BACKGROUND
fasting processes are evolutionarily highly conserved adaptive responses to food deprivation in all organisms.
mammals follow a day/night pattern that includes a daily resting period during which the body is in a fasted state and must adapt to the lack of nutrient intake by changing the metabolic state in several organs and at the systemic level.
when deprived of dietary nutrients, the body initially derives glucose from glycogen stores, which are quickly depleted  <cit> .
if fasting continues, peripheral organs switch from glucose to fatty acids as the primary energy source.
these fatty acids are mainly supplied from adipose tissue stores where they are released from triglyceride droplets by the process of lipolysis  <cit> .
however, the brain is not equipped to derive energy from fatty acids, but uninterrupted maintenance of its function is essential to organismal survival.
hence, the perhaps most astounding metabolic change in fasting is the massive increase in the hepatic production of ketone bodies, which the brain can use as a secondary energy source  <cit> .
the emergence of biochemical pathways that produce and then utilize ketone bodies is believed to be a major selective force in the evolutionary history of homo sapiens and this adaptive process, along with others, enables human adults to survive for more than two months without food intake  <cit> .
while this is crucial for survival of millions in poor, under-developed countries, western societies are facing a pandemic of obesity with more than one third of the population being obese in countries like the united states of america  <cit> .
in this context, fasting is still one of the most efficient weight loss measures and therefore a valuable therapeutic tool for the management of obesity  <cit> .
indeed, caloric restriction and alternate-day fasting have both been shown to increase longevity  <cit>  and reduce metabolic risk factors  <cit> .
further, a ketogenic state as in fasting can also be elicited by low-carbohydrate diets which are among the most efficient weight loss programs  <cit>  and are also used in clinical settings to treat forms of epilepsy  <cit> .
for these reasons it is imperative to add to the current knowledge about the molecular underpinnings and the systemic consequences of fasting.
in experimental animals, fasting is frequently used in studies where nutritional states need to be synchronized in order to keep biological variation  minimal and, thereby, to provide a steady baseline for all measured parameters.
the major problem with this approach is that there is no standard protocol that the research community adheres to and studies report fasting regimes in a wide range  and with different day-time starting points, often disregarding the intricate circadian regulation on the organ and the systemic level  <cit> .
consequently, a systematic meta-study identified fasting as one of the main sources of variation between different transcriptomic studies  <cit> .
hence, to estimate the impact on measured experimental parameters when animals are fasted, a deeper understanding of this process is needed.
for instance, whereas the fasting response of several mouse tissues  has been investigated at the transcriptome  <cit>  and proteome  <cit>  level, a comprehensive view on gene regulation in white adipose tissue of fasted mice is still missing.
in this work we investigated the dynamic and circadian responses to a fasting stimulus by measuring serum parameters and liver gene expression in fasted mice at several time points.
moreover, we measured the global transcriptome response to fasting in white adipose tissue, liver, and skeletal muscle utilizing microarray technology followed by a host of bioinformatic analyses.
interesting outcomes from the fasted adipose tissue data include a strong enrichment of upregulated genes coding for proteins that regulate transcription as well as apoptosis-related genes.
a major finding is the identification of the p <dig> signaling pathway as a common mediator of the fasting response in all three investigated tissues.
driven by these results we focused on the p53-target gene ddit <dig>  which was upregulated by fasting in all three tissues.
our experiments showed that, in cultured adipocytes, ddit <dig> is inducible by p <dig> activation and its ectopic expression is sufficient to augment lipolysis.
therefore we describe a new molecular component in the fasting-response downstream of p <dig>
RESULTS
kinetics of serum parameters and liver gene expression in mice during a 48 hour fasting period
despite a considerable amount of knowledge about the mechanisms of fasting response in different organisms , its exact timing, especially at time points earlier than 12 hours, is still unclear.
to assess the timely fasting response of serum metabolites we measured blood glucose, non-esterified fatty acids , glycerol, and β-hydroxybutyrate, as well as the hormones insulin and corticosterone, at the start of the experiment and at five time points during a 48 hour period  both in fasted and in control  male c57bl/6 j mice.
the experiment was started at the beginning of the light cycle  when mice are in their inactive phase.
for these time points we determined body weight and weight changes within each group  and measured the expression of genes in pathways central to the fasting response in liver, which is considered the main organ for maintaining systemic energy homeostasis.
significant differences were determined with a 2-way anova followed by a bonferroni posttest to determine significance for each time point .
zt: zeitgeber time .
immediate upregulation of hepatic gluconeogenesis by fasting
a well-known and essential transcriptional response to fasting is the upregulation of pck <dig> and g6pc mrnas  <cit> .
pck <dig>  participates in controlling the flux into the gluconeogenesis  pathway, while g6pc catalyzes the last step in this pathway converting glucose-6-phosphate to glucose.
activation of liver gng is essential in times of undernutrition to provide the brain with glucose, its primary energy source.
we found that this upregulation occurs already after 3 hours fasting and continues, at least in case of pck <dig> mrna, throughout 48 hours of fasting .
further, we measured continuous upregulation of pcx as well as gyk .
pcx converts pyruvate to oxaloacetate, which in turn serves as substrate for pck <dig>  gyk, which has already been shown to be upregulated by fasting in a ppara-dependent manner  <cit> , catalyzes the first step in the glycerol phosphate shuttle which utilizes glycerol released from triglyceride stores to be converted into lipids or shunted into the gng pathway.
in accordance with the early upregulation of gluconeogenic genes, serum levels of corticosterone  rose immediately after onset of fasting, showing >4-fold higher levels later in the fasting period .
however, during the first 6 hours of fasting, blood glucose levels are similar in fasted and control-fed mice despite an immediate drop in serum insulin in fasted mice .
this might be explained by an early activation of glycogenolysis in the major glucose storing tissues .
after 12 hours, glycogen stores seem to be depleted and serum glucose levels are significantly lower in fasted mice, suggesting that liver gng is compensating only partly for the lack of dietary glucose by keeping blood glucose levels around 100 mg/dl .
hence, our data on the expression profiles of gluconeogenic genes show an immediate upregulation of hepatic gng  <dig> to 6 hours after food withdrawal, despite a delayed reduction of serum glucose levels.
increase of liver ketogenesis by three hours after onset of fasting
one substrate for hepatic gng is glycerol, which is mainly derived from adipose tissue lipolysis where triglycerides are hydrolyzed to glycerol and nefa.
as indicated in figure 1b, this process results in an increase in serum glycerol and nefa after 6 hours of fasting.
serum nefa levels normalize to the control-fed levels at the 24 hour time point, presumably due to increased uptake and utilization of free fatty acids in several tissues  while glycogen stores are further depleted.
apart from being a major energy source in times of nutrient deprivation, adipose tissue-derived fatty acids serve as substrate for ketogenesis, the synthesis of ketone bodies  in the liver.
these ketone bodies can be used as secondary energy source by the brain, which cannot utilize fatty acids directly  <cit> .
in our data set, serum β-hydroxybutyrate levels are increasing steadily over 48 hours of fasting compared to ad libitum-fed control mice with the first significant increase at 3 hours .
this increase is concordant with the upregulation of liver hmgcs <dig> mrna which is a key enzyme in liver ketogenesis  <cit>  .
interestingly, liver ketogenesis and serum β-hydroxybutyrate are increased hours before blood glucose levels begin to drop .
