Differential Gene Expression Analysis of Bovine Macrophages after Exposure to the Penicillium Mycotoxins Citrinin and/or Ochratoxin A.

Mycotoxins produced by fungal species commonly contaminate livestock feedstuffs, jeopardizing their health and diminishing production. Citrinin (CIT) and ochratoxin A (OTA) are mycotoxins produced by Penicillium spp. and commonly co-occur. Both CIT and OTA can modulate immune response by inhibiting cell proliferation and differentiation, altering cell metabolism, and triggering programmed cell death. The objective of this study was to determine the effects of sublethal exposure (i.e., the concentration that inhibited cell proliferation by 25% (IC25)) to CIT, OTA or CIT + OTA on the bovine macrophage transcriptome. Gene expression was determined using the Affymetrix Bovine Genome Array. After 6 h of exposure to CIT, OTA or CIT + OTA, the number of differentially expressed genes (DEG), respectively, was as follows: 1471 genes (822 up-regulated, 649 down-regulated), 5094 genes (2611 up-regulated, 2483 down-regulated) and 7624 genes (3984 up-regulated, 3640 down-regulated). Of these, 179 genes (88 up-regulated, 91 down-regulated) were commonly expressed between treatments. After 24 h of exposure to CIT, OTA or CIT + OTA the number of DEG, respectively, was as follows: 3230 genes (1631 up-regulated, 1599 down-regulated), 8558 genes (4167 up-regulated, 4391 down-regulated), and 10,927 genes (6284 up-regulated, 4643 down-regulated). Of these, 770 genes (247 up-regulated, 523 down-regulated) were commonly expressed between treatments. The categorization of common biological functions and pathway analysis suggests that the IC25 of both CIT and OTA, or their combination, induces cellular oxidative stress, a slowing of cell cycle progression, and apoptosis. Collectively, these effects contribute to inhibiting bovine macrophage proliferation.

Effects of a docosahexaenoic acid-rich microalgae nutritional product on insulin sensitivity after prolonged dexamethasone treatment in healthy mature horses.

OBJECTIVE: To determine effects of a microalgae nutritional product on insulin sensitivity in horses. ANIMALS: 8 healthy mature horses. PROCEDURES :Horses (n = 4/group) received a basal diet without (control diet) or with docosahexaenoic acid-rich microalgae meal (150 g/d) for 49 days (day 0 = first day of diet). On day 28, an isoglycemic hyperinsulinemic clamp procedure was performed. Horses then received dexamethasone (0.04 mg/kg/d) for 21 days. On day 49, the clamp procedure was repeated. After a 60-day washout, horses received the alternate diet, and procedures were repeated. Plasma fatty acid, glucose, and insulin concentrations and glucose and insulin dynamics during the clamp procedure were measured on days 28 and 49. Two estimates of insulin sensitivity (reciprocal of the square root of the insulin concentration and the modified insulin-to-glucose ratio for ponies) were calculated. RESULTS: Baseline glucose and insulin concentrations or measures of insulin sensitivity on day 28 did not differ between horses when fed the control diet or the basal diet plus microalgae meal. On day 49 (ie, after dexamethasone administration), the microalgae meal was associated with lower baseline insulin and glucose concentrations and an improved modified insulin-to-glucose ratio for ponies, compared with results for the control diet. CONCLUSIONS AND CLINICAL RELEVANCE: Although the microalgae meal had no effect on clamp variables following dexamethasone treatment, it was associated with improved plasma glucose and insulin concentrations and insulin sensitivity estimates. A role for microalgae in the nutritional management of insulin-resistant horses warrants investigation.

Stress and inflammatory gene networks in bovine liver are altered by plane of dietary energy during late pregnancy.

