BACKGROUND
abstinence or absence of food requires the body to recruit metabolites from pre-existing stores.
based on the rate of weight loss, nitrogen excretion, concentration of plasma metabolites and resting metabolic rate, the body is thought to pass through three successive adaptive phases during fasting  <cit>  that have been associated with the primary fuel that is putatively available to the tissues .
during the brief postabsorptive period, the rate of weight loss is relatively high .
the decreasing insulin levels induce glycogenolysis  and lipolysis  <cit>  to support circulating glucose, triglyceride and cholesterol levels  <cit> .
during the subsequent "coping" phase, the loss of body mass is slower .
this state, which can be maintained for several weeks in humans  <cit> , for almost a week in rats  <cit> , and for 2– <dig> days in mice  <cit> , is thought to depend, at least in humans, on lipids as the main fuel source.
however, amino-acid oxidation and, hence, protein catabolism remains necessary for continuous anaplerosis of the tca cycle  <cit> .
it is widely accepted that muscle is a main source of amino acids from protein catabolism, that protein catabolism is maintained by an increased in the circulating levels of glucocorticosteroids, and that glutamine and alanine are the main carriers of this energy source  <cit>  to the intestine, liver and kidney  <cit> .
as a result, total splanchnic glucose production amounts to approximately  <dig> grams daily in humans after several weeks of starvation  <cit> .
despite this enhanced glucose production, but reflecting the enhanced fatty-acid oxidation and ketone-body synthesis in muscle and splanchnic region  <cit> , the brain gradually switches to ketone-body oxidation after several weeks of starvation  <cit> .
during the preterminal phase, finally, the rate of loss of body weight may increase again .
because the fat stores are depleted, proteolysis remains the sole, nonsustainable source of fuel.
the maintenance of the fuel supply during fasting requires an extensive exchange of metabolites from organs that break down the stores of fats or proteins to organs that consume these metabolites.
this exchange mainly occurs as glucose, lactate, amino acids, triglycerides and ketone bodies.
the question that arises from these global findings concerns the contribution of individual organs to the whole-body adaptive response to fasting.
our previous study of the effects of fasting on the small intestine  <cit>  suggested, in comparison with that of liver  <cit>  and muscle  <cit> , an organ-specific response to fasting.
our study  <cit>  included both shorter and longer periods of fasting than earlier published studies  <cit> .
the aim of the current study was, therefore, to determine the characteristics of gene-expression profile of mouse liver between  <dig> and  <dig> hours of fasting, using a genome-wide transcriptomics approach.
our findings show that the adaptive response of the liver peaks around  <dig> hours after food withdrawal and, unexpectedly, declines thereafter.
the major components of the response were fatty-acid β-oxidation and ketone-body synthesis, and oxidative and energy metabolism during the first  <dig> hours of fasting, and glycogen synthesis and the urea cycle throughout the entire fasting period.
RESULTS
effects of food withdrawal on liver structure
during the first  <dig> hours of fasting, mice lost ~12% of their body weight .
thereafter, weight loss remained steady at a rate of ~7% per day, so that mice had lost ~30% of their initial weight after  <dig> hours of fasting .
note that we expressed daily differences in the rate of weight loss on a per-day basis to define a common denominator for the  <dig> h- and  <dig> h-fasted animals.
liver wet weight declined more than body weight , especially during the first  <dig> hours of fasting, and amounted to ~ <dig>  ~ <dig>  ~ <dig> and ~10% per day after  <dig>   <dig>   <dig> and  <dig> hours of fasting, respectively.
after  <dig> hours, the liver had, therefore, lost almost 50% of its initial weight.
the basic architecture of the liver lobules  and the zonation of gene expression as studied by the expression of glutamine synthetase and carbamoylphosphate synthetase  remained unaffected.
staining for the appearance of active-caspase  <dig> revealed no changes in the number of apoptotic cells upon fasting, not even after  <dig> hours .
in agreement, the apoptotic genes that were represented on the microarrays showed no significant change in expression in fasted compared to fed mice.
since there was no reduction in the number of liver cells during fasting, we took two approaches to estimate the decrease in average cell size.
the summation of  <dig> hepatocyte diameters in three  <dig> hours fasted and three control animals, amounted to 25% reduction in cell diameter.
