BACKGROUND
the hypothalamic-pituitary-adrenal  axis and more particularly the adrenal gland constitute a principal node of the mammalian endocrine system.
the main function of the adrenal cortex is to produce glucocorticoids  and mineralocorticoids under the influence of pituitary adrenocorticotropic hormone .
adrenal hormones, essential for survival, play important roles in stress responses, metabolism regulation, immunity, reproduction, water and salt balance and various brain functions  <cit> .
large variations in hpa axis activity  have been related to genetic factors .
variations in hpa axis activity are also related to important metabolic differences.
for example, human abdominal obesity has been associated with alterations in hpa axis functioning  <cit> .
in a recent experiment comparing five genetic stocks of pigs , a positive relationship between cortisol levels in urine  and body fat content was found both within and across breeds  <cit> .
in addition, meishan pigs have been reported to exhibit higher basal cortisol levels, response to acth and body fat content than large white pigs  <cit> .
meishan and large white lines of pigs thus constitute a valuable biological model to investigate the relationship between hpa axis activity and metabolic regulation.
among the different genetic mechanisms involved in hpa axis activity variability  <cit> , several experimental findings suggest that sensitivity to acth is a major target in human  <cit>  and in rats  <cit> .
in the pig, genetic-based differences in cortisol secretion were shown in response to corticotropin-releasing hormone  although the acth response did not differ among individuals  <cit> .
moreover, metabolic clearance of cortisol bears no relationship to the cortisol response to acth  <cit> .
previous findings  <cit>  indicate that the difference in hpa axis activity between lw and ms pigs may originate from the adrenal gland although these breeds also differ in corticosteroid-binding globulin  levels that influence circulating levels of cortisol  <cit> .
in this study, we explore the molecular mechanism responsible for the difference in adrenal sensitivity to acth in ms and lw pigs.
the actions of acth in the adrenal cortex are mediated via two temporally distinct pathways.
acute and chronic regulation of steroidogenesis occur within minutes and hours, respectively  <cit> .
the acute response is initiated by the mobilization and delivery of the substrate, cholesterol, for steroid hormone biosynthesis from the outer to the inner mitochondrial membrane, where it is metabolized to pregnenolone by the cytochrome p <dig> cholesterol side chain cleavage enzyme   <cit> .
the slower, long-lasting response to acth directs transcription of genes encoding the steroidogenic enzymes  <cit> .
most studies of acth action have focused on the long term induction  of transcripts either by investigating transcripts with specific functions in steroidogenesis  <cit>  or by genome-wide analysis  <cit> .
however, the acute response to acth stimulation has been reported to require de novo protein synthesis .
the steroidogenic acute regulatory protein , that is responsible for the transfer of cholesterol from the outer to the inner mitochondrial membrane, has been proposed to be the rate-limiting and regulated step in steroidogenesis  <cit> .
changes in star gene expression induced by acth have been observed as early as  <dig> min and were maximally elevated between  <dig> and  <dig> h  <cit> .
on the other hand, cortisol responses to acth injections in meishan and large white pigs have been reported to be at maximum levels at  <dig> hour  <cit> .
taking together these findings, we hypothesized that transcriptional regulation may in fact take place in the acute response to acth, i.e.
within  <dig> hour, in both lines of pigs.
differences in transcriptional regulation at the adrenal gland could already exist between both genotypes in basal conditions because basal cortisol levels are typically greater in ms pigs.
considering the findings described above and the literature concerning acute regulation of transcripts by acth in adrenals, we undertook a microarray analysis of gene expression in the adrenal glands of ms and lw pigs under basal conditions and in response to acute stimulation by acth.
the aim of this study was to investigate the molecular bases of the genetic differences in adrenal sensitivity to acth.
we found that genotype and/or acth affected the expression of  <dig> genes which provide new potential candidate genes to explain genetic variations in the adrenal sensitivity to acth.
RESULTS
cortisol levels
comparison of plasma cortisol concentrations  in control and acth-injected pigs showed significant breed  and treatment  effects and significant interaction .
in control animals, basal cortisol levels were higher in meishan pigs than in large white pigs.
injection of acth increased cortisol levels in both genotypes but to a larger extent in large white than in meishan pigs.
gene transcript regulation by genotype and acth treatment
using the normalization process described in the methods section,  <dig> of the  <dig> transcripts present on the pig cdna array  were found to be expressed in adrenal glands in our experimental conditions.
using the criterion of < 5% fdr,  <dig> transcripts were identified to be significantly up-regulated or downregulated by genotype and/or acth challenge .
among these,  <dig> transcripts corresponded to unique annotated transcripts  and  <dig> remained unknown.
these  <dig> transcripts were categorized according to the genotype and/or treatment effect and ordered by absolute fold change.
