to NPY by injecting them with NPY and collecting adipose tissue for evaluation of adipogenesis (fat cell formation), triacylglycerol synthesis (fat synthesis), and lipolysis (fat breakdown), using biochemical and gene expression assays. Cells will be isolated from adipose and treated with NPY to determine effects on proliferation and differentiation into adipocytes. Receptor function will be blocked in order to identify the pathway through which NPY mediates its function in fat, and NPY mRNA will be suppressed in order to evaluate the role of adipose tissue-derived NPY in fat cell formation.This research is expected to provide insight into molecular mechanisms that govern adipose tissue deposition in chickens. This information can be used to direct selective fat deposition in chickens such that there is more lean muscle mass and relatively less waste in the form of carcass fat, and to also reduce excess fat accumulation in broiler breeders that develop metabolic disorders as a consequence of obesity.
Animal Health Component
0%
Research Effort Categories
Basic
100%
Applied
0%
Developmental
0%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
30532201020100%
Knowledge Area
305 - Animal Physiological Processes;

Subject Of Investigation
3220 - Meat-type chicken, live animal;

Field Of Science
1020 - Physiology;
Goals / Objectives
Our goal is to describe adipose tissue deposition in the broiler, with a focus on factors that influence both feed intake and adiposity. Understanding how long-term selection for low or high body weight affected neuropeptide systems involved with body fat deposition, with a focus on neuropeptide Y (NPY), provides a model to understand fat development in the broiler. Because preliminary data show an effect of NPY on fat development in chickens, with a difference in NPY responsiveness between low-weight (LWS) and high-weight (HWS)-selected chickens, there is strong rationale to elucidate mechanisms underlying NPY's effects on adipose tissue development in chickens. The objectives of this three year project are as follows:Determine the effect of intraperitoneal injection of NPY on LWS and HWS adipose tissue function, and elucidate underlying mechanisms.Determine the effects of NPY treatment on proliferation and differentiation in adipose tissue cells from LWS and HWS chicks.Determine if adipose-derived NPY plays a role in chick adipose tissue cell proliferation and differentiation, using a gene knockdown approach.Elucidate the signaling pathway(s) responsible for NPY's effects on chick fat cells using receptor sub-type-specific and secondary messenger antagonists.
Project Methods
Pedigreed chicks from lines selected for low or high body weight will be used for the studies. Chicks will be injected intraperitoneally with neuropeptide (NPY), and effects on adipose function measured. Cells will be isolated from the adipose of both lines and effects of NPY on proliferation and differentiation into adipocytes determined. The function of NPY and its receptors will be blocked in the adipose cells. These experiments are novel because the role of NPY in chicken adipose physiology is unknown.Efforts include incorporating research into teaching and disseminating findings via presentations at scientific conferences (Poultry Science) and refereed publications.Key milestones and indicators for success include annual publications in refereed journals and presentations at conferences. Benchmarks also include completing experiments according to the timeline provided in the grant proposal.