The prepartal dietary energy level is tightly correlated with the degree of tissue mobilization that the animal experiences around parturition (giving birth). To better understand the link between the dry period dietary energy management and the inflammatory status around parturition, 12 multiparous Holstein cows were fed for the entire dry period either a high-wheat straw/lower-energy diet to supply at least 100% of the calculated net energy for lactation (NEL) (control, CON) or a higher-energy diet to supply >140% of NEL (overfed, OVE). The blood was sampled throughout the transition period for biomarker analyses. Liver tissue samples were taken on days -14, 7, 14, and 30 relative to parturition for triacylglycerol (TAG) composition and gene expression analysis. Fifty genes involved in inflammation, endoplasmic reticulum (ER), and oxidative stress, and cell cycle and growth were evaluated. Although blood biomarkers did not reveal signs of a greater inflammatory status compared with OVE, CON cows had a greater activation of the intrahepatic unfolded protein response prepartum. However, postpartum mRNA profiling indicated that the OVE group experienced a mild but sustained level of ER stress, with higher oxidative stress and impairment of antioxidant mechanisms. After parturition, inflammation-related genes were upregulated in OVE cows compared with CON. However, CON cows experienced a gradual increase in expression of key inflammatory transcription regulators up to 30 days postpartum which agreed with the lower plasma albumin and cholesterol, suggesting an inflammatory state. Data underscored that ER stress is not necessarily linked with inflammation during the peripartal period. Gene expression data also suggest that prepartum overnutrition could have negative effects on normal cell cycle activity. Overall, allowing cows to overconsume energy prepartum increased the hepatic pro-inflammatory response prepartum and up to the point of parturition. Subsequently, cows fed the lower-energy diet experienced a gradual increase in the inflammatory response. The lack of differences between groups in voluntary feed intake and lactation capacity suggests that nutritional management prepartum triggers different mechanisms that affect ER and oxidative stress along with inflammation. Although no clinical disorders were detected, these alterations expose animals to the development of immuno-metabolic disorders.

Acute mammary and liver transcriptome responses after an intramammary Escherichia coli lipopolysaccharide challenge in postpartal dairy cows.

The study investigated the effect of an intramammary lipopolysaccharide (LPS) challenge on the bovine mammary and liver transcriptome and its consequences on metabolic biomarkers and liver tissue composition. At 7 days of lactation, 7 cows served as controls (CTR) and 7 cows (LPS) received an intramammary Escherichia coli LPS challenge. The mammary and liver tissues for transcriptomic profiling were biopsied at 2.5 h from challenge. Liver composition was evaluated at 2.5 h and 7 days after challenge, and blood biomarkers were analyzed at 2, 3, 7 and 14 days from challenge. In mammary tissue, the LPS challenge resulted in 189 differentially expressed genes (DEG), with 20 down-regulated and 169 up-regulated. In liver tissue, there were 107 DEG in LPS compared with CTR with 42 down-regulated and 65 up-regulated. In mammary, bioinformatics analysis highlighted that LPS led to activation of NOD-like receptor signaling, Toll-like receptor signaling, RIG-I-like receptor signaling and apoptosis pathways. In liver, LPS resulted in an overall inhibition of fatty acid elongation in mitochondria and activation of the p53 signaling pathway. The LPS challenge induced changes in liver lipid composition, a systemic inflammation (rise of blood ceruloplasmin and bilirubin), and an increase in body fat mobilization. The data suggest that cells within the inflamed mammary gland respond by activating mechanisms of pathogen recognition. However, in the liver the response likely depends on mediators originating from the udder that affect liver functionality and specifically fatty acid metabolism (ß-oxidation, ketogenesis, and lipoprotein synthesis).

Overfeeding Dairy Cattle During Late-Pregnancy Alters Hepatic PPARα-Regulated Pathways Including Hepatokines: Impact on Metabolism and Peripheral Insulin Sensitivity.

Hepatic metabolic gene networks were studied in dairy cattle fed control (CON, 1.34 Mcal/kg) or higher energy (overfed (OVE), 1.62 Mcal/kg) diets during the last 45 days of pregnancy. A total of 57 target genes encompassing PPARα-targets/co-regulators, hepatokines, growth hormone (GH)/insulin-like growth factor 1 (IGF-1) axis, lipogenesis, and lipoprotein metabolism were evaluated on -14, 7, 14, and 30 days around parturition. OVE versus CON cows were in more negative energy balance (NEB) postpartum and had greater serum non-esterified fatty acids (NEFA), ß-hydroxybutyrate (BHBA), and liver triacylglycerol (TAG) concentrations. Milk synthesis rate did not differ. Liver from OVE cows responded to postpartal NEB by up-regulating expression of PPARα-targets in the fatty acid oxidation and ketogenesis pathways, along with gluconeogenic genes. Hepatokines (fibroblast growth factor 21 (FGF21), angiopoietin-like 4 (ANGPTL4)) and apolipoprotein A-V (APOA5) were up-regulated postpartum to a greater extent in OVE than CON. OVE led to greater blood insulin prepartum, lower NEFA:insulin, and greater lipogenic gene expression suggesting insulin sensitivity was not impaired. A lack of change in APOB, MTTP, and PNPLA3 coupled with upregulation of PLIN2 postpartum in cows fed OVE contributed to TAG accumulation. Postpartal responses in NEFA and FGF21 with OVE support a role of this hepatokine in diminishing adipose insulin sensitivity.