table  <dig> lists transcripts most significantly affected by genotype and/or treatment at p <  <dig> .
among these,  <dig> transcripts were significantly affected by genotype ,  <dig> transcripts were significantly affected by acth treatment  and  <dig> transcripts were significantly affected by both genotype and acth treatment.
the major functional categories for these genes included transcription regulation, cell growth/maintenance, signal transduction, structural/cell adhesion/ecm and protein kinase activity.
genes differentially expressed between meishan  and large white  pigs and/or in response to acute stimulation by acth were identified using a pig cdna microarray made up of  <dig> cdna clones.
the most significant affected genes  were listed in the table, categorized on the basis of up or down-regulated by genotype and/or acth and sorting by decreasing fold changes.
complete list is given in the additional file  <dig>
quantitative analysis
eleven genes highly significantly affected by genotype and/or treatment factors were selected for further examination by quantitative real-time pcr .
changes in transcripts levels were confirmed for nine genes.
correlation coefficients between expression levels as measured with membrane hybridization and real-time pcr were significant  for  <dig> out of  <dig> genes tested .
the magnitudes of the changes were roughly similar between the array and the real-time pcr except for the largest fold-changes  that seemed dampened on the microarrays.
an exception was rnf <dig> gene, which showed no difference between genotypes of pigs or in response to acth when assayed by quantitative rt-pcr, but which exhibited a significant genotype difference when tested against the pig cdna array.
this disparity may have resulted from a false-discovery error or from cross-hybridization of transcripts to region of similarity in the arrayed rnf <dig> cdna.
the second exception was fxc <dig>  which showed no significant difference when assayed by quantitative rt-pcr, but which exhibited a significant genotype difference when tested against the pig cdna array.
however, the fold changes between breeds found on the array were of low magnitude .
the changes in expression levels for  <dig> genes shown by microarray analysis to be significantly regulated by genotype and/or acth were quantified by real-time pcr.
for comparison, relative expression values for each gene were determined in each animal .
changes in expression levels were also analyzed for  <dig> additional genes of great interest for our study whereas they were either shown by microarray analysis to be not significant  or not determined  since they were not included on the microarray or due to the data normalization process.
* ldlr gene was represented by  <dig> cdna clones on the microarray and changes in its expression were verified by real-time pcr using only the cdna sequence for which exons/introns structure could be deduced.
eleven additional genes involved in acth signaling, metabolism and mobilization of cholesterol, steroidogenesis and clock genes that were either not found to be significantly affected by genotype or acth treatment  or not present on the array  were selected for further examination by quantitative real-time pcr because they were interesting for our study .
the absence of significant changes in the microarray study was confirmed for six out of seven genes.
the mc2r gene that encodes the acth receptor was not present on the array.
quantitative real-time pcr showed that the mc2r gene was not significantly affected by genotype or by acth treatment.
inasmuch as the present data showed that two transcripts involved in cholesterol transport  were significantly affected by genotype and acth treatment, changes in the expression of scarb <dig> and bzrp genes, also involved in cholesterol transport, were measured by quantitative rt-pcr.
quantitative rt-pcr analysis showed that genotype and acth treatment did not affect the expression of scarb <dig>  however, the expression of bzrp gene was found to be greater in large white pigs whereas bzrp was not found expressed on the array probably due to the low expression level of this gene and to the data normalization process used in the microarray analysis.
sqle is a key gene involved in an early step of cholesterol biosynthesis de novo.
its expression was not significantly affected by genotype or acth on the array.
this was confirmed by real-time pcr.
cyp11a <dig>  hsd3b <dig>  cyp <dig> and cyp11b that encode enzymes involved in steroidogenesis were unaffected by genotype or acth on the microarrays.
quantitative rt-pcr confirmed that neither genotype nor acth affected the levels of those genes except for cyp <dig>  while no significant difference was found when tested against the pig cdna array, expression of cyp <dig> was found to be greater in lw pigs when assayed by quantitative rt-pcr, but the fold changes were of low magnitude.
finally,  <dig> clock genes per <dig>  cry <dig> and bmal <dig> reported to be involved in circadian corticosteroids biosynthesis or adrenal responsiveness to acth were further investigated by quantitative real-time pcr.
no significant change in cry <dig> expression on the microarray was confirmed by real-time pcr.
the two  <dig> other clock genes, per <dig> and bmal <dig>  not present on the microarray were found significantly affected by acth treatment and genotype, respectively.
per <dig> was down-regulated in response to acth treatment and expression of bmal <dig> was greater in lw pigs.
discussion
we used a comprehensive gene expression profiling by microarray analysis to identify groups of genes differentially expressed by genotype and/or by acute acth treatment.
this is the first gene array analysis investigating in vivo adrenal response to an acute acth stimulation and exploring genetic variability at the adrenal level by using different breeds of pigs .
