Progress 04/15/15 to 04/14/19
Outputs
Target Audience:Nutritionists, veterinarians, animal scientists, professional students (veterinary medicine), veterinary technicians, and undergraduate students (animal sciences and physiology). Our efforts to reach these audiences included lectures to students in veterinary medicine and animal science programs. Also, talks in regional and national conferences of bovine practitioners, dairy scientists, and animal nutritionists. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Postdoctoral Scientists: W. Raphael BVSc, MS, PhD Rahul Nelli BVSc, MVM, PhD Jenne De Koster, DVM, PhD Graduate Students: Clarissa Streider-Barboza DVM, MS, PhD Kyan Thelen MS Professional Students (DVM candidates) Ashley Zondlak DVM Situnyiwe Chirunga DVM Sarah LaTendresse DVM candidate Undergraduate Students Kyan Thelen, Animal Science 2014-2015 Stephanie Steele, Animal Science 2014 Aaron Corbett, Animal Science 2015 Connor Lewicki, Lyman Briggs 2015-2018 Whitney James, Animal Science 2016 Libby Greenberg, Animal Science 2017 Madison Smith, Animal Science 2017-2018 Joshua Geldersma, Biochemistry and Microbiology 2018 How have the results been disseminated to communities of interest?Results from the project have been disseminated through: 1. Two review manuscripts: Contreras GA, Strieder-Barboza C, De Koster J. Symposium review: Modulating adipose tissue lipolysis and remodeling to improve immune function during the transition period and early lactation of dairy cows. J Dairy Sci. 2018 Mar;101(3):2737-2752. doi: 10.3168/jds.2017-13340. Contreras GA, Strieder-Barboza C, Raphael W. Adipose tissue lipolysis and remodeling during the transition period of dairy cows. J Anim Sci Biotechnol. 2017 May 5;8:41. doi: 10.1186/s40104-017-0174-4. eCollection 2017. 2. Nine original papers: Contreras GA, Kabara E, Brester J, Neuder L, Kiupel M. Macrophage infiltration in the omental and subcutaneous adipose tissues of dairy cows with displaced abomasum. J Dairy Sci. 2015; 98(9): 6176-6187. doi: 10.3168/jds.2015-9370 Contreras GA, Thelen K, Schmidt SE, Strieder-Barboza C, Preseault CL, Raphael W, Kiupel M, Caron J, Lock AL.Adipose tissue remodeling in late-lactation dairy cows during feed-restriction-induced negative energy balance.J Dairy Sci. 2016 Dec;99(12):10009-10021. doi: 10.3168/jds.2016-11552. Epub 2016 Oct 5. Contreras GA, Strieder-Barboza C, de Souza J, Gandy J, Mavangira V, Lock AL, Sordillo LM.Periparturient lipolysis and oxylipid biosynthesis in bovine adipose tissues.PLoS One. 2017 Dec 5;12(12):e0188621. doi: 10.1371/journal.pone.0188621. eCollection 2017. PubMed PMID: 29206843; PubMed Central PMCID: PMC5716552. Strieder-Barboza C, de Souza J, Raphael W, Lock AL, Contreras GA.Fetuin-A: A negative acute-phase protein linked to adipose tissue function in periparturient dairy cows.J Dairy Sci. 2018 Mar;101(3):2602-2616. doi: 10.3168/jds.2017-13644. Epub 2017 Dec 21. PubMed PMID: 29274966. Depreester E, De Koster J, Van Poucke M, Hostens M, Van den Broeck W, Peelman L, Contreras GA, Opsomer G.Influence of adipocyte size and adipose depot on the number of adipose tissue macrophages and the expression of adipokines in dairy cows at the end of pregnancy.J Dairy Sci. 2018 Jul;101(7):6542-6555. doi: 10.3168/jds.2017-13777. Epub 2018 Apr 5. PubMed PMID: 29627241. De Koster J, Strieder-Barboza C, de Souza J, Lock AL, Contreras GA.Short communication: Effects of body fat mobilization on macrophage infiltration in adipose tissue of early lactation dairy cows.J Dairy Sci. 2018 Aug;101(8):7608-7613. doi: 10.3168/jds.2017-14318. Epub 2018 Jun 7. PubMed PMID: 29885887. De Koster J, Nelli RK, Strieder-Barboza C, de Souza J, Lock AL, Contreras GA.The contribution of hormone sensitive lipase to adipose tissue lipolysis and its regulation by insulin in periparturient dairy cows.Sci Rep. 2018 Sep 6;8(1):13378. doi: 10.1038/s41598-018-31582-4. PubMed PMID: 30190510; PubMed Central PMCID: PMC6127149. Strieder-Barboza C, Contreras GA.Fetuin-A modulates lipid mobilization in bovine adipose tissue by enhancing lipogenic activity of adipocytes.J Dairy Sci. 2019 Feb 28. pii: S0022-0302(19)30203-6. doi: 10.3168/jds.2018-15808. [Epub ahead of print] PubMed PMID: 30827564 Strieder-Barboza C, Thompson E, Thelen K, Contreras GA.Technical note: Bovine adipocyte and preadipocyte co-culture as an efficient adipogenic model.J Dairy Sci. 2019 Apr;102(4):3622-3629. doi: 10.3168/jds.2018-15626. Epub 2019 Feb 14. PubMed PMID: 30772027 3. Over 25 oral and poster presentations in regional and national dairy science meetings. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Results from our project demonstrate that adipose tissue lipolysis during the periparturient period of dairy cows not only involves the release of fatty acids but also induces a remodeling process of the organ. Through in vivo experiments, we showed for the first time that in periparturient cows, adipose tissue remodeling is characterized by an inflammatory response with macrophage infiltration. In periparturient cows with excessive lipolysis, displaced abomasum, and ketosis, we found that adipose tissue macrophages accumulate in aggregates within omental and subcutaneous depots. Remarkably, these macrophages are highly pro-inflammatory and inhibit the action of insulin. In contrast, during moderate lipolysis induced by a short 4 day feed restriction protocol in late-lactation cows, adipose macrophage infiltration into adipose depots still occurs, but without a change in phenotype. Thus when lipolysis is moderate, these macrophages remove FA, diglycerides, monoglycerides, and other lipolysis products that can cause lipotoxicity and therefore do not inhibit insulin action.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
De Koster J, Nelli RK, Strieder-Barboza C, de Souza J, Lock AL, Contreras GA. The contribution of hormone sensitive lipase to adipose tissue lipolysis and its regulation by insulin in periparturient dairy cows. Scientific Reports. Scientific reports. 2018; 8(1):13378.