Progress 05/01/15 to 04/30/19

Outputs
Target Audience:The target audience of the research is the scientific community, especially industry, academic, and government researchers that are interested in improving nutrient utilization efficiency in poultry. The research is particularly relevant to the study of adipose tissue development and body composition in poultry, and how energy is partitioned to adipose tissue deposition in different anatomical depots. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The data from this research will comprise the bulk of a Ph.D. dissertation (to be defended in March of 2020). During this past year, one original research article was published and one was submitted for publication (currently under review), and abstracts were submitted to two scientific conferences (Poultry Science Association and Experimental Biology; posters at both). The Ph.D. student, Helen Xiao, also presented these findings at local (university-level) conferences. During this time, Helen mentored at least one undergraduate student in the laboratory and also served as a teaching assistant for Cellular and Molecular Neuroscience and Animal Phyisiology and Anatomy, experiences that allowed her to use the research as a platform to teach about scholarship and discovery. Thus, this project has provided ample opportunities for training and professional development at both the undergraduate and graduate level. How have the results been disseminated to communities of interest?The results were disseminated as a local (university) oral presentation, two poster presentations at scientific conferences (Experimental Biology and Poultry Science association), one published original research article, and one original research article that is under review with the journal. Helen Xiao, the Ph.D. student responsible for this project, also delivered a seminar to the Molecular and Cellular Biology program (monthly seminar series) at Virginia Tech, an interdisciplinary Ph.D. program that fosters a transdiscipinary approach to research and education.Results have also been shared in undergraduate classes, including Cellular and Molecular Neuroscience and Animal Physiology and Anatomy. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? An original research article was published, describing differences in adipose tissue development between the lines during the first two weeks post-hatch. Such differences can explain differential adipogenic responses to exogenous factors and growth of adipose tissue-derived cells in culture. In addition to experiments that were conducted in the previous year that contributed to this manuscript (biochemical, morphological, and molecular features of different adipose tissue depots), additional experiments were carried out to strengthen the conclusions, including measurement of plasma non-esterified fatty acids and body composition (via nuclear magnetic resonance imaging) at different ages. The results showed that in low body weight-selected chicks, elevated rates of catabolic processes such as lipolysis likely determine the minimal amount of fat deposition that occurs in all depots post-hatch in this line. In the high body weight-selected line, in contrast, there is a much greater amount of lipid synthesis and cellular hypertrophy, leading to rapid expansion of all adipose tissue depots post-hatch, especially in the abdominal region. A second original research article was submitted forpublication, describing differences in gene expression in specific adipose tissue cellular fractions. This experiment was conducted because of results from the previous year showing that adipose tissue cells from low body weight-selected chicks do not proliferate or differentiate into adipocytes in culture. Measurement of gene expression at the cellular level revealed some striking and unexpected differences between low- and high- body weight-selected lines of chicks, including that apoptosis might be a contributing factor in the remodeling of adipose tissue and limit the adipogenic potential of cells in the low body weight chicks. Collectively, the results from these two papers provide important insights on mechanisms associated wtih fat deposition in chickens during the early post-hatch period.Understanding the disproportionate contribution of different metabolic processes to tissue growth is essential for providing optimal housing and nutritional conditions that maximize genetic potential and support animal health and well-being.

Publications

  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Xiao, Y., G. Wang, M. Gerrard, S. Wieland, M. Davis, M. Cline, P. Siegel, and E. Gilbert. 2019. Changes in adipose tissue physiology during the first two weeks post-hatch in chicks from lines selected for low or high body weight. American Journal of Physiology  Regulatory, Comparative and Integrative Physiology. In Press.
  • Type: Journal Articles Status: Under Review Year Published: 2019 Citation: Xiao, Y., P. Siegel, M. Cline, and E. Gilbert. 2019. Gene expression differs at hatch and day 14 in cellular fractions of adipose tissue of chicks from lines predisposed to be anorexic or obese. Under review.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Xiao, Y., P. Siegel, M. Cline, and E. Gilbert. 2019. Changes in adipose tissue physiology during the first two weeks post-hatch in chicks from lines selected for low or high body weight. Experimental Biology, Physiology Section. FASEB J.
  • Type: Conference Papers and Presentations Status: Awaiting Publication Year Published: 2019 Citation: Xiao, Y., P. Siegel, M. Cline, and E. Gilbert. 2019. Gene expression differs at hatch and day 14 in cellular fractions of adipose tissue of chicks from lines predisposed to be lean or have excess fat. Poult. Sci.


