Gene networks driving bovine milk fat synthesis during the lactation cycle

<p>Abstract</p> <p>Background</p> <p>The molecular events associated with regulation of milk fat synthesis in the bovine mammary gland remain largely unknown. Our objective was to study mammary tissue mRNA expression via quantitative PCR of 45 genes associated with lipid synthesis (triacylglycerol and phospholipids) and secretion from the late pre-partum/non-lactating period through the end of subsequent lactation. mRNA expression was coupled with milk fatty acid (FA) composition and calculated indexes of FA desaturation and <it>de novo </it>synthesis by the mammary gland.</p> <p>Results</p> <p>Marked up-regulation and/or % relative mRNA abundance during lactation were observed for genes associated with mammary FA uptake from blood (<it>LPL</it>, <it>CD36</it>), intracellular FA trafficking (<it>FABP3</it>), long-chain (<it>ACSL1</it>) and short-chain (<it>ACSS2</it>) intracellular FA activation, <it>de novo </it>FA synthesis (<it>ACACA</it>, <it>FASN</it>), desaturation (<it>SCD</it>, <it>FADS1</it>), triacylglycerol synthesis (<it>AGPAT6</it>, <it>GPAM</it>, <it>LPIN1</it>), lipid droplet formation (<it>BTN1A1</it>, <it>XDH</it>), ketone body utilization (<it>BDH1</it>), and transcription regulation (<it>INSIG1</it>, <it>PPARG</it>, <it>PPARGC1A</it>). Change in <it>SREBF1 </it>mRNA expression during lactation, thought to be central for milk fat synthesis regulation, was ≤2-fold in magnitude, while expression of <it>INSIG1</it>, which negatively regulates SREBP activation, was >12-fold and had a parallel pattern of expression to <it>PPARGC1A</it>. Genes involved in phospholipid synthesis had moderate up-regulation in expression and % relative mRNA abundance. The mRNA abundance and up-regulation in expression of <it>ABCG2 </it>during lactation was markedly high, suggesting a biological role of this gene in milk synthesis/secretion. Weak correlations were observed between both milk FA composition and desaturase indexes (i.e., apparent SCD activity) with mRNA expression pattern of genes measured.</p> <p>Conclusion</p> <p>A network of genes participates in coordinating milk fat synthesis and secretion. Results challenge the proposal that <it>SREBF1 </it>is central for milk fat synthesis regulation and highlight a pivotal role for a concerted action among <it>PPARG</it>, <it>PPARGC1A</it>, and <it>INSIG1</it>. Expression of <it>SCD</it>, the most abundant gene measured, appears to be key during milk fat synthesis. The lack of correlation between gene expression and calculated desaturase indexes does not support their use to infer mRNA expression or enzyme activity (e.g., <it>SCD</it>). Longitudinal mRNA expression allowed development of transcriptional regulation networks and an updated model of milk fat synthesis regulation.</p

Gene Networks Driving Bovine Mammary Protein Synthesis During the Lactation Cycle

A crucial role for both insulin and mTOR in the regulation of milk protein synthesis is emerging. Bovine mammary biopsies harvested during late-pregnancy through end of subsequent lactation were used to evaluate via quantitative PCR the expression of 44 genes involved in pathways of insulin, mTOR, AMPK, and Jak2-Stat5 signalling and also glucose and amino acid (AA) transporters. We observed an increased expression during lactation of ELF5, AA and glucose transporters, insulin signaling pathway components, MAPK14, FRAP1, EIF4EBP2, GSK3A and TSC1 among mTOR signaling-related genes. Among ribosomal components RPL22 was down-regulated. The overall data support a central role of AA and glucose transporters and insulin signaling through mTOR for the regulation of protein synthesis in bovine mammary gland. Furthermore, the existence of translational competition favoring the translation of milk protein transcripts was inferred from the combined dataset

Plasma paraoxonase, health, inflammatory conditions, and liver function in transition dairy cows.

