Progress 05/15/19 to 05/14/22
Outputs
Target Audience:Veterinarians -The knowledge gained can be used by veterinarians in understanding, diagnosing and treating the development of fatty liver in transition dairy cows. Feed consults - The knowledge gained can be used in understanding and developing nutritional interventions for the prevention and treatment of metabolic disease in dairy cattle and assessment of life cycle feeding strategies. Nutrition researchers - the basic knowledge gained for this work provides a platform for a greater understanding of metabolism and metabolic disease to build out models of the interaction of genes and nutrients in controlling metabolism Entrepreneurs - the basic concepts gain can be used by entrepreneurs in developing products and processes that reduce incidence of metabolic disease and/or boost productive efficiency of milk production and improve animal health and well-being. Changes/Problems:Delays during COVID and access issues to use of animals and laboratory faciities as well as supply chain issues in restarting research. No technical issues were experienced that delayed progress. What opportunities for training and professional development has the project provided?Training and research experiences for 6 undergraduate studentsthat has contributed to their degree completion and interests in pursuing postgraduate studies. Experience and training for graduate students, postdoctoral fellows, and research technicians. Expanded use of stable isotope tracer technology to the study of nutrition and metabolism in domestic livestock and link to gene expression profiles. How have the results been disseminated to communities of interest?Peer reviewed publications Scientific meetings Extension conferences What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Impacts of postruminal supply of palm oil or flaxseed oil in conjunction with propionate or acetate on hepatic PC and PCK1 expression in lactating dairy cows. This study utilized four cannulated early-to-mid lactation dairy cows to assess the effect of prolonged postruminal infusion of either palm oil or flaxseed oil in combination with sodium acetate or sodium propionate on PC and PCK gene expression. We hypothesized that the palm oil would decrease the expression of PC and that propionate would induce PCK expression, but when paired with palm oil the expected increase in expression would be negligible. The data indicate that palm oil infusion did not affect the plasma fatty acid profiles or PC or PCK1 expression, or the ratio of PC to PCK1 in liver. Conversely flaxseed oil infusion elevated α-linolenic acid in blood (8X control levels) but there was no impact on PC or PCK1 expression or the ratio of PC to PCK1. Taken together the studies indicate that the capacity exists to elevate α-linolenic acid, a recognized modulator of PC in vitro, but that does not translate to a change in the expression of PC, PCK or their ratio in lactating cows in vivo. The lack of effect on the PC and PCK may be a consequence of high levels of other circulating fatty acids, from diet and de novo synthesis, and the lack of a metabolic stress on gluconeogenesis and hepatic energy metabolism. Feeding milk fat, flaxseed oil, soy oil, or the combination of milk fat and oil to pre-ruminating dairy calves and the impact on liver metabolic flux To minimize the influencing effects of dietary fatty acids and rumen modification of fatty acid in vivo we employed a pre-ruminating calf model of metabolism to explore the effects of changes in dietary fatty acid on PC and PCK expression and metabolic flux. Forty 14 day old male dairy calves were allocated to receive a milk based diet containing either: milk fat, soy oil, flaxseed oil, milk fat + soy oil, or milk fat + flaxseed oil for 7 days. Liver biopsies collected after 7 days of feeding and used in explant culture. The data revealed an increased flux through phosphoenolpyruvate (PEPCK) for the MF treatments compared to MF-Soy and Soy, but no difference in flux through PEPCK when MF is compared to MF-Flax or Flax. Interestingly, when propionate was the tracer, flux through PC was increased due to Flax compared to MF-Flax and MF alone, but was not different from MF-Soy or Soy. Oxidation to carbon dioxide and acid soluble products were not affected by the diets. The data indicate that milk fat (high in palmitic acid), soy oil (high in oleic acid), and flaxseed oil (high in α-linolenic acid) impact the flux through PC and PCK differently. The data indicate that the activity of PC and PCK are sensitive to shifts in dietary fatty acid supply in cattle at least in cases where the dietary differences in fatty acids are extreme. Knockdown of pyruvate carboxylase in Madin Darby Bovine Kidney cells alters PCK2 expression and metabolic flux. In order to study the direct effects of changes in PC and PCK on metabolic flux we generated a pyruvate carboxylase gene knockdown model Madin Darby bovine kidney cells using short hairpin RNA. These cells were validated by gene and protein expression analysis, and it was confirmed that PC gene expression and protein expression was knocked down. We also generated PC overexpression cells, and PC overexpression was confirmed with both gene and protein expression analysis. PC gene expression was increased 9 times and protein expression was increased. We then used the knockdown and overexpression cells in metabolic flux experiments where we incubated the cells in stably labeled propionate and pyruvate and radiolabeled propionate, lactate, and palmitic acid. We also collected cells for further gene expression analysis to determine if other genes, like PCK, change due to changes in PC. We found that when PC is knocked down that PCK2 expression is equivalent to PCK2 expression in PC overexpression cells. We also found that there was less than half the amount of carbon dioxide and acid soluble products from palmitic acid oxidation from the PC knockdown cells compared to the PC overexpression cells. Overall we have determined that the ratio of PC to PCK is an important determinant of nutrient flux in energy metabolismin liver thatis impacted by nutritional and physiological statusin dairy cattle.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2022
Citation:
Beckett, L.M., S. S. Donkin, and T. Casey. 2022. Response of hepatic gluconeogenic flux and transcriptome to circadian rhythm disruption. J Dairy Sci 105, Suppl. 1, 168.