in microarrays, which included almost  <dig> transcripts, the present results indicate that genotype and/or acth treatment affected the levels of  <dig> genes in adrenals.
moreover, although previous gene array analyses of acth action have been conducted in vitro and/or focused on the effects of chronic stimulation  <cit> , our experiments demonstrate in vivo that acute acth treatment affects a large number of transcripts.
for the vast majority of affected transcripts, the changes were less than 2-fold excepted for some, probably of great interest, for which changes were as much as  <dig> -fold.
while some studies reported that small changes may be due to the tendency of microarray analysis to underestimate fold changes in transcripts accumulation  <cit> , comparison of fold changes between microarray and real-time pcr analysis showed the accuracy of nylon microarrays used, even if fold-changes higher than 2-fold seemed to be somewhat underestimated.
our results suggest that genotypic difference and acth action may produce relatively small changes in transcript accumulation but these small changes could well be of physiological significance  <cit> .
the dose of acth used in our study has been shown to maximally activate cortisol production within one hour  <cit>  and thus, may produce maximum effects on steroidogenesis.
cortisol concentrations measured in our study were consistent with previous results reporting that cortisol levels induced by acth were higher in meishans than in large whites, as well as basal cortisol levels  <cit> .
however, acth did not affect gene expression of steroidogenesis enzymes .
this result is consistent with the fact that acute stimulation by acth had little effect on adrenal p450s and steroidogenic enzymes while in contrast, long-term acth treatment provokes profound changes in the mrna levels of many adrenal steroidogenic enzymes  <cit> .
interestingly, no differences in expression of steroidogenesis genes were found between meishan and large white pigs.
these results suggest that the difference of corticosteroidogenesis between meishan and large white pigs is not triggered by changes in gene expression of adrenal p <dig> and 3βhsd enzymes under basal state or following acute acth stimulation.
nevertheless, we cannot exclude that steroidogenic activity might be higher in meishan pigs than in large white pigs.
indeed, expression of several genes  involved in the tricarboxylic acid  pathway was greater in ms pigs.
the main catalytic function of tca cycle is to provide reducing equivalents to the respiratory complexes  <cit> , for example, steroid hydroxylation  <cit> .
moreover, the tca cycle also contributes to the synthesis of heme  <cit> , necessary for the prosthetic groups of the steroidogenic cytochrome p450s  <cit> .
in this respect, it is worth noting that alas <dig>  the rate-limiting enzyme in heme biosynthesis  <cit> , shows a greater expression in ms than in lw pigs .
these mechanisms may together result in a higher steroidogenic activity by supplying more reducing equivalents and heme to steroidogenic enzymes.
this hypothesis is supported by previous observations indicating that heme availability limited adrenal corticosteroid biosynthesis  <cit>  and by recent data showing that acute stimulation of steroid production by acth was significantly increased when heme oxygenase activity was inhibited  <cit> .
the effects of acth are mediated through the acth receptor  belonging to the melanocortin receptor family .
the binding of acth to its cognate g protein-coupled receptor promotes the activation of protein kinase a and mapk-dependent signaling cascades that collectively initiate adrenal-specific steroidogenic transcriptional programs  <cit> .
findings from numerous in vitro studies support the notion that acth is a positive regulator of acth-r mrna expression  <cit> .
more particularly, winnay and hammer  <cit>  demonstrated in vitro that acth stimulation acutely activates the mc2r gene promoter .
in our study, we showed that acth did not affect mc2r gene expression within  <dig> hour and that this gene was not differentially expressed between ms and lw pigs.
similarly, no difference in gene expression encoding key regulator proteins of the acth signaling pathway  was found in our study.
on the other hand, the levels of crem , a camp-dependent transcription factor that functions to activate genes involved in steroidogenesis  <cit> , were increased in presence of acth and higher in ms than lw pigs.
moreover, trophic hormone stimulation of steroidogenic cells has been shown to result in the activation of g proteins that stimulate adenylate cyclase activity and produce increased intracellular levels of camp  <cit> .
thus, high levels of crem in ms compared to lw and in response to acth may result to a larger increase of signal transduction induced by acth.
a constant supply of cholesterol is required within adrenal cells for steroidogenesis.
the rate-limiting step for steroidogenesis is the movement of unesterified cholesterol into mitochondria where it can then be metabolized by cyp11a <dig> and other enzymes in the steroidogenic pathway .
the movement of cholesterol into the mitochondria is mediated by steroidogenic acute regulatory protein   <cit>  and other partners such as peripheral-type benzodiazepine receptor or translocator protein  <cit> .
interestingly, greater expression of the star gene in meishan than in large white pigs found in our study suggests that enhanced cholesterol transport into mitochondria may contribute to the higher corticosteroid biosynthesis found in ms pigs compared to lw pigs.
