BACKGROUND
fasting, the act of willingly abstaining from food, is a frequently occurring natural status in humans.
fasting is a popular strategy to manage overweight or obesity, it is a traditional habit in certain religions or societies, and it is an accepted pre-surgical procedure.
during fasting whole-body fuel utilization gradually shifts from carbohydrates and fat in the fed state to proteins and fat after a day of fasting  <cit> .
the nuclear receptor peroxisome proliferator-activated receptor alpha  plays an important role in the control of the hepatic metabolic response  <cit> .
during fasting, free fatty acid levels in plasma are elevated and can activate pparα, which regulates a large array of hepatic genes including those involved in fatty acid catabolism.
the small intestine is the primary organ for digestion and selective absorption of nutrients and other food constituents.
absorption of these molecules across the intestinal epithelium occurs mainly by multiple transmembrane transporters  <cit>  that principally belong to two superfamilies, namely the solute carrier  and the atp binding cassette  superfamily of transporters  <cit> .
slc transporters located at the apical membrane of the enterocyte are responsible for the selective uptake of macronutrients, such as di- and tripeptides, hexoses and fatty acids  <cit> .
in contrast, abc transporters are efflux transporters responsible for the active removal of substances, including nutrients such as cholesterol, limiting their intracellular concentrations.
besides their presence in plasma membranes, slc and abc transporters are also located in intracellular organelles, such as mitochondria or peroxisomes, in which they are responsible for uptake or secretion of metabolites.
in addition, it has become clear that the intestinal epithelium is an important metabolic site, to a great extend responsible for the first-pass metabolism of nutrients and xenobiotics  <cit> .
numerous metabolic reactions occur in enterocytes, including those typically referred to as phase i and phase ii metabolism.
phase i metabolism commonly refers to oxidative, peroxidative, and reductive metabolism of endogenous compounds and drugs, mediated by cytochrome p <dig> isoenzymes   <cit> .
phase ii metabolism often succeed phase i metabolism and is mediated by several enzymatic systems.
in general, phase ii metabolism yields conjugated metabolites, increasing the water solubility of lipophilic compounds.
the most important phase ii enzymes are sulfotransferases   <cit> , udp-glucuronosyltransferases   <cit> , glutathione s-transferases   <cit> , n-acetyltransferases   <cit> , and epoxide hydrolases   <cit> .
several abc transporters can secrete metabolites resulting from phase i and phase ii enzymatic transformations  <cit> .
previous studies showed that fasting has a dramatic effect on small intestinal transport function  <cit> .
however, little is known on the expression of transport and phase i/ii metabolism genes in small intestine during fasting and the role of pparα therein.
we therefore set out to investigate the effects of fasting on expression of these genes using microarrays and quantitative rt-pcr .
we conclude that the absorptive as well as the detoxification capacity of the small intestine is altered during fasting, and that pparα mediates a part of the adaptive response to fasting.
RESULTS
effect of  <dig> hours of fasting on expression of transporter and phase i/ii metabolism genes
the affymetrix genechip mouse genome 430a array comprises  <dig>  probesets, representing  <dig>  unique genes.
annotation information from affymetrix was queried to compile a list of transporter and phase i/ii metabolism genes present on the array .
this list consisted of  <dig> probesets, encoding for  <dig> unique genes, and was used in the remainder of our analyses .
under basal, chow-fed conditions, our filtering protocol identified  <dig>  significantly expressed genes in the small intestine , of which  <dig> were transporters and phase i/ii metabolism genes.
after  <dig> hours of fasting,  <dig> genes, including  <dig> transporter and phase i/ii metabolism genes, were differentially expressed , corresponding to 13% of the expressed transporter and phase i/ii metabolism genes.
for selected genes additional qrt-pcr analyses were performed, which confirmed the array results .
analysis of all genes on the moe430a array compared with transporters and phase i/ii metabolism genes.
microarray results were confirmed with qrt-pcr.
fc = fold change, qrt-pcr control samples have been set arbitrarily to  <dig>  qrt-pcr data are means ± standard error .
solute carrier transporters
we studied  <dig> slc transporters, which amounts to 68% of the total number transporters of this superfamily currently known .
after  <dig> hours of fasting,  <dig> slc transporters were differentially expressed , which corresponded to 15% of the expressed slc transporter genes in the small intestine.