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
De Koster J, Strieder-Barboza C, de Souza J, Lock AL, Contreras GA. Short communication: Effects of body fat mobilization on macrophage infiltration in adipose tissue of early lactation dairy cows. J Dairy Sci. 2018 Aug;101(8):7608-7613. doi: 10.3168/jds.2017-14318. PubMed PMID: 29885887.
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Depreester E, De Koster J, Van Poucke M, Hostens M, Van den Broeck W, Peelman L, Contreras GA, Opsomer G. Influence of adipocyte size and adipose depot on the number of adipose tissue macrophages and the expression of adipokines in dairy cows at the end of pregnancy. J Dairy Sci. 2018 Jul;101(7):6542-6555. doi: 10.3168/jds.2017-13777.
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Strieder-Barboza C, Thompson E, Thelen K, Contreras GA. Technical note: Bovine adipocyte and preadipocyte co-culture as an efficient adipogenic model. J Dairy Sci. 2019 Apr;102(4):3622-3629. doi: 10.3168/jds.2018-15626. Epub 2019 Feb 14. PubMed PMID: 30772027.
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Strieder-Barboza C, Contreras GA. Fetuin-A modulates lipid mobilization in bovine adipose tissue by enhancing lipogenic activity of adipocytes. J Dairy Sci. 2019 Feb 28. pii: S0022-0302(19)30203-6. doi: 10.3168/jds.2018-15808. [Epub ahead of print] PubMed PMID: 30827564.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2018
Citation:
Strieder-Barboza C, Contreras GA. Fetuin-a modulates lipid mobilization in bovine adipose tissue by enhancing lipogenic activity of adipocytes. American Dairy Science Association Annual Meeting, June 24-27, 2018, Knoxville, TN. T006.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2018
Citation:
Contreras GA, Strieder-Barboza C, de Souza J, Gandy J, Nelli RK, De Koster J, Lock AL, Sordillo LM. Hormone sensitive lipase activity modulates the biosynthesis of oxidized linoleic acid metabolites in adipose tissue of periparturient dairy cows. American Dairy Science Association Annual Meeting, June 24-27, 2018, Knoxville, TN. 423
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2018
Citation:
Strieder-Barboza C, Thompson E, Thelen K, Contreras GA. In vitro adipogenic differentiation of subcutaneous primary bovine preadipocytes: A coculture model. American Dairy Science Association Annual Meeting, June 24-27, 2018, Knoxville, TN. T174
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2018
Citation:
Thompson E, Strieder-Barboza C, de Souza J, Nelli RK., De Koster J., Lock AL, Contreras GA. Osteopontin expression dynamics link macrophage infiltration and lipolysis intensity in adipose tissues of eriparturient cows. American Dairy Science Association Annual Meeting, June 24-27, 2018, Knoxville, TN. M191.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2018
Citation:
Contreras GA, LaTendresse S, De Koster J., Strieder-Barboza C, de Souza J, Lock AL. The oxidized linoleic acid metabolite 13-hydroxyoctadecadienoic acid modulates lipolysis in bovine adipose tissue and adipocytes. American Dairy Science Association Annual Meeting, June 24-27, 2018, Knoxville, TN. M189.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2018
Citation:
De Koster J, Nelli RK, Strieder-Barboza C, de Souza J, Lock AL, Contreras GA. The contribution of hormone sensitive lipase to adipose tissue lipolysis and its regulation by insulin in periparturient dairy cows. American Dairy Science Association Annual Meeting, June 24-27, 2018, Knoxville, TN. M188
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2018
Citation:
Contreras GA, LaTendresse S, Strieder-Barboza C, de Souza J, De Koster J, Lock AL. Oxidized Linoleic acid metabolites modulate lipolysis and lipogenesis in adipocytes. Keystone Symposia meeting on Bioenergetics and Metabolic Disease, pp 43, January 21-25, 2018. Keystone Resort Colorado.
