BACKGROUND
progress in lactation biology of the bovine mammary gland advanced substantially during the 20th century .
early studies with ruminants  defined and quantified major metabolic aspects of mammary lipid metabolism, including de novo synthesis and fatty acid  uptake from blood  <cit> .
milk lipid synthesis as well as droplet formation and secretion  <cit>  received particular interest due to their influence on the manufacturing properties and organoleptic quality of milk and dairy products.
recent work has been more focused on qualitative aspects of lipid feeding to manipulate milk fa composition.
milk fa profiles and fat production are affected by stage of lactation and nutrition  <cit> .
the latter, however, is by far the predominant environmental factor affecting milk fat production and it represents a practical tool to alter the yield and composition of fa regarded as functional food components .
clearly, deep understanding of mammary physiology and molecular adaptations to diet and/or physiological state are required for efficient manipulation of milk component synthesis and development of dairy products with specific characteristics .
functional genomics studies highlighted the complexity and coordinated set of molecular events that encompass murine , bovine  <cit> , caprine  <cit> , and porcine  <cit>  mammary adaptations to lactation, revealing new insights about the underlying transcriptomic regulation  <cit> .
until recently bovine functional genomics studies were not feasible.
however, up-to-date bovine genome sequencing and annotation efforts combined with quantitative pcr  have become powerful tools for high-precision gene expression analysis.
genetic engineering studies in plants have revealed that an increase in metabolic flux requires manipulation of most of the enzymes in a biosynthetic pathway, challenging the idea of a "limiting enzyme"  <cit> .
therefore, measurement of mrna for multiple genes and their networks in a pathway/s is essential to enable conclusions about a metabolic process and its outputs.
previous work in functional genomics also has reinforced the view that transcriptional regulation of gene expression is crucial because it is one of the major long-term regulatory mechanisms of cellular metabolism.
we recognize, however, that mrna expression is one of multiple factors to be considered when studying the complex molecular networks working simultaneously in tissues.
in fact, the ratio between mrna abundance and abundance of the functional protein coded by the mrna is hardly 1: <dig>  this has been demonstrated in yeast, especially for the low abundant proteins  <cit> .
there are numerous post-transcriptional and post-translational regulatory steps that preclude from inferring precisely protein abundance from mrna.
numerous types of molecular and chemical relationships also exist which directly or indirectly  could affect protein activity.
the fact remains that post-transcriptional regulation pertains more to short- than long-term regulation  <cit> .
one of the long-term goals in our laboratory is to define gene networks involved in regulating mammary lipid synthesis in dairy cows.
as an initial step to characterize these networks and their behavior, we have studied mammary tissue mrna expression across changes in physiological state.
selected genes included those associated with fa uptake from blood, intracellular fa activation/channelling, de novo synthesis, desaturation, regulation of transcription, utilization of ketone bodies, phospholipid and triacylglycerol  synthesis, lipid globule membrane formation, as well as novel "lipogenic" genes .
most of the selected genes were chosen based on previous studies with mammary tissue  <cit> .
others have only recently been discovered and their initial functional characterization conducted in mammary  or other tissues .
specific isoforms for several families of genes involved in tag synthesis were chosen based on previous published data from our laboratory  <cit> .
the biological effect of changes in gene expression was evaluated via milk fatty acid secretion.
<dig> the % mrna abundance is calculated by .
see materials and methods for details
methods
animals, sampling, and diet
holstein dairy cows of high genetic merit were used .
details of the experimental design were reported previously  <cit> .
briefly, percutaneous biopsies from each of  <dig> cows were obtained from the right or left rear quarter of the mammary gland at - <dig> ,  <dig>   <dig>   <dig>   <dig>   <dig>  and  <dig> d relative to parturition.
rna extraction, pcr, and design and evaluation of primers
specific details of these procedures are presented in the additional file  <dig> .
data processing and statistical analysis
pcr-normalized data are presented as n-fold change relative to - <dig> d. to estimate standard errors at - <dig> d, and prevent biases in statistical analysis, normalized data were transformed to obtain a perfect average of  <dig>  at - <dig> d, leaving the proportional difference between the biological replicates.
the same proportional change was calculated at all other time points to obtain a fold change relative to - <dig> d. this final dataset was analyzed using a mixed model with repeated measures in sas  to evaluate the effect of time relative to parturition on gene expression.
compound symmetry was the most appropriate covariate structure used for repeated measures analysis.
the model included the fixed effect of time  and the random effect of cow.
relative mrna abundance among transcripts
efficiency of pcr amplification for each gene was calculated using the standard curve method  .
relative mrna abundance among measured genes was calculated as previously reported  <cit> , using the inverse of pcr efficiency raised to Δct .
overall mrna abundance for each gene among all samples measured was calculated using the median Δct.
use of this technique for estimating relative mrna abundance among genes was necessary because relative mrna quantification was performed using a standard curve , which precluded a direct comparison among genes.
together, use of ct values corrected for the efficiency of amplification plus internal control genes as baseline overcome this limitation.
description of genes measured and overall % relative mrna abundance are reported in table  <dig>
milk yield, composition and fatty acid analysis
specific details regarding measurement of milk yield, composition, and fatty acid analysis are presented in the additional file  <dig>  daily yield of fatty acids  synthesized de novo was calculated by the sum of fa 4-14-carbon fa, and yield of fa taken up from blood by the sum of 18-24-carbon fa.
the index of acetyl-coa incorporated during fa elongation  was calculated as suggested previously  <cit>  with modifications .
gene network analysis
gene networks were evaluated using ingenuity pathway analysis® .
this is a web-based application that enables the discovery, visualization, and exploration of interaction networks.
the software relies on currently known relationships  among human, mouse, and rat genes/proteins.
RESULTS
milk fatty acid composition: a functional analysis
lactation patterns of fa synthesized vs. fa taken up  suggest that uptake from blood predominated during the first mo of lactation.
calculation of synthesized fa  and ace data  suggest that synthesis of fa from acetate or butyrate began during the first  <dig> wk of lactation and increased rapidly thereafter reaching a peak at  <dig> d. thus, we concluded based also on ratio of synthesized/imported  that de novo fa synthesis predominated after 1st mo post-partum.
the simple sum of fa yield  to estimate amount of fa originating de novo suffers from the assumption that butyrate in milk is completely derived from de novo synthesis.
reports indicated that the major part of butyrate  is incorporated directly into de novo-synthesized fa   <cit> .
however, a considerable portion also is esterified directly into the sn- <dig> of tag  <cit> .
pattern of ace  corresponded with pattern of acaca  and fasn  expression , both of which are key enzymes regulating de novo synthesis.
additional discussion on milk fa composition is available in additional file  <dig> .
<dig> the complete fa datasets  are reported in additional file  <dig> .
2effect of day.
<dig> fatty acid with carbon length 4÷ <dig> /tot fa .
<dig> fatty acid with carbon length > <dig> /tot fa .
<dig> ace corrected .
6ace corrected without considering odd chain fa  .
<dig> overall Δ <dig> desaturase index, calculated from /
a,b,c denote p <  <dig>
fatty acid uptake by mammary cells
lpl and vldlr and exogenous fa utilization
mammary cells take up lcfa from albumin-bound fatty acids  and lipoproteins.
vldl or chylomicrons are anchored to mammary endothelium by lipoprotein lipase , which then hydrolyzes tag in the lipoprotein core to release fa  <cit> .
lpl has higher activity in mammary  <cit>  compared with other tissues, probably due to its high mrna abundance.
the observed up-regulation in lpl mrna as early as the onset of milk synthesis was remarkable  because mouse mammary tissue had only a 2-fold increase in lpl transcript between pregnancy and lactation.
in the mouse, the increase was accompanied by 2-fold up-regulation of enzymatic activity  <cit> .
in contrast with murine, bovine mammary lpl expression pattern was remarkably similar to the lactation curve , which might be indicative of an important role of this gene in maintenance of milk synthesis.
recent evidence points at vldl receptor  as an essential component of lpl activity  <cit> .
vldlr expression was up-regulated throughout lactation, particularly in the first mo post-partum .
the mrna abundance of lpl and vldlr accounted for ~10% and ~ <dig> % of total genes measured .
despite these differences, data suggest an important role for vldlr in concert with lpl  <cit>  in milk fat synthesis during lactation.
mammary vldlr could act on chylomicrons or intestinal vldl, which contain apo-b <dig>  <cit> .
in general, our data are in agreement with previous work reporting higher efficiency of mammary tag uptake from lipoproteins at the beginning of lactation  <cit> .
the pattern of mammary tissue expression of lpl during lactation was in accordance with the typical increase in blood ldl in dairy cows post-partum, which is an indirect index of vldl utilization  <cit> .
fat/cd <dig> and fa internalization
passive diffusion of fa across membranes plays a minor role compared with protein-mediated fa uptake and the flip-flop mechanism  <cit> .
the main proteins involved in fa uptake in non-ruminant cells include fatty acid translocator fat/cd <dig>  and fatty acid transport proteins   <cit> .
cd <dig> mrna in our study accounted for ~5% of total genes measured  and had a large increase in expression  during lactation .
this protein is believed to participate in the process of milk fat secretion  <cit>  because of its presence in the milk fat globule membranes   <cit> .
our data support an important role for cd <dig> in milk fat synthesis.
although a role for this gene in milk fat secretion cannot be excluded we believe that its involvement in fa import in bovine mammary cells is more important.
we previously showed that bovine mammary tissue expresses most of the known slc27a isoforms, but only expression of slc27a <dig> was up-regulated during the first mo of lactation suggesting a role in nefa uptake  <cit> .
up-regulation in expression of slc27a <dig> and cd <dig>  and the fact that their proteins co-localize in murine heart subcellular fractions, support the concept of cooperation between both proteins during fa uptake.
cd <dig> also co-localizes with acyl-coa synthetases  and fatty acid binding proteins   <cit> .
clearly, fa uptake by bovine mammary cells is a complex and coordinated mechanism requiring evaluation of multiple genes/proteins.
activation and intracellular channelling of fa
acsl <dig> and acss <dig> and fa activation for milk tag
long-chain fa  are esterified with coa in the inner face of the plasma membrane prior to participating in metabolic pathways.
fa activation occurs primarily via acyl-coa synthetase long-chain family member isoforms   <cit> .
acsl <dig> mrna is predominant among acsl isoforms in bovine mammary tissue  <cit> , and it increased >4-fold at the onset of lactation suggesting this isoform is important for copious milk fat synthesis .
among enzymes involved in activation of short chain fa , acyl-coa synthetase short-chain family member  <dig>  had greater mrna abundance and up-regulation in expression than acss <dig> .
mrna abundance of acss <dig>  acss <dig>  and acsl <dig> in each case was <1% of genes investigated .
bovine acss isoforms have been isolated and characterized in tissues other than mammary  <cit> .
in the mouse, acss isoforms only have  <dig> % amino acid similarity and are located in different cell compartments.
acss <dig>  is exclusively present in cytosol, while acss <dig>  is primarily found in mitochondria  <cit> .
both enzymes have high affinity for acetate, with acss <dig> showing greater affinity than acss <dig>  the latter also has modest affinity for propionate  <cit> .
bovine acss <dig> activated >4-fold more 14c-acetate into co <dig> than lipid, suggesting it targets acetate towards oxidation  <cit> .
human acss <dig> was shown to channel acetate towards fa synthesis  <cit> .
in our study, both acss <dig> and acss <dig> mrna increased substantially during lactation .
acss <dig> transcript pattern corresponded with bovine mammary acetyl-coa production throughout lactation  <cit> .
thus, its large increase at the onset of lactation along with the pattern of ace during the first  <dig> d post-partum, suggest the protein encoded by this gene provides activated acetate for de novo fa synthesis.
in addition to its use in fa synthesis, acetate is the chief carbon source for energy generation in mammary accounting for ~33% of total co <dig> produced by the tissue  <cit> .
lower mrna abundance and pattern of expression of acss <dig> throughout lactation is in agreement with acetate use for oxidation  <cit> .