- Type:
Journal Articles
Status:
Submitted
Year Published:
2022
Citation:
Beckett, L.M., S. S. Donkin, and T. Casey.2022. Circadian Disruption Decreases Gluconeogenic Flux in Late Gestation, Non-Lactating Dairy Cows. J Dairy Science (submitted)
|
Progress 05/15/20 to 05/14/21
Outputs
Target Audience:Veterinarians Feed consults Nutrition researchers Entrepreneurs Changes/Problems:COVID-19 slowed progress on the project. What opportunities for training and professional development has the project provided?1 graduate student, 1 postdoc, and 1 technician were trained on cell culture, metabolic flux measures, and molecular biology techniques. All individuals participated in professional development through online conferences. How have the results been disseminated to communities of interest?Yes in journal publication and abstract form What do you plan to do during the next reporting period to accomplish the goals?Continue to examine the role of PC and PCK expression on metabolite flux in MDBK cells and in vivo. Devlop knockdown and overexpression models of PCK1 and PCK2. Develop an overexpression model of PC. Test the effects of major shifts in dietary fatty acid profiles on liver metabolism in ruminants using neonatal calves as an experimental model.
Impacts
What was accomplished under these goals?
In order to determine the role of PC in directing carbon flux through the TCA cycle and impact of specific changes in PC expression on metabolic flux Madin-Darby bovine kidney epithelial cells (MDBK) were cultured and selected to contain shRNA for pyruvate carboxylase (PC) or a non-targeting DNA sequence (scramble control, SCRM). Following cell selection and induction of shRNA the PC diminished (PCd) cells and control cells (PCn) were incubated with 1.0 mM U-[13C] lactate or 2.0 mM U-[13C] propionate for 3 h. Cells and media were separated and cell homogenates were extracted, dried, subjected to derivatization, and analyzed by GC-MS analysis to generate the mass isotopomer distributions (MID) for each metabolite in the TCA cycle and ancillary reactions. Experiments were repeated in 3 separate cell preparations and triplicate wells. Mass isotopomer distribution analysis (MIDA) was performed for MID using METRAN software for 13C-metabolic flux and statistical analysis. METRAN is based on the modeling framework called Elementary Metabolite Units (EMU) developed by Maciek Antoniewicz. The PC knockdown cells resulted in a 70% reduction in PC expression based on Western blot analysis. Knockdown of PC appears to alter metabolism of U-[13C] lactate to PEP, triose phosphates (DHAP), aspartate, asparagine, and glutamine. Knockdown of PC appears to alter metabolism of U-[13C] propionate to triose phosphates (DHAP), aspartate, asparagine, glutamine, glutamate, malate, and citrate but not PEP or glutamine. Further analysis and modeling is pending but the data are pointing to an impact of PC knockdown that is dependent on the point of entry to the gluconeogenesis pathway.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Beckett, L., J. Thimmapuram, S. Xie, T. Casey, and S. S. Donkin. 2020. Mammary gland transcriptome profiling reveals an abundance of transcripts for cell maintenance and protein turnover. J. Dairy Sci. 103:21
Teeple, K., A. Suarez-Trujillo, C. McCabe , J. Townsend, S. Donkin, K. Plaut, J. Boerman, and T. Casey. 2020. Disruption of circadian clocks in the prepartum dry period negatively affect mammary development. J. Dairy Sci. 103: 21.
Boesche, K.E. , S. S. Donkin. 2020. Pretreatment with saturated and unsaturated fatty acids regulates fatty acid oxidation in Madin-Darby bovine kidney cells. J. Dairy Sci. 103: 8841-8852.
Boesche, K. E., and S. S. Donkin. 2020. Bovine Pyruvate Carboxylase Gene Proximal Promoter Activity is Regulated by Saturated and Unsaturated Fatty Acids in Madin-Darby Bovine Kidney Cells. J. Dairy Sci. (accepted).
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2021
Citation:
Pyruvate carboxylase knockdown alters lactate oxidation in Madin-Darby bovine kidney cells. L. M. Beckett*, J. Laguna, S. Hilger, and S. S. Donkin, Purdue University, West Lafayette, IN. J Dairy Sci. 104, Suppl. 1:301.
|
Progress 05/15/19 to 05/14/20
Outputs
Target Audience:Scientists, Professors, Teachers, Veterinarians Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Graduate student trained in using GC-MS and computer modeling of metabolism. Technical support trained on cell transformation and gen silencing. Graduate student trained on pathway analysis and use of Metran modeling software and Matlab to map flux coefficientsand changes due to alterations in expression ratio of PC to PCK1. 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?Continue work on usingknockout cell models. Analyze in vivo data forexpression ofpyruvate carboxylase (PC) and phosphoenolpyruvate carboxykinase (PCK) and effects on metabolic flux. Complete analysis of stable isotope tracer kinetics and model usingMetran to determine best fit parameters to explain the role of the ration of PC to PCK1 in healthy productive cows compared to animals experiencing metabolic disease (fatty liver, ketosi?s)
Impacts
What was accomplished under these goals?
Analytical methods for analysis of metabolic flux using stable isotopes were established. We have successfully established procedures for positional isotopomer analysis of TCA intermediates and have begun to generate models for in vivo metabolic flux when the ratio of PC and PCK1 differs due to nutritional or physiological status. We have completed objectives to augment PC expression using postruminal fatty acid infusions (Linolenic acid) and PCK1 expression using propionate. Flux analysis for liver biopsy samples collected during these experiments was used for stable isotopomer analysis. Knockout cell lines for bovinepyruvate carboxylase (PC) and metabolized by phosphoenolpyruvate carboxykinase (PCK) were established and verified. We have measured the flux of propionate and lactate using stable isotope tracers in these cells lines and controls. Finding for oxidation confirm a role of PC in determining overall flux of lactate but not propionate carbonto CO2.
Publications
|
|