with respect to apical transporters, expression of the short-chain fatty acid transporter smct <dig>   <cit> , the carboxylate transporter nadc <dig>   <cit> , the prostaglandin transporter pgt   <cit> , and the sulphate transporter dtd   <cit>  was increased, whereas the expression level of the zinc transporter zip <dig>   <cit>  was suppressed.
the basolaterally located monocarboxylate transporter mct <dig>   <cit>  and iron transporter ireg <dig>   <cit>  were downregulated, and the basolateral neurotransmitter serotonin transporter sert   <cit> , was upregulated.
seven intracellular slc transporters were induced ; znt <dig> , g6pt <dig> , fatp <dig> , papst <dig> , cact , mrs3/ <dig> , and slc25a <dig>  znt <dig>  <cit>  is responsible for storage of zinc in sub-apically-located vesicles.
g6pt <dig>  <cit>  and fatp <dig>  <cit>  are localized at the endoplasmic reticulum, transporting glucose- <dig> phosphate and fatty acyl-coa esters, respectively.
the sulphate donor 3'-phosphoadenosine 5'-phosphosulfate  transporter papst <dig>  <cit>  is localized in the golgi.
finally, cact, mrs3/ <dig> and slc25a <dig> are all three present in mitochondria shuttling metabolites across the inner mitochondrial membrane  <cit> .
cact and mrs3/ <dig> transport fatty acyl carnitines for fatty acid oxidation and iron, respectively.
the function of slc25a <dig> is currently not known.
finally, fuct <dig>   <cit>  an additional member of the slc <dig> family, transporting nucleotide sugars, was downregulated.
a = the average log <dig> transformed expression value of normal fed and  <dig> hours fasted mice , sd = standard deviation, wt = wild-type mice.
in addition to the fold change, the intracellular localization of the corresponding protein is given.
phase i and ii metabolic genes
next we evaluated the effects of fasting on expression of phase i/ii metabolism genes .
results are presented in table  <dig>  we studied  <dig> cypp450s, which corresponds to 66% of the currently known mouse cypp450s.
five cypp450s were differentially expressed, corresponding to 29% of the expressed cypp450s in small intestine.
cyp2j <dig> was upregulated.
members of the cyp <dig> family are well known to be responsible for the nadph-dependent oxidation of steroids and fatty acid as well as drugs  <cit> .
in addition, expression of cyp3a <dig>  typically known because of its drug-metabolizing capacity  <cit> , was increased.
finally, cyp4a <dig> and cyp4b <dig>  involved in peroxisomal oxidation of fatty acids, and cyp27a <dig>  involved in conversion of cholesterol into 27-hydroxycholesterol, were all induced upon fasting  <cit> .
with respect to phase ii metabolism, expression of in total  <dig> enzymes was analyzed .
except for gsts, fasting had no denoting effect on expression of phase ii metabolism genes , since only  <dig> non-gsts were changed.
apart from gstm <dig>  all changed gsts were downregulated.
the aldo-keto reductase akr1b <dig>  <cit> , involved in detoxification of lipid peroxides, and two sulfotransferases, sult1d <dig> and chst <dig>  were increased.
a = the average log <dig> transformed expression value of normal fed and  <dig> hours fasted mice , sd = standard deviation , wt = wild-type mice.
abc transporters
effects of fasting on expression of abc transporters are summarized in table  <dig>  we analyzed  <dig> of the  <dig> murine abc transporters .
the transporters abca <dig>  <cit> , involved in basolateral efflux of cholesterol, and abcg <dig>  <cit> , involved in transport of sterols and stanols across the apical membrane, were upregulated.
a = the average log <dig> transformed expression value of normal fed and  <dig> hours fasted mice , sd = standard deviation, wt = wild-type mice.
in addition to the fold change, the intracellular localization of the corresponding protein is given.
time dependent changes of gene expression during fasting
to get insight into the time-dependent effects of fasting, we performed an additional experiment in which mice were deprived of food for various time points up to  <dig> hours.
intestinal weight, blood glucose and plasma free fatty acid levels were measured .
as expected, blood glucose levels decreased until  <dig> hours of fasting, after which it stabilized  <cit> .
free fatty acid levels rapidly increased at the onset of fasting, and remained constant after  <dig> hours.
intestinal weight decreased already after  <dig> hours of fasting, significant changes were found as from  <dig> hours of fasting.