overall, acss isoforms expression reflected the need for activation of acetate in mammary tissue.
fabp <dig> and fa trafficking towards milk tag
free diffusion of lcfa into cells is too slow to account for the rapid transport and selective targeting towards specific organelles  <cit> , thus, lcfa require specific transporters.
fatty acid binding protein  and acyl-coa binding protein  are the main intracellular fa transporters in non-ruminant cells  <cit> .
the former has high affinity for lcfa but also can bind acyl-coa  <cit> .
acbp is the major intracellular transporter of acyl-coa in several mammalian tissues  <cit> .
we previously observed the presence of mrna of all fabp isoforms, except fabp <dig> mrna, in bovine mammary tissue with greater abundance and up-regulation of fabp <dig> mrna during lactation.
transcript of fabp <dig> and fabp <dig> also were up-regulated during lactation but were less abundant compared with fabp <dig>  <cit> .
in the present study, fabp <dig> was the second most abundant transcript  among all measured, in accord with the large cytosolic content of its protein in mammary epithelium  <cit> .
the large mrna abundance of this gene also was a consequence of the 80-fold up-regulation during lactation, whereas acbp mrna abundance was < <dig> % among all genes and had a small increase  during lactation .
low acbp mrna abundance agrees with protein abundance data in bovine mammary  <cit> .
our results suggest a minor role of acbp in bovine mammary lipid synthesis, also supported by murine data  <cit> .
in addition to a trafficking role, fabp <dig> through binding of activated acyl-coas could buffer cells from negative effects of activated fa and prevent inhibition of acaca and scd , roles usually attributed to acbp  <cit> .
a positive relationship between fabp and scd has been demonstrated in chickens  <cit> , indicating a coordinated function between both proteins in mammary tissue as also suggested previously from an evaluation of published data  <cit> .
based on our longitudinal mrna expression and fatty acid data we propose that an important function of fabp <dig> in bovine mammary is to provide fa for scd.
large affinity of fabp <dig> for oleic acid and up-regulation of its mrna during lactation in bovine mammary tissue  <cit> , led us to propose that fabp <dig> provides stearoyl-coa   <cit>  to scd which then releases oleic acid to fabp <dig>  the fa are then available to other enzymes involved in tag synthesis.
membrane-associated atp transporters
abcg <dig> and its potential role in milk synthesis
we observed a 30-fold increase in abcg <dig>  transcript during lactation .
mrna abundance of this gene accounted for ~9% of total genes measured .
abcg <dig> is a member of the large atp binding cassette family of membrane-spanning efflux pumps that actively extrude a wide range of xenobiotics  <cit> .
it is present in apical membrane of murine mammary alveolar epithelia and plays a role in active secretion of toxins into milk  <cit> .
it also is present in the milk fat globule membrane   <cit> , probably in the external bilayer originating from plasma membrane, due to its apical membrane localization.
the large abcg <dig> mrna abundance and up-regulation, both in lactating bovine and murine mammary tissue  <cit> , is biologically puzzling because the primary role of this transporter in other tissues is detoxification.
recently, it was reported that one amino acid substitution at position  <dig>  of abcg <dig> resulted in decreased milk production but increased milk fat and protein concentration and yield  <cit> .
those data clearly do not support a role of abcg <dig> in synthesis or secretion of milk fat.
what seems apparent based on current bovine data is that abcg <dig> plays an essential role in secretion of "some" important milk constituent  <cit> .
cholesterol transport was suggested  <cit>  but it is not supported by the low amount  <cit>  and pattern of cholesterol in bovine milk throughout lactation  <cit> .
the only demonstrated role of abcg <dig> in secretion of a milk component is for riboflavin, an essential, but quantitatively marginal, nutrient for the neonate  <cit> .
therefore, its large up-regulation is suggestive of other functions in milk synthesis besides riboflavin secretion.
the amount and pattern of cholesterol secretion into bovine milk agrees with the pattern of abca <dig>  transcript we observed .
abca <dig> mrna accounted for <1% of all genes measured .
this gene also has low expression in a number of other bovine tissues, and bovine mammary in particular  <cit> .
abca <dig> is crucial for efflux of cholesterol from cells  <cit> .
taken together, data suggest a minor role of abca <dig> in bovine mammary cholesterol flux.
concerted action between de novo fa synthesis and desaturation in mammary tag synthesis
acaca, fasn, and de novo fa synthesis
production of scfa and palmitate from acetate is under control of acaca, considered the rate-limiting step in de novo fa synthesis  <cit> .
in subsequent steps, both, acetyl-coa and butyryl-coa  are primers for the cytosolic multifunctional protein fatty acid synthase   <cit> .
the major product of fasn is palmitate but in ruminants the enzyme also produces scfa  <cit> .
in the present study, acaca mrna abundance accounted for <1% whereas fasn accounted for 7% of total genes measured .
however, acaca mrna had greater up-regulation during lactation compared with fasn .
these data are consistent with activity values of the two enzymes from pregnancy through lactation in dairy cows  <cit> .
despite differences in magnitude, expression patterns among both genes were similar , confirming previous findings summarized in a recent review article  <cit> .
in fact, this was the case for several genes involved in tag synthesis and transport.
clearly, bovine mammary lipid synthesis requires coordinate expression of several genes for tag synthesis and secretion.
this point was stressed in studies of rabbit mammary lipid metabolism several decades ago  <cit> .
however, the scope of enzymes studied previously was relatively small compared with our study.
stearoyl-coa desaturase and milk tag synthesis
only a fraction of fa taken up by the mammary gland is unsaturated owing to extensive ruminal biohydrogenation.
the primary enzyme involved in monounsaturated fa synthesis is stearoyl-coa desaturase , which introduces a double bond in the Δ <dig> position of myristoyl-, palmitoyl-, and stearoyl-coa, primarily  <cit> .
to date, two scd isoforms have been identified and characterized in bovine: scd <dig> and scd <dig>  scd <dig> was first characterized in bovine adipose  <cit>  and until the discovery of scd <dig>  it was believed to be the only scd present in this species.
thus, the bovine gene is simply referred to as scd.
bovine scd <dig> has been identified and characterized only recently  <cit>  and it is expressed almost exclusively in brain.
the nucleotide sequence for a bovine scd <dig> homolog a  has been deposited at ncbi  <cit> , but no characterization of this isoform has been performed to date.
scd <dig> has  <dig> % and  <dig> % global nucleotide alignment identity  <cit>  with scd <dig> and scd <dig>  respectively.
the primer pair used in the present study  is specific only for scd <dig>  the scd mrna abundance was the highest  among all genes measured.
the large scd mrna abundance, relative to other classical lipogenic genes , and the >40-fold up-regulation during lactation  agrees with the suggestion by kinsella, based on lactating mammary scd activity  <cit> , that it plays a crucial role in tag synthesis.
all indexes of "apparent" desaturase activity increased during lactation except for stearic acid and palmitic acid desaturation.
the latter index had a peak at the beginning of lactation followed by a dramatic decrease .
the pattern of scd mrna was not significantly correlated with any of the Δ <dig> desaturase indexes , and was nearly opposite to the overall Δ <dig> desaturase index .
a lack of correlation between desaturase indexes and scd desaturase activity was found previously in bovine intramuscular fat  <cit> , which along with our findings suggests that use of indexes is inappropriate for inferring scd gene expression/activity at least when considering the lactation cycle  <cit> .
previous single-time point studies observed a positive correlation between scd mrna in goat mammary tissue and milk fat oleic acid  <cit>  or desaturase indexes  <cit> .
in our study, relative % mrna abundance of scd  was related to the total amount of Δ <dig> fa , which accounted for 19% of total milk fa .
overall, our results support a central function of scd in milk fat synthesis as previously demonstrated by intravenous infusions of sterculic acid, an inhibitor of scd activity  <cit> .
this point is supported, to some extent, by the recent discovery that both scd and dgat <dig> polymorphisms affect saturation level of milk fat and largely explain genetic variance on the desaturase indexes.
the effect, however, was greater for scd and primarily on medium/long-chain fa .
polymorphism in dgat <dig> explained less variability but had a greater effect on long-chain fa   <cit> .
the lack of correlation between temporal desaturase indexes  and scd mrna expression is not surprising due to the many factors that likely play a role in determining milk fa output.
an important factor to consider is the selective uptake of stearic acid from blood vldl by the mammary gland  <cit> .
mammary tissue relies heavily on utilization of vldl-tag during lactation  <cit> .
another key aspect is the high concentration of oleic acid, both in plasma at mid-lactation  <cit>  and the nefa pool at early lactation  <cit> .
oleic acid is the predominant fa in er membranes  <cit>  and Δ <dig> desaturase is essential for their functional maintenance  <cit> .
fabp <dig> expression and activity also could play a role in preferential channelling of palmitic and stearic acid for desaturation.
bovine fabp <dig> has a high affinity for both stearic and palmitic acid  <cit> .
very long-chain fatty acid desaturases
synthesis of very-long-chain fa is carried out by fatty acid desaturase  <dig>  and  <dig> , which add double bonds at the Δ <dig> and Δ <dig> position of pufa.
arachidonic acid , eicosapentaenoic acid , and docosahexaenoic acid  are synthesized via fads <dig> and fads <dig>  <cit> .
fads <dig> and fads <dig> mrna abundance was <1% of total genes measured .
the greater relative mrna abundance of fads <dig> compared with fads <dig> is similar to the one reported in rat mammary  <cit> .
relative mrna abundance in bovine mammary tissue was in concordance with the amount of product of both enzymes, which accounted only for  <dig> % and  <dig> % of total milk fa .
fads <dig> mrna expression increased 18-fold by d  <dig> postpartum, whereas fads <dig> mrna increased only 3-fold .
expression pattern of fads <dig> mrna did not agree with the observed Δ <dig> desaturase index, whereas fads <dig> mrna had a similar pattern compared with its index of activity .
in practical terms, it appears that fads <dig> mrna abundance/activity might be more amenable to dietary manipulation in order to increase omega-3/omega- <dig> ratio in milk fat.
formation of tag and milk lipid droplets
role for agpat <dig> and lpin <dig> in tag synthesis
discrete steps in the pathway of tag synthesis  <cit>  have been discerned in classical lipogenic tissues  and, despite lack of functional studies of mammary lipin, the same steps likely apply to the mammary gland.
expression of gpam , agpat <dig> , dgat <dig> , and lpin <dig>  mrna accounted for >2%, >1%, ~ <dig> %, and ~ <dig> %, respectively, of total transcripts measured .
dgat <dig> mrna expression was nearly undetectable.
despite these differences, we observed that lpin <dig> mrna was up-regulated during lactation by 20-fold .
the more abundant gpam and agpat <dig> mrna increased by 10- and 15-fold by d  <dig> post-partum .
gpam expression agrees with the greater enzyme activity in mammary gland during lactation in non-ruminants , and confirms its crucial role in tag synthesis  <cit> .
agpat <dig> and lpin <dig> are the major isoforms within each gene family in bovine mammary tissue  <cit> .
when the former was knocked out in lactating mice, they failed to synthesize milk fat  <cit> .
recent characterization of agpat <dig> showed that this gene is in fact the ortholog of human gpat <dig>   <cit> .
the product of gpat <dig> did not have agpat activity but instead a clear glycerol-3-phosphate acyltransferase activity  <cit> .
however, authors failed to demonstrate an increase in tag synthesis after overexpression of gpat <dig>  despite these results in the mouse, our data support an important role of agpat <dig> in bovine mammary and very likely in tag synthesis, as previously discussed  <cit> .
our data seem to suggest that agpat <dig> has agpat activity.
this is inferred by the lower mrna abundance and temporal increase in the transcript of other agpat in bovine mammary tissue  <cit> , as well as the large up-regulation of gpam expression.
it seems unlikely that bovine mammary tissue would require a larger number of enzymes with gpat activity relative to other downstream fa acylating enzymes.
a potential function of lpin <dig> in regulation of transcription of other genes involved in milk fat synthesis cannot be excluded.