Progress 05/01/17 to 04/30/18

Outputs
Target Audience:The target audience of the research is the scientific community, especially industry, academic, and government researchers that are interested in improving nutrient utilization efficiency in poultry. The research is particularly relevant to the study of adipose tissue development and body composition in poultry, and how energy is partitioned to adipose tissue deposition in different anatomical depots. Changes/Problems:As explained in the previous progress report, cells isolated from the low body weight-selected line of chickens do not survive in culture, thus the remaining objectives (all involving in vitro models) are now focused on understanding the limited growth potential of the cells from this line rather than the role of neuropeptide Y in regulating their growth (although such experiments may reveal a role of neuropeptide Y in this capacity). Currently, experiements are underway to sort various fractions of cells, measure gene expression of different factors involved in metabolism and growth at various stages of the adipocyte life cycle, and culture cells after removal of certain cellular populations.These results are expected to provide insights on novel pathways that regulate cellular proliferation and differentiation in adipose tissue and pathways that are associated with leanness and obesity in chickens. What opportunities for training and professional development has the project provided?An international exchange Ph.D. student from China Agricultural University, Lingbin Liu, published an original research article as first-author. Yang Xiao, a Ph.D. student, presented findings as a seminar at a research symposium at Virginia Tech, for which she received a travel award to present a poster at the Society for Neuroscience meeting in Washington, D.C. in November. She is first-author of an original research article that will soon be submitted to a refereed journal and has another paper that is in preparation. Guoqing Wang, a Ph.D. student, served as co-author on the paper that was published (and the one that will soon be submitted) and presented some of the findings at the same local research symposium in May of 2017. Yang Xiao and Guoqing Wang served as mentors to undergraduate students in the laboratory each academic semester (including during the summer) and as teaching assistants for courses like Cellular and Molecular Neuroscience and Animal Physiology and Anatomy, duringwhich they were able to share these research findings in the context of teaching about research, discovery, and scholarship. Guoqing Wang defended the Ph.D. dissertation in May. How have the results been disseminated to communities of interest?The results were disseminated as local (university-level) poster and oral presentations, a poster presentation at a national scientific conference (published abstract; Society for Neuroscience), and an original research article (Comparative Biochemistry and Physiology, Part A: Molecular and Integrative Physiology). One paper has been prepared and will soon be submitted to a journal and another is in preparation. What do you plan to do during the next reporting period to accomplish the goals?1. Publish two original research manuscripts in refereed journals. 2. Publish a review on mesenchymal stem cell biology and adipose tissue cellular development, with an emphasis on poultry. 3. Complete experiments to elucidate mechanisms underlying differences in the proliferative potential of cells isolated from the stromal-vascular fraction of adipose tissue inlow and high body weight-selected lines of chicks.

Impacts
What was accomplished under these goals? Regarding the first objective of the research, an original research article was published in a refereed journal. The research was also disseminated in the form of two presentations by graduate students (an oral and a poster) at the Virginia Tech Animal and Poultry Sciences Annual Research Symposium in May and a poster at the Society for Neuroscience annual meeting in November in Washington, D.C. (under a travel award received for the oral presentation in May). Progress was made in understanding differences in adipogenesis in cells isolated from the adipose tissue of low and high body weight-selected lines of chicks. Specifically, cellular fractions were isolated (stromal-vascular and adipocytes) and the mRNA abundance of a variety of factors associated with adipogenesis, cellular proliferation, lipid synthesis, lipolysis, and apoptosis was quantified. Results are expected to be submitted to a journal and published in 2018. The developmental regulation of adipose tissue expansion was evaluated in different anatomical depots in chicks from the low and high body weight-selected lines. Specifically, students measured mRNA abundance of a variety of factors associated with preadipocyte and adipocyte function, lipid metabolic enzyme activity, and morphological changes in adipose tissue cellularity during the first two weeks post-hatch. A manuscript was prepared and will soon be submitted for publication.