Bioinformatics analysis of transcriptome dynamics during growth in angus cattle longissimus muscle.

Transcriptome dynamics in the longissimus muscle (LM) of young Angus cattle were evaluated at 0, 60, 120, and 220 days from early-weaning. Bioinformatic analysis was performed using the dynamic impact approach (DIA) by means of Kyoto Encyclopedia of Genes and Genomes (KEGG) and Database for Annotation, Visualization and Integrated Discovery (DAVID) databases. Between 0 to 120 days (growing phase) most of the highly-impacted pathways (eg, ascorbate and aldarate metabolism, drug metabolism, cytochrome P450 and Retinol metabolism) were inhibited. The phase between 120 to 220 days (finishing phase) was characterized by the most striking differences with 3,784 differentially expressed genes (DEGs). Analysis of those DEGs revealed that the most impacted KEGG canonical pathway was glycosylphosphatidylinositol (GPI)-anchor biosynthesis, which was inhibited. Furthermore, inhibition of calpastatin and activation of tyrosine aminotransferase ubiquitination at 220 days promotes proteasomal degradation, while the concurrent activation of ribosomal proteins promotes protein synthesis. Therefore, the balance of these processes likely results in a steady-state of protein turnover during the finishing phase. Results underscore the importance of transcriptome dynamics in LM during growth.

Bioinformatics and Gene Network Analyses of the Swine Mammary Gland Transcriptome during Late Gestation.

We used the newly-developed Dynamic Impact Approach (DIA) and gene network analysis to study the sow mammary transcriptome at 80, 100, and 110 days of pregnancy. A swine oligoarray with 13,290 inserts was used for transcriptome profiling. An ANOVA with false discovery rate (FDR < 0.15) correction resulted in 1,409 genes with a significant time effect across time comparisons. The DIA uncovered that Fatty acid biosynthesis, Interleukin-4 receptor binding, Galactose metabolism, and mTOR signaling were among the most-impacted pathways. IL-4 receptor binding, ABC transporters, cytokine-cytokine receptor interaction, and Jak-STAT signaling were markedly activated at 110 days compared with 80 and 100 days. Epigenetic and transcription factor regulatory mechanisms appear important in coordinating the final stages of mammary development during pregnancy. Network analysis revealed a crucial role for TP53, ARNT2, E2F4, and PPARG. The bioinformatics analyses revealed a number of pathways and functions that perform an irreplaceable role during late gestation to farrowing.

Functional and gene network analyses of transcriptional signatures characterizing pre-weaned bovine mammary parenchyma or fat pad uncovered novel inter-tissue signaling networks during development.