|
Progress 04/15/17 to 04/14/18
Outputs
Target Audience:Nutritionists, veterinarians, animal scientists, professional students (veterinary medicine), and undergraduate students (animal science and phisiolgy) Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Postdoctoral scientists: Rahul Nelli BVSC, AH, MVM, PhD worked in the project during 2017 and is completeing the coculture experiments in aim 1. Jenne De Koster, DVM, PhD. Worked in the project during 2017 and completed the experiments in aim 2. Graduate Students: Clarissa Streider Barboza (PhD candidate) and Kyan Thelen (Masters candidate). Students were trained on qPCR, flow cytometry, ELISA, and Western blotting techniques. Their dissertation and thesis research objectives will support in part the development of the major goals of this project. Undergraduate Student: Connor Lewick. Connor training was focused on cell culture techniques. How have the results been disseminated to communities of interest?Preliminary results of experiments performed during the development of objectives 1 and 2 were presented as abstracts (10) in national meetings. Two original papers and 2 reviews were pubilshed in Journal of Dairy Science (2), Journal of Animal Science (1) and Biotechnology, and PLoS One (1). 1. Strieder-Barboza C, de Souza J, Raphael W, Lock AL, Contreras GA. Fetuin-A: A negative acute-phase protein linked to adipose tissue function in periparturient dairy cows. J Dairy Sci. 2017 Dec 20. pii: S0022-0302(17)31184-0. doi: 10.3168/jds.2017-13644. [Epub ahead of print] PubMed PMID: 29274966. 2. Contreras GA, Strieder-Barboza C, De Koster J. Modulating adipose tissue lipolysis and remodeling to improve immune function during the transition period and early lactation of dairy cows. J Dairy Sci. 2017 Oct 25. pii: S0022-0302(17)30959-1. doi: 10.3168/jds.2017-13340. [Epub ahead of print] PubMed PMID: 29102145. 3. Contreras GA, Strieder-Barboza C, de Souza J, Gandy J, Mavangira V, Lock AL, Sordillo LM. Periparturient lipolysis and oxylipid biosynthesis in bovine adipose tissues. PLoS One. 2017 Dec 5;12(12):e0188621. doi: 10.1371/journal.pone.0188621. eCollection 2017. PubMed PMID: 29206843; PubMed Central PMCID: PMC5716552. 4. Contreras GA, Strieder-Barboza C, Raphael W. Adipose tissue lipolysis and remodeling during the transition period of dairy cows. J Anim Sci Biotechnol. 2017 May 5;8:41. doi: 10.1186/s40104-017-0174-4. eCollection 2017. Review. PubMed PMID: 28484594; PubMed Central PMCID: PMC5420123. Abstracts 1. Strieder-Barboza C, de Souza J, Lock AL, Contreras GA. Effect of Prepartum Adiposity and Lipolysis on Gestational and Postnatal Adipose Tissue Inflammation and Immune Cell Infiltration. Experimental Biology, April 22-26, 2017, Chicago IL. The FASEB Journal 31 (1 Supplement), 964.1-964.1. Poster 2. Contreras GA, Strieder-Barboza C, de Souza J, Gandy J, Lock AL, Sordillo LM. The Effect of Prepartum Adiposity and Lipolysis on Gestational and Postnatal Oxylipids Biosynthesis. Experimental Biology, April 22-26, 2017, Chicago IL. The FASEB Journal 31 (1 Supplement), 1b316-1b316. Poster 3. Contreras GA, Strieder-Barboza C, de Souza J, Gandy J, Lock AL, Sordillo LM. Prepartum Adiposity and Lipolysis alter Gestational and Postnatal Oxylipids Fourth Annual Lipids @ Wayne May 10th 2017, Detroit MI. Abstract 3. Poster 4. Contreras GA, Strieder-Barboza C, De Koster J. Modulating adipose tissue lipolysis and remodeling to improve immune function in early lactation. ADSA 2017, Pittsburgh, PA, J Dairy Sci. 2017 Dec;100 (Suppl 2): 179. Animal Health: Joint ADSA/NMC Symposium: Mastitis Control and Milk Quality Globally: Past, Present, and an Amazing Future. Invited Presentation 5. De Koster J, Strieder-Barboza C, de Souza J, James W, Lock AL, Contreras GA. Periparturient disease affects macrophage infiltration in subcutaneous adipose tissue of early lactation dairy cows. American Dairy Science Association Annual Meeting, June 25-28, 2017, Pittsburgh, PA, J Dairy Sci. 2017 Dec;100 (Suppl 2): LB8, iii. Oral 6. Strieder-Barboza C, de Souza J, Lock AL, Contreras GA. Fetuin-A as a marker of adipose tissue function in transition dairy cows. American Dairy Science Association Annual Meeting, June 25-28, 2017, Pittsburgh, PA, J Dairy Sci. 2017 Dec;100 (Suppl 2): 537, 214. Oral 7. Contreras GA, Strieder-Barboza C, Thelen K, de Souza J, De Koster J, Lock AL. Adipose tissue remodeling in transition dairy cows is affected by body condition score and lipolysis intensity. American Dairy Science Association Annual Meeting, June 25-28, 2017, Pittsburgh, PA, J Dairy Sci. 2017 Dec;100 (Suppl 2): M194, 76. Poster 9. Contreras GA, Raphael W. Nonesterified fatty acids induce proinflammatory macrophage phenotype. American Dairy Science Association Annual Meeting, June 25-28, 2017, Pittsburgh, PA, J Dairy Sci. 2017 Dec;100 (Suppl 2): T33, 238. Poster 10. Contreras GA, Strieder-Barboza C, de Souza J, Gandy J, Lock AL, Sordillo LM. The effect of body condition score and lipolysis intensity on the biosynthesis of oxylipids in periparturient dairy cows. American Dairy Science Association Annual Meeting, June 25-28, 2017, Pittsburgh, PA, J Dairy Sci. 2017 Dec;100 (Suppl 2): T161, 286. Poster What do you plan to do during the next reporting period to accomplish the goals?Objective 1: Data obtained from coculture experiments and are being analyzed. A publication is being prepared. Objective 2: Data on macrophage infiltration and phenotype are being analyzed. Two publications are being prepare. One focuses on lipolytic responses and a second one is focused on adipose tissue macrophage infiltration and phenotype polarization.
Impacts
What was accomplished under these goals?