recently, it was demonstrated that lpin <dig> is essential for pparα  <cit>  activation but it also interacts with pparγ  <cit> .
in addition, lpin <dig> is a target of insulin-stimulated phosphorylation through mtor  <cit> , which in turn seems to promote microsomal vs. cytosolic localization of the protein.
it could be possible that in bovine mammary tissue insulin signalling through insr  and irs <dig>  as well as mtor , all of which had a significant increase in mrna expression  through lactation  <cit> , induces lpin <dig> phosphorylation and localization to er for dag synthesis and tag formation.
relative mrna abundance of dgat <dig> was 17-fold greater compared with dgat <dig>  and had modest up-regulation particularly in early lactation .
the temporal pattern in expression of this gene was similar to butyrate yield .
dgat <dig> acylates the sn- <dig> position of dag and most butyrate in milk tag is found here  <cit> .
this protein has high affinity for butyryl-coa and even higher for palmitoyl-coa  <cit> .
however, dgat <dig> might favour use of butyrate for the sn- <dig> position of dag as indicated by the larger affinity of agpat for lcfa in mammary  <cit>  along with the preferential incorporation of palmitic acid in the sn- <dig> and sn- <dig> position  <cit> .
the pattern of dgat <dig> expression was unexpected because it is considered a qtl  <cit>  for milk production traits, and is essential for murine mammary gland development and milk synthesis  <cit> .
data from the present study  suggest that dgat <dig>  compared with other genes involved in tag synthesis, is of minor importance in the overall process of milk fat synthesis.
the temporal decrease or lack of increase in expression of dgat <dig> in mammary tissue of fvb mouse  <cit>  provides additional support.
we do not, however, believe our findings contradict previous functional studies  <cit> , demonstrating a pivotal role for dgat <dig> in increasing milk tag.
the fact remains that dgat <dig> is one of many proteins composing the tag synthesis pathway  <cit> .
a lack in functionality of any gene in this pathway can likely reduce the efficiency of tag synthesis.
protein expression and functional studies during the entire lactation should be conducted to clarify the importance of dgat, and others, in mammary lipid synthesis.
milk lipid droplet formation
milk fat globules are formed in the er membrane via incorporation of newly-formed tag, transported to the apical membrane, and eventually released  <cit> .
well-defined proteins involved in these processes in mammary include butyrophilin , xanthine dehydrogenase , and adipophilin   <cit> .
relative mrna abundance among these genes confirms the large amount of the respective protein product found in mfgm  <cit> .
adfp mrna abundance  relative to other genes measured was almost twice that of btn1a <dig>  .
expression of btn1a <dig>  xdh, and adfp increased during lactation and averaged 14-, 8-, and 3-fold by d  <dig> postpartum, respectively .
the larger increase in expression of btn1a <dig> seems to support a more crucial role for this gene, as recently suggested  <cit> , in milk fat secretion compared with xdh and adfp.
however, despite the greater increase in mrna expression for btn1a <dig> than adfp at  <dig> d post-partum relative to pre-partum, the larger overall abundance of adfp transcript resulted in similar mrna abundance for both genes at  <dig> d .
the similar proportion in relative mrna of btn1a <dig> and adfp is in accordance with their protein abundance in mammary tissue  <cit> .
our results highlight the limitations of reporting gene expression data exclusively as n-fold change.
relative mrna abundance also needs to be considered.
the precise mechanism of milk fat secretion is still debated  but our data confirmed a role for btn1a <dig>  xdh, and adfp.
the similar pattern of expression and large correlation observed  are indicative of a concerted action among the  <dig> genes and, thus, provides support for the tripartite model of murine milk lipid secretion  <cit> .
perilipins are a family of proteins localized in the periphery of intracellular lipid droplets and are essential for droplet formation as well as lipolysis in adipose tissue  <cit> .
adfp and the perilipin gene  are both part of the perilipin family.
relative plin mrna abundance was ~ <dig> % of total genes measured  and its expression was only numerically up-regulated  during lactation.
our data do not support a significant role for plin in mammary lipid droplet formation.
functional studies could clarify the involvement, if any, of plin in this process.
bdh <dig> and oxct <dig> and utilization of ketone bodies by mammary
β-hydroxybutyrate  is the major ketone body produced in bovine species under most circumstances.
bovine mammary gland takes up large amounts of bhba from blood  <cit> .
previous studies, focused mostly on de novo synthesis and oxidation of fa, concluded that use of bhba  by mammary cells is primarily for de novo fa synthesis.
a minor portion is used as energy source through the tca cycle, leaving unaccounted the fate of a substantial portion of the bhba taken up  <cit> .
bdh <dig>  and oxct <dig>  catalyze the initial and committed steps of bhba utilization in mitochondria  <cit> .
mrna abundance for bdh <dig> and oxct <dig> accounted for < <dig> % of genes measured .
the large increase in expression and relative mrna abundance during lactation  correspond with their enzymatic activity level in lactating rat mammary tissue  <cit> .
based on these data, and a previous microarray study  <cit> , we propose that the major fate of bhba in bovine mammary is the synthesis of citrate .
transcription factors and nuclear receptors during lactation: potential roles in mammary lipid metabolism
srebf-related networks and tag synthesis
a large body of evidence supports the suggestion that srebp <dig>  is pivotal in the regulation of milk fat synthesis in mouse  <cit>  and cow  <cit> .
srebp <dig> and  <dig> reside as inactive precursors in the er membrane and are transported to the golgi for proteolytic cleavage  prior to entering the nucleus and activation of sterol responsive element-containing genes .
the transport step to the golgi is blocked by sterols via the sterol-sensing protein scap .
scap is essential for the movement of srebp isofoms from the er to the golgi, essentially acting as gate keeper for movement of inactive srebp <dig> and  <dig>  <cit> .
insulin induced gene   <dig> and  <dig> are proteins that interact with scap in an oxysterol-dependent and independent fashion and regulate the responsiveness of srebp <dig> and  <dig> processing via scap, thus altering rates of lipogenesis  <cit> .
expression of srebp genes , and thyroid hormone responsive spot <dig>  averaged ~2-fold by day  <dig> postpartum  but relative mrna abundance was ~ <dig> % for srebf <dig> and srebf <dig>  and only  <dig> % of total genes measured for thrsp .
there are two isoforms of srebp <dig>  that can be expressed at different levels in tissues.
the two isoforms differ by only  <dig> nucleotides at the first exon  <cit>  and each appears to be specific in the control of transcription of genes involved in cholesterol  or tag synthesis   <cit> .
the primer pair used in our study is unable to differentiate between these because it amplifies mrna at the 14th exon.
insig <dig>  insig <dig>  and scap mrna abundance accounted for ~ <dig> %, ~ <dig> %, and ~ <dig> %, respectively, of total genes studied .
these genes had increased mrna expression during lactation, ranging from ~ <dig> -fold for scap to ~12-fold for insig <dig> at or close to peak lactation .
it is apparent from our comprehensive analysis of srebf <dig>  in concert with its co-factors , that the complete activation program of srebp is up-regulated in the bovine mammary gland during lactation.
more insig <dig> mrna was expressed in mammary tissue than scap or srebf <dig> and  <dig> , and its level of up-regulation reached 10-12-fold between peak through mid-lactation .
if this pattern of insig <dig> mrna expression extended to the protein level, more srebp might be retained in the er via scap-insig <dig> binding   <cit> .
insig <dig> binds oxysterols  specifically and this specificity is directly correlated with the ability of these compounds to inhibit srebp cleavage  <cit> .
data on oxysterol concentration/synthesis in bovine mammary tissue are not readily available but considering that mammary tissue uptake and synthesis of cholesterol is small we can infer low presence of these compounds.
in this regard, it is interesting that genes coding for oxysterol binding proteins were up-regulated during lactation .
cleavage inhibition of srebp also can occur in the absence of sterols, particularly when the ratio of insig1/scap increases as in the present study  <cit> .
therefore, it appears that an increase of insig <dig> alone can be enough to block srebp cleavage.
in fact, decreased srebp activity as a consequence of increased insig <dig> transcript has been observed in liver when insig <dig> is overexpressed  <cit>  or during high fat diet-induced insig <dig> up-regulation  <cit> .
based on previous results and our data we postulate that mammary lipid synthesis cannot rely solely on transcriptional regulation via srebp <dig> which is probably inhibited or controlled by insig <dig>
the observed up-regulation of insig <dig> during lactation apparently does not make teleological sense because mammary tissue requires substantial up-regulation of genes involved in de novo lipid synthesis .
it is striking, however, that insig <dig> mrna was at its peak  when the cows likely were in positive energy balance, i.e.
high blood insulin/glucagon, and the ratio of de novo-synthesized fa to imported fa was maximal .
insig <dig> expression was positively correlated with the ratio of synthesized/imported fa .
paradoxically, these data suggest some involvement of insig <dig> in inducing fa synthesis.
this suggestion is supported by recent data  <cit> , where a decrease of insig <dig> during experimentally-induced milk fat depression in lactating cows was observed.
several factors could drive the marked increase in insig <dig> mrna during lactation.
for example, up-regulation of insig <dig> expression might be a consequence of srebf isoform mrna up-regulation and increased activity  of the corresponding proteins.
srebp1a and srebp <dig> directly regulate insig <dig> gene expression  <cit> .
given the lipogenic capacity of mammary tissue, it is more likely that srebp1c is the more abundant isoform.
thus, insig <dig> up-regulation in bovine mammary tissue could be under control of srebf <dig> .
another reason for marked insig <dig> mrna up-regulation might be its very short half-life  <cit> , or as a necessary mechanism to sense low mammary cholesterol levels in order to regulate de novo fa synthesis.
our data support a need of insig <dig> in controlling the induction of gene expression by srebf isoforms.
therefore, insig <dig> could play a central role in orchestrating lipid metabolism  in bovine mammary tissue during lactation.
in support of this, it previously has been suggested that high levels of insig <dig> create a situation in which low levels of endogenous sterols can trigger scap binding to insig <dig> without the necessity for exogenous sterols  <cit> .
the "brake" effect of insig <dig> on tag accumulation has been clearly demonstrated in mouse adipose tissue  <cit> , when overexpression of insig <dig> in 3t3-l <dig> cells led to a decrease in mrna abundance of lipogenic genes .
our data, however, do not support a similar effect of insig <dig> on expression of mammary lipogenic genes in ruminant.
srebf <dig> expression was up-regulated to a greater extent than srebf <dig> expression during lactation .
the reason for up-regulation of srebf <dig> expression, and the similar mrna abundance compared with srebf <dig>  is not apparent because this gene is thought to be involved primarily in cholesterol biosynthesis  <cit>  and the amount of cholesterol in milk is low  <cit> .
cholesterol is almost exclusively synthesized de novo in bovine mammary tissue  <cit> , thus srebf <dig> expression also might be necessary to meet cholesterol requirements for mfgm formation  <cit> .
in support of this we observed, via microarray analysis, that expression of several genes involved in cholesterol synthesis was significantly up-regulated  during lactation  <cit> .
an additional function of srebp <dig> is to induce mrna expression of genes involved in fa synthesis  <cit> .
it could be possible that srebf <dig> mrna up-regulation during lactation might compensate for the potential inhibition of insig <dig> on both srebp.
nuclear receptors and the lipogenic program
genes involved in fa transport such as lpl, cd <dig>  and acsl <dig> are peroxisome proliferator-activated receptor gamma  target genes  <cit> .
in the lactating mouse, expression of lpl and acsl <dig> was up-regulated significantly despite the fact that expression of pparγ was down-regulated  <cit> .
however, it has recently been demonstrated that changes in abundance of adipocytes at several stages of pregnancy in murine mammary tissue  could account for the decrease in pparγ gene  mrna abundance  <cit> .
in bovine mammary, pparg mrna accounted only for  <dig> % of total genes measured  but was consistently up-regulated during lactation .
the low mrna abundance of "adipocyte-specific" genes , particularly at the end of pregnancy , clearly indicates that biopsied cow mammary tissue contained low amounts of adipocytes.