this dynamic argues for the involvement of other sensors  detecting the absence of nutritional carbohydrates and signaling to liver to upregulate ketogenesis early in fasting.
a recent report introduced an intriguing, novel functional role for ketone bodies.
by inhibiting histone deacetylases, β-hydroxybutyrate was shown to induce the expression of genes that protect against oxidative damage in a variety of tissues  <cit> .
in that study, a change in histone deacetylation in kidney was shown at serum β-hydroxybutyrate concentrations greater than  <dig>  mm, a level we measured already after 3 hours of food withdrawal.
thus it is conceivable that this β-hydroxybutyrate-mediated mechanism could be activated early on, to set the stage for transcriptional regulation in response to fasting.
early fasting-onset upregulation of ppara target genes
fgf <dig> is a recently discovered hormone shown to be a major hub in the hepatic response to fasting by regulating fatty acid and glucose metabolism  <cit> .
in our data fgf <dig> mrna was elevated in liver of fasted animals throughout the 48 hours, peaking at 24 hours .
furthermore, fgf <dig> was shown to mediate its effect partly via upregulation of ppargc1a <cit> , a transcriptional coactivator we found to be highly increased by fasting in accordance with fgf <dig> levels .
ppargc1a in turn increases expression of many fasting response genes by binding and coactivating transcription factors such as ppara and glucocorticoid receptor  <cit> .
along these lines, most genes shown in figure  <dig> are ppara target genes  arguing for the central role of this transcription factor during fasting, evident from the phenotype of fasted ppara knock-out mice  <cit> .
however, the modest changes of liver ppara mrna levels  are unlikely to cause the strong alterations in ppara targets .
rather the transactivation of ppara by endogenous ligands , coactivation by ppargc1a, and synergistic regulation by other fasting-regulated transcription factors  could lead to the magnitude of increase of its target genes.
in summary, comparing expression of key liver fasting genes to serum parameters shows a coherent picture suggesting, in accordance with other recent studies  <cit> , a parallel activation of fasting-induced pathways rather than a sequential response as historically believed  <cit> .
this response is activated as early as  <dig> to 6 hours after food withdrawal and reaches a steady state between  <dig> and 24 hours.
our data further underlines that ppara acts as one major fasting hub, by coordinating expression of its target genes.
hence, we provide a detailed view of molecular response kinetics during a 48 hour fasting period in mice allowing one to extrapolate on the timely regulation of the fasting response in liver and in the whole organism.
global changes in transcriptome signatures of white adipose tissue, liver, and skeletal muscle in fasted mice
next, we aimed to elucidate rna abundance responses to fasting in a systematic and genome-wide manner.
most of the parameters determined in figure  <dig> show the highest difference between fasted and fed states at 24 hours initiating the experiment, suggesting that the metabolic adaption to fasting has reached a first steady state.
for this reason we chose this time point for transcriptome analyses of epididymal white adipose tissue , liver , and skeletal muscle  of fasted and control mice .
tissue-derived mrna from five fasted and five control mice were hybridized to affymetrix genechip arrays .
two hybridizations  were outliers as determined by principal component analysis and therefore excluded from further analysis.
hierarchical clustering of the remaining microarray data sets showed that experiments strongly cluster by tissue type .
the heatmap contains about  <dig> probe sets differentially expressed by ≥  <dig> -fold at a false discovery rate < 5% after multiple testing adjustment  in at least one condition.
wat showed the highest number of differentially expressed probe sets between fasting and fed states , more than double the number of genes regulated in liver .
additional file  <dig> provides the lists of probes expressed more than  <dig> -fold with an fdr <dig> for each of the three tissues.
functional annotation clustering of genes regulated in fasted white adipose tissue
although the transcriptome signatures of other mouse organs, including liver and muscle, during fasting were extensively investigated  <cit>  and analyses of adipose tissue transcriptomes during fasting are available  the current study is, to our knowledge, the first to focus on characterization of the global gene expression response of adipose tissue in fasted mice.
hence, we focused our initial analysis on the wat dataset and performed functional annotation of the genes up - or downregulated in fasted wat  by mapping these lists onto gene ontology  and kegg pathways  <cit>  utilizing the david tool  <cit> .
to visualize the results we employed the functional clustering option, which combines redundant entities, based on similarity of gene lists, into clusters.
figure 3a and b respectively show the resulting clusters from the lists of “wat downregulated” or “wat upregulated” genes together with contributing go domains in parentheses.
the x-axis shows the cluster group enrichment scores representing a geometric mean  of p-values of entities in each cluster.
we only show clusters with an enrichment score larger than  <dig> because they contain at least one entity with a significant p-value after multiple testing correction .
the detailed results of the functional clustering including terms/pathways contributing to each cluster are given in additional file  <dig>
functional clustering of genes downregulated by fasting in wat  yields terms related to “mitochondria” in the cellular component  go category, indicating suppression of mitochondrial processes in times of energy scarcity.
in the go domain molecular function  we find “gtp-associated” terms and go terms related to “nucleotide binding”.
go biological processes  and kegg pathways are found in clusters related to “steroid and sterol metabolism”, “carbohydrate metabolism” and “biosynthesis of fatty acids and amino acids”.
downregulation of steroid and sterol metabolism is analyzed and discussed in more detail later.
the cluster of carbohydrate metabolism is mainly comprised of go terms referring to catabolism of monosaccharides, like hexose or glucose, and might reflect the shift from glucose usage to fatty acid β-oxidation in prolonged fasting.
also the downregulation of fatty acid and amino acid biosynthesis is a plausible reaction of a fasting cell or tissue that needs to suppress energy consuming processes which increase anaplerotic pressure.
genes upregulated in fasted white adipose tissue are strongly connected to transcriptional regulation
genes that are upregulated in fasted wat  show the highest enrichment score for the go mf category related to “ion binding”, which is a rather general and unspecific term.
however, we find a cluster containing  <dig> genes that relate to “apoptosis” and “cell death” indicating that apoptotic pathways are activated during fasting in wat.
finally, we find high enrichment in a cluster containing “nucleus”-related go cc terms as well as a cluster with terms referring to “regulation of transcription” , both of which indicate a strong positive influence of fasting on the transcriptional program of adipose tissue.
this is consistent with wat being the tissue with the highest number of differentially regulated genes when compared to liv and sm .
to substantiate this finding we tested whether the genes in the upregulated list are enriched for transcriptional regulator molecules such as transcription factors, cofactors and chromatin remodelers as defined by the manually curated animaltfdb  <cit> .
as a control we also tested for enrichment in the list of genes downregulated by fasting in wat which did not map to “regulation of transcription”-associated go terms in our david analysis.
as shown in additional file  <dig> the wat upregulated list contains at least double the number of transcriptional regulators, compared to the wat downregulated list , and comprises 9%, 12%, and 16% of annotated mouse transcription factors, cofactors, and chromatin remodelers, respectively.
additional file  <dig> also lists the expression values and the annotations of regulated genes in wat according to the transcription factor subclasses defined in animaltfdb.
based on ranking of the entire data set, the gene-set enrichment analysis tool  <cit>  performs a similar analysis and yields a significant enrichment for the sub-categories  of transcription factors from animaltfdb for upregulated genes .
these transcription factor families contained  <dig> genes encoding zinc finger transcription factors ,  <dig> genes encoding basic leucin zippers , and  <dig> forkhead box genes that are upregulated in fasted wat .