all studies on star function agree that this enzyme mediates acute stimulation of steroidogenesis in response to acth administration and requires de novo protein synthesis .
however, we did not find changes in transcript levels of star  <dig> h following acth treatment while changes in star gene expression induced by acth have been previously observed as early as  <dig> min and levels were maximally elevated between  <dig> and  <dig> h in the rat  <cit> .
nevertheless, phosphorylation of more preexisting star protein in ms pigs, a mechanism involved in the acute response to acth stimulation  <cit> , could contribute to the higher cortisol levels induced in response to acth in ms than in lw pigs.
the unesterified cholesterol needed for steroidogenesis can be derived from several different sources .
in our study we did not find differences in gene expression of enzymes involved in endogenous cellular cholesterol synthesis, such as sqle or hmgcr, between genotypes or in response to acth while those genes have been reported to be regulated by acth  <cit> .
on the other hand, cellular cholesterol delivery for steroidogenesis includes uptake of lipoprotein-derived cholesterol via low density lipoprotein  receptor mediated endocytic pathways and srb <dig>  mediated "selective" pathways .
interestingly we found higher ldlr expression in ms than in lw pigs and in response to acth but no changes were observed in scarb <dig> expression.
changes in ldlr expression in response to acth found in our study are consistent with ldlr up-regulation reported in vitro following  <dig> h acth treatment in y <dig> mouse adrenal cells  <cit> .
conversely, while scarb <dig> have also been reported to be up-regulated by  <dig> h acth treatment in y <dig> mouse adrenal cells  <cit> , and to a larger extent than ldlr, we did not find change in scarb <dig> expression.
our results indicate that receptor-mediated endocytic uptake of ldl-cholesterol may be a more important source of cholesterol for adrenal steroidogenesis in pigs as is the case in humans  <cit> , while it appears to play a negligible role in mouse  <cit> .
moreover, this is the first evidence indicating in vivo that the acute response to acth may involve cellular cholesterol supply for steroidogenesis via ldlr regulation.
a large number of genes found to be differentially expressed in our study encode transcription factors.
most of them have not been yet described as requisite in transcription networks involved in adrenal steroidogenesis.
nevertheless, they are potential interesting candidates, particularly those that were affected by both genotype and acth treatment.
we were particularly interested in peripheral clock genes , because recent studies reported that in the adrenal they regulate a large number of genes involved in general cellular processes  as well as in pathways related to major organ-specific function  and probably adjust adrenal sensitivity to acth  <cit> .
interestingly, while per <dig> and cry <dig> were not differentially expressed in our study, bmal <dig> gene showed less expression in ms pigs.
thus, we can not exclude that differences in the expression level of some clock genes may be involved in differences in basal cortisol levels and adrenal reactivity to acth between ms and lw pigs.
further studies are needed to investigate other clock genes and to clarify how clock-controlled transcriptional rhythms in adrenals could contribute to the differences observed between both lines of pigs.
phosphorylation and dephosphorylation mechanisms might be also involved in the difference of adrenal function between lw and ms pigs because a large number of differentially expressed genes encode diverse protein kinases and protein phosphatases.
among them snf1lk  constitute a valuable candidate since it was reported to be an important regulator in the early phase of acth-signaling in the adrenal cortex  <cit> .
CONCLUSIONS
in conclusion, we report differential gene expression in adrenal in two lines of pigs in basal conditions and following acute acth treatment.
some of the genes have been already reported to be implicated in adrenal physiology, but the majority has not been documented as directly involved in steroidogenesis regulation or as acutely acth-responsive.
although their contributions to adrenal function merit further investigation, we may speculate on the involvement of a few of them.
the higher cortisol levels in basal state and in response to acth in ms than in lw pigs was probably not triggered by changes in gene expression of known key regulator proteins of the acth signaling pathway and steroidogenic enzymes.
however, a higher tca cycle activity in ms pigs than in lw pigs may explain the higher steroidogenic activity by supplying more reducing equivalents and heme to steroidogenic enzymes.
alternately, differences in the adrenal function between ms and lw pigs involve likely mechanisms requisite for cholesterol supply to steroidogenesis.
the genes described in this report are thus excellent potential candidates to mediate the genetic differences in adrenal steroidogenesis, particularly those affected by genotype and acth.
because dysregulation of glucocorticoid production results in diverse diseases, elucidation of the function of these genes in adrenals will lead to better understanding the molecular basis of such pathological conditions.