thus, our longitudinal data on pparg expression should represent that of epithelial cells.
with this premise and despite the low mrna abundance, up-regulation of pparg mrna during lactation suggests a potential role of this nuclear receptor in milk fat synthesis.
a recent study with pparγ-knockout mice indicated that absence of pparg increased utilization of fa for synthesis of inflammatory lipids due to reduced tag synthesis  <cit> .
a role of pparg in regulating the entire bovine milk fat synthesis machinery also is supported by recent results from our laboratory where treatment of mact cells  with rosiglitazone, a specific pparγ agonist, resulted in coordinated up-regulation in expression of genes involved in fa import , de novo fa synthesis , and tag synthesis   <cit> .
the relative mrna abundance for the ppar gamma coactivators, ppargc1a and ppargc1b, was  <dig> % and  <dig> % of total genes measured .
whereas expression of ppargc1a was substantially up-regulated through d  <dig> post-partum , expression of ppargc1b was consistently down-regulated during the entire lactation .
differences in relative mrna abundance, large temporal up-regulation of ppargc1a mrna, and down-regulation of ppargc1b transcript, suggest an important role of the former in bovine milk fat synthesis.
the importance of ppargc1a in the overall process of mammary lipid synthesis likely is more related to its well-defined role in regulating mitochondrial biogenesis and energy metabolism  <cit> .
up-regulation of ppargc1a during lactation agrees with reported increases in numbers and turn-over rate of mitochondria in lactating mammary tissue  <cit> .
furthermore, our combined data  also point to a concerted action of ppargc1a and insig <dig>  this last observation is captivating based on previous observations in murine  <cit>  and bovine mammary epithelial cells  <cit>  where insig <dig> was demonstrated to be a pparγ responsive gene, suggesting that pparg in mammary tissue could serve as regulator of srebp activity.
ceramide-synthesis genes in bovine mammary tissue
synthesis of ceramide in bovine mammary
ceramide, which is involved in cell signaling, cell cycle, and regulation of protein transport from er to golgi, is one of the most studied sphingolipids in nature  <cit> .
other sphingolipids with signaling roles include sphingosine  and sphingosine-1-phosphate   <cit> .
sphingomyelin synthesis from ceramide is important for milk quality because this compound is considered a functional food component  <cit> .
although minor compared with tag, sphingolipids are the third most important lipid component  <cit>  in bovine milk fat.
mfgm formation relies on sphingolipid and cholesterol availability  <cit> , thus coordinated synthesis of both compounds is pivotal to milk lipid droplet formation/secretion.
sphingolipids are involved in lipid synthesis regulation through their action on srebp  <cit> .
mammary tissue synthesizes sphingolipids de novo  <cit>  from palmitoyl-coa, leading to ceramide formation and incorporation into sphingomyelin  <cit> .
thus, palmitic acid used for ceramide synthesis in mammary appears a required step and also might represent a regulatory point for fa synthesis because ceramides can inhibit this process by blocking the activity of akt/pkb  <cit> .
regulation of fa synthesis by sphingolipids in mammary tissue has never been investigated.
thus, we selected genes crucial in ceramide synthesis/degradation, as well as enzymes involved in sph and s1p synthesis  to explore further their role in lipid synthesis regulation.
in accordance with the minor concentration of sphingolipids in milk, genes in this pathway had low mrna abundance ranging from  <dig> %  to  <dig> %  of total genes examined .
lass <dig> was the most abundant among sphingolipid-related genes and the only one with >2-fold up-regulation during lactation .
lass isoforms are orthologues of the yeast longevity-assurance gene.
the enzyme is localized in the er  <cit>  and isoforms appear to have specific tissue distribution, suggesting they perform "specialized" functions  <cit> .
lass <dig> had peak expression at  <dig> d postpartum, in agreement with previous data on milk sphingolipid concentration  <cit> .
combined data on genes associated with ceramide synthesis suggest an increase in synthesis coupled with decreased degradation throughout lactation .
an increase in mammary ceramide synthesis might potentially serve as a positive signal for proteins involved in lipid synthesis through activation of srebp <dig> as suggested previously  <cit> .
increased sphingolipid synthesis during lactation also could affect availability of cholesterol for mfgm, and might explain the inverse pattern between milk cholesterol ester and sphingolipid  <cit> .
sterol and ceramides and their role in mammary lipid synthesis
transport of ceramide from er to golgi is achieved by oxysterol binding proteins , which also act as sterol sensors whose function is to integrate cellular sterol status with sphingomyelin metabolism  <cit> .
a novel role for osbp in regulation of lipid synthesis was demonstrated when overexpression of osbp led to increases in tag synthesis in mouse liver and concomitant up-regulation of srebf <dig> and insig <dig>  <cit> .
mrna abundance of osbp and related genes  in mammary was comparable with that of srebf <dig> and  <dig>  and their expression was up-regulated ~ <dig> -fold during lactation  suggesting a functional role, likely involving the regulation of srebf <dig> and the coordination of sphingolipid and cholesterol synthesis.
transcription regulation networks and proposed milk fat synthesis model
we used our data set to develop gene networks using ipa .
details of networks are available in additional file  <dig>  and the legend of figure  <dig>  the resulting networks clearly underscore a central role for srebf <dig>  srebf <dig>  and pparg in controlling transcription of most of the genes assessed in the present study which coordinately regulate milk fat synthesis.
the network also highlights a putative role of pparg, in coordination with ppargc1a and insig <dig>  in controlling function/expression of srebf <dig> .
thus, our data challenge the notion that srebf <dig> is the central player regulating lipid synthesis in bovine mammary tissue.
we propose that a network of transcription regulators and nuclear receptors, including srebf <dig>  srebf <dig>  pparg, insig <dig>  and ppargc1a, coordinate activation of the genes driving the lipid synthesizing machinery .
more functional studies are clearly needed to determine whether long-term up-regulation of transcription factors and nuclear receptors are determinant in inducing and maintaining milk fat synthesis.
the specific roles of insig <dig> and pparg during lactation need to be determined.
besides transcriptional regulation, other regulatory steps can determine the activity of the protein.
for example, short term regulation of acaca activity also occurs at the post-translational level  <cit> .
taken together, our findings allowed for the development of an up-to-date model of milk fat synthesis regulation in bovine mammary tissue .
the model incorporates the most recent information available, including our data, on enzymes involved in milk fat synthesis .
following is the description of the model presented in figure 7:
◦ endothelial long-chain fa  transport: the vldl tag core  is hydrolyzed by lpl in cooperation with vldlr, which anchor the lipoprotein molecule and releases lcfa for transport across the endothelium into the extracellular space.
lcfa are imported into mammary cells via flip-flop  and protein-mediated mechanisms.
cd <dig> appears to be the most important protein regulating lcfa import, and it might work in concert with fatp <dig> .
once lcfa cross the membrane, most are activated into an lc-acyl-coa  primarily via acsl <dig> .
formation of lcacoa essentially traps lcfa inside the cell and activates them for subsequent utilization.
lcacoa are captured primarily by fabp <dig>  which transports them to specific intracellular organelles for utilization.
◦ fa channelling and metabolic fates: lcfa or lcacoa bound to fabp <dig> likely have three primary fates: 1) serve as substrate for scd, which inserts a double bound at Δ <dig> position of the lcfa , and the endogenous fa  is subsequently transported by fabp <dig> to enzymes involved in tag synthesis; 2) serve directly as substrate for tag synthesis via sequential reactions carried out by gpam , gpam in the present model is located on the er instead in the mitochondria, where the proper location for the product of this gene is.
previous studies have demonstrated larger microsomal compared with mitochondrial gpat activity in lactating bovine mammary tissue  <cit> .
to date, however, only the mammalian mitochondrial gpat have been annotated and characterized.
the insertion of gpam in the er attempts to account for this limitation and to simplify the model.
other enzymes important for tag synthesis include agpat <dig> , lpin <dig>  and to a less extent, dgat <dig> .
the tag pathway is denoted by black arrows and encircled by an orange line; 3) can regulate transcription via pparg .
◦ lipid droplet formation: once tag is formed it is inserted into the intra-leaflet of the er membrane to form lipid droplet.
in bovine mammary, adfp  is central for the formation of lipid droplets and for the secretory pathway involving btn1a <dig> .
the xdh  also seems to play a role in the mechanism encompassing adfp and btn1a <dig>
◦ de novo fa synthesis, activation, channelling, and formation of tag and phospholipid: de novo fa synthesis  is carried out by acaca and fasn utilizing acetyl-coa and butyryl-coa.
formation of acetyl-coa from acetate is carried out by acss <dig>  once fa with >10-carbons are formed they are activated by acsl <dig> and bound to fabp <dig>  which allows the fa to enter into the tag synthesis pathway.
short-chain fa  are inserted into tag via dgat <dig>  a portion of palmitate  can be utilized for sphingolipid synthesis through ceramide .
◦ transcriptional regulation: denoted by red arrows.
we propose a role for fabp <dig> in activation of gene expression by fa through pparγ.
besides up-regulation during lactation, a chief role of pparg in mammary lipid metabolism is supported by the large up-regulation in expression of ppargc1a and lpin <dig> two important pparγ co-factors.
regulation of genes involved in de novo synthesis might partly be under control of srebp <dig> , which is bound to scap in the er and it is transported to the golgi where it is cleaved.
activated srebp <dig> then enters the nucleus and could regulate gene expression.
however, the observed up-regulation of insig <dig>  could dampen srebp activity in bovine mammary during lactation or at the very least elicit tight regulation of srebp activity.
our data, together with previous findings, highlight a possible role of pparg in regulating srebp activity through regulation of insig <dig> expression .
results support an overlapping role of srebp <dig> in regulation of expression of gene involved in de novo fa synthesis.
alternate routes of acetate and butyrate utilization are denoted by green  and orange  arrows.
up-regulation in expression of acss <dig> during lactation allow utilization of acetate in mitochondria for energy generation primarily; whereas, mrna up-regulation of bdh <dig> and oxct <dig> allow bhba entry into mitochondria.
primary routes of bhba use are citrate synthesis  and mitochondrial fa synthesis .
◦ membrane-associated transporters: the marked mrna abundance and up-regulation of abcg <dig> during lactation suggest a pivotal role of this gene in milk synthesis/secretion.
demonstrated roles of abcg <dig> protein in mammary tissue include secretion of riboflavin  and xenobiotics .