based on the liver wet weight, the average cell diameter decreased 20% in the fasted animals.
effects of fasting on metabolism
ammonia levels had increased  <dig> -,  <dig> - and  <dig> -fold after  <dig>   <dig> and  <dig> hours of fasting, respectively .
glucose and lactate concentrations remained stable until  <dig> h of fasting, but decreased  <dig> and 43%, respectively  in the next  <dig> hours.
the plasma concentration of many amino acids changed at some time point of fasting, but only the changes in the concentration of taurine showed a trend with time .
accumulation of taurine helps protect cells from hypertonicity  <cit> , as may occur during shrinkage of fasting hepatocytes.
canonical pathways represent a set of about  <dig> signalling and metabolic maps in the metacore suit.
all maps are drawn from scratch by genego annotators and manually curated.
global gene-expression profile in the liver
to gain a comprehensive overview of the physiological response of the liver to fasting, whole-genome measurements were made.
compared to the fed group,  <dig>   <dig> and  <dig> transcripts, including expressed sequence tags and riken sequences, met our boundary condition for significance  after  <dig>   <dig>  and  <dig> hours of fasting, respectively .
the dendrogram generated by supervised hierarchical clustering  shows a clear separation between fed and fasted conditions.
among the arrays coming from fasted animals, those from  <dig> hours stand out, while the branches of the two earlier time points are intertwined, indicating that expression profiles are rather similar after  <dig> and  <dig> hours of fasting.
this is also reflected in the venn diagrams where the overlap between  <dig> and  <dig> hours is larger than the overlap with  <dig> hours.
global analysis reveals a strong early and an abated late response to fasting
we used genmapp and, in particular, metacore™ software to deduce the biological processes that change with an increasing duration of fasting from the liver transcriptome data.
in metacore, the degree of association of the uploaded datasets with predefined metabolic pathways is defined by p-values, with lower p-values being more relevant.
the expression of  <dig> genes that met our thresholds  could be linked to the metacore™ suite.
their distribution across time points is shown in figure 4a.
the graphs show the numbers of unique, similar and common genes for all three, and for two initial time points separately, showing that the response to fasting at  <dig> hours was similar to, but more pronounced than that at  <dig> hours.
we performed gene-set enrichment analysis in three different functional ontologies using metacore™: cellular processes, biological processes and canonical pathways.
based on the gene ontology categorization of cellular processes, fasting predominantly affected the metabolic processes, in particular the carboxylic-acid metabolizing processes, lipid and glucose metabolism.
the enrichment analysis for biological processes showed, more specifically, that genes involved in amino-acid, lipid, carbohydrate and energy metabolism responded most significantly to fasting .
the graph presents p-values as parameter of the likelihood that coordinate changes in the pathways shown were indeed present at the different time points of fasting.
as statistical parameter, the p-value encompasses no variation.
the changes in all processes except amino-acid metabolism showed a response that peaked at  <dig> hours after food withdrawal and declined thereafter.
the response during the late phase of fasting was dominated by amino-acid metabolism, although lipid and carbohydrate metabolism remained significantly regulated.
the figure further reveals that the changes in energy metabolism were significant at  <dig> hours of fasting only.
the common denominator of the overall fasting response was, therefore, metabolism of amino acids, carbohydrates, and lipids.
regulated pathways
since the global analysis does not reveal a direction in the changes and lacks functional detail, we scrutinized the pathways with most pronounced regulation for functional implications.
a list of the  <dig> top-scoring canonical pathways, shown in table  <dig>  points to gluconeogenesis, urea synthesis, and pparα-regulated fatty-acid oxidation as the major characteristics in the response of the liver to fasting.
amino-acid catabolism and urea synthesis
of all the pathways studied in the liver, the adaptive changes in amino-acid metabolism persisted throughout the fasting period .
of the enzymes in this group, those of the urea cycle were upregulated at all three time points .
among the genes consistently affected were argininosuccinate synthetase  <dig>  and argininosuccinate lyase , was not represented on the microarrays, but its expression level, as estimated by qpcr, was increased  <dig> -fold at all  <dig> time points .
urea synthesis occurs in periportal hepatocytes, whereas ammonia detoxification via glutamine synthesis occurs pericentrally.