for selected transporters and phase i/ii metabolism enzymes gene expression was measured using qrt-pcr .
genes were selected based on function and array data.
several genes were regulated gradually in time and seemed to follow the same pattern as the free fatty acid concentration .
other genes were acutely regulated  in this group nadc <dig> and gstm <dig> were most drastic regulated after  <dig> hours fasting.
finally, a group of genes responded only after prolonged fasting .
these data indicate that different mechanisms underlay the response to fasting.
role of pparα during fasting
it has been shown that pparα is an important mediator of the hepatic adaptive response to fasting  <cit> .
therefore we evaluated the role of this transcription factor in regulating transporter and phase i/ii metabolism genes in small intestine during fasting.
to this end, the effect of  <dig> hours fasting was compared in wild-type and pparα knockout mice .
eight of the genes identified in wild-type mice were pparα-dependently regulated, which corresponded to 24% of all genes regulated.
no genes were found to be suppressed in a pparα dependent manner.
qrt-pcr was used to confirm the differential expression of cyp4a <dig>  abca <dig>  and slc25a <dig> .
fc = fold change, wt = wild-type mice.
ko = pparα-null mice, a = the average log <dig> transformed expression value of normal fed  and  <dig> hours  fasted mice .
discussion
in this study we set out to determine the effect of fasting on several aspects directly related to the primary function of the small intestine; the selective absorption and metabolism of food components.
we find that of all genes encoding transport proteins and phase i/ii metabolic enzymes, approximately 13% were changed due to  <dig> hours of fasting.
effects on gene expression are dependent on the length of food deprivation, and pparα is required for the adaptive response of a subset of genes.
a summary of hypothetical functional outcomes of fasting in the murine small intestine is presented in figure  <dig>  and detailed below.
although in this study we only determined mrna levels, it has been reported that for the majority of genes the mrna levels reflect protein abundance very well  <cit> .
we therefore allow ourselves to speculate about the functional consequences of fasting.
nevertheless, these implications should ultimately be evaluated in follow-up studies.
changes in intestinal structure upon fasting differ between species  <cit> .
although we did not investigate this in the current study, both lenaerts et al.
<cit>  and chappell et al.
<cit>  observed only minor changes on murine mucosal structure upon fasting.
in addition, if occurring, we believe that changes on tissue base do not affect our gene expression data, because otherwise we should have seen more pronounced differences on gene expression level.
an array of intestinal transporters and phase i and ii metabolic enzymes are required to handle adequately various endogenous molecules, food components including nutrients and xenobiotics.
our finding that  <dig> hours of fasting results in the differential expression of 13% of the transporter and phase i/ii metabolism genes has implications for metabolism of the different types of substrates.
regulation of genes involved in intestinal motility
expression of the serotonin transporter sert was increased after  <dig> hours of fasting.
serotonin, a neurotransmitter secreted by enterochromaffin cells, is considered to play a key role in normal functioning of the gut, initiating peristaltic reflex pathways and facilitating propulsive activity.
sert-mediated uptake of serotonin in enterocytes is responsible for the termination of the action of serotonin in the intestine  <cit> .
after a meal, the small intestine exhibits a pattern of phasic contractions of various amplitudes  <cit> .
during fasting, these phasic contractions are replaced by a cyclic pattern with less frequent contractions, enough to propel undigested food residues and sloughed enterocytes.
as it is known that serotonin increases the frequency of intestinal contractions  <cit> , removal of serotonin by sert preserves these lower motility reflexes during fasting.
sult1d <dig> is involved in the sulfation of serotonin for the serotonin removal in enterocytes  <cit> .
for this conjugation the activated form of sulphate, paps, is needed.
sult1d <dig>  the apical sulphate transporter dtd, and the paps transporter papst <dig> were all upregulated during fasting.
we believe that the coordinated induction of these enzymes is required to maintain the lower peristaltic reflexes during fasting .
regulation of genes involved in metabolism of energy-yielding substrates
fasting increased the expression of cact and fatp <dig>  and g6pt <dig>  three intracellular slc transporters involved in transport of substrates of mitochondrial β-oxidation and glycogenolysis, respectively  <cit> .
changes in gene expression of these transporters coincided with differential gene expression of enzymes involved in both processes .