CONCLUSIONS
lactation was characterized by dramatic up-regulation in expression of genes associated with fa uptake from blood  and intracellular transport/channelling .
these adaptations were mirrored in milk fa profiles, showing that mammary uptake relative to de novo synthesis predominated in early lactation.
although of lower magnitude, lactation also induced up-regulation of mrna of genes involved in activation of fa , de novo synthesis , desaturation , synthesis of tag , lipid droplet formation , and ketone body utilization .
temporal expression of genes with well-defined roles in mammary lipid metabolism peaked at  <dig> d post-partum and to some extent followed the lactation curve.
we could deduce a central role in endogenous oleic acid synthesis via scd for mammary tag synthesis.
however, there was no statistical correlation between expression patterns of genes involved in desaturation and  desaturase indexes rendering their use to infer temporal enzyme expression/activity meaningless.
furthermore, expression data highlighted the importance of ketone body utilization, mitochondrial biogenesis and pparγ activity , and lipid droplet formation  in the global scheme of milk fat synthesis and secretion .
novel findings included a likely role for pparg, lass <dig>  insig <dig>  srebf <dig>  and osbp in regulating lipid synthesis and mammary intracellular equilibrium between cholesterol and sphingolipids.
the complexity of mammary molecular adaptations over time was underscored by gene network analysis  as well as the apparent interrelationships that must coordinate the overall process of milk fat synthesis and secretion .
this point is further exemplified by the large number of annotated transcripts, among them several transcription factors, whose expression is markedly up-regulated during lactation  <cit>  most of which have currently unknown functions in bovine mammary tissue.
abbreviations
ace: acetyl-coa incorporation during fa elongation; bhba: β-hydroxybutyrate; dag: diacylglycerol; fa: fatty acid; lcfa: long-chain fatty acid; lpa: lysophosphatidic acid; mgfm: milk fat globule membrane; pa: phosphatidic acid; qpcr: real-time rt-pcr; scfa: short-chain fatty acid; tag: triacylglycerol
authors' contributions
mb conducted qpcr analysis, data transformation, handling and statistical analysis of data, and drafted the manuscript.
jjl collected mammary biopsies and milk samples, conceived and designed the study, participated in its coordination, and helped draft the manuscript.
all authors read and approved the final manuscript.
supplementary material
additional file 1
supplementary materials and methods and results and discussion.
the file contain additional materials and methods  accompanied by  <dig> tables which include cows features , qpcr primers information  and validation ; the file contain also additional results and discussion about milk fa composition, accompanied by  <dig> tables  and  <dig> figures which include: the entire curve of lactation and milk fat yield and % , Δ <dig> and Δ <dig> calculated indexes , possible model of utilization of bhba in bovine mammary ; possible utilization of bhba for mitochondrial fa synthesis , and possible model of phospholipid metabolism in bovine mammary during lactation .
for each figure a detailed discussion is also provided in the caption.
click here for file
additional file 2
excel file with correlation.
the file contains pearson correlations among all measurements .
correlations were analyzed using proc corr of sas .
the file contains  <dig> sheets with correlations and a legend.
click here for file
acknowledgements
partial financial support for the gene expression work and milk fatty acid analysis was provided by the cooperative state research, education and extension service, u. s. department of agriculture, under project no.
illu-538- <dig> .
we gratefully acknowledge the help from the staff of the university of illinois dairy research and teaching unit for animal care, dr. robin e. everts  for his continued support with annotation of the bovine microarray platform, dr. harris a. lewin  for the continued collaborations using the bovine oligonucleotide microarray, and dr. walter l. hurley for helpful suggestions during development of the manuscript.

 progress in lactation biology of the bovine mammary gland advanced substantially during the 20th century. early studies with ruminants defined and quantified major metabolic aspects of mammary lipid metabolism, including de novo synthesis and fatty acid uptake from blood. milk lipid synthesis as well as droplet formation and secretion received particular interest due to their influence on the manufacturing properties and organoleptic quality of milk and dairy products. recent work has been more focused on qualitative aspects of lipid feeding to manipulate milk fa composition. milk fa profiles and fat production are affected by stage of lactation and nutrition. the latter, however, is by far the predominant environmental factor affecting milk fat production and it represents a practical tool to alter the yield and composition of fa regarded as functional food components. clearly, deep understanding of mammary physiology and molecular adaptations to diet and/or physiological state are required for efficient manipulation of milk component synthesis and development of dairy products with specific characteristics. functional genomics studies highlighted the complexity and coordinated set of molecular events that encompass murine, bovine, caprine, and porcine mammary adaptations to lactation, revealing new insights about the underlying transcriptomic regulation. until recently bovine functional genomics studies were not feasible. however, up-to-date bovine genome sequencing and annotation efforts combined with quantitative pcr have become powerful tools for high-precision gene expression analysis. genetic engineering studies in plants have revealed that an increase in metabolic flux requires manipulation of most of the enzymes in a biosynthetic pathway, challenging the idea of a "limiting enzyme". therefore, measurement of mrna for multiple genes and their networks in a pathway/s is essential to enable conclusions about a metabolic process and its outputs. previous work in functional genomics also has reinforced the view that transcriptional regulation of gene expression is crucial because it is one of the major long-term regulatory mechanisms of cellular metabolism. we recognize, however, that mrna expression is one of multiple factors to be considered when studying the complex molecular networks working simultaneously in tissues. in fact, the ratio between mrna abundance and abundance of the functional protein coded by the mrna is hardly 1: this has been demonstrated in yeast, especially for the low abundant proteins. there are numerous post-transcriptional and post-translational regulatory steps that preclude from inferring precisely protein abundance from mrna. numerous types of molecular and chemical relationships also exist which directly or indirectly could affect protein activity. the fact remains that post-transcriptional regulation pertains more to short- than long-term regulation. one of the long-term goals in our laboratory is to define gene networks involved in regulating mammary lipid synthesis in dairy cows. as an initial step to characterize these networks and their behavior, we have studied mammary tissue mrna expression across changes in physiological state. selected genes included those associated with fa uptake from blood, intracellular fa activation/channelling, de novo synthesis, desaturation, regulation of transcription, utilization of ketone bodies, phospholipid and triacylglycerol synthesis, lipid globule membrane formation, as well as novel "lipogenic" genes. most of the selected genes were chosen based on previous studies with mammary tissue. others have only recently been discovered and their initial functional characterization conducted in mammary or other tissues. specific isoforms for several families of genes involved in tag synthesis were chosen based on previous published data from our laboratory. the biological effect of changes in gene expression was evaluated via milk fatty acid secretion. the % mrna abundance is calculated by. see materials and methods for details methods animals, sampling, and diet holstein dairy cows of high genetic merit were used. details of the experimental design were reported previously. briefly, percutaneous biopsies from each of cows were obtained from the right or left rear quarter of the mammary gland at -, and d relative to parturition. rna extraction, pcr, and design and evaluation of primers specific details of these procedures are presented in the additional file. data processing and statistical analysis pcr-normalized data are presented as n-fold change relative to - d. to estimate standard errors at - d, and prevent biases in statistical analysis, normalized data were transformed to obtain a perfect average of at - d, leaving the proportional difference between the biological replicates. the same proportional change was calculated at all other time points to obtain a fold change relative to - d. this final dataset was analyzed using a mixed model with repeated measures in sas to evaluate the effect of time relative to parturition on gene expression. compound symmetry was the most appropriate covariate structure used for repeated measures analysis. the model included the fixed effect of time and the random effect of cow. relative mrna abundance among transcripts efficiency of pcr amplification for each gene was calculated using the standard curve method. relative mrna abundance among measured genes was calculated as previously reported, using the inverse of pcr efficiency raised to Δct. overall mrna abundance for each gene among all samples measured was calculated using the median Δct. use of this technique for estimating relative mrna abundance among genes was necessary because relative mrna quantification was performed using a standard curve, which precluded a direct comparison among genes. together, use of ct values corrected for the efficiency of amplification plus internal control genes as baseline overcome this limitation. description of genes measured and overall % relative mrna abundance are reported in table milk yield, composition and fatty acid analysis specific details regarding measurement of milk yield, composition, and fatty acid analysis are presented in the additional file daily yield of fatty acids synthesized de novo was calculated by the sum of fa 4-14-carbon fa, and yield of fa taken up from blood by the sum of 18-24-carbon fa. the index of acetyl-coa incorporated during fa elongation was calculated as suggested previously with modifications. gene network analysis gene networks were evaluated using ingenuity pathway analysis®. this is a web-based application that enables the discovery, visualization, and exploration of interaction networks. the software relies on currently known relationships among human, mouse, and rat genes/proteins.  milk fatty acid composition: a functional analysis lactation patterns of fa synthesized vs. fa taken up suggest that uptake from blood predominated during the first mo of lactation. calculation of synthesized fa and ace data suggest that synthesis of fa from acetate or butyrate began during the first wk of lactation and increased rapidly thereafter reaching a peak at d. thus, we concluded based also on ratio of synthesized/imported that de novo fa synthesis predominated after 1st mo post-partum. the simple sum of fa yield to estimate amount of fa originating de novo suffers from the assumption that butyrate in milk is completely derived from de novo synthesis. reports indicated that the major part of butyrate is incorporated directly into de novo-synthesized fa. however, a considerable portion also is esterified directly into the sn- of tag. pattern of ace corresponded with pattern of acaca and fasn expression, both of which are key enzymes regulating de novo synthesis. additional discussion on milk fa composition is available in additional file. the complete fa datasets are reported in additional file. 2effect of day. fatty acid with carbon length 4÷ /tot fa. fatty acid with carbon length > /tot fa. ace corrected. 6ace corrected without considering odd chain fa. overall Δ desaturase index, calculated from / a,b,c denote p < fatty acid uptake by mammary cells lpl and vldlr and exogenous fa utilization mammary cells take up lcfa from albumin-bound fatty acids and lipoproteins. vldl or chylomicrons are anchored to mammary endothelium by lipoprotein lipase, which then hydrolyzes tag in the lipoprotein core to release fa. lpl has higher activity in mammary compared with other tissues, probably due to its high mrna abundance. the observed up-regulation in lpl mrna as early as the onset of milk synthesis was remarkable because mouse mammary tissue had only a 2-fold increase in lpl transcript between pregnancy and lactation. in the mouse, the increase was accompanied by 2-fold up-regulation of enzymatic activity. in contrast with murine, bovine mammary lpl expression pattern was remarkably similar to the lactation curve, which might be indicative of an important role of this gene in maintenance of milk synthesis. recent evidence points at vldl receptor as an essential component of lpl activity. vldlr expression was up-regulated throughout lactation, particularly in the first mo post-partum. the mrna abundance of lpl and vldlr accounted for ~10% and ~ % of total genes measured. despite these differences, data suggest an important role for vldlr in concert with lpl in milk fat synthesis during lactation. mammary vldlr could act on chylomicrons or intestinal vldl, which contain apo-b. in general, our data are in agreement with previous work reporting higher efficiency of mammary tag uptake from lipoproteins at the beginning of lactation. the pattern of mammary tissue expression of lpl during lactation was in accordance with the typical increase in blood ldl in dairy cows post-partum, which is an indirect index of vldl utilization. fat/cd and fa internalization passive diffusion of fa across membranes plays a minor role compared with protein-mediated fa uptake and the flip-flop mechanism. the main proteins involved in fa uptake in non-ruminant cells include fatty acid translocator fat/cd and fatty acid transport proteins. cd mrna in our study accounted for ~5% of total genes measured and had a large increase in expression during lactation. this protein is believed to participate in the process of milk fat secretion because of its presence in the milk fat globule membranes. our data support an important role for cd in milk fat synthesis. although a role for this gene in milk fat secretion cannot be excluded we believe that its involvement in fa import in bovine mammary cells is more important. we previously showed that bovine mammary tissue expresses most of the known slc27a isoforms, but only expression of slc27a was up-regulated during the first mo of lactation suggesting a role in nefa uptake. up-regulation in expression of slc27a and cd and the fact that their proteins co-localize in murine heart subcellular fractions, support the concept of cooperation between both proteins during fa uptake. cd also co-localizes with acyl-coa synthetases and fatty acid binding