among the transcription factor-encoding genes with the highest upregulation by fasting  we find irf <dig>  which has been shown to tip the scales between lipogenesis and lipolysis in the latter direction in a fasting-induced manner  <cit> .
consequently, fat-specific irf <dig> knock-out mice are deficient in lipolysis and show increased adiposity  <cit> .
furthermore, genes encoding the transcription factors zim <dig> and peg <dig> are massively upregulated by fasting .
interestingly, these two genes are adjacently located at an imprinted region on mouse chromosome  <dig>  the peg <dig> knock-out mouse model develops increased adiposity despite lower food intake.
this was attributed to developmental deficiencies that lead to aberrant leptin signaling in the hypothalamus  <cit> .
however, our data suggest a direct involvement of the peg3/zim <dig> locus in adipose tissue biology.
the present study is, to our knowledge, the first to focus on the characterization of the transcriptome response to fasting of wat in mice.
surprisingly, we do not find go mapping to the biological process “lipid catabolism” which would be expected to be prominent during fasting in wat.
the absence thereof could be explained by abundant posttranscriptional regulation of lipolysis by kinases such as protein kinase a  <cit>  and amp-activated protein kinase  <cit>  which are not reflected at the transcript level.
instead our analyses specifically reveal an unexpected upregulation of cell death pathways as well as a strong enrichment of transcriptional regulators among genes activated by fasting in wat.
p <dig> signaling as top ranking pathway in the fasting response of major metabolic tissues
by producing and exporting glucose, fatty acids, glycerol, and ketone bodies wat, liv, and sm represent the organs mainly responsible for energy homeostasis during a fasting period in mammals  <cit> .
to reveal common pathways we focused on  <dig> genes that were regulated by fasting in all three tissues .
table  <dig> lists these genes ranked by decreasing average expression level.
mapping the common list to go biological processes and kegg using david yielded one kegg pathway as significantly enriched after multiple testing correction: the p <dig> signaling pathway .
additionally, an independent analysis with metacore  focusing on networks overrepresented in the common list reveals the p <dig> node as the second highest scoring network hub .
the p <dig> transcription factor is known to regulate a number of tumor suppressor pathways including cellular senescence, apoptosis, and dna repair in response to various stressors  <cit> .
about 50% of human cancers carry mutations in the p <dig> gene  <cit> .
however, evidence is now accumulating that p <dig> plays a prominent role in  metabolism as well  <cit> .
there is no direct evidence of a physiological importance of p <dig> signaling in fasting, but some studies report an activation of p <dig>  and an induction of its target genes, upon glucose deprivation in cultured cells  <cit> .
in our dataset we observe a host of p <dig> target genes apparently regulated by fasting, and a heatmap of the genes described below is shown in figure 4d.
for instance, the canonical p <dig> target cdkn1a <cit> , a cell cycle regulator better known as p <dig>  is one of the top ranked genes in table  <dig> and is the gene with the highest fasting-induced upregulation in liver .
another example is lipin- <dig> encoded by the gene lpin <dig>  which has recently been described as a novel p <dig> target  <cit> .
in our data lpin <dig> is upregulated in wat, liv, and sm by fasting.
depending on its subcellular location lipin- <dig> can act to enhance fatty acid oxidation by interacting with ppara and ppargc1a   <cit>  or, as a phosphatidate phosphatase in the cytosol, to perform a key step in triglyceride biosynthesis  <cit> .
being upregulated by fasting suggests activity of its nuclear form to drive oxidation of fatty acids in lipid storing tissues.
other p <dig> targets are the sestrins, sesn <dig>  and sesn <dig> , which have recently been shown to be induced by fasting in liver and to protect it from oxidative damage in a fasting/refeeding scenario  <cit> .
interestingly, sen et al.
report that p <dig> physically interacts with ppargc1a  <cit> , which we show to be strongly upregulated in liver  by qpcr as well as in our microarray study in wat and liv .
in their study sen et al.
show that ppargc1a can bind and thereby direct p <dig> to the promoters/enhancers of pro-arrest as well as metabolic target genes.
thus, this interaction of p <dig> and its coactivator ppargc1a could lead to a tissue-specific coordination of p <dig> to target genes relevant for a proper response.
genes with > <dig> x change  in all investigated tissues.
fasting-mediated downregulation of srebp pathways is common to major metabolic tissues
it was reported that p <dig> is capable of suppressing the promoter of the gene encoding the transcription factor sterol-regulatory element binding protein  <dig> .
the srebp family consists of three members: srebp1a and srebp1c, both transcribed from the same gene srebf <dig>  with srebp-1c being the predominant isoform to regulate lipogenesis in metabolic tissues, such as wat and liv; and srebp <dig>  transcribed from the srebf <dig> locus and responsible for regulation of sterol metabolism  <cit> .
consistent with these reports, a metacore analysis on the common list delivers “regulation of lipid metabolism” as top ranking pathways with the srebp family of transcription factors in its center .
as mapped in figure 4c, we find srebf <dig> and srebf <dig> downregulated in all three tissues .
concordantly, established srebp <dig> downstream genes, coding for enzymes which encompass the fatty acid biosynthesis pathway like acss <dig>  acaca, fasn, scd <dig>  and scd <dig> <cit> , are robustly down-regulated by fasting in our data .
further, david analyses for the set of commonly regulated genes  as well as for regulated liver genes  yields go biological processes that refer to steroid metabolism and, more specifically, to cholesterol biosynthesis.
as a pivotal regulator of cholesterol homeostasis in cells, srepb <dig> mediates its effects by control of de novo synthesis and/or by regulation of cholesterol import  <cit> .
to regulate de novo synthesis, srebp <dig>  binds to promoters of most of the enzymes in the cholesterol biosynthesis pathway  <cit> .
we find that transcripts for most of these enzymes are significantly downregulated or show at least a trend to downregulation .
the internalization of cholesterol is regulated by srebp-mediated transcription of the ldl-receptor ldlr .
figure 4f shows a heatmap of these srebp <dig> target genes  <cit>  and impressively demonstrates reduction of transcripts for nearly all components of the cholesterol biosynthesis and uptake pathway in the investigated tissues.
given the extensive and intricate networks that, depending on sterol or nutrient availability, post-translationally regulate processing and activation of membrane-bound srebps  <cit> , our finding that fasting broadly regulates srebp-dependent pathways already at the transcriptional level is rather surprising.
especially in liver, this downregulation of cholesterol biosynthesis in combination with the upregulation of hgmcs <dig> , which condenses acetoacetyl coa and acetyl coa to β-hydroxy β-methylglutaryl coa, hints to a diversion of substrate from sterol synthesis to ketone body synthesis.
hence, based on our analyses on genes regulated in wat, liv, and sm, we hypothesize a shared mechanism that responds to the fasting stimulus in all three tissues: the p <dig> signaling pathway is activated by fasting in wat, liv, and sm .
coactivators like ppargc1a direct p <dig> to promoters/enhancers of genes targeted for transcription activation or repression when nutrients are lacking.
upregulation of p <dig> targets such as lpin <dig> might contribute to the shift of fasted tissues to fatty acid oxidation to provide energy substrates.
the observation that p <dig> knock-out mice are incapable of inducing liver fatty acid oxidation upon fasting  <cit>  underlines this hypothesis.
in addition, a p53-mediated downregulation of srebp <dig> is followed by a repression of fatty acid biosynthesis .
