Recipient Organization
PURDUE UNIVERSITY
(N/A)
WEST LAFAYETTE,IN 47907
Performing Department
(N/A)
Non Technical Summary
Ketosis is a well-known metabolic disorder ofdairy cows characterized by the inability of cows to cope with the excessive negative energy balancehappening soon after parturition. Clinically ketotic cows havesignificant milk production losses andan increased risk to suffer from other detrimental health conditions. It is estimated that 5 to 15% of dairy cows in the U.S. sufferfrom the condition, totalingU$110-330million in annual losses to the dairy industry.The involvement of a dysregulated skeletal muscle metabolismin the etiology ofketosis in dairy cows remains unknown.This project willdelineate mechanisms by which skeletal musclemay help modulate whole-body energy metabolism and homeostasis. Identifying the involvement of skeletal musclein the etiology of ketosis will be fundamental for the development of innovative nutritional and therapeutic interventions to prevent metabolic disorders in dairy cowswhile improving animal production and welfare.The goals are to identify the effects of ketosis on skeletal muscletranscriptome and to characterize changes in tissue oxidative stress, triglyceride content, intramyocellular fat abundance, and fatty acid oxidative capacity. In addition, we will characterize how 2 major lipokinesthat are associated with health in postpartum cowsimpact the transcriptional profileand fatty acid metabolism of differentiated bovine myotubes in vitro. We expect to demonstrate the role of skeletal muscle fatty acid metabolism in the overall metabolic fitness of postpartum dairy cows and in the etiology of ketosis. Results of this study will address a significantknowledge gap in dairy sciencesand allow furtherinvestigations ofnovelstrategiesin the modulation of excessive negative energy balanceof postpartum cows.
Animal Health Component
10%
Research Effort Categories
Basic
90%
Applied
10%
Developmental
(N/A)
Goals / Objectives
The goals of this projectare to identify the effects of ketosis on skeletal muscletranscriptome and to characterize changes in tissue oxidative stress, triglyceride content, intramyocellular fat abundance, and fatty acid oxidative capacity. In addition, we will characterize how 2 lipokines that are associated with health in postpartum cows (based on our preliminary data) impact the transcriptional profile and fatty acid metabolism of differentiated bovine myotubes in vitro. Our central hypothesis is that skeletal muscledysfunction is a key component in the etiology of ketosis characterized by a signature muscle transcriptional profile change, and linked to a lower concentration of adipose tissue lipokines that promote fatty acid uptake and oxidation in myotubes.
Project Methods
For the first aimof this study, multiparous Holstein dairy cows from 1 large commercial farmwill be used. Clinical ketosis will be defined ascows with a blood BHB concentration ≥3.0 mmol/Lthat are detected in the first 7 days-in-milk (DIM), accompanied by a decrease in milk production. For each clinical ketosis case, a control cow of the same parity and within 1 ± 1 DIM of the ketosis case will be enrolled. Healthy cows will be defined as cows with a blood BHB <1.2 mmol/L, without any other clinical abnormalities in a physical exam. Muscle and subcutaneous adipose tissue samples will be aseptically collected from the paralumbar fossa via surgical biopsy. One muscle aliquot will be immersed in isopentane after harvesting, snap frozen, and stored at-80o C for cryostat sectioning, Oil Red O (ORO) staining, and intramyocellular fat estimation. All other samples will be snap-frozen in liquid nitrogen after harvesting and storedat-80o C until further analyses. One SM aliquot will be submitted to GENEWIZ (Azenta Life Sciences, Chelmsford, MA) for isolation of total RNA and next-generation sequencing using their standard RNA-Seq Data Analysis Package program. Sequencing will be performed using an Illumina HiSeq 4000 (Illumina, San Diego, CA). At least 50 million 150-bp paired-end reads will be sequenced for each sample. Sequence quality will be assessed using FastQC and quality trimming will be performed using Trim Galore (v 0.6.7) to trim adapters and reads; only reads with a quality cutoff of Phred33 above 30 and of at least 75 reads will be retained. Quality controlled reads will be mapped against the Bos taurus reference genome using STAR. STAR-derived mapping results and annotation (GTF/GFF) file for reference genome will be used as input for the HTSeq packageto obtain the read counts for