genes for the pericentral enzymes ornithine-aminotransferase  and proline dehydrogenase , which provide glutamate for glutamine synthesis, were upregulated  <dig> -,  <dig> - and  <dig> -fold and  <dig> -,  <dig> - and  <dig> - fold at  <dig>   <dig> and  <dig> hours, respectively.
glutamine synthetase  itself was, however, not regulated.
remarkably, the expression of amino-acid catabolizing enzymes themselves was barely affected by fasting.
only the degradation of branched-chain keto-acids  was upregulated, as shown by the upregulation of acetyl-coenzyme a dehydrogenase, medium chain , enoyl-coenzyme a, hydratase/3-hydroxyacyl-coenzyme , hydroxyacyl-coenzyme a dehydrogenase, short chain , acetyl-coenzyme a acyltransferase  <dig> , and 3-hydroxy-3-methylglutaryl-coenzyme a lyase  – all within first the  <dig> hours .
this finding suggests that the adaptations in amino-acid catabolism during fasting mainly occur outside the liver.
since neither glutamate-pyruvate transaminase nor ammonia-inducible liver glutaminase were upregulated, the capacity of the liver to deaminate the amino-carriers alanine and glutamine must have been sufficient.
tca cycle and electron-transport chain
the strong induction of the urea cycle suggests a strong stimulation of amino-acid oxidation or gluconeogenesis.
in agreement with this hypothesis, both the expression of enzymes of the tricarboxylic-acid  cycle and oxidative phosphorylation were induced in fasted liver, again mainly at  <dig> hours.
aconitase  <dig> , isocitrate dehydrogenase 3β  , oxoglutarate dehydrogenase , dihydrolipoamide s-succinyltransferase , fumarate hydratase  <dig>  and malate dehydrogenase  <dig>  were all upregulated at  <dig> hours of fasting , indicating an increased capacity of the cycle.
dlst and fh <dig> were  <dig>  and  <dig>  times induced at  <dig> hours of fasting, while aco <dig> expression was also  <dig> -fold increased at  <dig> hours.
in agreement with an increased capacity of the tca cycle, the expression of the genes of the electron-transport chain was strongly stimulated .
four genes belonging to nadh-ubiquinone oxidoreductase complex: nadh dehydrogenase  1α subcomplex subunit  <dig> , nadh dehydrogenase  1α subcomplex subunit  <dig> , nadh dehydrogenase  flavoprotein  <dig>  and nadh dehydrogenase  flavoprotein  <dig> , were all approximately  <dig> -fold upregulated.
expression of the genes of the atp synthase complex, atp synthase subunit α , atp synthase δ chain  and atp synthase lipid-binding protein , was  <dig> – <dig> -fold induced at  <dig> hours.
ubiquinol-cytochrome-c reductase complex core protein  <dig>  was  <dig> -fold upregulated after  <dig> hours, whereas the energy-dissipating uncoupling protein  <dig>  was  <dig> -fold downregulated at this time point .
taken together, these data indicate that the capacity for atp synthesis in the liver is strongly upregulated during the first day of food deprivation.
gluconeogenesis
phosphoenolpyruvate carboxykinase  <dig> , a key enzyme in the gluconeogenic route, was upregulated  <dig> -,  <dig> - and  <dig> -fold on the microarrays and  <dig> -,  <dig> , and  <dig> -fold in the qpcr measurements at  <dig>   <dig> and  <dig> hours of fasting, respectively .
cytosolic glutamate oxaloacetate transaminase  <dig>  was also strongly upregulated at all three time points .
in addition, malate dehydrogenase  and mitochondrial glutamate oxaloacetate transaminase  were induced .
together, these data suggest an increased capacity of the malate-aspartate shuttle across the mitochondrial membrane, which would accommodate an enhanced carbon flux from the mitochondria.
all other steps that were affected by fasting were shared by the glycolytic and gluconeogenic pathways and were regulated during the first day of fasting only .
phosphoglycerate mutase  <dig>  was  <dig> -fold upregulated at  <dig> hours of fasting, while glucosephosphate isomerase  <dig> , aldolase 1a isoform , triosephosphate isomerase  <dig> , glyceraldehyde-3-phosphate dehydrogenase  and enolase 1α  were  <dig> -,  <dig> -,  <dig> -,  <dig> -,  <dig> - fold upregulated at  <dig> hours of fasting, respectively.