however, we note that other  pathways that are regulated at the post-translational level, and therefore not reflected in rna levels, could be responsible for some of the observed effects, such as downregulation of srebp transcripts.
one conceivable example is the activation of ampk by fasting which, via subsequent deactivation of mechanistic target of rapamycin complex  <dig> , could be responsible for the decrease in srebp mrna  <cit> .
however, based on our analyses we propose a novel and potentially crucial role for p <dig> in fasting, which eventually could manifest in profound transcriptional changes in several metabolic pathways.
although functional proof of this mechanism is necessary, several reports support our model  <cit> .
validation of expression of top-ranked genes commonly regulated by fasting in wat, liv, and sm
to validate the microarray data by means of qpcr we selected the top  <dig> genes from table  <dig> , none of which have previously been functionally described in the context of fasting in mice, as well as per <dig> and fasn, known responders to food deprivation and thereby positive controls .
per <dig> is an essential regulator in the core clock machinery of circadian rhythm  <cit>  and, in liv and at at the beginning of the light phase, it was previously shown that its expression is higher in fasting compared to control fed mice  <cit> .
this difference is due to a circadian phase shift that takes place during an extended fasting period  <cit> .
as mentioned above fasn has long been known to be a downstream target gene of srebp <dig> and is downregulated due to the drop in srebp <dig> levels during fasting  <cit> .
the basic-leucin zipper transcription factor cebpd has been described in a number of cellular contexts, such as osteogenesis and adipogenesis .
cebpd expression is known to respond to glucocorticoids and to increased camp levels, both of which could explain its upregulation upon fasting.
cdkn1a  as a major p <dig> target gene, is mainly described as a cell cycle and apoptosis regulator that inhibits cyclin-dependent kinases  <cit>  and has no known role in fasting.
finally, ddit <dig>  a gene initially reported to be readily induced by dexamethasone  <cit>  as well as upon certain cellular stresses , shows the highest extent of upregulation in wat and sm in the common list .
interestingly, it has also been described as a p <dig> target gene  <cit> , which led us to further investigate it .
hence, we confirmed fasting-mediated regulation of all genes selected for qpcr validation in all three tissues  and show a strong correlation with the microarray measurements for all these genes .
this introduces three intriguing and novel players in the response to fasting.
ddit <dig> is fasting-induced in wat, liv, and sm and is inducible by p <dig> activation in cultured adipocytes
to investigate p <dig> signaling as a common fasting regulator in wat, liv, and sm, we focused on dna damage-induced transcript  <dig> , the top ranking gene in table  <dig> which has been described as functional p <dig> target gene  <cit> .
in all three tissues investigated, ddit <dig> mrna is upregulated at latest by 24 hours after onset of fasting and overnight fasting is sufficient to increase ddit <dig> protein levels , which has been shown by others in gastrocnemius muscle of rats  <cit> .
in our data, differences in the magnitude of fasting-induction between the mrna and protein level  could be explained by the fact that ddit <dig> protein stability is highly regulated in different cell systems  <cit> .
however, especially in adipose tissue ddit <dig> protein seems to be stably induced when mice are fasted.
to show that ddit <dig> can be directly induced by p <dig> in adipocytes, we treated mature c3h10t1/ <dig> adipocytes for 6 hours with nutlin- <dig>  a specific p <dig> activator  <cit> .
nutlin- <dig> treatment led to an increase of ddit <dig> mrna similar to the canonical target cdkn1a .
most importantly, ddit <dig> protein was also stably induced in all replicates .
in addition, the p <dig> targets sesn <dig> and srebf <dig> were regulated by nutlin- <dig> in a way similar to the in vivo fasting situation .
hence, ddit <dig> is stably induced by fasting and upregulated by p <dig> activation in cultured adipocytes.
overexpression of ddit <dig> is sufficient to increase lipolysis in cultured adipocytes
in a recent report ddit <dig> was shown to be involved in lipid metabolism in adipocytes signaling via the mtorc <dig> pathway  <cit> .
also in other studies, ddit <dig> has been repeatedly described as a negative regulator of mtorc <dig> in a variety of cell types  <cit> .
interestingly, in the context of starvation, the nutrient-sensitive mtorc <dig> pathway needs to be suppressed for the proper fasting response in liver  <cit>  and its suppression induces lipolysis in adipocytes  <cit> .
hence, we examined whether upregulation of ddit <dig> promotes lipolysis in adipocytes by inhibiting mtorc <dig> activity.
for this we transiently overexpressed ddit <dig> in differentiated c3h10t1/ <dig> adipocytes  and determined glycerol and ffa in the medium as a measure of lipolysis.
indeed, we observed a ~30% increased glycerol release and a ~40% increased ffa release from ddit <dig> overexpressing cells compared to the empty vector control , while expression of genes in the lipolytic pathway remained unchanged .
upon β-adrenergic stimulation  the increase in lipolysis upon ddit <dig> overexpression was still evident, although not statistically significant .
however, as assayed by phosphorylation of the downstream target s6k <dig> at threonine  <dig>  mtorc <dig> activity was unchanged despite effective overexpression of ddit <dig> .
an antibody against total s6k <dig> protein served as loading control and cells treated with rapamycin, a potent exogenous mtorc <dig> inhibitor, as control for phosphorylation-specific s6k <dig> antibody.
others have reported that, in sm, dexamethasone-mediated ddit <dig> increase leads to reduced mtorc <dig> signaling  <cit> , but, judging from our data, in adipocytes ddit4-mediated lipolysis seems to be independent of mtorc <dig> activity.
this is consistent with the observation that the ddit4-mtorc <dig> axis is functional in some cell types but not in others  <cit> .
further, as knock-down of ddit <dig> was reported to decrease insulin-stimulated de-novo lipogenesis in adipocytes  <cit> , we wanted to rule out that the observed increase in lipolysis upon ddit <dig> overexpression is merely an effect of increased lipogenesis and with that higher lipid content per se.
in addition to an unchanged phenotype as shown by mrna expression of adipocyte-specific genes , we did not detect an increase in the incorporation of radio-labeled glucose into total lipids in ddit <dig> overexpressing cells compared to control cells .
rather, a small decrease in lipogenesis was observed , which is consistent with a potential role of ddit <dig> in regulating fasting responses in adipocytes.
hence, our data on the p53-target ddit <dig> presents a valuable example for hypothesis generation from a large-scale data set by suggesting a new role in the fine-tuning of the fasting-response in adipose tissue.
however, further investigation is needed to elucidate the exact mechanism by which ddit <dig> is enhancing lipolysis in adipocytes, because we show that this is independent form mtorc <dig> and de-novo lipogenesis.
CONCLUSIONS
in this study, we took a comprehensive view on the fasting process in mice.
our combination of focused and genome-wide approaches reveals several fasting-related findings:  we provide a novel view on the immediate and dynamic response to fasting in mice during a 48 hour period.
these experiments focus on the timely regulation of liver genes relayed through the ppara signaling pathway, which manifests in coordinated changes of serum parameters.
the observed responses occur very early  after onset of fasting and show simultaneous activation of different pathways.
to our knowledge this is the first study focusing on the transcriptome response of white adipose tissue in fasted mice.
with our bioinformatic analyses we identify an upregulation of apoptosis-related transcripts as well as a strong enrichment of transcriptional control components in the set of upregulated genes.
concentrating our analyses on genes regulated in the three tissues mainly responsible for energy homeostasis during fasting , the p <dig> signaling pathway appears to be a common and central regulator of fasting, possibly partly mediating its effect by down-regulation of the srepb pathway.
finally, we performed experiments that prove that ddit <dig>  a p <dig> target gene upregulated by fasting in all three tissues, is induced by p <dig> activation and sufficient to increase lipolysis in cultured adipocytes.
in conclusion, our transcriptome study of three tissues combined with bioinformatic analyses and mechanistic in vitro experiments, suggests the p53-ddit <dig> axis as a novel mechanism in the fine-tuning of fasting common to major metabolic tissues.