Objective 1: Determine the mechanisms by which adipose tissue macrophage proinflammatory polarization during lipolysis reduces bovine adipocyte insulin sensitivity Major activities completed / experiments conducted Cooculture experiments: Adipocytes developed under a coculture model (optimized in the previous year) and adipose tissue explants were exposed to monocyte derived macrophages (protocol optimized in the previous year) that were polarized to M1 or M2 or remained untreated (M0). 2. Summary statistics and discussion of results Adipocytes and explants that were exposed to M1 had a greater response to lipolytic stimulus with a beta adrenergic agonist compared to those exposed to M2 or M0. These in vitro and ex vivo experiments demonstrate that excessive macrophage infiltration into adipose tissue and polarization towards M1 will enhance lipolytic responses in adipose tissues. Objective 2: Assess the impact of lipolysis inhibition on adipose tissue macrophage inflammatory phenotype and adipose insulin sensitivity in lactating dairy cows. 1. Major activities completed / experiments conducted Recent experiments by other groups (Havlin et al 2016,doi.org/10.1071/AN15419) demonstrated variabitlity in the effects of niacin supplementation to periparturient cows. To avoid the possibility of this variability in our experiments, we opted for using fatty acid supplementation during the transition period to minimize lipolytic responses while enhancing lipogenic activity in adipose tissues. 2. Data Collected Twenty-two multiparous (3.14 ± 1.28 lactations at the moment of selection, mean ± SD) Holstein dairy cows from the Michigan State University Dairy Teaching and Research Center (East Lansing, MI) were enrolled in this study. At the moment of selection, cows were non-lactating and pregnant (> 235 d of gestation). Cows were housed in tie-stall barns bedded with sawdust and fed a dry period TMR diet from 60 d to 21 d pre-partum, a close-up diet from 21 d up till calving and an early lactation diet following parturition. Samples of adipose tissue and plasma were collected at(-10d, DRY) and 11 ± 0.2 d (+10d, FRESH) and 24 ± 0.4 d (+24d, LACT). In this study we evaluated the effect of time relative to calving and FA supplementation on: a) insulin sensitivity of adipose tissues; b) lipolytic responses; c) macrophage infiltratoin and polarization in adipose tissues. 3. Summary statistics and discussion of results Our initial analysis focused on the effect of time on insulin sensitivity in adipose tissues. Using an explant model we found that basal lipolytic activity was higher in adipose explants at DRY compared with FRESH . Independent of time, explants with larger adipocytes demonstrated higher basal lipolytic activity. To evaluate the activity of hormone sensitive lipase (HSL) during the transition period, we used a chemical inhibitor (CAY10499, CAY) and found that HSL activity was very low in samples taken at DRY. In FRESH and LACT, CAY inhibited basal lipolytic activity 36.05 ± 4.51 % and 43.05 ± 4.83 %, respectively. We also report that insulin inhibited beta adrenergic agonist stimulated lipolytic activity and the inhibition was more pronounced for an insulin dose of 1 µg/L (late lactation levels) compared with 0.2 µg/L (FRESH level). Inhibition of stimulated lipolysis was more pronounced in DRY compared with the FRESH for both insulin doses. Size of the adipocytes had no influence on the inhibitory effect of insulin. We conclude that AT lipolytic responses during the transition period are characterized by a shift from adipose triglyceride lipase dependent basal lipolysis during positive energy balance (DRY) to HSL dependent basal lipolysis during negative energy balance (FRESH and LACT). The decrease in basal lipolytic activity and a broadly decreased transcriptional regulation of the lipogenic gene network suggests that decreased lipogenesis is an important contributor to FFA release from adipose tissues during early lactation. In addition, insulin resistance of the lipolytic activity develops during the early lactation period and is characterized by a decreased activation of AKT.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Strieder-Barboza C, de Souza J, Raphael W, Lock AL, Contreras GA. Fetuin-A: A negative acute-phase protein linked to adipose tissue function in periparturient dairy cows. J Dairy Sci. 2017 Dec 20. pii: S0022-0302(17)31184-0. doi: 10.3168/jds.2017-13644. [Epub ahead of print] PubMed PMID: 29274966.
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Contreras GA, Strieder-Barboza C, De Koster J. Modulating adipose tissue lipolysis and remodeling to improve immune function during the transition period and early lactation of dairy cows. J Dairy Sci. 2017 Oct 25. pii: S0022-0302(17)30959-1. doi: 10.3168/jds.2017-13340. [Epub ahead of print] PubMed PMID: 29102145.
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Contreras GA, Strieder-Barboza C, de Souza J, Gandy J, Mavangira V, Lock AL, Sordillo LM. Periparturient lipolysis and oxylipid biosynthesis in bovine adipose tissues. PLoS One. 2017 Dec 5;12(12):e0188621. doi: 10.1371/journal.pone.0188621. eCollection 2017. PubMed PMID: 29206843; PubMed Central PMCID: PMC5716552.