Publications

  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Liu, L., G. Wang, Y. Xiao, S. Shipp, P. Siegel, M. Cline, and E. Gilbert. 2017. Peripheral neuropeptide Y differentially influences adipogenesis and lipolysis in chicks from lines selected for low or high body weight. Comparative Biochemistry and Physiology, Part A: Molecular and Integrative Physiology. 213:1-10.
  • Type: Journal Articles Status: Other Year Published: 2018 Citation: Xiao, Y., G. Wang, M. Gerrard, S. Wieland, M. Davis, P. Siegel, M. Cline, and E. Gilbert. Changes in adipose tissue physiology during the first two weeks post-hatch in chicks from lines selected for low or high body weight. Poultry Science. To be submitted.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: 4.Xiao, Y., L. Liu, G. Wang, S. Shipp, P. Siegel, M. Cline, and E. Gilbert. 2017. Peripheral neuropeptide Y differentially influences adipogenesis and lipolysis in chicks from lines selected for low or high body weight. Program No. 416.12. Washington, D.C.: Society for Neuroscience, 2017, Online.


Progress 05/01/16 to 04/30/17

Outputs
Target Audience:The target audience of the research is the scientific community, especially researchers that are interested in improving nutrient utilization efficiency in poultry. The research is particularly relevant to the study of adipose tissue development and body composition in poultry, and how energy is partitioned to adipose tissue deposition in different anatomical depots. Changes/Problems:The in-vivo experiments were successful and revealed striking differences between the lines in their adipose tissue physiological responses to exogenous neuropeptide Y. However, the cells in the stromal-vascular fraction isolated from the abdominal adipose tissue of chickens from the low weight-selected linedo not expand in culture. Preliminary cell sorting and immunohistochemical assays indicate that there are differences in the proportion of adipose tissue-derived mesenchymal stems cells between low and high weight-selected lines of chicks. Thus, rather than characterizing differences in neuropeptide Y-induced adipogenesis in cell culture, remaining experiments (at the cellular level) will focus on differences in the adipogenic potential of adipose tissue-derived progenitor cells that may explain why low-weight chicks are resistant to fat deposition and are very lean relative to the high-weight line. What opportunities for training and professional development has the project provided?A Master's student, Steven Shipp, optimized conditions for the cell culture experiments, and defended the thesis in December of 2016. A Ph.D. student, Yang Xiao, assumed responsibility for the project in August of 2016 and has since performed cell culture, cell sorting, immunohistochemical, and gene expression experiments to address Objectives 2-4 of the project. An international exchange Ph.D. student from Sichuan Agricultural University, Lingbin Liu, has performed gene expression, plasma non-esterified fatty acid, and enzyme activity assays in collaboration with Ph.D. student Guoqing Wang, to complete the first objective of the project. These students have presented findings at laboratory meetings and this year will present at a national conference and publish their results in a refereed journal. This project has also provided opportunities for mentoring undergraduate students in the laboratory. How have the results been disseminated to communities of interest?The results have been assembled into a manuscript that is to be submitted to a refereed journal. Results will also be disseminated in the form of a poster or oral presentation at a national scientific conference and at university- and state-level research symposiums. What do you plan to do during the next reporting period to accomplish the goals?1. Publish at least one manuscript in a refereed journal 2. Present findings at local and national conferences 3. Complete gene knockdown experiments and elucidate signal transduction pathways underlying effects of neuropeptide Y on adipogenesis 4. Elucidate mechanisms underlying differences in the proliferative potential of cells isolated from the stromal-vascular fraction of adipose tissue from low and high weight-selected lines of chicks