BACKGROUND: The neonatal bovine mammary fat pad (MFP) surrounding the mammary parenchyma (PAR) is thought to exert proliferative effects on the PAR through secretion of local modulators of growth induced by systemic hormones. We used bioinformatics to characterize transcriptomics differences between PAR and MFP from approximately 65 d old Holstein heifers. Data were mined to uncover potential crosstalk through the analyses of signaling molecules preferentially expressed in one tissue relative to the other. RESULTS: Over 9,000 differentially expressed genes (DEG; False discovery rate or=1.5-fold difference between PAR and MFP. Within the DEG highly-expressed in PAR vs. MFP (n = 736) we noted significant enrichment of functions related to cell cycle, structural organization, signaling, and DNA/RNA metabolism. Only actin cytoskeletal signaling was significant among canonical pathways. DEG more highly-expressed in MFP vs. PAR (n = 742) belong to lipid metabolism, signaling, cell movement, and immune-related functions. Canonical pathways associated with metabolism and signaling, particularly immune- and metabolism-related were significantly-enriched. Network analysis uncovered a central role of MYC, TP53, and CTNNB1 in controlling expression of DEG highly-expressed in PAR vs. MFP. Similar analysis suggested a central role for PPARG, KLF2, EGR2, and EPAS1 in regulating expression of more highly-expressed DEG in MFP vs. PAR. Gene network analyses revealed putative inter-tissue crosstalk between cytokines and growth factors preferentially expressed in one tissue (e.g., ANGPTL1, SPP1, IL1B in PAR vs. MFP; ADIPOQ, IL13, FGF2, LEP in MFP vs. PAR) with DEG preferentially expressed in the other tissue, particularly transcription factors or pathways (e.g., MYC, TP53, and actin cytoskeletal signaling in PAR vs. MFP; PPARG and LXR/RXR Signaling in MFP vs. PAR). CONCLUSIONS: Functional analyses underscored a reciprocal influence in determining the biological features of MFP and PAR during neonatal development. This was exemplified by the potential effect that the signaling molecules (cytokines, growth factors) released preferentially (i.e., more highly-expressed) by PAR or MFP could have on molecular functions or signaling pathways enriched in the MFP or PAR. These bidirectional interactions might be required to coordinate mammary tissue development under normal circumstances or in response to nutrition.

High-starch diets induce precocious adipogenic gene network up-regulation in longissimus lumborum of early-weaned Angus cattle.

Adipocyte differentiation is probably controlled by transcriptional and post-transcriptional regulation. Longissimus lumborum from Angus steers (aged 155 d; seven animals per diet) fed high-starch or low-starch diets for 112 d (growing phase) followed by a common high-starch diet for an additional 112 d (finishing phase) was biopsied at 0, 56, 112 and 224 d for transcript profiling via quantitative PCR of twenty genes associated with adipogenesis and energy metabolism. At 56 d steers fed high starch had greater expression of PPARgamma as well as the lipogenic enzymes ATP citrate lyase (ACLY), glucose-6-phosphate dehydrogenase (G6PD), fatty acid synthase (FASN), fatty acid binding protein 4 (FABP4), stearoyl-CoA desaturase (SCD), glycerol-3-phosphate acyltransferase, mitochondrial (GPAM), and diacylglycerol O-acyltransferase homologue 2 (DGAT2), and the adipokine adiponectin (ADIPOQ). Expression of insulin-induced gene 1 (INSIG1) was also greater with high starch at 56 d. Steers fed low starch experienced a marked increase in FASN, FABP4, SCD, DGAT2 and thyroid hormone-responsive (SPOT14 homologue, rat) (THRSP) between 56 and 112 d of feeding. A greater expression of the transcription factors sterol regulatory element-binding transcription factor 1 (SREBF1) and MLX interacting protein-like (MLXIPL) was observed at 224 d in steers fed high starch, suggesting a nutritional imprinting effect. Carryover effects of low starch feeding were discerned by greater expression at 224 d of THRSP, FABP4, SCD and DGAT2. These steers also had greater PPARgamma at 224 d. Despite these responses, low starch led to greater expression at 224 d of nuclear receptor subfamily 2, group F, member 2 (NR2F2), a known repressor of rodent adipocyte differentiation through its negative effects on PPARgamma, ADIPOQ and FABP4. Results suggested that early exposure to high starch induced precocious intramuscular adipocyte proliferation and metabolic imprinting of lipogenic transcription regulators. Low starch might have blunted the PPARgamma-driven adipogenic response through up-regulation of NR2F2 but the endogenous ligand for this nuclear receptor remains unknown.

Adipogenic and energy metabolism gene networks in longissimus lumborum during rapid post-weaning growth in Angus and Angus x Simmental cattle fed high-starch or low-starch diets.