 fasting processes are evolutionarily highly conserved adaptive responses to food deprivation in all organisms. mammals follow a day/night pattern that includes a daily resting period during which the body is in a fasted state and must adapt to the lack of nutrient intake by changing the metabolic state in several organs and at the systemic level. when deprived of dietary nutrients, the body initially derives glucose from glycogen stores, which are quickly depleted. if fasting continues, peripheral organs switch from glucose to fatty acids as the primary energy source. these fatty acids are mainly supplied from adipose tissue stores where they are released from triglyceride droplets by the process of lipolysis. however, the brain is not equipped to derive energy from fatty acids, but uninterrupted maintenance of its function is essential to organismal survival. hence, the perhaps most astounding metabolic change in fasting is the massive increase in the hepatic production of ketone bodies, which the brain can use as a secondary energy source. the emergence of biochemical pathways that produce and then utilize ketone bodies is believed to be a major selective force in the evolutionary history of homo sapiens and this adaptive process, along with others, enables human adults to survive for more than two months without food intake. while this is crucial for survival of millions in poor, under-developed countries, western societies are facing a pandemic of obesity with more than one third of the population being obese in countries like the united states of america. in this context, fasting is still one of the most efficient weight loss measures and therefore a valuable therapeutic tool for the management of obesity. indeed, caloric restriction and alternate-day fasting have both been shown to increase longevity and reduce metabolic risk factors. further, a ketogenic state as in fasting can also be elicited by low-carbohydrate diets which are among the most efficient weight loss programs and are also used in clinical settings to treat forms of epilepsy. for these reasons it is imperative to add to the current knowledge about the molecular underpinnings and the systemic consequences of fasting. in experimental animals, fasting is frequently used in studies where nutritional states need to be synchronized in order to keep biological variation minimal and, thereby, to provide a steady baseline for all measured parameters. the major problem with this approach is that there is no standard protocol that the research community adheres to and studies report fasting regimes in a wide range and with different day-time starting points, often disregarding the intricate circadian regulation on the organ and the systemic level. consequently, a systematic meta-study identified fasting as one of the main sources of variation between different transcriptomic studies. hence, to estimate the impact on measured experimental parameters when animals are fasted, a deeper understanding of this process is needed. for instance, whereas the fasting response of several mouse tissues has been investigated at the transcriptome and proteome level, a comprehensive view on gene regulation in white adipose tissue of fasted mice is still missing. in this work we investigated the dynamic and circadian responses to a fasting stimulus by measuring serum parameters and liver gene expression in fasted mice at several time points. moreover, we measured the global transcriptome response to fasting in white adipose tissue, liver, and skeletal muscle utilizing microarray technology followed by a host of bioinformatic analyses. interesting outcomes from the fasted adipose tissue data include a strong enrichment of upregulated genes coding for proteins that regulate transcription as well as apoptosis-related genes. a major finding is the identification of the p signaling pathway as a common mediator of the fasting response in all three investigated tissues. driven by these results we focused on the p53-target gene ddit which was upregulated by fasting in all three tissues. our experiments showed that, in cultured adipocytes, ddit is inducible by p activation and its ectopic expression is sufficient to augment lipolysis. therefore we describe a new molecular component in the fasting-response downstream of p  kinetics of serum parameters and liver gene expression in mice during a 48 hour fasting period despite a considerable amount of knowledge about the mechanisms of fasting response in different organisms, its exact timing, especially at time points earlier than 12 hours, is still unclear. to assess the timely fasting response of serum metabolites we measured blood glucose, non-esterified fatty acids, glycerol, and β-hydroxybutyrate, as well as the hormones insulin and corticosterone, at the start of the experiment and at five time points during a 48 hour period both in fasted and in control male c57bl/6 j mice. the experiment was started at the beginning of the light cycle when mice are in their inactive phase. for these time points we determined body weight and weight changes within each group and measured the expression of genes in pathways central to the fasting response in liver, which is considered the main organ for maintaining systemic energy homeostasis. significant differences were determined with a 2-way anova followed by a bonferroni posttest to determine significance for each time point. zt: zeitgeber time. immediate upregulation of hepatic gluconeogenesis by fasting a well-known and essential transcriptional response to fasting is the upregulation of pck and g6pc mrnas. pck participates in controlling the flux into the gluconeogenesis pathway, while g6pc catalyzes the last step in this pathway converting glucose-6-phosphate to glucose. activation of liver gng is essential in times of undernutrition to provide the brain with glucose, its primary energy source. we found that this upregulation occurs already after 3 hours fasting and continues, at least in case of pck mrna, throughout 48 hours of fasting. further, we measured continuous upregulation of pcx as well as gyk. pcx converts pyruvate to oxaloacetate, which in turn serves as substrate for pck gyk, which has already been shown to be upregulated by fasting in a ppara-dependent manner, catalyzes the first step in the glycerol phosphate shuttle which utilizes glycerol released from triglyceride stores to be converted into lipids or shunted into the gng pathway. in accordance with the early upregulation of gluconeogenic genes, serum levels of corticosterone rose immediately after onset of fasting, showing >4-fold higher levels later in the fasting period. however, during the first 6 hours of fasting, blood glucose levels are similar in fasted and control-fed mice despite an immediate drop in serum insulin in fasted mice. this might be explained by an early activation of glycogenolysis in the major glucose storing tissues. after 12 hours, glycogen stores seem to be depleted and serum glucose levels are significantly lower in fasted mice, suggesting that liver gng is compensating only partly for the lack of dietary glucose by keeping blood glucose levels around 100 mg/dl. hence, our data on the expression profiles of gluconeogenic genes show an immediate upregulation of hepatic gng to 6 hours after food withdrawal, despite a delayed reduction of serum glucose levels. increase of liver ketogenesis by three hours after onset of fasting one substrate for hepatic gng is glycerol, which is mainly derived from adipose tissue lipolysis where triglycerides are hydrolyzed to glycerol and nefa. as indicated in figure 1b, this process results in an increase in serum glycerol and nefa after 6 hours of fasting. serum nefa levels normalize to the control-fed levels at the 24 hour time point, presumably due to increased uptake and utilization of free fatty acids in several tissues while glycogen stores are further depleted. apart from being a major energy source in times of nutrient deprivation, adipose tissue-derived fatty acids serve as substrate for ketogenesis, the synthesis of ketone bodies in the liver. these ketone bodies can be used as secondary energy source by the brain, which cannot utilize fatty acids directly. in our data set, serum β-hydroxybutyrate levels are increasing steadily over 48 hours of fasting compared to ad libitum-fed control mice with the first significant increase at 3 hours. this increase is concordant with the upregulation of liver hmgcs mrna which is a key enzyme in liver ketogenesis. interestingly, liver ketogenesis and serum β-hydroxybutyrate are increased hours before blood glucose levels begin to drop. this dynamic argues for the involvement of other sensors detecting the absence of nutritional carbohydrates and signaling to liver to upregulate ketogenesis early in fasting. a recent report introduced an intriguing, novel functional role for ketone bodies. by inhibiting histone deacetylases, β-hydroxybutyrate was shown to induce the expression of genes that protect against oxidative damage in a variety of tissues. in that study, a change in