in addition, the increased expression of cyp4a <dig> and cyp4b <dig> points to enhanced peroxisomal oxidation of fatty acids  <cit> , and an elevated level of nadc <dig> implies increased uptake from luminal dicarboxylates  as well as citrate secreted by pancreatic and gastric juice  <cit> .
thus, these changes reflect the molecular events caused by the switch of fuel utilization from carbohydrates to fatty acid oxidation .
regulation of genes involved in cholesterol efflux
cholesterol can be secreted from enterocytes by chylomicrons and by the efflux transporters abca <dig> and abcg5/g <dig>  expression of the cholesterol efflux transporters abca <dig> and abcg <dig> was increased upon fasting.
since both carriers are lxr target genes  <cit>  we believe this is due to the profoundly increased expression levels of cyp27a <dig>  which results in enhanced levels of the potent lxr agonist 27-hydroxycholesterol  <cit> .
since during fasting no cholesterol is required for chylomicron formation, we speculate this may be a compensatory mechanism for the enterocyte to balance its intracellular cholesterol concentrations .
regulation of genes involved in metabolism of xenobiotics
various cypp450s with well-known drug-metabolizing capacity were differentially expressed after fasting .
increased expression of cyp3a <dig> after fasting has also been observed in rat liver  <cit> .
although cyp3a <dig> does not have a direct human ortholog, it has similar substrate specificity as human cyp3a <dig>  cyp3a <dig> is considered to be the major metabolizing enzyme of approximately half of the drugs in use today  <cit> .
our results indicate that upon a fasting period drugs may be more efficiently detoxified , which is of relevance e.g.
during surgical interventions in patients.
except for gsts, which were expressed at reduced levels, fasting had no denoting effect on expression of phase ii metabolism genes.
gsh plays an important role in the defense against oxidative stress  <cit> , and is required as cofactor for glutathione peroxidase  and glutathione s-transferase  activity.
gpx detoxifies peroxides using gsh as an electron donor, producing gssg as end product which in turn is converted back into gsh by glutathione reductase .
gsts catalyze the conjugation of gsh to a wide variety of endogenous and exogenous electrophilic compounds.
it is known that upon fasting levels of gsh are reduced in small intestine and liver  <cit> , likely as a result of increased oxidation and decreased presence of its dietary precursory amino acids  <cit> .
we believe these reduced gsh levels may be responsible for the observed reduced mrna levels of the glutathione s-transferase , since it is known that expression levels of gsts are directly dependent on the presence of gsh  <cit> .
in addition, it is known that dietary electrophiles are able to activate the transcription factor nrf <dig>  which can activate among others gsts  <cit> .
during fasting these dietary electrophiles are not available, which may be an additional explanation for the reduced expression levels of gsts.
the time dependent fasting experiment showed that the reduction of gstm <dig> is a very acute process.
thus, regardless the underlying mechanism, the reduced gsh and gsts levels will render the fasting gut more sensitive towards electrophilic stressors and other gst substrates, which may have implications for humans on drug therapy during fasting and the period directly thereafter .
role of pparα during fasting
we showed that eight of the transporters and phase i/ii metabolism genes were pparα dependently regulated during fasting .
genes that were most prominently regulated by pparα were involved in lipid metabolism .
in liver cyp4a <dig> and abca <dig> are known to be regulated via pparα during fasting  <cit> .
furthermore, cyp4a <dig> is a known pparα target gene  <cit> .
it has also been reported that abca <dig> is regulated by pparα  <cit> , although no ppar responsive elements have been identified in its promoter region  <cit> .
sert and dtd were pparα dependently regulated, which indicates that pparα may link nutritional status to peristaltic movement.
sert, dtd, slc25a <dig>  smct <dig>  chst <dig>  and mgst <dig> have all not been identified yet as pparα target genes.
we conclude that pparα is required for the adaptive response of a subset of genes.
CONCLUSIONS
in this study we provide an overview of the effects of fasting on expression of transporter and phase i/ii metabolism genes in the small intestine.
twenty-four hours of fasting had a high impact on gene expression of murine small intestinal transporter and phase i/ii metabolism genes.
in addition, we demonstrated that the effects on gene expression are dependent on the length of food deprivation.
affected processes can functionally be summarized as a) increased oxidation of fat and xenobiotics, b) increased activation of pparα, c) increased cholesterol secretion, d) increased susceptibility to electrophilic stressors, and e) reduced intestinal motility.
finally, we showed that pparα mediates a part of the adaptive response to fasting.