Paraoxonase (PON) is a liver protein with hydrolase activity that is released into the blood stream. Paraoxonase may serve as an index of liver function because it is drastically reduced in chronic liver damage. Sixty-seven periparturient dairy cows were used to evaluate the relationship between plasma PON, health problems, inflammatory conditions, and liver function. Baseline plasma PON concentrations during the first 30 d in milk (DIM) were retrospectively used to group cows into quartiles. Metabolic profile, lipid metabolites (e.g., nonesterified fatty acids, beta-hydroxybutyrate), inflammatory indices (haptoglobin, ceruloplasmin), low and high density lipoprotein cholesterol, vitamin A, vitamin E, reactive oxygen metabolites, total antioxidants, and PON in plasma were measured 2 wk before to 8 wk after calving. Weekly milk yield, body condition score, and all health problems were recorded. After parturition (7 DIM), cows in the lower PON group had the lowest plasma concentrations of negative acute phase proteins compared with the higher PON group for retinol binding protein (23.2 +/- 2.86 vs. 36.0 +/- 2.96 microg/dL of vitamin A), albumin (31.6 +/- 0.73 vs. 33.9 +/- 0.75 g/L), total cholesterol (2.04 +/- 0.30 vs. 2.45 +/- 0.42 mmol/L), and the highest concentrations of haptoglobin (0.67 vs. 0.24 +/- 0.03 g/L; positive acute phase protein) and globulins (37.2 vs. 32.3 +/- 1.4 g/L). Plasma bilirubin was highest in the cows (10.1 vs. 6.2 +/- 0.6 micromol/L) in the lowest PON quartile. Plasma PON was negatively correlated with haptoglobin (r = -0.39) and bilirubin (r = -0.42) and positively correlated with retinol binding protein (r = 0.54), albumin (r = 0.38), and cholesterol (r = 0.55) fractions. A total of 82.3% of cows in the lower quartile and no cows in the upper quartile experienced serious inflammation. Lower quartile cows produced 28.1 +/- 10.3 kg of milk/d; whereas upper quartile cows produced 38.3 +/- 7.7 kg of milk/d during the first 30 DIM. A reduction in the ability of the liver to cope with the increased metabolic demand near parturition in dairy cows can be diagnosed using changes in baseline plasma PON

In vitro–In vivo Hybrid Approach for Studying Modulation of NRF2 in Immortalized Bovine Mammary Cells

Nuclear factor erythroid 2-related factor 2 (NRF2) plays a key role in the response to oxidative stress. Diets containing known NRF2 modulators could be used to minimize oxidative stress in dairy cows. Currently, studies evaluating the activity of NRF2 in bovine have used the classical in vitro approach using synthetic media, which is very different than in vivo conditions. Furthermore, studies carried out in vivo cannot capture the short-term and dynamic response of NRF2. Thus, there is a need to develop new approaches to study NRF2 modulation. The aim of the present study was to establish an in vitro–in vivo hybrid system to investigate activation of NRF2 in bovine cells that can serve as an intermediate model with results closer to what is expected in vivo. To accomplish the aim, we used a combination of a gene reporter assay in immortalized bovine mammary cells, synthetic NRF2 modulators, and blood serum from periparturient cows. Synthetic agonist tert-butylhydroquinone and sulforaphane confirmed to be effective activators of bovine NRF2 with acute and large effect at 30 and 5 μM, respectively, with null response after the above doses due to cytotoxicity. When the agonists were added to blood serum the response was more linear with maximum activation of NRF2 at 100 and 30 μM, respectively, and the cytotoxicity was prevented. High concentration of albumin in blood serum plays an important role in such an effect. Brusatol (100 nM) was observed to be an effective NRF2 inhibitor while also displaying general protein synthesis inhibition and cytotoxicity when added to synthetic media. A consistent inhibition of NRF2 was observed when brusatol was added to the blood serum but the cytotoxicity was reduced. The synthetic inhibitor ML385 had no effect on modulation of bovine NRF2. Hydrogen peroxide activates NRF2 in bovine mammary cells starting from 100 μM; however, strong cytotoxicity was detected starting at 250 μM when cells were cultivated in the synthetic media, while blood serum prevented cytotoxicity. Overall, our data indicated that the use of synthetic media can be misleading in the study of NRF2 in bovine and the use of blood serum appears necessary