histone deacetylation in kidney was shown at serum β-hydroxybutyrate concentrations greater than  mm, a level we measured already after 3 hours of food withdrawal. thus it is conceivable that this β-hydroxybutyrate-mediated mechanism could be activated early on, to set the stage for transcriptional regulation in response to fasting. early fasting-onset upregulation of ppara target genes fgf is a recently discovered hormone shown to be a major hub in the hepatic response to fasting by regulating fatty acid and glucose metabolism. in our data fgf mrna was elevated in liver of fasted animals throughout the 48 hours, peaking at 24 hours. furthermore, fgf was shown to mediate its effect partly via upregulation of ppargc1a, a transcriptional coactivator we found to be highly increased by fasting in accordance with fgf levels. ppargc1a in turn increases expression of many fasting response genes by binding and coactivating transcription factors such as ppara and glucocorticoid receptor. along these lines, most genes shown in figure  are ppara target genes arguing for the central role of this transcription factor during fasting, evident from the phenotype of fasted ppara knock-out mice. however, the modest changes of liver ppara mrna levels are unlikely to cause the strong alterations in ppara targets. rather the transactivation of ppara by endogenous ligands, coactivation by ppargc1a, and synergistic regulation by other fasting-regulated transcription factors could lead to the magnitude of increase of its target genes. in summary, comparing expression of key liver fasting genes to serum parameters shows a coherent picture suggesting, in accordance with other recent studies, a parallel activation of fasting-induced pathways rather than a sequential response as historically believed. this response is activated as early as to 6 hours after food withdrawal and reaches a steady state between and 24 hours. our data further underlines that ppara acts as one major fasting hub, by coordinating expression of its target genes. hence, we provide a detailed view of molecular response kinetics during a 48 hour fasting period in mice allowing one to extrapolate on the timely regulation of the fasting response in liver and in the whole organism. global changes in transcriptome signatures of white adipose tissue, liver, and skeletal muscle in fasted mice next, we aimed to elucidate rna abundance responses to fasting in a systematic and genome-wide manner. most of the parameters determined in figure  show the highest difference between fasted and fed states at 24 hours initiating the experiment, suggesting that the metabolic adaption to fasting has reached a first steady state. for this reason we chose this time point for transcriptome analyses of epididymal white adipose tissue, liver, and skeletal muscle of fasted and control mice. tissue-derived mrna from five fasted and five control mice were hybridized to affymetrix genechip arrays. two hybridizations were outliers as determined by principal component analysis and therefore excluded from further analysis. hierarchical clustering of the remaining microarray data sets showed that experiments strongly cluster by tissue type. the heatmap contains about probe sets differentially expressed by ≥  -fold at a false discovery rate < 5% after multiple testing adjustment in at least one condition. wat showed the highest number of differentially expressed probe sets between fasting and fed states, more than double the number of genes regulated in liver. additional file provides the lists of probes expressed more than -fold with an fdr for each of the three tissues. functional annotation clustering of genes regulated in fasted white adipose tissue although the transcriptome signatures of other mouse organs, including liver and muscle, during fasting were extensively investigated and analyses of adipose tissue transcriptomes during fasting are available the current study is, to our knowledge, the first to focus on characterization of the global gene expression response of adipose tissue in fasted mice. hence, we focused our initial analysis on the wat dataset and performed functional annotation of the genes up - or downregulated in fasted wat by mapping these lists onto gene ontology and kegg pathways utilizing the david tool. to visualize the results we employed the functional clustering option, which combines redundant entities, based on similarity of gene lists, into clusters. figure 3a and b respectively show the resulting clusters from the lists of “wat downregulated” or “wat upregulated” genes together with contributing go domains in parentheses. the x-axis shows the cluster group enrichment scores representing a geometric mean of p-values of entities in each cluster. we only show clusters with an enrichment score larger than because they contain at least one entity with a significant p-value after multiple testing correction. the detailed results of the functional clustering including terms/pathways contributing to each cluster are given in additional file functional clustering of genes downregulated by fasting in wat yields terms related to “mitochondria” in the cellular component go category, indicating suppression of mitochondrial processes in times of energy scarcity. in the go domain molecular function we find “gtp-associated” terms and go terms related to “nucleotide binding”. go biological processes and kegg pathways are found in clusters related to “steroid and sterol metabolism”, “carbohydrate metabolism” and “biosynthesis of fatty acids and amino acids”. downregulation of steroid and sterol metabolism is analyzed and discussed in more detail later. the cluster of carbohydrate metabolism is mainly comprised of go terms referring to catabolism of monosaccharides, like hexose or glucose, and might reflect the shift from glucose usage to fatty acid β-oxidation in prolonged fasting. also the downregulation of fatty acid and amino acid biosynthesis is a plausible reaction of a fasting cell or tissue that needs to suppress energy consuming processes which increase anaplerotic pressure. genes upregulated in fasted white adipose tissue are strongly connected to transcriptional regulation genes that are upregulated in fasted wat show the highest enrichment score for the go mf category related to “ion binding”, which is a rather general and unspecific term. however, we find a cluster containing genes that relate to “apoptosis” and “cell death” indicating that apoptotic pathways are activated during fasting in wat. finally, we find high enrichment in a cluster containing “nucleus”-related go cc terms as well as a cluster with terms referring to “regulation of transcription”, both of which indicate a strong positive influence of fasting on the transcriptional program of adipose tissue. this is consistent with wat being the tissue with the highest number of differentially regulated genes when compared to liv and sm. to substantiate this finding we tested whether the genes in the upregulated list are enriched for transcriptional regulator molecules such as transcription factors, cofactors and chromatin remodelers as defined by the manually curated animaltfdb. as a control we also tested for enrichment in the list of genes downregulated by fasting in wat which did not map to “regulation of transcription”-associated go terms in our david analysis. as shown in additional file the wat upregulated list contains at least double the number of transcriptional regulators, compared to the wat downregulated list, and comprises 9%, 12%, and 16% of annotated mouse transcription factors, cofactors, and chromatin remodelers, respectively. additional file also lists the expression values and the annotations of regulated genes in wat according to the transcription factor subclasses defined in animaltfdb. based on ranking of the entire data set, the gene-set enrichment analysis tool performs a similar analysis and yields a significant enrichment for the sub-categories of transcription factors from animaltfdb for upregulated genes. these transcription factor families contained genes encoding zinc finger transcription factors, genes encoding basic leucin zippers, and forkhead box genes that are upregulated in fasted wat. among the transcription factor-encoding genes with the highest upregulation by fasting we find irf which has been shown to tip the scales between lipogenesis and lipolysis in the latter direction in a fasting-induced manner. consequently, fat-specific irf knock-out mice are deficient in lipolysis and show increased adiposity. furthermore, genes encoding the transcription factors zim and peg are massively upregulated by fasting. interestingly, these two genes are adjacently located at an imprinted region on mouse chromosome the peg knock-out mouse model develops increased adiposity despite lower food intake. this was attributed to developmental deficiencies that lead to aberrant leptin signaling in the hypothalamus. however, our data suggest a direct involvement of the peg3/zim locus in adipose tissue biology. the present study is, to our knowledge, the first to