Source: WASHINGTON STATE UNIVERSITY submitted to NRP
VITAMIN A, ZFP423 AND INTRAMUSCULAR ADIPOGENESIS IN BEEF CATTLE
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1005681
Grant No.
2015-67015-23219
Cumulative Award Amt.
$500,000.00
Proposal No.
2014-04923
Multistate No.
(N/A)
Project Start Date
Apr 1, 2015
Project End Date
Mar 31, 2020
Grant Year
2015
Program Code
[A1231]- Animal Health and Production and Animal Products: Improved Nutritional Performance, Growth, and Lactation of Animals
Recipient Organization
WASHINGTON STATE UNIVERSITY
240 FRENCH ADMINISTRATION BLDG
PULLMAN,WA 99164-0001
Performing Department
Animal Sciences
Non Technical Summary
The palatability of beef is determined by its marbling and tenderness. Marbling, or the fat flecks and streaks distributed inside the lean, makes beef juicy and flavorful. Therefore, it is critically important to enhance marbling, which requires the formation of abundant fat cells inside the muscle during development. Recently, we identified a pool of cells which are the source of new fat cells in beef muscle. We further observed that vitamin A enhances the formation of new fat cells through enhancing the expression of Zfp423, a key regulator of fat cell formation. Our hypothesis is that vitamin A supplementation during the critical stage for the formation of fat cells inside muscle will enhance marbling through increasing the expression of Zfp423. We will examine the impact of vitamin A administration to neonatal calves on fat cell formation and marbling in the resulting beef, and further explore the mechanisms behind fat cell formation and marbleing. We plan to conduct both animal and cell culture studies, with the objective to enhance our understanding of the impact of vitamin A on fat cell formation inside muscle. Due to the ease of manipulating vitamin A levels, defining the role of vitamin A in beef marbling will provide a practical target to improve beef quality, which will dramatically improve quality and efficiency of beef production.
Animal Health Component
20%
Research Effort Categories
Basic
80%
Applied
20%
Developmental
0%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
3053310105080%
3053320105020%
Knowledge Area
305 - Animal Physiological Processes;

Subject Of Investigation
3320 - Meat, beef cattle; 3310 - Beef cattle, live animal;