 the hypothalamic-pituitary-adrenal axis and more particularly the adrenal gland constitute a principal node of the mammalian endocrine system. the main function of the adrenal cortex is to produce glucocorticoids and mineralocorticoids under the influence of pituitary adrenocorticotropic hormone. adrenal hormones, essential for survival, play important roles in stress responses, metabolism regulation, immunity, reproduction, water and salt balance and various brain functions. large variations in hpa axis activity have been related to genetic factors. variations in hpa axis activity are also related to important metabolic differences. for example, human abdominal obesity has been associated with alterations in hpa axis functioning. in a recent experiment comparing five genetic stocks of pigs, a positive relationship between cortisol levels in urine and body fat content was found both within and across breeds. in addition, meishan pigs have been reported to exhibit higher basal cortisol levels, response to acth and body fat content than large white pigs. meishan and large white lines of pigs thus constitute a valuable biological model to investigate the relationship between hpa axis activity and metabolic regulation. among the different genetic mechanisms involved in hpa axis activity variability, several experimental findings suggest that sensitivity to acth is a major target in human and in rats. in the pig, genetic-based differences in cortisol secretion were shown in response to corticotropin-releasing hormone although the acth response did not differ among individuals. moreover, metabolic clearance of cortisol bears no relationship to the cortisol response to acth. previous findings indicate that the difference in hpa axis activity between lw and ms pigs may originate from the adrenal gland although these breeds also differ in corticosteroid-binding globulin levels that influence circulating levels of cortisol. in this study, we explore the molecular mechanism responsible for the difference in adrenal sensitivity to acth in ms and lw pigs. the actions of acth in the adrenal cortex are mediated via two temporally distinct pathways. acute and chronic regulation of steroidogenesis occur within minutes and hours, respectively. the acute response is initiated by the mobilization and delivery of the substrate, cholesterol, for steroid hormone biosynthesis from the outer to the inner mitochondrial membrane, where it is metabolized to pregnenolone by the cytochrome p cholesterol side chain cleavage enzyme. the slower, long-lasting response to acth directs transcription of genes encoding the steroidogenic enzymes. most studies of acth action have focused on the long term induction of transcripts either by investigating transcripts with specific functions in steroidogenesis or by genome-wide analysis. however, the acute response to acth stimulation has been reported to require de novo protein synthesis. the steroidogenic acute regulatory protein, that is responsible for the transfer of cholesterol from the outer to the inner mitochondrial membrane, has been proposed to be the rate-limiting and regulated step in steroidogenesis. changes in star gene expression induced by acth have been observed as early as min and were maximally elevated between and h. on the other hand, cortisol responses to acth injections in meishan and large white pigs have been reported to be at maximum levels at hour. taking together these findings, we hypothesized that transcriptional regulation may in fact take place in the acute response to acth, i.e. within hour, in both lines of pigs. differences in transcriptional regulation at the adrenal gland could already exist between both genotypes in basal conditions because basal cortisol levels are typically greater in ms pigs. considering the findings described above and the literature concerning acute regulation of transcripts by acth in adrenals, we undertook a microarray analysis of gene expression in the adrenal glands of ms and lw pigs under basal conditions and in response to acute stimulation by acth. the aim of this study was to investigate the molecular bases of the genetic differences in adrenal sensitivity to acth. we found that genotype and/or acth affected the expression of genes which provide new potential candidate genes to explain genetic variations in the adrenal sensitivity to acth.  cortisol levels comparison of plasma cortisol concentrations in control and acth-injected pigs showed significant breed and treatment effects and significant interaction. in control animals, basal cortisol levels were higher in meishan pigs than in large white pigs. injection of acth increased cortisol levels in both genotypes but to a larger extent in large white than in meishan pigs. gene transcript regulation by genotype and acth treatment using the normalization process described in the methods section, of the transcripts present on the pig cdna array were found to be expressed in adrenal glands in our experimental conditions. using the criterion of < 5% fdr, transcripts were identified to be significantly up-regulated or downregulated by genotype and/or acth challenge. among these, transcripts corresponded to unique annotated transcripts and remained unknown. these transcripts were categorized according to the genotype and/or treatment effect and ordered by absolute fold change. table lists transcripts most significantly affected by genotype and/or treatment at p <. among these, transcripts were significantly affected by genotype, transcripts were significantly affected by acth treatment and transcripts were significantly affected by both genotype and acth treatment. the major functional categories for these genes included transcription regulation, cell growth/maintenance, signal transduction, structural/cell adhesion/ecm and protein kinase activity. genes differentially expressed between meishan and large white pigs and/or in response to acute stimulation by acth were identified using a pig cdna microarray made up of cdna clones. the most significant affected genes were listed in the table, categorized on the basis of up or down-regulated by genotype and/or acth and sorting by decreasing fold changes. complete