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Contreras GA, Strieder-Barboza C, Raphael W. Adipose tissue lipolysis and remodeling during the transition period of dairy cows. J Anim Sci Biotechnol. 2017 May 5;8:41. doi: 10.1186/s40104-017-0174-4. eCollection 2017.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2017
Citation:
Strieder-Barboza C, de Souza J, Lock AL, Contreras GA. Effect of Prepartum Adiposity and Lipolysis on Gestational and Postnatal Adipose Tissue Inflammation and Immune Cell Infiltration. Experimental Biology, April 22-26, 2017, Chicago IL. The FASEB Journal 31 (1 Supplement), 964.1-964.1.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2017
Citation:
Contreras GA, Strieder-Barboza C, de Souza J, Gandy J, Lock AL, Sordillo LM. The Effect of Prepartum Adiposity and Lipolysis on Gestational and Postnatal Oxylipids Biosynthesis. Experimental Biology, April 22-26, 2017, Chicago IL. The FASEB Journal 31 (1 Supplement), 1b316-1b316.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2017
Citation:
Contreras GA, Strieder-Barboza C, de Souza J, Gandy J, Lock AL, Sordillo LM. Prepartum Adiposity and Lipolysis alter Gestational and Postnatal Oxylipids Fourth Annual Lipids @ Wayne May 10th 2017, Detroit MI.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2017
Citation:
Contreras GA, Strieder-Barboza C, De Koster J. Modulating adipose tissue lipolysis and remodeling to improve immune function in early lactation. ADSA 2017, Pittsburgh, PA, J Dairy Sci. 2017 Dec;100 (Suppl 2): 179. Animal Health: Joint ADSA/NMC Symposium: Mastitis Control and Milk Quality Globally: Past, Present, and an Amazing Future
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2017
Citation:
De Koster J, Strieder-Barboza C, de Souza J, James W, Lock AL, Contreras GA. Periparturient disease affects macrophage infiltration in subcutaneous adipose tissue of early lactation dairy cows. American Dairy Science Association Annual Meeting, June 25-28, 2017, Pittsburgh, PA, J Dairy Sci. 2017 Dec;100 (Suppl 2): LB8, iii. Oral
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2017
Citation:
Strieder-Barboza C, de Souza J, Lock AL, Contreras GA. Fetuin-A as a marker of adipose tissue function in transition dairy cows. American Dairy Science Association Annual Meeting, June 25-28, 2017, Pittsburgh, PA, J Dairy Sci. 2017 Dec;100 (Suppl 2): 537, 214.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2017
Citation:
Contreras GA, Strieder-Barboza C, Thelen K, de Souza J, De Koster J, Lock AL. Adipose tissue remodeling in transition dairy cows is affected by body condition score and lipolysis intensity. American Dairy Science Association Annual Meeting, June 25-28, 2017, Pittsburgh, PA, J Dairy Sci. 2017 Dec;100 (Suppl 2): M194, 76.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2017
Citation:
Contreras GA, Raphael W. Nonesterified fatty acids induce proinflammatory macrophage phenotype. American Dairy Science Association Annual Meeting, June 25-28, 2017, Pittsburgh, PA, J Dairy Sci. 2017 Dec;100 (Suppl 2): T33, 238.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2017
Citation:
Contreras GA, Strieder-Barboza C, de Souza J, Gandy J, Lock AL, Sordillo LM. The effect of body condition score and lipolysis intensity on the biosynthesis of oxylipids in periparturient dairy cows. American Dairy Science Association Annual Meeting, June 25-28, 2017, Pittsburgh, PA, J Dairy Sci. 2017 Dec;100 (Suppl 2): T161, 286.
|
Progress 04/15/16 to 04/14/17
Outputs
Target Audience:Nutritionists, veterinarians, animal scientists, professional students (veterinary medicine), and undergraduate students (animal sciences and physiology) Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Postdoctoral scientists: W. Raphael BVSc, MS, PhD. Worked in the project from March 2015 until July 2016. Dr. Raphael conducted the monocyte / macrophage experiments developing isolation and polarization protocols. Rahul Nelli BVSc & AH, MVM, PhD. was hired in November 2016. Dr. Nelli is completing coculture experiments (macrophage/adipocytes). Jenne De Koster, DVM, PhD. was hired in January 2017. Dr. De Koster work focuses on in vivo studies evaluating adipose tissue remodeling and lipolysis inhibition. Graduate Students: Clarissa Streider Barboza (PhD candidate) and Kyan Thelen (Masters candidate). Students were trained on qPCR, flow cytometry, ELISA, and Western blotting techniques. Their dissertation and thesis research objectives will support in part the development of the major goals of this project. Professional Student: Sarah Hammar (DVM candidate). Sarah is being trained in surgical procedures for adipose tissue collection and in evaluation of lipolytic responses in adipocytes and adipose tissue explants. Undergraduate Students: Connor Lewick and Whitney James. Their training was focused on cell culture techniques. How have the results been disseminated to communities of interest?Preliminary results of experiments performed during the development of objectives 1 and 2 were presented as abstracts in national meetings. A publication was accepted for publication in Journal of Dairy Science and two reviews on adipose tissue biology are being reviewed for publication in the Journal of Dairy Science and the Journal of Animal Science and Biotechnology. 1. Contreras GA, Thelen K, Schmidt SE, Strieder-Barboza C, Preseault CL, Raphael W, Kiupel M, Caron J, Lock AL. Adipose tissue remodeling in late-lactation dairy cows during feed-restriction-induced negative energy balance. J Dairy Sci. 2016 Dec;99(12):10009-10021. doi: 10.3168/jds.2016-11552. PubMed PMID: 27720147. 2. Strieder-Barboza C, Raphael W, Schmidt SE, Lock AL, Sordillo LM, Contreras GA. Fetuin-A: A novel biomarker for lipolysis-induced metabolic stress in transition dairy cows. ADSA 2016, Salt Lake City, UT, J. Anim Sci. Vol. 94 No. supplement 5, p. 352-353. Third place PhD students oral presentation ADSA 2016. 3. Schmidt SE, Thelen K, Preseault CL, Contreras GA, Lock AL. Feed restriction-induced negative energy balance alters the fatty acid profiles of adipose tissue and milk fat of dairy cows. ADSA 2016, Salt Lake City, UT, J. Anim Sci. Vol. 94 No. supplement 5, p. 353-554. 4. Schmidt SE, Thelen K, Contreras GA, Lock AL. Effects of-10,-12 conjugated linoleic acid on gene expression and lipid content of adipocytes derived from lactating dairy cows. ADSA 2016, Salt Lake City, UT, J. Anim Sci. Vol. 94 No. supplement 5, p. 370-371. 5. Raphael W. Contreras GA. Bovine macrophage phenotype influences inflammatory response to lipopolysaccharide. ADSA 2016, Salt Lake City, UT, J. Anim Sci. Vol. 94 No. supplement 5, p. 85-86. 6. Contreras GA. Adipose Tissue Metabolism in Relation to Transition Cow Disease. American College of Veterinary Internal Medicine Forum 2016, Denver, CO, June 11, 2016 What do you plan to do during the next reporting period to accomplish the goals?Objective 1. We will test the effect of lipolysis and its inhibition on macrophage phenotype using the optimized flow cytometry protocols that we developed during the previous reporting period. We are also going to evaluate the effect of macrophage phenotype on the responses of adipocytes to insulin signaling including a functional assay for fatty acid uptake. This assay was adapted and optimized for ruminant adipocytes during the present reporting period. Objective 2. We will evaluate the effects of lipolysis modulation during the periparturient period and early lactation on adipose tissue remodeling, adipocyte insulin signaling, and macrophage phenotype.