Field Of Science
1050 - Developmental biology;
Goals / Objectives
Our overarching hypothesis is that elevation of intramuscular preadipocyte formation during early postnatal development dramatically increases intramuscular adipocytes, forming sites for intramuscular fat accumulation (marbling). As a foundational step, the overallobjective of this proposal is to test the hypothesis that vitamin A supplementation during the critical stage for intramuscular adipogenic commitment programs preadipocyte formation in muscle via Zfp423.We have two sub-objectives:1) To examine the impact ofvitamin Aintake during early calf development on intramuscular adipogenesis and marbling in the resulting beef. The neonatal to early weaning stages are critical for intramuscular adipocyte formation. Neonatal calves will be administered with/without vitamin A supplementation by injection (200,000 IU as retinyl palmitate per calf at birth, and 300,000 IU at one month of age). At 2 months of age, calves will be subjected to biopsy to obtain muscle samplesfor analyzing intramuscular adipogenesis. The subsequent steers will be harvested ataround 18 months of age formarbling and meat quality analyses.We anticipate that vitamin A administration during the early animal development stimulates intramuscular fat accumulation and marbling in the resulting steers.2) To further explore the mechanistic link among retinoic acid, Zfp423 and adipogenic differentiation. We will separate fibro/adipogenic progenitor cells from beef cattle, which will be treated withretinoic acid to elucidate the interactions among retinoic acid, Zfp423, and intramuscular adipogenesis. We anticipate that retinoic acid,the bioactive metabolite of vitamin A, will reduce Polycomb proteins binding to the Zfp423 promoter, which will be correlated with reduced DNA methylation in the Zfp423 promoter and enhanced adpogenesis. We will also analyze the involvement of histone modifications in adipogenic differentiation. Our objective is to establish the molecular links between vitamin A, Zfp423 and PPARG expression, and the subsequent adipogenic differentiation of progenitor cells of beef cattle.
Project Methods
Aim 1: To examine the impact of vitamin A intake during early calf development on intramuscular adipogenesis and marbling in the resulting beef.Animal treatments: After birth, 20 calves will be separated into two groups receiving either vehicle only or vitamin A intramuscular injection (200,000 IU as retinyl palmitate), which will be repeated after one month at 300,000 IU to accommodate calf weight gain. The injection method fully utilizes the enormous ability of the liver to store excessive vitamin A. During vitamin A treatment to calves, cows will be assigned to a conventional diet consisting of native grass hay fortified with vitamins and minerals fed at NRC (2000) recommendations. According to power analyses, only 8 animals per treatment are needed. Here, we propose to use 10 animals per treatment in case of incidentals, which could result in having to withdraw animals from the experiment. During pregnancy, lactating cows will be managed to maintain a body condition score of 5 (scale is 1-9), and calves will be kept with their mothers. After 2 months of vitamin A treatments, biopsy samples of Longissimus dorsi (LD) muscle will be obtained from calves.Biopsy: Biopsies of LD muscle will be obtained from calves at the end of vitamin A treatment. Biopsies will be taken at a constant location, between the 10th and 11th rib of the left side at 1 inch depth and 4 cm lateral to the midline of the thoracic vertebrae. In addition, one blood sample will be taken from the jugular vein while the animals are in the chute for biopsy, and the serum will be used for analyzing vitamin A metabolites. Glucose, fatty acids and insulin are related to adipogenic differentiation, and will be analyzed.Muscle structure and biochemical analyses: Muscle samples will be further processed immediately after biopsy. One small piece of muscle biopsy sample (0.2 g) will be frozen in OCT compound (Tissue-Tek, Sakura Finetek USA, Inc., Torrance, CA), and another piece (0.2 g) will be placed in fresh 4% paraformaldehyde before being embedded in paraffin for further analysis. The remaining muscle samples will be frozen in liquid nitrogen for biochemical analyses. Biochemical analyses of muscle samples include markers of adipogenesis (PPARG, FABP4 and others) and fibrogenesis (collagen 1, fibronectin and TGFb), which will provide information regarding adipogenic differentiation and adipose tissue formation inside muscle. In addition, because Zfp423 is a transcription factor regulating adipogenic gene expression, higher Zfp423 in vitamin A-treated steers is expected to also propel the expression of genes regulating lipid accumulation. Therefore, we plan to analyze the mRNA expression of a number of genes related to lipogenesis and lipolysis.Steer harvest and biochemical analyses: Following vitamin A treatments, calves and cows will be managed per WSU herd management procedures, including castration within 1 month after birth and weaning at 6 months of age and then fed a hay-based diet. The daily dry matter (DM) intake and growth performance of calves in the feedlot will be measured. At market weight, steers will be slaughtered in the Meat Laboratory at WSU according to USDA regulations. A slice of the LD muscle between the 13th to 14th rib on the left side of the carcass will be sampled and used for biochemical and structural analyses. Intramuscular adipocytes will be collected from LD muscle; in addition, we will also obtain kidney fat and subcutaneous fat (above the 13th to 14th rib on the left side of the carcasses). Both kidney fat and subcutaneous fat will be embedded in paraffin for structural analyses as described above. Moreover, biochemical analyses and determination of expression of enzymes involved in lipogenesis and lipolysis will be conducted.Carcass composition and beef quality analyses: Hot carcass weight will be obtained. In addition, kidney, pelvic and heart (KPH) fat willalso bemeasured. Carcasses will be halved and hanged in the cooler (4 °C) for 24 hr (solidifying fat and muscle for accurate measurement) and the right side of carcasses will be ribbed between the 12th and13th ribs for measuring backfat thickness, ribeye area and marbling, and carcass grading according to USDA standard methods.Aim 2: To further explore the mechanistic link among retinoic acid, Zfp423 and adipogenic differentiation.Retinoic acid regulates adipogenic commitment via Zfp423: To analyze whether vitamin A treatment alters the adipogenic differentiation and DNA methylation in fibro/adipogenic progenitor cells (FAPs), we will separate FAPs from both calves and steer muscle samples using PDGFRa as a surface marker. These cells will be used for studying the effect of retinoic acid on the adipogenic commitment of FAPs, focusing on Zfp423. We expect to demonstrate that Zfp423 is a key mediator linking retinoic acid to FAP adipogenic commitment.Retinoic acid reduces Polycomb group binding to the Zfp423 promoter: We will further explore mechanisms linking retinoic acid and Zfp423 expression, focusing on Polycomb group proteins. We expect that retinoic acid will reduce Polycomb group protein binding to the Zfp423 promoter but increase Trithorax protein binding, and that these effects will be correlated with lower levels of both H3K27me3 and DNA methylation in the Zfp423 promoter and enhanced Zfp423 expression, all leading to elevated adipogenic commitment and generation of preadipocytes.Trascription factors regulate epigenetic modificationsof Zfp423 promoter induced by retinoic acid: In addition, we will explore possible transcription factors regulating epigenetic modifications in the Zfp423 promoter and its expression affected by retinoic acid. Overall, we anticipate that retinoic acid treatment will reduce Polycomb proteins binding to the Zfp423 promoter, which will be correlated with reduced DNA methylation in FAPs. Thus, DNA methylation will be implicated as a major mechanism linking retinoic acid to Zfp423 expression and adipogenic commitment. These studies will establish a molecular link between vitamin A and Zfp423 expression, and the subsequent adipogenic differentiation.