 fasting, the act of willingly abstaining from food, is a frequently occurring natural status in humans. fasting is a popular strategy to manage overweight or obesity, it is a traditional habit in certain religions or societies, and it is an accepted pre-surgical procedure. during fasting whole-body fuel utilization gradually shifts from carbohydrates and fat in the fed state to proteins and fat after a day of fasting. the nuclear receptor peroxisome proliferator-activated receptor alpha plays an important role in the control of the hepatic metabolic response. during fasting, free fatty acid levels in plasma are elevated and can activate pparα, which regulates a large array of hepatic genes including those involved in fatty acid catabolism. the small intestine is the primary organ for digestion and selective absorption of nutrients and other food constituents. absorption of these molecules across the intestinal epithelium occurs mainly by multiple transmembrane transporters that principally belong to two superfamilies, namely the solute carrier and the atp binding cassette superfamily of transporters. slc transporters located at the apical membrane of the enterocyte are responsible for the selective uptake of macronutrients, such as di- and tripeptides, hexoses and fatty acids. in contrast, abc transporters are efflux transporters responsible for the active removal of substances, including nutrients such as cholesterol, limiting their intracellular concentrations. besides their presence in plasma membranes, slc and abc transporters are also located in intracellular organelles, such as mitochondria or peroxisomes, in which they are responsible for uptake or secretion of metabolites. in addition, it has become clear that the intestinal epithelium is an important metabolic site, to a great extend responsible for the first-pass metabolism of nutrients and xenobiotics. numerous metabolic reactions occur in enterocytes, including those typically referred to as phase i and phase ii metabolism. phase i metabolism commonly refers to oxidative, peroxidative, and reductive metabolism of endogenous compounds and drugs, mediated by cytochrome p isoenzymes. phase ii metabolism often succeed phase i metabolism and is mediated by several enzymatic systems. in general, phase ii metabolism yields conjugated metabolites, increasing the water solubility of lipophilic compounds. the most important phase ii enzymes are sulfotransferases, udp-glucuronosyltransferases, glutathione s-transferases, n-acetyltransferases, and epoxide hydrolases. several abc transporters can secrete metabolites resulting from phase i and phase ii enzymatic transformations. previous studies showed that fasting has a dramatic effect on small intestinal transport function. however, little is known on the expression of transport and phase i/ii metabolism genes in small intestine during fasting and the role of pparα therein. we therefore set out to investigate the effects of fasting on expression of these genes using microarrays and quantitative rt-pcr. we conclude that the absorptive as well as the detoxification capacity of the small intestine is altered during fasting, and that pparα mediates a part of the adaptive response to fasting.  effect of hours of fasting on expression of transporter and phase i/ii metabolism genes the affymetrix genechip mouse genome 430a array comprises probesets, representing unique genes. annotation information from affymetrix was queried to compile a list of transporter and phase i/ii metabolism genes present on the array. this list consisted of probesets, encoding for unique genes, and was used in the remainder of our analyses. under basal, chow-fed conditions, our filtering protocol identified significantly expressed genes in the small intestine, of which were transporters and phase i/ii metabolism genes. after hours of fasting, genes, including transporter and phase i/ii metabolism genes, were differentially expressed, corresponding to 13% of the expressed transporter and phase i/ii metabolism genes. for selected genes additional qrt-pcr analyses were performed, which confirmed the array results. analysis of all genes on the moe430a array compared with transporters and phase i/ii metabolism genes. microarray results were confirmed with qrt-pcr. fc = fold change, qrt-pcr control samples have been set arbitrarily to qrt-pcr data are means ± standard error. solute carrier transporters we studied slc transporters, which amounts to 68% of the total number transporters of this superfamily currently known. after hours of fasting, slc transporters were differentially expressed, which corresponded to 15% of the expressed slc transporter genes in the small intestine. with respect to apical transporters, expression of the short-chain fatty acid transporter smct, the carboxylate transporter nadc, the prostaglandin transporter pgt, and the sulphate transporter dtd was increased, whereas the expression level of the zinc transporter zip was suppressed. the basolaterally located monocarboxylate transporter mct and iron transporter ireg were downregulated, and the basolateral neurotransmitter serotonin transporter sert, was upregulated. seven intracellular slc transporters were induced; znt, g6pt, fatp, papst, cact, mrs3/, and slc25a znt is responsible for storage