list is given in the additional file quantitative analysis eleven genes highly significantly affected by genotype and/or treatment factors were selected for further examination by quantitative real-time pcr. changes in transcripts levels were confirmed for nine genes. correlation coefficients between expression levels as measured with membrane hybridization and real-time pcr were significant for out of genes tested. the magnitudes of the changes were roughly similar between the array and the real-time pcr except for the largest fold-changes that seemed dampened on the microarrays. an exception was rnf gene, which showed no difference between genotypes of pigs or in response to acth when assayed by quantitative rt-pcr, but which exhibited a significant genotype difference when tested against the pig cdna array. this disparity may have resulted from a false-discovery error or from cross-hybridization of transcripts to region of similarity in the arrayed rnf cdna. the second exception was fxc which showed no significant difference when assayed by quantitative rt-pcr, but which exhibited a significant genotype difference when tested against the pig cdna array. however, the fold changes between breeds found on the array were of low magnitude. the changes in expression levels for genes shown by microarray analysis to be significantly regulated by genotype and/or acth were quantified by real-time pcr. for comparison, relative expression values for each gene were determined in each animal. changes in expression levels were also analyzed for additional genes of great interest for our study whereas they were either shown by microarray analysis to be not significant or not determined since they were not included on the microarray or due to the data normalization process. * ldlr gene was represented by cdna clones on the microarray and changes in its expression were verified by real-time pcr using only the cdna sequence for which exons/introns structure could be deduced. eleven additional genes involved in acth signaling, metabolism and mobilization of cholesterol, steroidogenesis and clock genes that were either not found to be significantly affected by genotype or acth treatment or not present on the array were selected for further examination by quantitative real-time pcr because they were interesting for our study. the absence of significant changes in the microarray study was confirmed for six out of seven genes. the mc2r gene that encodes the acth receptor was not present on the array. quantitative real-time pcr showed that the mc2r gene was not significantly affected by genotype or by acth treatment. inasmuch as the present data showed that two transcripts involved in cholesterol transport were significantly affected by genotype and acth treatment, changes in the expression of scarb and bzrp genes, also involved in cholesterol transport, were measured by quantitative rt-pcr. quantitative rt-pcr analysis showed that genotype and acth treatment did not affect the expression of scarb however, the expression of bzrp gene was found to be greater in large white pigs whereas bzrp was not found expressed on the array probably due to the low expression level of this gene and to the data normalization process used in the microarray analysis. sqle is a key gene involved in an early step of cholesterol biosynthesis de novo. its expression was not significantly affected by genotype or acth on the array. this was confirmed by real-time pcr. cyp11a hsd3b cyp and cyp11b that encode enzymes involved in steroidogenesis were unaffected by genotype or acth on the microarrays. quantitative rt-pcr confirmed that neither genotype nor acth affected the levels of those genes except for cyp while no significant difference was found when tested against the pig cdna array, expression of cyp was found to be greater in lw pigs when assayed by quantitative rt-pcr, but the fold changes were of low magnitude. finally, clock genes per cry and bmal reported to be involved in circadian corticosteroids biosynthesis or adrenal responsiveness to acth were further investigated by quantitative real-time pcr. no significant change in cry expression on the microarray was confirmed by real-time pcr. the two other clock genes, per and bmal not present on the microarray were found significantly affected by acth treatment and genotype, respectively. per was down-regulated in response to acth treatment and expression of bmal was greater in lw pigs. discussion we used a comprehensive gene expression profiling by microarray analysis to identify groups of genes differentially expressed by genotype and/or by acute acth treatment. this is the first gene array analysis investigating in vivo adrenal response to an acute acth stimulation and exploring genetic variability at the adrenal level by using different breeds of pigs. in microarrays, which included almost transcripts, the present results indicate that genotype and/or acth treatment affected the levels of genes in adrenals. moreover, although previous gene array analyses of acth action have been conducted in vitro and/or focused on the effects of chronic stimulation, our experiments demonstrate in vivo that acute acth treatment affects a large number of transcripts. for the vast majority of affected transcripts, the changes were less than 2-fold excepted for some, probably of great interest, for which changes were as much as -fold. while some studies reported that small changes may be due to the tendency of microarray analysis to underestimate fold changes in transcripts accumulation, comparison of fold changes between microarray and real-time pcr analysis showed the accuracy of nylon microarrays used, even if fold-changes higher than 2-fold seemed to be somewhat underestimated. our results suggest that genotypic difference and acth action may produce relatively small changes in transcript accumulation but these small changes could well be of physiological significance. the dose of acth used in our study has been shown to maximally activate cortisol production within one hour and thus, may produce maximum effects on steroidogenesis. cortisol concentrations measured in our study were consistent with previous results reporting that