each gene feature for each sample. Counts from all replicates will be then merged using custom Perl scripts to generate a read count matrix for all samples. Differentially expressed genes between ketotic and healthy cows will be performed using DESeq2, and used to execute GO and KEGG enrichment analysis using clusterProfiler. The top 10 differentially expressed genes will be verified via real-time quantitative PCR analysis. Muscle triglyceride and fatty acid analysis will be performed at the National Mouse Metabolic Phenotyping Center, University of Cincinnati Medical Center. Lipid extraction will be performed following the Folch method, and fatty acid analysis undertaken using gas chromatography. SM triglyceride will be quantified using a colorimetric assay (GOP-PAP method; Randox Laboratories). Super-resolution microscopy for high-resolution intracellular imaging and image processing for ORO intensity analysis will be performed at the Purdue Imaging Facility, Bindley Bioscience Center. Muscle oxidative stress (OS) will be assessed by the direct measurement of hydroperoxides using a commercially available kit (Item #: 705003; Cayman Chemical, MI) following manufacturer's recommendation.Muscle fatty acid beta-oxidation will be measured in tissue extract using a commercial kit (Cat #: E-141; Biomedical Research Service, State University of New York).For the second study aim,muscle satellite cell-derived primary myoblasts will be isolated from the external oblique muscle collected from prepubertal dairy heifers following a standard protocol utilized in the PD's laboratory.To investigate the impact of 9,10- and 12,13-DiHOME on the bovine muscle transcriptome, bovine myoblasts will be seeded at 0.3 x 106 cells per well into Matrigel-coated 6-well plates on day 1. Myoblasts will be differentiated into myotubes for 3 days. After, cells will be treated with 1.75 mM of 9,10- or 12,13-diHOME (to resemble plasma concentration) or vehicle control (methyl acetate) and incubated for 18 h at 37oC, 5% CO2. Cells will be maintained in DMEM media containing 2% horse serum and 1% P/S as to prevent potential myotube delamination during the experiment. After 18 h, cells will be harvested, snap-frozen, and submitted to GENEWIZ for RNA-Seq analysis, and will follow analyses similarly as described in aim1. Myotube fatty acid uptake will be assessed by using a validated real-time uptake kinetics assay of a dodecanoic acid fluorescent fatty acid substrate (QBT Fatty Acid Uptake Assay Kit, Molecular Devices Corp.). The capacity of 12,13-diHOME and 9,10-diHOME in increasing fatty acid oxidation of bovine myotubes will be investigated using the XF Palmitate-BSA FAO substrate and Cell Mito Stress Test by monitoring mitochondrial respiration rates (i.e., oxidation of exogenous palmitate) in a Seahorse XF24 (Agilent Technologies). Concentrations of sc adipose tissue 9,10- and 12,13-diHOME will be analyzed by liquid chromatography-tandem mass spectrometry. Adipose tissue oxylipid extraction will follow the same method as described in Contreras et al. (2017). Briefly, adipose tissue will be weighed for normalization, homogenized in a solution containing 500 µL methanol, 2 µL antioxidant reducing agent containing (50% methanol, 25% ethanol, and 25% HPLC-grade water with 0.9 mM butylated hydroxy toluene, 0.54 mM EDTA, 3.2 mM triphenylphosphine, and 5.6 mM indomethacin), and 15 µL of deuterated internal standards (Catalog numbers: 10009993 and 10009994; Cayman Chemical). Samples will be centrifuged at 14,000 x g at 4oC for 5 min and the supernatant collected. Measurements of 9.10- and 12,13-diHOME will follow a similar protocol used in our preliminary study using an Agilent 6470 QQQ LC/MS/MS.Statistical analyses will be performed in SAS v9.4. RNA-Seq data analysis has been described previously. Linear mixed models will be built to determine the effect of ketosis (ketosis/healthy) on muscle total triglyceride content, intramyocellular adipose abundance, oxidative stress, and fatty acid oxidation activity (Aim 1), and 9,10- and 12,13-diHOME adipose concentrations (Aim 2). Cow will be included as a random effect in all models. Model residuals will be assessed for homoscedasticity and variable transformations performed if needed. Linear mixed models will be used to assess the effect of treatment (9,10- or 12,13-diHOME versus controls) for fatty acid oxidative capacity. Repeated measures model will be used to assess fatty acid uptake as previously described; different covariance structures (e.g., AR1) will be tested and the one yielding the lowest model AIC used.