proteins. clearly, fa uptake by bovine mammary cells is a complex and coordinated mechanism requiring evaluation of multiple genes/proteins. activation and intracellular channelling of fa acsl and acss and fa activation for milk tag long-chain fa are esterified with coa in the inner face of the plasma membrane prior to participating in metabolic pathways. fa activation occurs primarily via acyl-coa synthetase long-chain family member isoforms. acsl mrna is predominant among acsl isoforms in bovine mammary tissue, and it increased >4-fold at the onset of lactation suggesting this isoform is important for copious milk fat synthesis. among enzymes involved in activation of short chain fa, acyl-coa synthetase short-chain family member had greater mrna abundance and up-regulation in expression than acss. mrna abundance of acss acss and acsl in each case was <1% of genes investigated. bovine acss isoforms have been isolated and characterized in tissues other than mammary. in the mouse, acss isoforms only have % amino acid similarity and are located in different cell compartments. acss is exclusively present in cytosol, while acss is primarily found in mitochondria. both enzymes have high affinity for acetate, with acss showing greater affinity than acss the latter also has modest affinity for propionate. bovine acss activated >4-fold more 14c-acetate into co than lipid, suggesting it targets acetate towards oxidation. human acss was shown to channel acetate towards fa synthesis. in our study, both acss and acss mrna increased substantially during lactation. acss transcript pattern corresponded with bovine mammary acetyl-coa production throughout lactation. thus, its large increase at the onset of lactation along with the pattern of ace during the first d post-partum, suggest the protein encoded by this gene provides activated acetate for de novo fa synthesis. in addition to its use in fa synthesis, acetate is the chief carbon source for energy generation in mammary accounting for ~33% of total co produced by the tissue. lower mrna abundance and pattern of expression of acss throughout lactation is in agreement with acetate use for oxidation. overall, acss isoforms expression reflected the need for activation of acetate in mammary tissue. fabp and fa trafficking towards milk tag free diffusion of lcfa into cells is too slow to account for the rapid transport and selective targeting towards specific organelles, thus, lcfa require specific transporters. fatty acid binding protein and acyl-coa binding protein are the main intracellular fa transporters in non-ruminant cells. the former has high affinity for lcfa but also can bind acyl-coa. acbp is the major intracellular transporter of acyl-coa in several mammalian tissues. we previously observed the presence of mrna of all fabp isoforms, except fabp mrna, in bovine mammary tissue with greater abundance and up-regulation of fabp mrna during lactation. transcript of fabp and fabp also were up-regulated during lactation but were less abundant compared with fabp. in the present study, fabp was the second most abundant transcript among all measured, in accord with the large cytosolic content of its protein in mammary epithelium. the large mrna abundance of this gene also was a consequence of the 80-fold up-regulation during lactation, whereas acbp mrna abundance was < % among all genes and had a small increase during lactation. low acbp mrna abundance agrees with protein abundance data in bovine mammary. our results suggest a minor role of acbp in bovine mammary lipid synthesis, also supported by murine data. in addition to a trafficking role, fabp through binding of activated acyl-coas could buffer cells from negative effects of activated fa and prevent inhibition of acaca and scd, roles usually attributed to acbp. a positive relationship between fabp and scd has been demonstrated in chickens, indicating a coordinated function between both proteins in mammary tissue as also suggested previously from an evaluation of published data. based on our longitudinal mrna expression and fatty acid data we propose that an important function of fabp in bovine mammary is to provide fa for scd. large affinity of fabp for oleic acid and up-regulation of its mrna during lactation in bovine mammary tissue, led us to propose that fabp provides stearoyl-coa to scd which then releases oleic acid to fabp the fa are then available to other enzymes involved in tag synthesis. membrane-associated atp transporters abcg and its potential role in milk synthesis we observed a 30-fold increase in abcg transcript during lactation. mrna abundance of this gene accounted for ~9% of total genes measured. abcg is a member of the large atp binding cassette family of membrane-spanning efflux pumps that actively extrude a wide range of xenobiotics. it is present in apical membrane of murine mammary alveolar epithelia and plays a role in active secretion of toxins into milk. it also is present in the milk fat globule membrane, probably in the external bilayer originating from plasma membrane, due to its apical membrane localization. the large abcg mrna abundance and up-regulation, both in lactating bovine and murine mammary tissue, is biologically puzzling because the primary role of this transporter in other tissues is detoxification. recently, it was reported that one amino acid substitution at position of abcg resulted in decreased milk production but increased milk fat and protein concentration and yield. those data clearly do not support a role of abcg in synthesis or secretion of milk fat. what seems apparent based on current bovine data is that abcg plays an essential role in secretion of "some" important milk constituent. cholesterol transport was suggested but it is not supported by the low amount and pattern of cholesterol in bovine milk throughout lactation. the only demonstrated role of abcg in secretion of a milk component is for riboflavin, an essential, but quantitatively marginal, nutrient for the neonate. therefore, its large up-regulation is suggestive of other functions in milk synthesis besides riboflavin secretion. the amount and pattern of cholesterol secretion into bovine milk agrees with the pattern of abca transcript we observed. abca mrna accounted for <1% of all genes measured. this gene also has low expression in a number of other bovine tissues, and bovine mammary in particular. abca is crucial for efflux of cholesterol from cells. taken together, data suggest a minor role of abca in bovine mammary cholesterol flux. concerted action between de novo fa synthesis and desaturation in mammary tag synthesis acaca, fasn, and de novo fa synthesis production of scfa and palmitate from acetate is under control of acaca, considered the rate-limiting step in de novo fa synthesis. in subsequent steps, both, acetyl-coa and butyryl-coa are primers for the cytosolic multifunctional protein fatty acid synthase. the major product of fasn is palmitate but in ruminants the enzyme also produces scfa. in the present study, acaca mrna abundance accounted for <1% whereas fasn accounted for 7% of total genes measured. however, acaca mrna had greater up-regulation during lactation compared with fasn. these data are consistent with activity values of the two enzymes from pregnancy through lactation in dairy cows. despite differences in magnitude, expression patterns among both genes were similar, confirming previous findings summarized in a recent review article. in fact, this was the case for several genes involved in tag synthesis and transport. clearly, bovine mammary lipid synthesis requires coordinate expression of several genes for tag synthesis and secretion. this point was stressed in studies of rabbit mammary lipid metabolism several decades ago. however, the scope of enzymes studied previously was relatively small compared with our study. stearoyl-coa desaturase and milk tag synthesis only a fraction of fa taken up by the mammary gland is unsaturated owing to extensive ruminal biohydrogenation. the primary enzyme involved in monounsaturated fa synthesis is stearoyl-coa desaturase, which introduces a double bond in the Δ position of myristoyl-, palmitoyl-, and stearoyl-coa, primarily. to date, two scd isoforms have been identified and characterized in bovine: scd and scd scd was first characterized in bovine adipose and until the discovery of scd it was believed to be the only scd present in this species. thus, the bovine gene is simply referred to as scd. bovine scd has been identified and characterized only recently and it is expressed almost exclusively in brain. the nucleotide sequence for a bovine scd homolog a has been deposited at ncbi, but no characterization of this isoform has been performed to date. scd has % and % global nucleotide alignment identity with scd and scd respectively. the primer pair used in the present study is specific only for scd the scd mrna abundance was the highest among all genes measured. the large scd mrna abundance, relative to other classical lipogenic genes, and the >40-fold up-regulation during lactation agrees with the suggestion by kinsella, based on lactating mammary scd activity, that it plays a crucial role in tag synthesis. all indexes of "apparent" desaturase activity increased during lactation except for stearic acid and palmitic acid desaturation. the latter index had a peak at the beginning of lactation followed by a dramatic decrease. the pattern of scd mrna was not significantly correlated with any of the Δ desaturase indexes, and was nearly opposite to the overall Δ desaturase index. a lack of correlation between desaturase indexes and scd desaturase activity was found previously in bovine intramuscular fat, which along with our findings suggests that use of indexes is inappropriate for inferring scd gene expression/activity at least when considering the lactation cycle. previous single-time point studies observed a positive correlation between scd mrna in goat mammary tissue and milk fat oleic acid or desaturase indexes. in our study, relative % mrna abundance of scd was related to the total amount of Δ fa, which accounted for 19% of total milk fa. overall, our results support a central function of scd in milk fat synthesis as previously demonstrated by intravenous infusions of sterculic acid, an inhibitor of scd activity. this point is supported, to some extent, by the recent discovery that both scd and dgat polymorphisms affect saturation level of milk fat and largely explain genetic variance on the desaturase indexes. the effect, however, was greater for scd and primarily on medium/long-chain fa. polymorphism in dgat explained less variability but had a greater effect on long-chain fa. the lack of correlation between temporal desaturase indexes and scd mrna expression is not surprising due to the many factors that likely play a role in determining milk fa output. an important factor to consider is the selective uptake of stearic acid from blood vldl by the mammary gland. mammary tissue relies heavily on utilization of vldl-tag during lactation. another key aspect is the high concentration of oleic acid, both in plasma at mid-lactation and the nefa pool at early lactation. oleic acid is the predominant fa in er membranes and Δ desaturase is essential for their functional maintenance. fabp expression and activity also could play a role in preferential channelling of palmitic and stearic acid for desaturation. bovine fabp has a high affinity for both stearic and palmitic acid. very long-chain fatty acid desaturases synthesis of very-long-chain fa is carried out by fatty acid desaturase and, which add double bonds at the Δ and Δ position of pufa. arachidonic acid, eicosapentaenoic acid, and docosahexaenoic acid are synthesized via fads and fads. fads and fads mrna abundance was <1% of total genes measured. the greater relative mrna abundance of fads compared with fads is similar to the one reported in rat mammary. relative mrna abundance in bovine mammary tissue was in concordance with the amount of product of both enzymes, which accounted only for % and % of total milk fa. fads mrna expression increased 18-fold by d postpartum, whereas fads mrna increased only 3-fold. expression pattern of fads mrna did not agree with the observed Δ desaturase index, whereas fads mrna had a similar pattern compared with its index of activity. in practical terms, it appears that fads mrna abundance/activity might be more amenable to dietary manipulation in order to increase omega-3/omega- ratio in milk fat. formation of tag and milk lipid droplets role for agpat and lpin in tag synthesis discrete steps in the pathway of tag synthesis have been discerned in classical lipogenic tissues and, despite lack of functional studies of mammary lipin, the same steps likely apply to the mammary gland. expression of gpam, agpat, dgat, and lpin mrna accounted for >2%, >1%, ~ %, and ~ %, respectively, of total transcripts measured. dgat mrna expression was nearly undetectable. despite these differences, we observed that lpin mrna was up-regulated during lactation by 20-fold. the more abundant gpam and agpat mrna increased by 10- and 15-fold by d post-partum. gpam expression agrees with the greater enzyme activity in mammary gland during lactation in non-ruminants, and confirms its crucial role in tag synthesis. agpat and lpin are the major isoforms within each gene family in bovine mammary tissue. when the former was knocked out in lactating mice, they failed to synthesize milk fat. recent characterization of agpat showed that this gene is in fact the ortholog of human gpat. the product of gpat did not have agpat activity but instead a clear glycerol-3-phosphate acyltransferase activity. however, authors failed to demonstrate an increase in tag synthesis after overexpression of gpat despite these results in the mouse, our data support an important role of agpat in bovine mammary and very likely in tag synthesis, as previously discussed. our data seem to suggest that agpat has agpat activity. this is inferred by the lower mrna abundance and temporal increase in the transcript of other agpat in bovine mammary tissue, as well as the large up-regulation of gpam expression. it seems unlikely that bovine mammary tissue would require a larger number of enzymes with gpat activity relative to other downstream fa acylating enzymes. a potential function of lpin in regulation of transcription of other genes involved in milk fat synthesis cannot be excluded. recently, it was demonstrated that lpin is essential for pparα activation but it also interacts with pparγ. in addition, lpin is a target of insulin-stimulated phosphorylation through mtor, which in turn seems to promote microsomal vs. cytosolic localization of the protein. it could be possible that in bovine mammary tissue insulin signalling through insr and irs as well as mtor, all of which had a significant increase in mrna expression through lactation, induces lpin phosphorylation and localization to er for dag synthesis and tag formation. relative mrna abundance of dgat was 17-fold greater compared with dgat and had modest up-regulation particularly in early lactation. the temporal pattern in expression of this gene was similar to butyrate yield. dgat acylates the sn- position of dag and most butyrate in milk tag is found here. this protein has high affinity for butyryl-coa and even higher for palmitoyl-coa. however, dgat