Impacts
What was accomplished under these goals?
Objective 1. Determine the mechanisms by which adipose tissue macrophage proinflammatory polarization during lipolysis reduces bovine adipocyte insulin sensitivity. Major activities completed / experiments conducted Optimization of adipocyte culture protocol: We have improved standard adipogenic protocols by including a supplementation of a fatty acid mixture that mimics the composition of the NEFA fraction in circultating plasma during the first week of lactation. With this improved protocol we obtain adipocytes the have a very similar fatty acid compostion to that of adipose tissues in vivo. This assures that the coculture experiments (adipocyte/macrophage) proposed in this specific aim will replicate in vivo conditions very closely. Optimization of flow cytometry protocol for bovine macrophages: Using antibodies against surface markers CD14, CD16, CD38, CD163, and CD206 and intracellular protein CD68 we are able to categorize polarization towards the M1 or M2 phenotype in macrophages cultured in vitro or harvested from stromal vascular cells from adipose tissue biopsies. This protocol will assure an adequate characterization of macrophage phenotype in the coculture experiments (adipocyte/macrophage) proposed in this specific aim. Fatty acid uptake assay: Lipid uptake by adipocytes is reduced by insulin signaling dysregulation. To evaluate adipocyte function during coculture with ATM we adapted a fatty acid uptake assay used in adipocytes from monogastric animals. This assay uses a fluorescent fatty acid that emits signal when is incorporated into the cell. Optimization of the protocol required fine tuning of seeding density, serum deprivation times, and passage of the cells prior to the assay. Data collection in progress Coculture experiments are being performed. Monocyte derived macrophages are cultured for 24 hours with conditioned media (ACM) from adipocytes stimulated with 10M of isoproterenol to induce lipolysis. The effect of ACM on macrophage phenotype is being evaluated by flow cytometry and gene expression profiling. Objective 2: Assess the impact of lipolysis inhibition on adipose tissue macrophage inflammatory phenotype and adipose insulin sensitivity in lactating dairy cows. Major activities completed / experiments conducted Experiments performed in the previous year of this project demonstrated that using a model of caloric restriction in late-lactation dairy cows that induced a NEB similar to that observed in transition dairy cows induced adipose tissue macrophage (ATM) infiltration in adipose tissue (AT) but with only minimal changes in inflammatory phenotype. These results underscore the need for evaluating the effects of physiological changes related to parturition and the onset of lactation or extended periods of lipolysis (or both) on ATM inflammatory phenotype and their impact on AT insulin sensitivity. Therefore, it was necessary to evaluated these changes in periparturient dairy cows before performing experiments with lipolysis inhibitors. A group of periparturient dairy cows was followed during the periparturient period from dry off until 3 weeks into lactation. An evaluation of adipose tissue remodeling in AT including immune cell trafficking was performed and the effect of body condition score (BCS) and lipolysis intensity was assessed. Data collected Blood and subcutaneous AT samples were collected from multiparous Holstein cows with high (HB; n=12; BCS>3.75) or moderate (MB; n=9; BCS<3.5) BCS at 27±7 (FO) and 10±5 (CU) d prepartum and at 8±3 (PP) d postpartum. Expression of genes related to AT remodeling was analyzed by RT-PCR, and immune cell trafficking in AT SVF was evaluated by flow cytometry. 3. Summary statistics and discussion of results Lipolysis increased at CU and reached its peak at PP compared to FO as reflected in circulating free fatty acid (FFA) concentrations (FO: 0.27±0.05, CU: 0.39±0.05, PP:0.99±0.05 mEq/L, P<0.05). FFA were higher in HB (0.63±0.02 mEq/L) compared to MB (0.47±0.02 mEq/L) cows reflecting an effect of BCS on lipolysis rate during gestation and after parturition. Gene expression indicated that osteopontin (SPP1), an inflammatory cytokine that triggers macrophage infiltration during AT remodeling, its receptor CD44, and signal regulatory protein α (SIRPA), a marker of mononuclear immune cells, were higher at PP compared with FO and CU. Serum FFA concentrations were positively associated with AT expression of SIRPA (r=0.59; P<0.001), and negatively associated with expression of IL10 (r=-0.54; P<0.001), an anti-inflammatory cytokine. Immune cell trafficking showed that BCS had no effect on the expression of macrophage markers CD14, CD16, and CD163. However, compared to MB, HB had fewer SVF cells expressing T cell markers CD8, CD4, and CD3, and B cells (all P<0.01). Collectively these data indicate that during the transition period, lipolysis induces macrophage infiltration into AT by enhancing the expression of chemoattractant adipokines independently of prepartum BCS.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
Contreras GA, Thelen K, Schmidt SE, Strieder-Barboza C, Preseault CL, Raphael W, Kiupel M, Caron J, Lock AL. Adipose tissue remodeling in late-lactation dairy cows during feed-restriction-induced negative energy balance. J Dairy Sci. 2016 Dec;99(12):10009-10021. doi: 10.3168/jds.2016-11552. PubMed PMID: 27720147
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2016
Citation:
Raphael W. Contreras GA. Bovine macrophage phenotype influences inflammatory response to lipopolysaccharide. ADSA 2016, Salt Lake City, UT, J. Anim Sci. Vol. 94 No. supplement 5, p. 85-86
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2016
Citation:
Schmidt SE, Thelen K, Contreras GA, Lock AL. Effects of-10,-12 conjugated linoleic acid on gene expression and lipid content of adipocytes derived from lactating dairy cows. ADSA 2016, Salt Lake City, UT, J. Anim Sci. Vol. 94 No. supplement 5, p. 370-371.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2016
Citation:
Schmidt SE, *1Thelen K, Preseault CL, Contreras GA, Lock AL. Feed restriction-induced negative energy balance alters the fatty acid profiles of adipose tissue and milk fat of dairy cows. ADSA 2016, Salt Lake City, UT, J. Anim Sci. Vol. 94 No. supplement 5, p. 353-554.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2016
Citation:
Contreras GA. Adipose Tissue Metabolism in Relation to Transition Cow Disease. American College of Veterinary Internal Medicine Forum 2016, Denver, CO, June 11, 2016
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2016
Citation:
Strieder-Barboza C, Raphael W, Schmidt SE, Lock AL, Sordillo LM, Contreras GA. Fetuin-A: A novel biomarker for lipolysis-induced metabolic stress in transition dairy cows. ADSA 2016, Salt Lake City, UT, J. Anim Sci. Vol. 94 No. supplement 5, p. 352-353.