cortisol levels induced by acth were higher in meishans than in large whites, as well as basal cortisol levels. however, acth did not affect gene expression of steroidogenesis enzymes. this result is consistent with the fact that acute stimulation by acth had little effect on adrenal p450s and steroidogenic enzymes while in contrast, long-term acth treatment provokes profound changes in the mrna levels of many adrenal steroidogenic enzymes. interestingly, no differences in expression of steroidogenesis genes were found between meishan and large white pigs. these results suggest that the difference of corticosteroidogenesis between meishan and large white pigs is not triggered by changes in gene expression of adrenal p and 3βhsd enzymes under basal state or following acute acth stimulation. nevertheless, we cannot exclude that steroidogenic activity might be higher in meishan pigs than in large white pigs. indeed, expression of several genes involved in the tricarboxylic acid pathway was greater in ms pigs. the main catalytic function of tca cycle is to provide reducing equivalents to the respiratory complexes, for example, steroid hydroxylation. moreover, the tca cycle also contributes to the synthesis of heme, necessary for the prosthetic groups of the steroidogenic cytochrome p450s. in this respect, it is worth noting that alas the rate-limiting enzyme in heme biosynthesis, shows a greater expression in ms than in lw pigs. these mechanisms may together result in a higher steroidogenic activity by supplying more reducing equivalents and heme to steroidogenic enzymes. this hypothesis is supported by previous observations indicating that heme availability limited adrenal corticosteroid biosynthesis and by recent data showing that acute stimulation of steroid production by acth was significantly increased when heme oxygenase activity was inhibited. the effects of acth are mediated through the acth receptor belonging to the melanocortin receptor family. the binding of acth to its cognate g protein-coupled receptor promotes the activation of protein kinase a and mapk-dependent signaling cascades that collectively initiate adrenal-specific steroidogenic transcriptional programs. findings from numerous in vitro studies support the notion that acth is a positive regulator of acth-r mrna expression. more particularly, winnay and hammer demonstrated in vitro that acth stimulation acutely activates the mc2r gene promoter. in our study, we showed that acth did not affect mc2r gene expression within hour and that this gene was not differentially expressed between ms and lw pigs. similarly, no difference in gene expression encoding key regulator proteins of the acth signaling pathway was found in our study. on the other hand, the levels of crem, a camp-dependent transcription factor that functions to activate genes involved in steroidogenesis, were increased in presence of acth and higher in ms than lw pigs. moreover, trophic hormone stimulation of steroidogenic cells has been shown to result in the activation of g proteins that stimulate adenylate cyclase activity and produce increased intracellular levels of camp. thus, high levels of crem in ms compared to lw and in response to acth may result to a larger increase of signal transduction induced by acth. a constant supply of cholesterol is required within adrenal cells for steroidogenesis. the rate-limiting step for steroidogenesis is the movement of unesterified cholesterol into mitochondria where it can then be metabolized by cyp11a and other enzymes in the steroidogenic pathway. the movement of cholesterol into the mitochondria is mediated by steroidogenic acute regulatory protein and other partners such as peripheral-type benzodiazepine receptor or translocator protein. interestingly, greater expression of the star gene in meishan than in large white pigs found in our study suggests that enhanced cholesterol transport into mitochondria may contribute to the higher corticosteroid biosynthesis found in ms pigs compared to lw pigs. all studies on star function agree that this enzyme mediates acute stimulation of steroidogenesis in response to acth administration and requires de novo protein synthesis. however, we did not find changes in transcript levels of star h following acth treatment while changes in star gene expression induced by acth have been previously observed as early as min and levels were maximally elevated between and h in the rat. nevertheless, phosphorylation of more preexisting star protein in ms pigs, a mechanism involved in the acute response to acth stimulation, could contribute to the higher cortisol levels induced in response to acth in ms than in lw pigs. the unesterified cholesterol needed for steroidogenesis can be derived from several different sources. in our study we did not find differences in gene expression of enzymes involved in endogenous cellular cholesterol synthesis, such as sqle or hmgcr, between genotypes or in response to acth while those genes have been reported to be regulated by acth. on the other hand, cellular cholesterol delivery for steroidogenesis includes uptake of lipoprotein-derived cholesterol via low density lipoprotein receptor mediated endocytic pathways and srb mediated "selective" pathways. interestingly we found higher ldlr expression in ms than in lw pigs and in response to acth but no changes were observed in scarb expression. changes in ldlr expression in response to acth found in our study are consistent with ldlr up-regulation reported in vitro following h acth treatment in y mouse adrenal cells. conversely, while scarb have also been reported to be up-regulated by h acth treatment in y mouse adrenal cells, and to a larger extent than ldlr, we did not find change in scarb expression. our results indicate that receptor-mediated endocytic uptake of ldl-cholesterol may be a more important source of cholesterol for adrenal steroidogenesis in pigs as is the case in humans, while it appears to play a negligible role in mouse. moreover, this is the first evidence indicating in vivo that the acute response to acth may involve cellular cholesterol supply for steroidogenesis via ldlr regulation. a large number of genes found to be