of zinc in sub-apically-located vesicles. g6pt and fatp are localized at the endoplasmic reticulum, transporting glucose- phosphate and fatty acyl-coa esters, respectively. the sulphate donor 3'-phosphoadenosine 5'-phosphosulfate transporter papst is localized in the golgi. finally, cact, mrs3/ and slc25a are all three present in mitochondria shuttling metabolites across the inner mitochondrial membrane. cact and mrs3/ transport fatty acyl carnitines for fatty acid oxidation and iron, respectively. the function of slc25a is currently not known. finally, fuct an additional member of the slc family, transporting nucleotide sugars, was downregulated. a = the average log transformed expression value of normal fed and hours fasted mice, sd = standard deviation, wt = wild-type mice. in addition to the fold change, the intracellular localization of the corresponding protein is given. phase i and ii metabolic genes next we evaluated the effects of fasting on expression of phase i/ii metabolism genes. results are presented in table we studied cypp450s, which corresponds to 66% of the currently known mouse cypp450s. five cypp450s were differentially expressed, corresponding to 29% of the expressed cypp450s in small intestine. cyp2j was upregulated. members of the cyp family are well known to be responsible for the nadph-dependent oxidation of steroids and fatty acid as well as drugs. in addition, expression of cyp3a typically known because of its drug-metabolizing capacity, was increased. finally, cyp4a and cyp4b involved in peroxisomal oxidation of fatty acids, and cyp27a involved in conversion of cholesterol into 27-hydroxycholesterol, were all induced upon fasting. with respect to phase ii metabolism, expression of in total enzymes was analyzed. except for gsts, fasting had no denoting effect on expression of phase ii metabolism genes, since only non-gsts were changed. apart from gstm all changed gsts were downregulated. the aldo-keto reductase akr1b, involved in detoxification of lipid peroxides, and two sulfotransferases, sult1d and chst were increased. a = the average log transformed expression value of normal fed and hours fasted mice, sd = standard deviation, wt = wild-type mice. abc transporters effects of fasting on expression of abc transporters are summarized in table we analyzed of the murine abc transporters. the transporters abca, involved in basolateral efflux of cholesterol, and abcg, involved in transport of sterols and stanols across the apical membrane, were upregulated. a = the average log transformed expression value of normal fed and hours fasted mice, sd = standard deviation, wt = wild-type mice. in addition to the fold change, the intracellular localization of the corresponding protein is given. time dependent changes of gene expression during fasting to get insight into the time-dependent effects of fasting, we performed an additional experiment in which mice were deprived of food for various time points up to hours. intestinal weight, blood glucose and plasma free fatty acid levels were measured. as expected, blood glucose levels decreased until hours of fasting, after which it stabilized. free fatty acid levels rapidly increased at the onset of fasting, and remained constant after hours. intestinal weight decreased already after hours of fasting, significant changes were found as from hours of fasting. for selected transporters and phase i/ii metabolism enzymes gene expression was measured using qrt-pcr. genes were selected based on function and array data. several genes were regulated gradually in time and seemed to follow the same pattern as the free fatty acid concentration. other genes were acutely regulated in this group nadc and gstm were most drastic regulated after hours fasting. finally, a group of genes responded only after prolonged fasting. these data indicate that different mechanisms underlay the response to fasting. role of pparα during fasting it has been shown that pparα is an important mediator of the hepatic adaptive response to fasting. therefore we evaluated the role of this transcription factor in regulating transporter and phase i/ii metabolism genes in small intestine during fasting. to this end, the effect of hours fasting was compared in wild-type and pparα knockout mice. eight of the genes identified in wild-type mice were pparα-dependently regulated, which corresponded to 24% of all genes regulated. no genes were found to be suppressed in a pparα dependent manner. qrt-pcr was used to confirm the differential expression of cyp4a abca and slc25a. fc = fold change, wt = wild-type mice. ko = pparα-null mice, a = the average log transformed expression value of normal fed and hours fasted mice. discussion in this study we set out to determine the effect of fasting on several aspects directly related to the primary function of the small intestine; the selective absorption and metabolism of food components. we find that of all genes encoding transport proteins and phase i/ii metabolic enzymes, approximately 13% were changed due to hours of fasting. effects on gene expression are dependent on the length of food deprivation, and pparα is required for the adaptive response of a subset of genes. a summary of hypothetical functional outcomes of fasting in the murine small intestine is presented