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Progress 04/15/15 to 04/14/16
Outputs
Target Audience:Target audiences: Nutritionists, veterinarians, animal scientists, professional students (veterinary medicine), and undergraduate students (animal sciences and physiology). Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Postdoctoral scientist: W. Raphael BVSc, MS, PhD. was hired in March 2015 and conducted the monocyte / macrophage experiments. Graduate Students: Clarissa Streider Barboza (PhD candidate) and Kyan Thelen (Masters candidate). Students were trained on qPCR, flow cytometry, ELISA, and Western blotting techniques. Their dissertation and thesis research objectives will support in part the development of the major goals of this project. Professional Student: Situnyiwe Chirunga Thembi (DVM candidate). Thembi was trained on cell culture and collaborated on the development of macrophage polarization protocols. Undergraduate Student: Connor Lewick. Connor training was focused on cell culture techniques. How have the results been disseminated to communities of interest?Preliminary results of experiments performed during the development of objectives 1 and 2 were presented as abstracts in national meetings. A publication was accepted for publication in Journal of Dairy Science. Contreras GA, Kabara ED, Brester J, Neuder L, Kiupel M. Macrophage infiltration in the omental and subcutaneous adipose tissues of dairy cows with displaced abomasum. 2015. J Dairy Sci. 98;9;6176-87 Thelen L, Preseault C, Schmidt S, Caron J, Lock AL, Contreras GA. Adipose Tissue Macrophages Infiltration During Negative Energy Balance Tri-State Dairy Nutrition Conference. Fort Wayne IN. In Press. First Place Undergraduate Oral Presentation. Contreras GA, Thelen K, Raphael W, Caron J, Preseault CL, Schmidt SE, Lock AL. Lipolysis induces adipose tissue macrophage infiltration in lactating dairy cows. Poster presentation at American Dairy Science Association 2015 Annual Meeting. Chirunga S, Raphael W, Contreras GA. In vitro polarization of bovine macrophages: determining the impact of adipocyte lipolysis on macrophage phenotype in dairy cattle. Merial-NIH National Veterinary Scholars Symposium, Davis CA, 2015, pp 115. Poster presentation. Strieder-Barboza C, Contreras GA. Fetuin-A Dynamics in Transition Cows Plasma. AABP, 50th Annual Convention Proceedings, New Orleans, LA, September 17-19, 2015. In press. Oral presentation. What do you plan to do during the next reporting period to accomplish the goals?Objective 1. Having defined the phenotypes of classical and alternative macrophages, the next logical set of experiments will evaluate potential effects that macrophages and adipocytes have on each other during the transition period. To emulate in vivo transition cow conditions, macrophages will be cultured in media conditioned to adipocytes undergoing lipolysis. The impact of adipocyte lipolysis on macrophage phenotype will be measured by qPCR, flow cytometry, and Western blot. Based on our recent published results, we expect lipolysis to polarize macrophage phenotype similar to classical phenotype macrophages stimulated with interferon-?. The final experiments for objective 1 will measure non-esterified fatty acid release from cultured adipocytes in response to polarized macrophages, and the impact of insulin on macrophage-induced lipolysis. Objective 2. Using the minimally invasive techniques developed during year 1. We plan to evaluate key markers of adipose tissue remodeling at the gene, protein, and functional level in transition dairy cows.
Impacts
What was accomplished under these goals?