differentially expressed in our study encode transcription factors. most of them have not been yet described as requisite in transcription networks involved in adrenal steroidogenesis. nevertheless, they are potential interesting candidates, particularly those that were affected by both genotype and acth treatment. we were particularly interested in peripheral clock genes, because recent studies reported that in the adrenal they regulate a large number of genes involved in general cellular processes as well as in pathways related to major organ-specific function and probably adjust adrenal sensitivity to acth. interestingly, while per and cry were not differentially expressed in our study, bmal gene showed less expression in ms pigs. thus, we can not exclude that differences in the expression level of some clock genes may be involved in differences in basal cortisol levels and adrenal reactivity to acth between ms and lw pigs. further studies are needed to investigate other clock genes and to clarify how clock-controlled transcriptional rhythms in adrenals could contribute to the differences observed between both lines of pigs. phosphorylation and dephosphorylation mechanisms might be also involved in the difference of adrenal function between lw and ms pigs because a large number of differentially expressed genes encode diverse protein kinases and protein phosphatases. among them snf1lk constitute a valuable candidate since it was reported to be an important regulator in the early phase of acth-signaling in the adrenal cortex.  in conclusion, we report differential gene expression in adrenal in two lines of pigs in basal conditions and following acute acth treatment. some of the genes have been already reported to be implicated in adrenal physiology, but the majority has not been documented as directly involved in steroidogenesis regulation or as acutely acth-responsive. although their contributions to adrenal function merit further investigation, we may speculate on the involvement of a few of them. the higher cortisol levels in basal state and in response to acth in ms than in lw pigs was probably not triggered by changes in gene expression of known key regulator proteins of the acth signaling pathway and steroidogenic enzymes. however, a higher tca cycle activity in ms pigs than in lw pigs may explain the higher steroidogenic activity by supplying more reducing equivalents and heme to steroidogenic enzymes. alternately, differences in the adrenal function between ms and lw pigs involve likely mechanisms requisite for cholesterol supply to steroidogenesis. the genes described in this report are thus excellent potential candidates to mediate the genetic differences in adrenal steroidogenesis, particularly those affected by genotype and acth. because dysregulation of glucocorticoid production results in diverse diseases, elucidation of the function of these genes in adrenals will lead to better understanding the molecular basis of such pathological conditions. 
moreover, up-regulation of star and ldlr genes in ms and/or in response to acth suggest that differences in the adrenal function between ms and lw may also involve mechanisms requisite for cholesterol supply to steroidogenesis. a pig cdna microarray was used to identify changes in gene expression in basal conditions and in response to acth stimulation. higher tca cycle activity in ms than in lw may thus result in higher steroidogenic activity and thus explain the typically higher cortisol levels in ms compared to lw. the aim of this study was to investigate molecular bases of genetic variability of the adrenal sensitivity to acth, a major source of variability, in meishan and large white pigs, ms being reported to exhibit higher basal cortisol levels, response to acth and fatness than lw. variability in hypothalamic-pituitary-adrenal axis activity has been shown to be influenced by genetic factors and related to great metabolic differences such as obesity. genotype and/or acth affected the expression of genes related to transcription, cell growth/maintenance, signal transduction, cell structure/adhesion/extra cellular matrix and protein kinase/phosphatase activity. no change in the expression of known key regulator proteins of the acth signaling pathway or of steroidogenic enzymes was found. the present study provides new potential candidate genes to explain genetic variations in the adrenal sensitivity to acth and better understand relationship between hpa axis activity and obesity. however, mdh sdha, suclg genes involved in the tricarboxylic acid pathway, were over-expressed in ms pigs.
 variability in hypothalamic-pituitary-adrenal axis activity has been shown to be influenced by genetic factors and related to great metabolic differences such as obesity. the aim of this study was to investigate molecular bases of genetic variability of the adrenal sensitivity to acth, a major source of variability, in meishan and large white pigs, ms being reported to exhibit higher basal cortisol levels, response to acth and fatness than lw. a pig cdna microarray was used to identify changes in gene expression in basal conditions and in response to acth stimulation.  genotype and/or acth affected the expression of genes related to transcription, cell growth/maintenance, signal transduction, cell structure/adhesion/extra cellular matrix and protein kinase/phosphatase activity. no change in the expression of known key regulator proteins of the acth signaling pathway or of steroidogenic enzymes was found. however, mdh sdha, suclg genes involved in the tricarboxylic acid pathway, were over-expressed in ms pigs. higher tca cycle activity in ms than in lw may thus result in higher steroidogenic activity and thus explain the typically higher cortisol levels in ms compared to lw. moreover, up-regulation of star and ldlr genes in ms and/or in response to acth suggest that differences in the adrenal function between ms and lw may also involve mechanisms requisite for cholesterol supply to steroidogenesis.  the present study provides new potential candidate genes to explain genetic variations in the adrenal sensitivity to acth and better understand relationship between hpa axis activity and obesity. 