Progress 04/01/15 to 03/31/20

Outputs
Target Audience:Scientific community, beef cattle producers Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?M.S. student, Mrs. Natasha Moffitt-Hemmer, graduated in April, 2020. Ph.D. student, Mr. Xiangdong Liu and Postdoc, Dr. Yanting Chen, partcipiated in this project. How have the results been disseminated to communities of interest?The importance of vitamin A in early beef cattle development has been desseminated through shortcourses for producers,scientific presentations in conferences andpeer-reviewed journal articles. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? We have finished animal studies and all data analyses associated with this project. We found that neonatal vitamin A supplementation not only enhanced the intramuscular adipocyte development which resultedin higher marbling fat content in steers, but also increased muscle cell formation and the lean/fat ratio of steers at harvest. As a result of vitamin A treatment during the neonatal stage, the growth efficiency and beef quality of cattle can be improved. Mechanistically, we developed an in vitro 3-dimensional culture system to study adipocyte formation and muscle development in beef cattle, and found that vitamin A increases adipocyte progenitor cell proliferatioin, which explains the increase in intramuscular adipocyte number in cattle received vitamin A treatment during the early development. In addition, vitamin A promotes the proliferation of satellite cells, which explains the enhanced muscle growth in beef cattle treated with vitamin A. Therefore, vitamin A supplementation during the early development appears to be an effective method to enhance cattle growthefficiency.

Publications

  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Li, X., X. Fu, G. Yang, and M. Du. (2019). Review: Enhancing intramuscular fat development via targeting fibro-adipogenic progenitor cells in meat animals. Animal, 14: 312-321.
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Zhao, L., Y. Huang, and M. Du. (2019). Farm Animals for Studying Muscle Development and Metabolism: dual purposes for animal production and human health. Animal Frontiers, 9:3.
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Wei, S., A. Li, L. Zhang, and M. Du. (2019). Long noncoding RNAs in adipogenesis and adipose development of meat animals. Journal of Animal Science, 97: 2644-2657.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Du, M., Invited Speaker. Awardee Talk - Metabolites in stem cell differentiation and fetal development. Physiology Session I, the Annual Meeting of American Society of Animal Science, Austin, TX. July 9 to 12, 2019.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Du, M., Invited Speaker. Molecular pathways regulating myogenesis. Aspen/Snowmass Perinatal Biology Symposium, Snowmass, CO. August 24 to 27, 2019


Progress 04/01/18 to 03/31/19

Outputs
Target Audience:Scientific community, and beef and other livestock producers. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Two graduate students, Mrs. Natasha Moffitt-Hemmer and Xiangdong Liu, and a postdoc, Dr. Yanting Chen received training. How have the results been disseminated to communities of interest?Journal articles and presentations in scientific conferences,symposiums and short-courses. What do you plan to do during the next reporting period to accomplish the goals?We will continue to define mechanisms leading to the proliferation of satellite cells and fibro/adipogenic progenitor cells due to vitamin A supplementation.

Impacts
What was accomplished under these goals? We have finished all animal studies and almost analyses of collected tissues. We found that neonatal vitamin A supplementation enhances overall cattle growth, more prominently the intramuscular fatand muscle growth, which improves production efficiency and beef quality.We continued to explore the mechanisms leading to the enhanced muscle and adipose development, and found thatvitamin A stimulates theproliferation of fibro/adipogenic progenitor cells and satellite cells, which can explain the increase in overall growth. Currently, we are exploring mechanisms leading to the proliferation ofthese progenitor cells.