BACKGROUND: Transcriptional networks coordinate adipocyte differentiation and energy metabolism in rodents. The level of fiber and starch in diets with adequate energy content fed to young cattle has the potential to alter intramuscular adipose tissue development in skeletal muscle. Post-weaning alterations in gene expression networks driving adipogenesis, lipid filling, and intracellular energy metabolism provide a means to evaluate long-term effects of nutrition on longissimus muscle development across cattle types. RESULTS: Longissimus lumborum (LL) from Angus (n = 6) and Angus x Simmental (A x S; n = 6) steer calves (155 +/- 10 days age) fed isonitrogenous high-starch (HiS; 1.43 Mcal/kg diet dry matter; n = 6) or low-starch (LoS; 1.19 Mcal/kg diet dry matter; n = 6) diets was biopsied at 0, 56, and 112 days of feeding for transcript profiling of 31 genes associated with aspects of adipogenesis and energy metabolism. Intake of dietary energy (9.44 +/- 0.57 Mcal/d) across groups during the study did not differ but feed efficiency (weight gain/feed intake) during the first 56 days was greater for steers fed HiS. Expression of PPARG increased ca. 2-fold by day 56 primarily due to HiS in A x S steers. Several potential PPARG-target genes (e.g., ACACA, FASN, FABP4, SCD) increased 2.5-to-25-fold by day 56 across all groups, with responses (e.g., FASN, FABP4) being less pronounced in A x S steers fed LoS. This latter group of steers had markedly greater blood plasma glucose (0.99 vs. 0.79 g/L) and insulin (2.95 vs. 1.17 microg/L) by day 112, all of which were suggestive of insulin resistance. Interactions were observed for FABP4, FASN, GPAM, SCD, and DGAT2, such that feeding A x S steers high-starch and Angus steers low-starch resulted in greater fold-changes by day 56 or 112 (GPAM). Marked up-regulation of INSIG1 (4-to-8-fold) occurred throughout the study across all groups. SREBF1 expression, however, was only greater on day 112 namely due to LoS in A x S steers. The lipogenic transcription factor THRSP was 6-to-60-fold greater by day 56 primarily due to HiS in A x S steers, constituting the greatest response among all genes. CONCLUSION: Results involving gene markers of mature adipocytes (e.g., PPARG, THRSP, SCD) provided evidence of intramuscular adipose tissue differentiation during the early portion of the growing phase. The resulting gene networks underscored a central role for PPARG in controlling transcription of genes which are known to co-ordinately regulate adipocyte differentiation and lipid filling in non-ruminants. Unlike rodents, INSIG1 appears to play an important role in cattle muscle adipogenesis. We propose that a network of transcription regulators and nuclear receptors including PPARG-target genes, INSIG1, and THRSP, coordinate activation of adipocyte differentiation and lipid filling at an early age.

Gene expression ratio stability evaluation in prepubertal bovine mammary tissue from calves fed different milk replacers reveals novel internal controls for quantitative polymerase chain reaction.

Prepubertal mammary development can be affected by nutrition partly through alterations in gene network expression. Quantitative PCR (qPCR) remains the most accurate method to measure mRNA expression but is subject to analytical errors that introduce variation. Thus, qPCR data normalization through the use of internal control genes (ICG) is required. The objective of this study was to mine microarray data (> 10,000 genes) from prepubertal mammary parenchyma and stroma to identify the most suitable ICG for normalization of qPCR. Tissue for RNA extraction was obtained from calves ( approximately 63 d old; n = 5/diet) fed a control (200 g/kg crude protein, 210 g/kg crude fat, fed at 441 g/d dry matter) or a high-protein milk replacer (280 g/kg crude protein, 200 g/kg crude fat, fed at 951 g/d dry matter). ICG were selected based on both absence of expression variation across treatment and of coregulation (gene network analysis). Genes evaluated were ubiquitously expressed transcript, protein phosphatase 1 regulatory (inhibitor) subunit 11 (PPP1R11), matrix metallopeptidase 14 (MMP14), ClpB caseinolytic peptidase B, SAPS domain family member 1 (SAPS1), mitochondrial GTPase 1 (MTG1), mitochondrial ribosomal protein L39, ribosomal protein S15a (RPS15A), and actin beta (ACTB). Network analysis demonstrated that MMP14 and ACTB are coregulated by v-myc myelocytomatosis viral oncogene, tumor protein p53, and potentially insulin-like growth factor 1. Pairwise comparison of expression ratios showed that ACTB, MMP14, and SAPS1 had the lowest stability and were unsuitable as ICG. PPP1R11, RPS15A, and MTG1 were the most stable among ICG tested. We conclude that the geometric mean of PPP1R11, RPS15A, and MTG1 is ideal for normalization of qPCR data in prepubertal bovine mammary tissue. This study provides a list of candidate ICG that could be used by researchers working in bovine mammary development and allied fields.