Impacts
What was accomplished under these goals? 1. We determined the effect of intraperitoneal injection of neuropeptide Y on low (LWS) and high (HWS) weight-selected line chick adipose tissue function, and elucidated some of the underlying mechanisms. Amanuscript was prepared for submission to a journal. Results will also be disseminated in the form of an abstract to a local (university) and national scientific conference.At 5 days post-hatch, LWS and HWS chicks were intraperitoneally injected with 0 (vehicle), 60, or 120 µg/kg BW NPY and subcutaneous adipose tissue and plasma were collected at 1, 3, 6, 12, and 24 h (n=12). NPY injection increased glycerol-3-phosphate dehydrogenase (G3PDH) activity at 1 and 3 h and reduced plasma NEFAs at 1 and 12 h. G3PDH activity was greater in HWS than LWS while NEFAs were greater in LWS. At 1 h, peroxisome proliferator-activated receptorgamma (PPARg), CCAAT/enhancer binding protein (C/EBP)α, and microsomal triglyceride transfer protein (MTTP) mRNAs were reduced in NPY-injected chicks whereas NPY receptor 1 (NPYR1) was increased. Expression of stearoyl-CoA desaturase (SCD1) was increased by NPY at 1 h in HWS but not LWS. PPARg (3 and 6 h), C/EBPβ (3 h), C/EBPα (6 h) and NPYR1 and 2 (24 h) mRNAs were greater in NPY- than vehicle-injected chicks. At several times, adipose triglyceride lipase, MTTP, perilipin 1, NPYR1, and NPYR2 mRNAs were greater in LWS than HWS, while expression of SCD1, glycerol-3-phosphate acyltransferase 3 and lipoprotein lipase was greater in HWS than LWS. Thus, NPY promotes fat deposition and inhibits lipolysis in chicks, with line differences indicative of greater rates of lipolysis in LWS and adipogenesis in HWS. 2. We optimized conditions for culturing the stromal-vascular fraction of cells from HWS chicks for proliferation and differentiation assays. The cells from LWS chicks do not expand. Cell sorting, immunohistochemical, and gene expression assays have revealed differences between LWS and HWS in their mesenchymal stem cell populations and proliferative capacity in abdominal adipose tissue. These studies are ongoing. 3. We are optimizing conditions for lentiviral-mediated RNA interference to evaluate the effects of NPY receptor knockdown on HWS chickadipose tissue cell proliferation and differentiation.

Publications


    Progress 05/01/15 to 04/30/16

    Outputs
    Target Audience:The target audience of the research is the scientificcommunity, especially researchers that are interested inimproving nutrient utilization efficiency in poultry. The research is particularly relevent to the study of body composition in poultry and how energy is partitioned to adipose tissue deposition in different anatomical depots. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?A graduate student, Steven Shipp has gained experience in carrying out animal studies (poultry husbandry, intraperitoneal injections, removing blood, tissue and other organs from chicks), molecular biology techniques (total RNA isolation, reverse transcription, and real time PCR),performing primary cell culture experiments, and the workflow of cloning and producing lentiviruses expressing shRNA for knocking down expression of the neuropeptide Y receptor sub-types via RNA interference. Steven has also mentored an undergraduate volunteer in the laboratory. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?During the next reporting period: 1) a new graduate student will assume responsibility for the samples collected from the injection studies and isolate RNA, perform reverse transcription, and carry out real time PCR to measure mRNA abundance of NPY signling and lipid metabolism-associated genes in adipose tissue. The student will also measure neuropeptide Y abundance in the plasma samples. 2) Steven Shipp will optimize conditions for knocking down the NPY receptors in chick preadipocytes and prepare to carry out the cell culture experiments with cells isolated from the low and high body weight-selected lines. 3) Abstracts will be generated and presented at a national meeting, such as Experimental Biology or Poultry Science.

    Impacts
    What was accomplished under these goals? Since the start date we have: 1) Collected subcutaneous adipose tissue and plasma from low and high body weight-selected line chickens that were injected intraperitoneally with one of several doses of neuropeptide Y. Fat samples were collected at 1, 3, 6, 12, and 24 hours post-injection and are awaiting total RNA extraction. Non-esterified fatty acid concentrations were measured in the plasma samples. There were differences between low and high body weight chickens in their NEFA concentrations after NPY injection, suggesting that NPY affects lipolysis differently in the lines. 2) Generated lentiviral constructs expressing shRNA targeting the NPY R1 and R2 receptors, and particles for transduction studies in cell culture.

    Publications