in figure and detailed below. although in this study we only determined mrna levels, it has been reported that for the majority of genes the mrna levels reflect protein abundance very well. we therefore allow ourselves to speculate about the functional consequences of fasting. nevertheless, these implications should ultimately be evaluated in follow-up studies. changes in intestinal structure upon fasting differ between species. although we did not investigate this in the current study, both lenaerts et al. and chappell et al. observed only minor changes on murine mucosal structure upon fasting. in addition, if occurring, we believe that changes on tissue base do not affect our gene expression data, because otherwise we should have seen more pronounced differences on gene expression level. an array of intestinal transporters and phase i and ii metabolic enzymes are required to handle adequately various endogenous molecules, food components including nutrients and xenobiotics. our finding that hours of fasting results in the differential expression of 13% of the transporter and phase i/ii metabolism genes has implications for metabolism of the different types of substrates. regulation of genes involved in intestinal motility expression of the serotonin transporter sert was increased after hours of fasting. serotonin, a neurotransmitter secreted by enterochromaffin cells, is considered to play a key role in normal functioning of the gut, initiating peristaltic reflex pathways and facilitating propulsive activity. sert-mediated uptake of serotonin in enterocytes is responsible for the termination of the action of serotonin in the intestine. after a meal, the small intestine exhibits a pattern of phasic contractions of various amplitudes. during fasting, these phasic contractions are replaced by a cyclic pattern with less frequent contractions, enough to propel undigested food residues and sloughed enterocytes. as it is known that serotonin increases the frequency of intestinal contractions, removal of serotonin by sert preserves these lower motility reflexes during fasting. sult1d is involved in the sulfation of serotonin for the serotonin removal in enterocytes. for this conjugation the activated form of sulphate, paps, is needed. sult1d the apical sulphate transporter dtd, and the paps transporter papst were all upregulated during fasting. we believe that the coordinated induction of these enzymes is required to maintain the lower peristaltic reflexes during fasting. regulation of genes involved in metabolism of energy-yielding substrates fasting increased the expression of cact and fatp and g6pt three intracellular slc transporters involved in transport of substrates of mitochondrial β-oxidation and glycogenolysis, respectively. changes in gene expression of these transporters coincided with differential gene expression of enzymes involved in both processes. in addition, the increased expression of cyp4a and cyp4b points to enhanced peroxisomal oxidation of fatty acids, and an elevated level of nadc implies increased uptake from luminal dicarboxylates as well as citrate secreted by pancreatic and gastric juice. thus, these changes reflect the molecular events caused by the switch of fuel utilization from carbohydrates to fatty acid oxidation. regulation of genes involved in cholesterol efflux cholesterol can be secreted from enterocytes by chylomicrons and by the efflux transporters abca and abcg5/g expression of the cholesterol efflux transporters abca and abcg was increased upon fasting. since both carriers are lxr target genes we believe this is due to the profoundly increased expression levels of cyp27a which results in enhanced levels of the potent lxr agonist 27-hydroxycholesterol. since during fasting no cholesterol is required for chylomicron formation, we speculate this may be a compensatory mechanism for the enterocyte to balance its intracellular cholesterol concentrations. regulation of genes involved in metabolism of xenobiotics various cypp450s with well-known drug-metabolizing capacity were differentially expressed after fasting. increased expression of cyp3a after fasting has also been observed in rat liver. although cyp3a does not have a direct human ortholog, it has similar substrate specificity as human cyp3a cyp3a is considered to be the major metabolizing enzyme of approximately half of the drugs in use today. our results indicate that upon a fasting period drugs may be more efficiently detoxified, which is of relevance e.g. during surgical interventions in patients. except for gsts, which were expressed at reduced levels, fasting had no denoting effect on expression of phase ii metabolism genes. gsh plays an important role in the defense against oxidative stress, and is required as cofactor for glutathione peroxidase and glutathione s-transferase activity. gpx detoxifies peroxides using gsh as an electron donor, producing gssg as end product which in turn is converted back into gsh by glutathione reductase. gsts catalyze the conjugation of gsh to a wide variety of endogenous and exogenous electrophilic compounds. it is known that upon fasting levels of gsh are reduced in small intestine and liver, likely as a result of increased oxidation and decreased presence of its dietary precursory amino acids. we believe