might favour use of butyrate for the sn- position of dag as indicated by the larger affinity of agpat for lcfa in mammary along with the preferential incorporation of palmitic acid in the sn- and sn- position. the pattern of dgat expression was unexpected because it is considered a qtl for milk production traits, and is essential for murine mammary gland development and milk synthesis. data from the present study suggest that dgat compared with other genes involved in tag synthesis, is of minor importance in the overall process of milk fat synthesis. the temporal decrease or lack of increase in expression of dgat in mammary tissue of fvb mouse provides additional support. we do not, however, believe our findings contradict previous functional studies, demonstrating a pivotal role for dgat in increasing milk tag. the fact remains that dgat is one of many proteins composing the tag synthesis pathway. a lack in functionality of any gene in this pathway can likely reduce the efficiency of tag synthesis. protein expression and functional studies during the entire lactation should be conducted to clarify the importance of dgat, and others, in mammary lipid synthesis. milk lipid droplet formation milk fat globules are formed in the er membrane via incorporation of newly-formed tag, transported to the apical membrane, and eventually released. well-defined proteins involved in these processes in mammary include butyrophilin, xanthine dehydrogenase, and adipophilin. relative mrna abundance among these genes confirms the large amount of the respective protein product found in mfgm. adfp mrna abundance relative to other genes measured was almost twice that of btn1a. expression of btn1a xdh, and adfp increased during lactation and averaged 14-, 8-, and 3-fold by d postpartum, respectively. the larger increase in expression of btn1a seems to support a more crucial role for this gene, as recently suggested, in milk fat secretion compared with xdh and adfp. however, despite the greater increase in mrna expression for btn1a than adfp at d post-partum relative to pre-partum, the larger overall abundance of adfp transcript resulted in similar mrna abundance for both genes at d. the similar proportion in relative mrna of btn1a and adfp is in accordance with their protein abundance in mammary tissue. our results highlight the limitations of reporting gene expression data exclusively as n-fold change. relative mrna abundance also needs to be considered. the precise mechanism of milk fat secretion is still debated but our data confirmed a role for btn1a xdh, and adfp. the similar pattern of expression and large correlation observed are indicative of a concerted action among the genes and, thus, provides support for the tripartite model of murine milk lipid secretion. perilipins are a family of proteins localized in the periphery of intracellular lipid droplets and are essential for droplet formation as well as lipolysis in adipose tissue. adfp and the perilipin gene are both part of the perilipin family. relative plin mrna abundance was ~ % of total genes measured and its expression was only numerically up-regulated during lactation. our data do not support a significant role for plin in mammary lipid droplet formation. functional studies could clarify the involvement, if any, of plin in this process. bdh and oxct and utilization of ketone bodies by mammary β-hydroxybutyrate is the major ketone body produced in bovine species under most circumstances. bovine mammary gland takes up large amounts of bhba from blood. previous studies, focused mostly on de novo synthesis and oxidation of fa, concluded that use of bhba by mammary cells is primarily for de novo fa synthesis. a minor portion is used as energy source through the tca cycle, leaving unaccounted the fate of a substantial portion of the bhba taken up. bdh and oxct catalyze the initial and committed steps of bhba utilization in mitochondria. mrna abundance for bdh and oxct accounted for < % of genes measured. the large increase in expression and relative mrna abundance during lactation correspond with their enzymatic activity level in lactating rat mammary tissue. based on these data, and a previous microarray study, we propose that the major fate of bhba in bovine mammary is the synthesis of citrate. transcription factors and nuclear receptors during lactation: potential roles in mammary lipid metabolism srebf-related networks and tag synthesis a large body of evidence supports the suggestion that srebp is pivotal in the regulation of milk fat synthesis in mouse and cow. srebp and reside as inactive precursors in the er membrane and are transported to the golgi for proteolytic cleavage prior to entering the nucleus and activation of sterol responsive element-containing genes. the transport step to the golgi is blocked by sterols via the sterol-sensing protein scap. scap is essential for the movement of srebp isofoms from the er to the golgi, essentially acting as gate keeper for movement of inactive srebp and. insulin induced gene and are proteins that interact with scap in an oxysterol-dependent and independent fashion and regulate the responsiveness of srebp and processing via scap, thus altering rates of lipogenesis. expression of srebp genes, and thyroid hormone responsive spot averaged ~2-fold by day postpartum but relative mrna abundance was ~ % for srebf and srebf and only % of total genes measured for thrsp. there are two isoforms of srebp that can be expressed at different levels in tissues. the two isoforms differ by only nucleotides at the first exon and each appears to be specific in the control of transcription of genes involved in cholesterol or tag synthesis. the primer pair used in our study is unable to differentiate between these because it amplifies mrna at the 14th exon. insig insig and scap mrna abundance accounted for ~ %, ~ %, and ~ %, respectively, of total genes studied. these genes had increased mrna expression during lactation, ranging from ~ -fold for scap to ~12-fold for insig at or close to peak lactation. it is apparent from our comprehensive analysis of srebf in concert with its co-factors, that the complete activation program of srebp is up-regulated in the bovine mammary gland during lactation. more insig mrna was expressed in mammary tissue than scap or srebf and, and its level of up-regulation reached 10-12-fold between peak through mid-lactation. if this pattern of insig mrna expression extended to the protein level, more srebp might be retained in the er via scap-insig binding. insig binds oxysterols specifically and this specificity is directly correlated with the ability of these compounds to inhibit srebp cleavage. data on oxysterol concentration/synthesis in bovine mammary tissue are not readily available but considering that mammary tissue uptake and synthesis of cholesterol is small we can infer low presence of these compounds. in this regard, it is interesting that genes coding for oxysterol binding proteins were up-regulated during lactation. cleavage inhibition of srebp also can occur in the absence of sterols, particularly when the ratio of insig1/scap increases as in the present study. therefore, it appears that an increase of insig alone can be enough to block srebp cleavage. in fact, decreased srebp activity as a consequence of increased insig transcript has been observed in liver when insig is overexpressed or during high fat diet-induced insig up-regulation. based on previous results and our data we postulate that mammary lipid synthesis cannot rely solely on transcriptional regulation via srebp which is probably inhibited or controlled by insig the observed up-regulation of insig during lactation apparently does not make teleological sense because mammary tissue requires substantial up-regulation of genes involved in de novo lipid synthesis. it is striking, however, that insig mrna was at its peak when the cows likely were in positive energy balance, i.e. high blood insulin/glucagon, and the ratio of de novo-synthesized fa to imported fa was maximal. insig expression was positively correlated with the ratio of synthesized/imported fa. paradoxically, these data suggest some involvement of insig in inducing fa synthesis. this suggestion is supported by recent data, where a decrease of insig during experimentally-induced milk fat depression in lactating cows was observed. several factors could drive the marked increase in insig mrna during lactation. for example, up-regulation of insig expression might be a consequence of srebf isoform mrna up-regulation and increased activity of the corresponding proteins. srebp1a and srebp directly regulate insig gene expression. given the lipogenic capacity of mammary tissue, it is more likely that srebp1c is the more abundant isoform. thus, insig up-regulation in bovine mammary tissue could be under control of srebf. another reason for marked insig mrna up-regulation might be its very short half-life, or as a necessary mechanism to sense low mammary cholesterol levels in order to regulate de novo fa synthesis. our data support a need of insig in controlling the induction of gene expression by srebf isoforms. therefore, insig could play a central role in orchestrating lipid metabolism in bovine mammary tissue during lactation. in support of this, it previously has been suggested that high levels of insig create a situation in which low levels of endogenous sterols can trigger scap binding to insig without the necessity for exogenous sterols. the "brake" effect of insig on tag accumulation has been clearly demonstrated in mouse adipose tissue, when overexpression of insig in 3t3-l cells led to a decrease in mrna abundance of lipogenic genes. our data, however, do not support a similar effect of insig on expression of mammary lipogenic genes in ruminant. srebf expression was up-regulated to a greater extent than srebf expression during lactation. the reason for up-regulation of srebf expression, and the similar mrna abundance compared with srebf is not apparent because this gene is thought to be involved primarily in cholesterol biosynthesis and the amount of cholesterol in milk is low. cholesterol is almost exclusively synthesized de novo in bovine mammary tissue, thus srebf expression also might be necessary to meet cholesterol requirements for mfgm formation. in support of this we observed, via microarray analysis, that expression of several genes involved in cholesterol synthesis was significantly up-regulated during lactation. an additional function of srebp is to induce mrna expression of genes involved in fa synthesis. it could be possible that srebf mrna up-regulation during lactation might compensate for the potential inhibition of insig on both srebp. nuclear receptors and the lipogenic program genes involved in fa transport such as lpl, cd and acsl are peroxisome proliferator-activated receptor gamma target genes. in the lactating mouse, expression of lpl and acsl was up-regulated significantly despite the fact that expression of pparγ was down-regulated. however, it has recently been demonstrated that changes in abundance of adipocytes at several stages of pregnancy in murine mammary tissue could account for the decrease in pparγ gene mrna abundance. in bovine mammary, pparg mrna accounted only for % of total genes measured but was consistently up-regulated during lactation. the low mrna abundance of "adipocyte-specific" genes, particularly at the end of pregnancy, clearly indicates that biopsied cow mammary tissue contained low amounts of adipocytes. thus, our longitudinal data on pparg expression should represent that of epithelial cells. with this premise and despite the low mrna abundance, up-regulation of pparg mrna during lactation suggests a potential role of this nuclear receptor in milk fat synthesis. a recent study with pparγ-knockout mice indicated that absence of pparg increased utilization of fa for synthesis of inflammatory lipids due to reduced tag synthesis. a role of pparg in regulating the entire bovine milk fat synthesis machinery also is supported by recent results from our laboratory where treatment of mact cells with rosiglitazone, a specific pparγ agonist, resulted in coordinated up-regulation in expression of genes involved in fa import, de novo fa synthesis, and tag synthesis. the relative mrna abundance for the ppar gamma coactivators, ppargc1a and ppargc1b, was % and % of total genes measured. whereas expression of ppargc1a was substantially up-regulated through d post-partum, expression of ppargc1b was consistently down-regulated during the entire lactation. differences in relative mrna abundance, large temporal up-regulation of ppargc1a mrna, and down-regulation of ppargc1b transcript, suggest an important role of the former in bovine milk fat synthesis. the importance of ppargc1a in the overall process of mammary lipid synthesis likely is more related to its well-defined role in regulating mitochondrial biogenesis and energy metabolism. up-regulation of ppargc1a during lactation agrees with reported increases in numbers and turn-over rate of mitochondria in lactating mammary tissue. furthermore, our combined data also point to a concerted action of ppargc1a and insig this last observation is captivating based on previous observations in murine and bovine mammary epithelial cells where insig was demonstrated to be a pparγ responsive gene, suggesting that pparg in mammary tissue could serve as regulator of srebp activity. ceramide-synthesis genes in bovine mammary tissue synthesis of ceramide in bovine mammary ceramide, which is involved in cell signaling, cell cycle, and regulation of protein transport from er to golgi, is one of the most studied sphingolipids in nature. other sphingolipids with signaling roles include sphingosine and sphingosine-1-phosphate. sphingomyelin synthesis from ceramide is important for milk quality because this compound is considered a functional food component. although minor compared with tag, sphingolipids are the third most important lipid component in bovine milk fat. mfgm formation relies on sphingolipid and cholesterol availability, thus coordinated synthesis of both compounds is pivotal to milk lipid droplet formation/secretion. sphingolipids are involved in lipid synthesis regulation through their action on srebp. mammary tissue synthesizes sphingolipids de novo from palmitoyl-coa, leading to ceramide formation and incorporation into sphingomyelin. thus, palmitic acid used for ceramide synthesis in mammary appears a required step and also might represent a regulatory point for fa synthesis because ceramides can inhibit this process by blocking the activity of akt/pkb. regulation of fa synthesis by sphingolipids in mammary tissue has never been investigated. thus, we selected genes crucial in ceramide synthesis/degradation, as well as enzymes involved in sph and s1p synthesis to explore further their role in lipid synthesis regulation. in accordance with the minor concentration of sphingolipids in milk, genes in this pathway had low mrna abundance ranging from % to % of total genes examined. lass was the most abundant among sphingolipid-related genes and the only one with >2-fold up-regulation during lactation. lass isoforms are orthologues of the yeast longevity-assurance gene. the enzyme is localized in the er and isoforms appear to have specific tissue distribution, suggesting