Peroxisome proliferator-activated receptor / does not regulate glucose uptake and lactose synthesis in bovine mammary epithelial cells cultivated in vitro

The hypothesis of the study was that inhibition of PPAR/ increases glucose uptake and lactose synthesis in bovine mammary epithelial cells by reducing the expression of the glucose transporter mRNA destabiliser calreticulin. Three experiments were conducted to test the hypothesis using immortalised bovine mammary alveolar (MACT) and primary bovine mammary (PBMC) cells. In Experiment 1, the most effective dose to inhibit PPAR/ activity among two synthetic antagonists (GSK-3787 and PT-s58) was assessed using a gene reporter assay. In Experiment 2, the effect on glucose uptake and lactose synthesis was evaluated by measuring glucose and lactose in the media and expression of related key genes upon modulation of PPAR/ using GSK-3787, the synthetic PPAR/ agonist GW-501516, or a combination of the two in cells cultivated in plastic. In Experiment 3, the same treatments were applied to cells cultivated in Matrigel and glucose and lactose in media were measured. In Experiment 1 it was determined that a significant inhibition of PPAR/ in the presence or absence of fetal bovine serum was achieved with 1000 nm GSK-3787 but no significant inhibition was observed with PT-s58. In Experiment 2, inhibition of PPAR/ had no effect on glucose uptake and lactose synthesis but they were both increased by GW-501516 in PBMC. The mRNA abundance of PPAR/ target gene pyruvate dehydrogenase kinase 4 was increased but transcription of calreticulin was decreased (only in MACT cells) by GW-501516. Treatment with GSK-3787 did not affect the transcription of measured genes. No effects on glucose uptake or lactose synthesis were detected by modulation of PPAR/ activity on cells cultivated in Matrigel. The above data do not provide support for the original hypothesis and suggest that PPAR/ does not play a major role in glucose uptake and lactose synthesis in bovine mammary epithelial cells

Nutrigenomic effect of saturated and unsaturated long chain fatty acids on lipid-related genes in goat mammary epithelial cells:What is the role of PPARγ?

A prior study in bovine mammary (MACT) cells indicated that long-chain fatty acids (LCFA) C16:0 and C18:0, but not unsaturated LCFA, control transcription of milk fat-related genes partly via the activation of peroxisome proliferator-activated receptor gamma (PPAR&gamma;). However, in that study, the activation of PPAR&gamma; by LCFA was not demonstrated but only inferred. Prior data support a lower response of PPAR&gamma; to agonists in goat mammary cells compared to bovine mammary cells. The present study aimed to examine the hypothesis that LCFA alter the mRNA abundance of lipogenic genes in goat mammary epithelial cells (GMEC) at least in part via PPAR&gamma;. Triplicate cultures of GMEC were treated with a PPAR&gamma; agonist (rosiglitazone), a PPAR&gamma; inhibitor (GW9662), several LCFA (C16:0, C18:0, t10,c12-CLA, DHA, and EPA), or a combination of GW9662 with each LCFA. Transcription of 28 genes involved in milk fat synthesis was measured using RT-qPCR. The data indicated that a few measured genes were targets of PPAR&gamma; in GMEC (SCD1, FASN, and NR1H3) while more genes required a basal activation of PPAR&gamma; to be transcribed (e.g., LPIN1, FABP3, LPL, and PPARG). Among the tested LCFA, C16:0 had the strongest effect on upregulating transcription of measured genes followed by C18:0; however, for the latter most of the effect was via the activation of PPAR&gamma;. Unsaturated LCFA downregulated transcription of measured genes, with a lesser effect by t10,c12-CLA and a stronger effect by DHA and EPA; however, a basal activation of PPAR&gamma; was essential for the effect of t10,c12-CLA while the activation of PPAR&gamma; blocked the effect of DHA. The transcriptomic effect of EPA was independent from the activation of PPAR&gamma;. Data from the present study suggest that saturated LCFA, especially C18:0, can modulate milk fat synthesis partly via PPAR&gamma; in goats. The nutrigenomic effect of C16:0 is not via PPAR&gamma; but likely via unknown transcription factor(s) while PPAR&gamma; plays an indirect role on the nutrigenomic effect of polyunsaturated LCFA (PUFA) on milk fat related genes, particularly for CLA (permitting effect) and DHA (blocking effect)