these reduced gsh levels may be responsible for the observed reduced mrna levels of the glutathione s-transferase, since it is known that expression levels of gsts are directly dependent on the presence of gsh. in addition, it is known that dietary electrophiles are able to activate the transcription factor nrf which can activate among others gsts. during fasting these dietary electrophiles are not available, which may be an additional explanation for the reduced expression levels of gsts. the time dependent fasting experiment showed that the reduction of gstm is a very acute process. thus, regardless the underlying mechanism, the reduced gsh and gsts levels will render the fasting gut more sensitive towards electrophilic stressors and other gst substrates, which may have implications for humans on drug therapy during fasting and the period directly thereafter. role of pparα during fasting we showed that eight of the transporters and phase i/ii metabolism genes were pparα dependently regulated during fasting. genes that were most prominently regulated by pparα were involved in lipid metabolism. in liver cyp4a and abca are known to be regulated via pparα during fasting. furthermore, cyp4a is a known pparα target gene. it has also been reported that abca is regulated by pparα, although no ppar responsive elements have been identified in its promoter region. sert and dtd were pparα dependently regulated, which indicates that pparα may link nutritional status to peristaltic movement. sert, dtd, slc25a smct chst and mgst have all not been identified yet as pparα target genes. we conclude that pparα is required for the adaptive response of a subset of genes.  in this study we provide an overview of the effects of fasting on expression of transporter and phase i/ii metabolism genes in the small intestine. twenty-four hours of fasting had a high impact on gene expression of murine small intestinal transporter and phase i/ii metabolism genes. in addition, we demonstrated that the effects on gene expression are dependent on the length of food deprivation. affected processes can functionally be summarized as a) increased oxidation of fat and xenobiotics, b) increased activation of pparα, c) increased cholesterol secretion, d) increased susceptibility to electrophilic stressors, and e) reduced intestinal motility. finally, we showed that pparα mediates a part of the adaptive response to fasting. 
however, little is known on expression of intestinal transport and phase i/ii metabolism genes during fasting and the role the fatty acid-activated transcription factor pparα may play herein. after hours of fasting, expression levels of of the analyzed transporter and phase i/ii metabolism genes were changed. upregulated genes were involved in transport of energy-yielding molecules in processes such as glycogenolysis and mitochondrial and peroxisomal oxidation of fatty acids. differentially expressed genes are involved in a variety of processes, which functionally can be summarized as a) increased oxidation of fat and xenobiotics, b) increased cholesterol secretion, c) increased susceptibility to electrophilic stressors, and d) reduced intestinal motility. we therefore investigated the effects of fasting on expression of these genes using affymetrix genechip moe430a arrays and quantitative rt-pcr. we have characterized the response to fasting on expression of transporters and phase i/ii metabolic enzymes in murine small intestine.
 fasting has dramatic effects on small intestinal transport function. however, little is known on expression of intestinal transport and phase i/ii metabolism genes during fasting and the role the fatty acid-activated transcription factor pparα may play herein. we therefore investigated the effects of fasting on expression of these genes using affymetrix genechip moe430a arrays and quantitative rt-pcr.  after hours of fasting, expression levels of of the analyzed transporter and phase i/ii metabolism genes were changed. upregulated genes were involved in transport of energy-yielding molecules in processes such as glycogenolysis and mitochondrial and peroxisomal oxidation of fatty acids. other induced genes were responsible for the inactivation of the neurotransmitter serotonin, formation of eicosanoids, or for secretion of cholesterol. cyp3a typically known because of its drug metabolizing capacity, was also increased. fasting had no pronounced effect on expression of phase ii metabolic enzymes, except for glutathione s-transferases which were down-regulated. time course studies revealed that some genes were acutely regulated, whereas expression of other genes was only affected after prolonged fasting. finally, we identified genes that were pparα-dependently upregulated upon fasting.  we have characterized the response to fasting on expression of transporters and phase i/ii metabolic enzymes in murine small intestine. differentially expressed genes are involved in a variety of processes, which functionally can be summarized as a) increased oxidation of fat and xenobiotics, b) increased cholesterol secretion, c) increased susceptibility to electrophilic stressors, and d) reduced intestinal motility. this knowledge increases our understanding of gut physiology, and may be of relevance for e.g. pre-surgery regimen of patients. 