they perform "specialized" functions. lass had peak expression at d postpartum, in agreement with previous data on milk sphingolipid concentration. combined data on genes associated with ceramide synthesis suggest an increase in synthesis coupled with decreased degradation throughout lactation. an increase in mammary ceramide synthesis might potentially serve as a positive signal for proteins involved in lipid synthesis through activation of srebp as suggested previously. increased sphingolipid synthesis during lactation also could affect availability of cholesterol for mfgm, and might explain the inverse pattern between milk cholesterol ester and sphingolipid. sterol and ceramides and their role in mammary lipid synthesis transport of ceramide from er to golgi is achieved by oxysterol binding proteins, which also act as sterol sensors whose function is to integrate cellular sterol status with sphingomyelin metabolism. a novel role for osbp in regulation of lipid synthesis was demonstrated when overexpression of osbp led to increases in tag synthesis in mouse liver and concomitant up-regulation of srebf and insig. mrna abundance of osbp and related genes in mammary was comparable with that of srebf and and their expression was up-regulated ~ -fold during lactation suggesting a functional role, likely involving the regulation of srebf and the coordination of sphingolipid and cholesterol synthesis. transcription regulation networks and proposed milk fat synthesis model we used our data set to develop gene networks using ipa. details of networks are available in additional file and the legend of figure the resulting networks clearly underscore a central role for srebf srebf and pparg in controlling transcription of most of the genes assessed in the present study which coordinately regulate milk fat synthesis. the network also highlights a putative role of pparg, in coordination with ppargc1a and insig in controlling function/expression of srebf. thus, our data challenge the notion that srebf is the central player regulating lipid synthesis in bovine mammary tissue. we propose that a network of transcription regulators and nuclear receptors, including srebf srebf pparg, insig and ppargc1a, coordinate activation of the genes driving the lipid synthesizing machinery. more functional studies are clearly needed to determine whether long-term up-regulation of transcription factors and nuclear receptors are determinant in inducing and maintaining milk fat synthesis. the specific roles of insig and pparg during lactation need to be determined. besides transcriptional regulation, other regulatory steps can determine the activity of the protein. for example, short term regulation of acaca activity also occurs at the post-translational level. taken together, our findings allowed for the development of an up-to-date model of milk fat synthesis regulation in bovine mammary tissue. the model incorporates the most recent information available, including our data, on enzymes involved in milk fat synthesis. following is the description of the model presented in figure 7: ◦ endothelial long-chain fa transport: the vldl tag core is hydrolyzed by lpl in cooperation with vldlr, which anchor the lipoprotein molecule and releases lcfa for transport across the endothelium into the extracellular space. lcfa are imported into mammary cells via flip-flop and protein-mediated mechanisms. cd appears to be the most important protein regulating lcfa import, and it might work in concert with fatp. once lcfa cross the membrane, most are activated into an lc-acyl-coa primarily via acsl. formation of lcacoa essentially traps lcfa inside the cell and activates them for subsequent utilization. lcacoa are captured primarily by fabp which transports them to specific intracellular organelles for utilization. ◦ fa channelling and metabolic fates: lcfa or lcacoa bound to fabp likely have three primary fates: 1) serve as substrate for scd, which inserts a double bound at Δ position of the lcfa, and the endogenous fa is subsequently transported by fabp to enzymes involved in tag synthesis; 2) serve directly as substrate for tag synthesis via sequential reactions carried out by gpam, gpam in the present model is located on the er instead in the mitochondria, where the proper location for the product of this gene is. previous studies have demonstrated larger microsomal compared with mitochondrial gpat activity in lactating bovine mammary tissue. to date, however, only the mammalian mitochondrial gpat have been annotated and characterized. the insertion of gpam in the er attempts to account for this limitation and to simplify the model. other enzymes important for tag synthesis include agpat, lpin and to a less extent, dgat. the tag pathway is denoted by black arrows and encircled by an orange line; 3) can regulate transcription via pparg. ◦ lipid droplet formation: once tag is formed it is inserted into the intra-leaflet of the er membrane to form lipid droplet. in bovine mammary, adfp is central for the formation of lipid droplets and for the secretory pathway involving btn1a. the xdh also seems to play a role in the mechanism encompassing adfp and btn1a ◦ de novo fa synthesis, activation, channelling, and formation of tag and phospholipid: de novo fa synthesis is carried out by acaca and fasn utilizing acetyl-coa and butyryl-coa. formation of acetyl-coa from acetate is carried out by acss once fa with >10-carbons are formed they are activated by acsl and bound to fabp which allows the fa to enter into the tag synthesis pathway. short-chain fa are inserted into tag via dgat a portion of palmitate can be utilized for sphingolipid synthesis through ceramide. ◦ transcriptional regulation: denoted by red arrows. we propose a role for fabp in activation of gene expression by fa through pparγ. besides up-regulation during lactation, a chief role of pparg in mammary lipid metabolism is supported by the large up-regulation in expression of ppargc1a and lpin two important pparγ co-factors. regulation of genes involved in de novo synthesis might partly be under control of srebp, which is bound to scap in the er and it is transported to the golgi where it is cleaved. activated srebp then enters the nucleus and could regulate gene expression. however, the observed up-regulation of insig could dampen srebp activity in bovine mammary during lactation or at the very least elicit tight regulation of srebp activity. our data, together with previous findings, highlight a possible role of pparg in regulating srebp activity through regulation of insig expression. results support an overlapping role of srebp in regulation of expression of gene involved in de novo fa synthesis. alternate routes of acetate and butyrate utilization are denoted by green and orange arrows. up-regulation in expression of acss during lactation allow utilization of acetate in mitochondria for energy generation primarily; whereas, mrna up-regulation of bdh and oxct allow bhba entry into mitochondria. primary routes of bhba use are citrate synthesis and mitochondrial fa synthesis. ◦ membrane-associated transporters: the marked mrna abundance and up-regulation of abcg during lactation suggest a pivotal role of this gene in milk synthesis/secretion. demonstrated roles of abcg protein in mammary tissue include secretion of riboflavin and xenobiotics.  lactation was characterized by dramatic up-regulation in expression of genes associated with fa uptake from blood and intracellular transport/channelling. these adaptations were mirrored in milk fa profiles, showing that mammary uptake relative to de novo synthesis predominated in early lactation. although of lower magnitude, lactation also induced up-regulation of mrna of genes involved in activation of fa, de novo synthesis, desaturation, synthesis of tag, lipid droplet formation, and ketone body utilization. temporal expression of genes with well-defined roles in mammary lipid metabolism peaked at d post-partum and to some extent followed the lactation curve. we could deduce a central role in endogenous oleic acid synthesis via scd for mammary tag synthesis. however, there was no statistical correlation between expression patterns of genes involved in desaturation and desaturase indexes rendering their use to infer temporal enzyme expression/activity meaningless. furthermore, expression data highlighted the importance of ketone body utilization, mitochondrial biogenesis and pparγ activity, and lipid droplet formation in the global scheme of milk fat synthesis and secretion. novel findings included a likely role for pparg, lass insig srebf and osbp in regulating lipid synthesis and mammary intracellular equilibrium between cholesterol and sphingolipids. the complexity of mammary molecular adaptations over time was underscored by gene network analysis as well as the apparent interrelationships that must coordinate the overall process of milk fat synthesis and secretion. this point is further exemplified by the large number of annotated transcripts, among them several transcription factors, whose expression is markedly up-regulated during lactation most of which have currently unknown functions in bovine mammary tissue. abbreviations ace: acetyl-coa incorporation during fa elongation; bhba: β-hydroxybutyrate; dag: diacylglycerol; fa: fatty acid; lcfa: long-chain fatty acid; lpa: lysophosphatidic acid; mgfm: milk fat globule membrane; pa: phosphatidic acid; qpcr: real-time rt-pcr; scfa: short-chain fatty acid; tag: triacylglycerol authors' contributions mb conducted qpcr analysis, data transformation, handling and statistical analysis of data, and drafted the manuscript. jjl collected mammary biopsies and milk samples, conceived and designed the study, participated in its coordination, and helped draft the manuscript. all authors read and approved the final manuscript. supplementary material additional file 1 supplementary materials and methods and results and discussion. the file contain additional materials and methods accompanied by tables which include cows features, qpcr primers information and validation; the file contain also additional results and discussion about milk fa composition, accompanied by tables and figures which include: the entire curve of lactation and milk fat yield and %, Δ and Δ calculated indexes, possible model of utilization of bhba in bovine mammary; possible utilization of bhba for mitochondrial fa synthesis, and possible model of phospholipid metabolism in bovine mammary during lactation. for each figure a detailed discussion is also provided in the caption. click here for file additional file 2 excel file with correlation. the file contains pearson correlations among all measurements. correlations were analyzed using proc corr of sas. the file contains sheets with correlations and a legend. click here for file acknowledgements partial financial support for the gene expression work and milk fatty acid analysis was provided by the cooperative state research, education and extension service, u. s. department of agriculture, under project no. illu-538-. we gratefully acknowledge the help from the staff of the university of illinois dairy research and teaching unit for animal care, dr. robin e. everts for his continued support with annotation of the bovine microarray platform, dr. harris a. lewin for the continued collaborations using the bovine oligonucleotide microarray, and dr. walter l. hurley for helpful suggestions during development of the manuscript.
change in srebf mrna expression during lactation, thought to be central for milk fat synthesis regulation, was ≤2-fold in magnitude, while expression of insig which negatively regulates srebp activation, was >12-fold and had a parallel pattern of expression to ppargc1a. results challenge the proposal that srebf is central for milk fat synthesis regulation and highlight a pivotal role for a concerted action among pparg, ppargc1a, and insig expression of scd, the most abundant gene measured, appears to be key during milk fat synthesis. longitudinal mrna expression allowed development of transcriptional regulation networks and an updated model of milk fat synthesis regulation. the molecular events associated with regulation of milk fat synthesis in the bovine mammary gland remain largely unknown. marked up-regulation and/or % relative mrna abundance during lactation were observed for genes associated with mammary fa uptake from blood, intracellular fa trafficking, long-chain and short-chain intracellular fa activation, de novo fa synthesis, desaturation, triacylglycerol synthesis, lipid droplet formation, ketone body utilization, and transcription regulation. our objective was to study mammary tissue mrna expression via quantitative pcr of genes associated with lipid synthesis and secretion from the late pre-partum/non-lactating period through the end of subsequent lactation. genes involved in phospholipid synthesis had moderate up-regulation in expression and % relative mrna abundance. the mrna abundance and up-regulation in expression of abcg during lactation was markedly high, suggesting a biological role of this gene in milk synthesis/secretion. mrna expression was coupled with milk fatty acid composition and calculated indexes of fa desaturation and de novo synthesis by the mammary gland. the lack of correlation between gene expression and calculated desaturase indexes does not support their use to infer mrna expression or enzyme activity. weak correlations were observed between both milk fa composition and desaturase indexes with mrna expression pattern of genes measured. a network of genes participates in coordinating milk fat synthesis and secretion.
 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 genes associated with lipid synthesis and secretion from the late pre-partum/non-lactating period through the end of subsequent lactation. mrna expression was coupled with milk fatty acid composition and calculated indexes of fa desaturation and de novo synthesis by the mammary gland.  marked up-regulation and/or % relative mrna abundance during lactation were observed for genes associated with mammary fa uptake from blood, intracellular fa trafficking, long-chain and short-chain intracellular fa activation, de novo fa synthesis, desaturation, triacylglycerol synthesis, lipid droplet formation, ketone body utilization, and transcription regulation. change in srebf mrna expression during lactation, thought to be central for milk fat synthesis regulation, was ≤2-fold in magnitude, while expression of insig which negatively regulates srebp activation, was >12-fold and had a parallel pattern of expression to ppargc1a. genes involved in phospholipid synthesis had moderate up-regulation in expression and % relative mrna abundance. the mrna abundance and up-regulation in expression of abcg 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 with mrna expression pattern of genes measured.  a network of genes participates in coordinating milk fat synthesis and secretion. results challenge the proposal that srebf is central for milk fat synthesis regulation and highlight a pivotal role for a concerted action among pparg, ppargc1a, and insig expression of scd, 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. longitudinal mrna expression allowed development of transcriptional regulation networks and an updated model of milk fat synthesis regulation. 