A Novel Dynamic Impact Approach (DIA) for Functional Analysis of Time-Course Omics Studies: Validation Using the Bovine Mammary Transcriptome

The overrepresented approach (ORA) is the most widely-accepted method for functional analysis of microarray datasets. The ORA is computationally-efficient and robust; however, it suffers from the inability of comparing results from multiple gene lists particularly with time-course experiments or those involving multiple treatments. To overcome such limitation a novel method termed Dynamic Impact Approach (DIA) is proposed. The DIA provides an estimate of the biological impact of the experimental conditions and the direction of the impact. The impact is obtained by combining the proportion of differentially expressed genes (DEG) with the log2 mean fold change and mean –log P-value of genes associated with the biological term. The direction of the impact is calculated as the difference of the impact of up-regulated DEG and down-regulated DEG associated with the biological term. The DIA was validated using microarray data from a time-course experiment of bovine mammary gland across the lactation cycle. Several annotation databases were analyzed with DIA and compared to the same analysis performed by the ORA. The DIA highlighted that during lactation both BTA6 and BTA14 were the most impacted chromosomes; among Uniprot tissues those related with lactating mammary gland were the most positively-impacted; within KEGG pathways ‘Galactose metabolism’ and several metabolism categories related to lipid synthesis were among the most impacted and induced; within Gene Ontology “lactose biosynthesis” among Biological processes and “Lactose synthase activity” and “Stearoyl-CoA 9-desaturase activity” among Molecular processes were the most impacted and induced. With the exception of the terms ‘Milk’, ‘Milk protein’ and ‘Mammary gland’ among Uniprot tissues and SP_PIR_Keyword, the use of ORA failed to capture as significantly-enriched (i.e., biologically relevant) any term known to be associated with lactating mammary gland. Results indicate the DIA is a biologically-sound approach for analysis of time-course experiments. This tool represents an alternative to ORA for functional analysis

Cell Rep

Decades of research have focused on the circuit connectivity between retinal neurons, but only a handful of amacrine cells have been described functionally and placed in the context of a specific retinal circuit. Here, we identify a circuit where inhibition from a specific amacrine cell plays a vital role in shaping the feature selectivity of a postsynaptic ganglion cell. We record from transgenically labeled CRH-1 amacrine cells and identify a postsynaptic target for CRH-1 amacrine cell inhibition in an atypical retinal ganglion cell (RGC) in mouse retina, the Suppressed-by-Contrast (SbC) RGC. Unlike other RGC types, SbC RGCs spike tonically in steady illumination and are suppressed by both increases and decreases in illumination. Inhibition from GABAergic CRH-1 amacrine cells shapes this unique contrast response profile to positive contrast. We show the existence and impact of this circuit, with both paired recordings and cell-type-specific ablation.1F32EY025930-01/EY/NEI NIH HHS/United StatesDP2 EY026770/EY/NEI NIH HHS/United StatesF32 EY025930/EY/NEI NIH HHS/United StatesNIH DP2-DEY026770A/DP/NCCDPHP CDC HHS/United StatesR01 EY018204/EY/NEI NIH HHS/United States2016-01-08T00:00:00Z26711334PMC469800