Objective 1: Determine the mechanisms by which adipose tissue macrophage proinflammatory polarization during lipolysis reduces bovine adipocyte insulin sensitivity 1. Major activities completed / experiments conducted Protocols were developed for monocyte isolation from whole blood of cows, and differentiation of monocytes in vitro into classical and alternative phenotype macrophages. Several features of the in vitro culture system were evaluated in order to optimize monocyte differentiation, including plate adhesion, duration of culture, and growth media composition. At the same time, methods were developed for successful isolation of pre-adipocytes from stromal vascular cells in bovine adipose tissue and differentiation of pre-adipocytes into adipocytes. 2. Data collected Classical and alternative macrophage phenotypes were induced using cytokines (interferon-gamma, interleukins-4 and 13) and defined using gene and protein expression profiles measured by qPCR, flow cytometry, Western blots and novel chromatographic methods of protein separation (WES by Protein Simple). Efficiency of qPCR was verified for a comprehensive panel of genes involved with lipogenesis, lipolysis, and inflammation. Detection and quantification of proteins were validated using WES and Western blot techniques. 3. Summary statistics and discussion of results Monocytes differentiated into macrophages after 48 hours of culture, featuring visible dendrites (light microscopy) and increased CD68 expression (n = 3). Classical macrophage phenotype was defined as enhanced IL6, TNF, and CD16 expression with interferon-gamma stimulation (n = 8). Alternative macrophage phenotype was defined as abrogated IL6 and IL10 expression with LPS stimulation and interleukin-4 and 13 priming. Objective 2: Assess the impact of lipolysis inhibition on adipose tissue macrophage inflammatory phenotype and adipose insulin sensitivity in lactating dairy cows. 1. Major activities completed / experiments conducted Before evaluating anti-lipolytic agents in vivo, it is necessary to first characterize adipose tissue remodeling as a result of lipolysis and/or periparturient pysiological adapatations. To address this, a protocol for induction of negative energy balance (NEB) in mid lactation dairy cows was developed and tested to emulate NEB observed in transition dairy cattle. Adipose tissue remodeling as a result of NEB induced lipolysis was evaluated independently of confounding factors related to calving and the onset of lactation. 2. Data collected Multiparous mid lactation cows (DIM 119-210) were fed a common diet (d-14 to -1) and then randomly assigned to one of two 4 d (d1 to 4) feeding protocols: FR=feed restricted, AL=ad libitum. Cows in this feeding protocol reached a NEB of -13.5±1.9 Mcal/d and exhibit a strong lipolytic response. Blood, urine, and milk were collected on d -7, -3 and -2, and then daily on d1-d4. Plasma total NEFA concentrations were measured. Omental (OM) and subcutaneous (SC) adipose tissue samples were collected on d -3 and d4 using minimally invasive surgical techniques. Immediately after harvesting, adipose tissues were aliquoted for further processing including RNA extraction, immunohistochemistry, flow cytometry, and protein analysis 3. Summary statistics and discussion of results FR group showed a lipolytic response characterized by a marked increase in plasma NEFA concentrations. By d4, NEFA in FR animals was 0.52 mEq/L compared to 0.17 mEq/L in cows fed ad libitum (P<0.01). Lipolytic activity was demonstrated in OM and SC adipose depots by increased phosphorylation of hormone sensitive lipase. FR drastically reduced the concentrations of plasma insulin, however circulating glucose concentrations remained unaltered. In response to 4 days of caloric restriction, OM and SC significantly reduced the expression of lipogenic genes Fasn, Scd1, and Elovl6, lipolytic gene Abhd5, and tended to inhibit the transcription of the gene encoding ATGL (Pnpla2). At the same time FR tended to increase the expression of fatty acid transporter Fabp4. FR had no effect on the transcription of Lipe that encodes HSL and the adipogenic regulator gene Pparg. NEB reduces milk production and alters composition. Production and composition values at d4 were calculated and compared to those obtained on d-1. As expected by d4 of limiting feed intake in the FR group, milk yield, energy corrected milk, and fat corrected milk were reduced. In contrast FR increased fat yield significantly and tended to elevate milk fat concentration. Remarkably, cows with higher ECM at d-1 exhibited a stronger increase in fat yield and milk fat concentration. Reducing caloric intake negatively affected milk protein yield and tended to reduce MUN, however no effects were observed in milk protein content. Similarly, lactose and total solids yield and concentration were significantly reduced in FR cows. NEB alters ATM trafficking but does not affect their phenotype. RT-qPCR analysis of OM and SC revealed that the expression of classical macrophages (M1) related gene TNFa tended to be upregulated by FR (P=0.06). In contrast, there was no effect of caloric restriction on other M1 related pro-inflammatory genes including CCL2, and ITGAX. Transcription of alternative macrophage (M2) related anti-inflammatory genes Arg1 and Il10 was numerically higher in FR cows but not significant (P=0.11 and P=0.22 respectively). No FR or site effectts were observed in the expression of M2 related gene CCL22. The expression of SIRPA, a marker of mononuclear, dendritic, and granulocytes was significantly decreased by FR and its expression was lower in OM of both AL and FR cows. Flow cytometry data indicates that FR increased the number of cells expressing CD172a, a mononuclear cell marker, in OM but not in SC. More B cells were detected in OM than in SC however there was no FR effect. When analyzing tissue sections, we detected a higher number of cells immuno reactive to CD172a in OM of FR cows compared to SC in the same group of cows and to OM and SC in the AL group. ECM gene expression varies by AT site but is not affected by NEB. Expression of extracellular matrix protein genes Col1a1 and Col6a2 was higher in SC than OM, however was not affected by caloric restriction. In contrast, the expression of THBS1 was similar in both sites and remain unaffected after FR. Finally, no effect of caloric restriction was observed on the expression of cell cycle genes Ccna2, Ccnb2, and Mki67, indicating minimal mitogenic activity. In line with this finding, no changes were observed in the expression of SPP1 (encoding osteopontin) and its receptor CD44 as a result of caloric restriction or anatomical location.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Contreras GA, Kabara ED, Brester J, Neuder L, Kiupel M. Macrophage infiltration in the omental and subcutaneous adipose tissues of dairy cows with displaced abomasum. 2015. J Dairy Sci. 98;9;6176-87
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2015
Citation:
Contreras GA, Thelen K, Raphael W, Caron J, Preseault CL, Schmidt SE, Lock AL. Lipolysis induces adipose tissue macrophage infiltration in lactating dairy cows. 2015. J. Dairy Sci. 98(Suppl. 2):707.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2015
Citation:
Chirunga S, Raphael W, Contreras GA. In vitro polarization of bovine macrophages: determining the impact of adipocyte lipolysis on macrophage phenotype in dairy cattle. Merial-NIH National Veterinary Scholars Symposium, Davis CA, July 30 - August 2, 2015, pp 115.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2015
Citation:
Barboza C, Contreras GA. Fetuin-A Dynamics in Transition Cows Plasma. AABP, 50th Annual Convention Proceedings, New Orleans, LA, September 17-19, 2015. In press.
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