Publications

  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Ma, Y.N., B. Wang, Z.X. Wang, N. A. Gomez, M. J. Zhu, and M. Du. (2018). Three dimensional spheroid culture of adipose stromal vascular cells for studying adipogenesis in beef cattle. Animal, 12: 2123-2129.
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Harris, C. L., B. Wang, J. M. Deavila, J. R. Busboom, M. Maquivar, S. M. Parish, B. McCann, M. L. Nelson, and M. Du. (2018). Vitamin A administration at birth promotes calf growth and marbling fat development in Angus beef cattle. Journal of Animal Science and Biotechnology, 9: 55.
  • Type: Journal Articles Status: Awaiting Publication Year Published: 2019 Citation: Zhao, L., Y. Huang, and M. Du. (2019). Farm Animals for Studying Muscle Development and Metabolism: dual purposes for animal production and human health. Animal Frontiers, In press.
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Wang, B., C. L. Harris, W. Nie, X. Fu, J. M. Deavila, M. J. Zhu, M. Maquivar, S. M. Parish, J. R. Busboom, M. L. Nelson, and M. Du. (2018). Neonatal vitamin A injection promotes cattle muscle development and increase oxidative muscle fibers. Journal of Animal Science and Biotechnology, 9: 82.
  • Type: Journal Articles Status: Awaiting Publication Year Published: 2019 Citation: Wei, S., A. Li, L. Zhang, and M. Du. (2019). Long noncoding RNAs in adipogenesis and adipose development of meat animals. Journal of Animal Science, In press.


Progress 04/01/17 to 03/31/18

Outputs
Target Audience:Scientific community, and beef and other livestock producers. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Two graduate students,Corrine Harris, Bo Wang, finished their graduate training. Eric Testroet, a postdoc involved in this project, has obtained a faculty position. How have the results been disseminated to communities of interest?Journal articles and presentations in scientific conferences What do you plan to do during the next reporting period to accomplish the goals?We will continue to define mechanisms responsible for the observed changes in adipogenesis and marbling fat development in neonatal calves due to vitamin A treatment.

Impacts
What was accomplished under these goals? We have finished most analyses of collected tissues. Based on data obtained, we found that neonatal vitamin A supplementation enhances adipogenesis and myogenesis during neonatal development, which result in improved marbling and weight gain of the subsequent steers. In addition, compared to Wagyu cattle, the satellite cells are more abundant in Angus, which also show higher myogenic capacity. To effectively analyze adipogenesis, we established a three-dimensional spheroid culture system, which can mimic adipose tissue development in vivo.

Publications

  • Type: Journal Articles Status: Awaiting Publication Year Published: 2018 Citation: Ma, Y.N., B. Wang, Z.X. Wang, N. A. Gomez, M. J. Zhu, and M. Du. (2018). Three dimensional spheroid culture of adipose stromal vascular cells for studying adipogenesis in beef cattle. Animal, In press.
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Fu, X., Q. Yang, B. Wang, J. Zhao, M. Zhu, S. M. Parish, and M. Du. (2017). Reduced satellite cell density and myogenesis in Wagyu compared to Angus cattle as a possible explanation of its high marbling. Animal, 9: 1-8.
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Du, M., S.P. Ford, and M. J. Zhu. (2017). Optimizing livestock production efficiency through maternal nutritional management and fetal developmental programming. Animal Frontiers, 7: 5-11.
  • Type: Journal Articles Status: Under Review Year Published: 2018 Citation: Harris, C. L., M. L. Nelson, B. Wang, J. M. Deavila, J. R. Busboom, M. Maquivar, S. M. Parish, B. McCann, and M. Du. (2018). Vitamin A administration at birth promotes calf growth and marbling fat development in Angus beef cattle. Journal of Animal Science and Biotechnology, Revision submitted.


Progress 04/01/16 to 03/31/17

Outputs
Target Audience:Scientific community, and beef and other livestock producers Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Graduate students: Corrine Harris, Bo Wang, Natasha Moffitt, and Xing Fu How have the results been disseminated to communities of interest?Journal articles and presentations in scientific conferences What do you plan to do during the next reporting period to accomplish the goals?We will continue to define mechanisms responsible for the observed changes in myogenesis and adipo/fibrogenesis in neonatal calves due to vitamin A treatment.