focus on the characterization of the transcriptome response to fasting of wat in mice. surprisingly, we do not find go mapping to the biological process “lipid catabolism” which would be expected to be prominent during fasting in wat. the absence thereof could be explained by abundant posttranscriptional regulation of lipolysis by kinases such as protein kinase a and amp-activated protein kinase which are not reflected at the transcript level. instead our analyses specifically reveal an unexpected upregulation of cell death pathways as well as a strong enrichment of transcriptional regulators among genes activated by fasting in wat. p signaling as top ranking pathway in the fasting response of major metabolic tissues by producing and exporting glucose, fatty acids, glycerol, and ketone bodies wat, liv, and sm represent the organs mainly responsible for energy homeostasis during a fasting period in mammals. to reveal common pathways we focused on genes that were regulated by fasting in all three tissues. table  lists these genes ranked by decreasing average expression level. mapping the common list to go biological processes and kegg using david yielded one kegg pathway as significantly enriched after multiple testing correction: the p signaling pathway. additionally, an independent analysis with metacore focusing on networks overrepresented in the common list reveals the p node as the second highest scoring network hub. the p transcription factor is known to regulate a number of tumor suppressor pathways including cellular senescence, apoptosis, and dna repair in response to various stressors. about 50% of human cancers carry mutations in the p gene. however, evidence is now accumulating that p plays a prominent role in metabolism as well. there is no direct evidence of a physiological importance of p signaling in fasting, but some studies report an activation of p and an induction of its target genes, upon glucose deprivation in cultured cells. in our dataset we observe a host of p target genes apparently regulated by fasting, and a heatmap of the genes described below is shown in figure 4d. for instance, the canonical p target cdkn1a, a cell cycle regulator better known as p is one of the top ranked genes in table  and is the gene with the highest fasting-induced upregulation in liver. another example is lipin- encoded by the gene lpin which has recently been described as a novel p target. in our data lpin is upregulated in wat, liv, and sm by fasting. depending on its subcellular location lipin- can act to enhance fatty acid oxidation by interacting with ppara and ppargc1a or, as a phosphatidate phosphatase in the cytosol, to perform a key step in triglyceride biosynthesis. being upregulated by fasting suggests activity of its nuclear form to drive oxidation of fatty acids in lipid storing tissues. other p targets are the sestrins, sesn and sesn, which have recently been shown to be induced by fasting in liver and to protect it from oxidative damage in a fasting/refeeding scenario. interestingly, sen et al. report that p physically interacts with ppargc1a, which we show to be strongly upregulated in liver by qpcr as well as in our microarray study in wat and liv. in their study sen et al. show that ppargc1a can bind and thereby direct p to the promoters/enhancers of pro-arrest as well as metabolic target genes. thus, this interaction of p and its coactivator ppargc1a could lead to a tissue-specific coordination of p to target genes relevant for a proper response. genes with > x change in all investigated tissues. fasting-mediated downregulation of srebp pathways is common to major metabolic tissues it was reported that p is capable of suppressing the promoter of the gene encoding the transcription factor sterol-regulatory element binding protein. the srebp family consists of three members: srebp1a and srebp1c, both transcribed from the same gene srebf with srebp-1c being the predominant isoform to regulate lipogenesis in metabolic tissues, such as wat and liv; and srebp transcribed from the srebf locus and responsible for regulation of sterol metabolism. consistent with these reports, a metacore analysis on the common list delivers “regulation of lipid metabolism” as top ranking pathways with the srebp family of transcription factors in its center. as mapped in figure 4c, we find srebf and srebf downregulated in all three tissues. concordantly, established srebp downstream genes, coding for enzymes which encompass the fatty acid biosynthesis pathway like acss acaca, fasn, scd and scd, are robustly down-regulated by fasting in our data. further, david analyses for the set of commonly regulated genes as well as for regulated liver genes yields go biological processes that refer to steroid metabolism and, more specifically, to cholesterol biosynthesis. as a pivotal regulator of cholesterol homeostasis in cells, srepb mediates its effects by control of de novo synthesis and/or by regulation of cholesterol import. to regulate de novo synthesis, srebp binds to promoters of most of the enzymes in the cholesterol biosynthesis pathway. we find that transcripts for most of these enzymes are significantly downregulated or show at least a trend to downregulation. the internalization of cholesterol is regulated by srebp-mediated transcription of the ldl-receptor ldlr. figure 4f shows a heatmap of these srebp target genes and impressively demonstrates reduction of transcripts for nearly all components of the cholesterol biosynthesis and uptake pathway in the investigated tissues. given the extensive and intricate networks that, depending on sterol or nutrient availability, post-translationally regulate processing and activation of membrane-bound srebps, our finding that fasting broadly regulates srebp-dependent pathways already at the transcriptional level is rather surprising. especially in liver, this downregulation of cholesterol biosynthesis in combination with the upregulation of hgmcs, which condenses acetoacetyl coa and acetyl coa to β-hydroxy β-methylglutaryl coa, hints to a diversion of substrate from sterol synthesis to ketone body synthesis. hence, based on our analyses on genes regulated in wat, liv, and sm, we hypothesize a shared mechanism that responds to the fasting stimulus in all three tissues: the p signaling pathway is activated by fasting in wat, liv, and sm. coactivators like ppargc1a direct p to promoters/enhancers of genes targeted for transcription activation or repression when nutrients are lacking. upregulation of p targets such as lpin might contribute to the shift of fasted tissues to fatty acid oxidation to provide energy substrates. the observation that p knock-out mice are incapable of inducing liver fatty acid oxidation upon fasting underlines this hypothesis. in addition, a p53-mediated downregulation of srebp is followed by a repression of fatty acid biosynthesis. however, we note that other pathways that are regulated at the post-translational level, and therefore not reflected in rna levels, could be responsible for some of the observed effects, such as downregulation of srebp transcripts. one conceivable example is the activation of ampk by fasting which, via subsequent deactivation of mechanistic target of rapamycin complex, could be responsible for the decrease in srebp mrna. however, based on our analyses we propose a novel and potentially crucial role for p in fasting, which eventually could manifest in profound transcriptional changes in several metabolic pathways. although functional proof of this mechanism is necessary, several reports support our model. validation of expression of top-ranked genes commonly regulated by fasting in wat, liv, and sm to validate the microarray data by means of qpcr we selected the top genes from table , none of which have previously been functionally described in the context of fasting in mice, as well as per and fasn, known responders to food deprivation and thereby positive controls. per is an essential regulator in the core clock machinery of circadian rhythm and, in liv and at at the beginning of the light phase, it was previously shown that its expression is higher in fasting compared to control fed mice. this difference is due to a circadian phase shift that takes place during an extended fasting period. as mentioned above fasn has long been known to be a downstream target gene of srebp and is downregulated due to the drop in srebp levels during fasting. the basic-leucin zipper transcription factor cebpd has been described in a number of cellular contexts, such as osteogenesis and adipogenesis. cebpd expression is known to respond to glucocorticoids and to increased camp levels, both of which could explain its upregulation upon fasting. cdkn1a as a major p target gene, is mainly described as a cell cycle and apoptosis regulator that inhibits cyclin-dependent kinases and has no known role in fasting. finally, ddit a gene initially reported to be readily induced by dexamethasone as well as upon certain cellular stresses, shows the highest extent of upregulation in wat and sm in the common list. interestingly, it has also been described as a p target gene, which led us to further investigate it. hence, we confirmed fasting-mediated regulation of all genes selected for qpcr validation in all three tissues and show a strong correlation with the microarray measurements for all these genes. this introduces three intriguing and novel players in the response to fasting. ddit is fasting-induced in wat, liv, and sm and is inducible by p activation in cultured adipocytes to investigate p signaling as a common fasting regulator in wat, liv, and sm, we focused on dna damage-induced transcript, the top ranking gene in table  which has been described as functional p target gene. in all three tissues investigated, ddit mrna is upregulated at latest by 24 hours after onset of fasting and overnight fasting is sufficient to increase ddit protein levels, which has been shown by others in gastrocnemius muscle of rats. in our data, differences in the magnitude of fasting-induction between the mrna and protein level could be explained by the fact that ddit protein stability is highly regulated in different cell systems. however, especially in adipose tissue ddit protein seems to be stably induced when mice are fasted. to show that ddit can be directly induced by p in adipocytes, we treated mature c3h10t1/ adipocytes for 6 hours with nutlin- a specific p activator. nutlin- treatment led to an increase of ddit mrna similar to the canonical target cdkn1a. most importantly, ddit protein was also stably induced in all replicates. in addition, the p targets sesn and srebf were regulated by nutlin- in a way similar to the in vivo fasting situation. hence, ddit is stably induced by fasting and upregulated by p activation in cultured adipocytes. overexpression of ddit is sufficient to increase lipolysis in cultured adipocytes in a recent report ddit was shown to be involved in lipid metabolism in adipocytes signaling via the mtorc pathway. also in other studies, ddit has been repeatedly described as a negative regulator of mtorc in a variety of cell types. interestingly, in the context of starvation, the nutrient-sensitive mtorc pathway needs to be suppressed for the proper fasting response in liver and its suppression induces lipolysis in adipocytes. hence, we examined whether upregulation of ddit promotes lipolysis in adipocytes by inhibiting mtorc activity. for this we transiently overexpressed ddit in differentiated c3h10t1/ adipocytes and determined glycerol and ffa in the medium as a measure of lipolysis. indeed, we observed a ~30% increased glycerol release and a ~40% increased ffa release from ddit overexpressing cells compared to the empty vector control, while expression of genes in the lipolytic pathway remained unchanged. upon β-adrenergic stimulation the increase in lipolysis upon ddit overexpression was still evident, although not statistically significant. however, as assayed by phosphorylation of the downstream target s6k at threonine mtorc activity was unchanged despite effective overexpression of ddit. an antibody against total s6k protein served as loading control and cells treated with rapamycin, a potent exogenous mtorc inhibitor, as control for phosphorylation-specific s6k antibody. others have reported that, in sm, dexamethasone-mediated ddit increase leads to reduced mtorc signaling, but, judging from our data, in adipocytes ddit4-mediated lipolysis seems to be independent of mtorc activity. this is consistent with the observation that the ddit4-mtorc axis is functional in some cell types but not in others. further, as knock-down of ddit was reported to decrease insulin-stimulated de-novo lipogenesis in adipocytes, we wanted to rule out that the observed increase in lipolysis upon ddit overexpression is merely an effect of increased lipogenesis and with that higher lipid content per se. in addition to an unchanged phenotype as shown by mrna expression of adipocyte-specific genes, we did not detect an increase in the incorporation of radio-labeled glucose into total lipids in ddit overexpressing cells compared to control cells. rather, a small decrease in lipogenesis was observed, which is consistent with a potential role of ddit in regulating fasting responses in adipocytes. hence, our data on the p53-target ddit presents a valuable example for hypothesis generation from a large-scale data set by suggesting a new role in the fine-tuning of the fasting-response in adipose tissue. however, further investigation is needed to elucidate the exact mechanism by which ddit is enhancing lipolysis in adipocytes, because we show that this is independent form mtorc and de-novo lipogenesis.  in this study, we took a comprehensive view on the fasting process in mice. our combination of focused and genome-wide approaches reveals several fasting-related findings: we provide a novel view on the immediate and dynamic response to fasting in mice during a 48 hour period. these experiments focus on the timely regulation of liver genes relayed through the ppara signaling pathway, which manifests in coordinated changes of serum parameters. the observed responses occur very early after onset of fasting and show simultaneous activation of different pathways. to our knowledge this is the first study focusing on the transcriptome response of white adipose tissue in fasted mice. with our bioinformatic analyses we identify an upregulation of apoptosis-related transcripts as well as a strong enrichment of transcriptional control components in the set of upregulated genes. concentrating our analyses on genes regulated in the three tissues mainly responsible for energy homeostasis during fasting, the p signaling pathway appears to be a common and central regulator of fasting, possibly partly mediating its effect by down-regulation of the srepb pathway. finally, we performed experiments that prove that ddit a p target gene upregulated by fasting in all three tissues, is induced by p activation and sufficient to increase lipolysis in cultured adipocytes. in conclusion, our transcriptome study of three tissues combined with bioinformatic analyses and mechanistic in vitro experiments, suggests the p53-ddit axis as a novel mechanism in the fine-tuning of fasting common to major metabolic tissues. 
in summary, this combination of focused and global profiling approaches provides a comprehensive molecular characterization of the processes operating during fasting in mice and suggests a role for p and its downstream target ddit as novel components in the transcriptional response to food deprivation. we investigated the dynamic changes of liver gene expression and serum parameters of mice at several time points during a 48 hour fasting experiment and then focused on the global gene expression changes in epididymal white adipose tissue as well as on pathways common to wat, liver, and skeletal muscle. this approach produced several intriguing insights: rather than a sequential activation of biochemical pathways in fasted liver, as current knowledge dictates, our data indicates a concerted parallel response; this first characterization of the transcriptome signature of wat of fasted mice reveals a remarkable activation of components of the transcription apparatus; most importantly, our bioinformatic analyses indicate p as central node in the regulation of fasting in major metabolic tissues; and forced expression of ddit a fasting-regulated p target gene, is sufficient to augment lipolysis in cultured adipocytes.
 fasting induces specific molecular and metabolic adaptions in most organisms. in biomedical research fasting is used in metabolic studies to synchronize nutritional states of study subjects. because there is a lack of standardization for this procedure, we need a deeper understanding of the dynamics and the molecular mechanisms in fasting.  we investigated the dynamic changes of liver gene expression and serum parameters of mice at several time points during a 48 hour fasting experiment and then focused on the global gene expression changes in epididymal white adipose tissue as well as on pathways common to wat, liver, and skeletal muscle. this approach produced several intriguing insights: rather than a sequential activation of biochemical pathways in fasted liver, as current knowledge dictates, our data indicates a concerted parallel response; this first characterization of the transcriptome signature of wat of fasted mice reveals a remarkable activation of components of the transcription apparatus; most importantly, our bioinformatic analyses indicate p as central node in the regulation of fasting in major metabolic tissues; and forced expression of ddit a fasting-regulated p target gene, is sufficient to augment lipolysis in cultured adipocytes.  in summary, this combination of focused and global profiling approaches provides a comprehensive molecular characterization of the processes operating during fasting in mice and suggests a role for p and its downstream target ddit as novel components in the transcriptional response to food deprivation. fastingstarvationnutrient deprivationadipose tissuep signalingddit4lipolysis 