Impacts
What was accomplished under these goals? We have finished the cattle studies and are currently analyzing changes in collected tissues. Based on carcass data obtained, we found that neonatal vitamin A supplementation enhances muscle growth and also improves marbling of the subsequent steers. We found that the satellite cells are activated in vitamin A treated calves, which contributes to enhanced muscle growth in these cattle. Currently, we are analyzing changes in adipose tissue development in beef cattle due to vitamin A treatment.

Publications

  • Type: Journal Articles Status: Submitted Year Published: 2017 Citation: Wang, B., C. L. Harris, W. Nie, X. Fu, J. M. Deavila, M. J. Zhu, M. Maquivar, S. M. Parish, J. R. Busboom, M. L. Nelson, and M. Du. (2017). Neonatal vitamin A administration promotes cattle muscle development. Journal of Animal Science, Submitted.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Wang, B., Q. Yang, C. L. Harris, M. L. Nelson, J. R. Busboom, M. J. Zhu, and M. Du. (2016). Nutrigenomic regulation of adipose tissue development  role of retinoic acid. Meat Science, 120: 100-106.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Miao, Z. G., L. P. Zhang, X. Fu, Q. Y. Yang, M. J. Zhu, M. V. Dodson, and M. Du. (2016). Invited review: Mesenchymal progenitor cells in intramuscular connective tissue development. Animal, 10: 75-81.


Progress 04/01/15 to 03/31/16

Outputs
Target Audience:Scientific community, beef cattle producers Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Currently, one Ph.D. student, Bo Wang, and one M. S. student, Corrine Harris, are involved in this project. In addition, undergraduate students are also involved in feeding calves. Thus, the funding of this proposal provide training opportunities for them. How have the results been disseminated to communities of interest?Yes, we are writing meeting abstracts and journal articles. There are review papers published. What do you plan to do during the next reporting period to accomplish the goals?We plan to harvest animals and conduct carcass evaluation and meat quality analyses. We plan to continue biochemical and structural analyses of muscle and adipose samples.

Impacts
What was accomplished under these goals? The overall objective of this proposal is to test the hypothesis that vitamin A supplementation during the critical stage for intramuscular adipogenic commitment programs preadipocyte formation in muscle via Zfp423. There have two sub-objectives: 1) Examine the impact of vitamin A intake during early calf development on intramuscular adipogenesis and marbling in the resulting beef; and 2) further explore the mechanistic link among retinoic acid, Zfp423 and adipogenic differentiation. We have initiated the calf study, where calves were injected with Vitamin A. We found that Vitamin A promoted calf growth by increasing average body weight. In addition, based on ultrasound data, the marbling appeared to be enhanced by Vitamin A treatment. Also, we did a muscle biopsy in calves at the end of Vitamin A supplementation, which were used for structural and biochemical analyses. We also separated stromal vascular cells from biopsy muscle samples. Based on preliminary analyses, we found that Vitamin A increased the density of adipogenic progenitor cells. Currently, we are conducting comprehensive analyses.

Publications

  • Type: Journal Articles Status: Accepted Year Published: 2016 Citation: Miao, Z. G., L. P. Zhang, X. Fu, Q. Y. Yang, M. J. Zhu, M. V. Dodson, and M. Du. (2016). Invited review: Mesenchymal progenitor cells in intramuscular connective tissue development. Animal, 10: 75-81.
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Du, M., W. Bo, M.J. Zhu. (2015). Fetal programming in meat production. Meat Science, 109: 40-47.
  • Type: Journal Articles Status: Published Year Published: 2015 Citation: Wei, S., X. Fu, X. Liang, M. Zhu, Z. Jiang, S. M. Parish, M. V. Dodson, L. Zan, and M. Du. (2015). Enhanced mitogenesis in stromal vascular cells derived from subcutaneous adipose tissue of Wagyu compared with those of Angus cattle. Journal of Animal Science, 93: 1015-1024.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2016 Citation: Wang, B., X. Fu, S. M. Zhang, X. W. Liang, M. J. Zhu, and M. Du. (2016). Maternal Vitamin A Supplementation Expands Adipose Progenitor Population through Promoting Vascular Development. Experimental Biology Meeting, San Diego, California, April 1-2, 2016.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2016 Citation: Wang, B., M. J. Zhu, and M. Du. (2016). All-trans-Retinoic Acid Inhibits Adipogenesis by Interrupting Gadd45a Induced Zfp423 Demethylation. Experimental Biology Meeting, San Diego, California, April 1-2, 2016.