these data indicate that the enhanced capacity of the gluconeogenic pathway would largely depend on enhanced tca and malate-aspartate cycling, and that this adaptive response in gene expression might be restricted to a single day in the mouse.
liver glycogen accumulation upon prolonged fasting
the near total return to "normalcy" of gene expression at  <dig> hours  was striking.
because glucose-6-phosphatase expression was not upregulated, we explored the possibility that glucose precursors were channelled into glycogen.
as expected,  periodic acid-schiff  staining showed the complete disappearance of glycogen from the liver after  <dig> hours of fasting , but some staining had returned at  <dig> hours and intense staining was seen in 72-hours fasted liver.
whereas glycogen was localized around the portal veins in fed liver, it was deposited exclusively around the central veins after  <dig> hours of fasting, with sharp borders towards the empty cells.
fatty-acid catabolism and ketone-body synthesis
the enhanced expression of fatty-acid catabolizing enzymes was also limited to the initial phase of fasting.
the expression of the transcription factor pparα, a major regulator of fatty-acid oxidation, was  <dig> -fold upregulated at  <dig> hours of fasting .
furthermore, the mitochondrial carnitine/acylcarnitine fatty-acid translocase  was  <dig> -fold upregulated at  <dig> hours, while carnitine palmitoyltransferase  <dig>  on the inner mitochondrial membrane was  <dig> -fold upregulated at both  <dig> and  <dig> hours of fasting.
however, the expression of cpt <dig>  which is present on the outer mitochondrial membrane and is sensitive to malonyl-coa inhibition, remained unchanged.
the  <dig> acyl-coenzyme a dehydrogenases , involved in oxidation of very long-, long-, medium- and short-chain fatty acids, were all upregulated in the first  <dig> hours of fasting .
the β-subunit of the trifunctional protein  was 2- and  <dig> -fold upregulated at  <dig> and  <dig> hours, respectively, while another subunit, hydroxyacyl-coenzyme a dehydrogenase, short chain  showed increased expression after  <dig> hours of fasting only, indicating altogether a strong stimulation of fatty-acid oxidation at the gene-expression level during the first day of fasting.
the expression of hmgcoa synthase  <dig>  was also strongly stimulated during the first day of fasting , indicating an increased capacity of the synthesis of ketone bodies from acetyl-coa.
this process is further facilitated by increased expression of genes involved in branched-chain keto-acid degradation  at  <dig> and  <dig> hours.
interestingly, neither the cytoplasmic hmgcoa synthase  nor hmgcoa reductase , the key enzyme in de novo cholesterol synthesis pathway  <cit> , have changed the expression levels in fasted liver.
among the genes involved in fatty-acid synthesis, enoyl coenzyme a hydratase domain containing  <dig>  was  <dig>  and  <dig> -fold downregulated at  <dig> and  <dig> hours, while stearoyl-coenzyme a desaturase  <dig>  showed a  <dig> -fold decrease in expression at  <dig> hours of fasting.
these data underscore the importance of enhanced lipid catabolism in the liver, which, in the mouse, apparently occurs during the first day of fasting only.
oxidative stress and unfolded protein response
the enhanced expression of tca cycle and oxidative-phosphorylation enzymes often causes oxidative stress.
indeed, cytosolic superoxide dismutase  was  <dig> -fold upregulated after  <dig> hours, and the early growth response protein  <dig> , its transcriptional regulator  <cit> ,  <dig> -fold.
furthermore, catalase  and stress-regulated mitogen-activated protein kinase  <dig>  were both  <dig> -fold upregulated at this time point.
in addition, metallothionein  <dig> gene, known to be involved in protection against oxidative stress and metal toxicity  <cit> , was intensely upregulated .
interestingly, the  <dig> top-scoring processes obtained from a biological-process enrichment analysis all belonged to the unfolded-protein response  stress).
to present the relevant data in a single figure, we created a network using the shortest-path algorithm .
the resulting network provides links based on the known interaction data between the nodes from the query data set, and also between the nodes that regulate the given genes or metabolites.
it shows  <dig> heat-shock and  <dig> other proteins, all upregulated  <dig> – <dig>  fold, indicating upregulation of this stress-response pathway in fasted liver.
downstream of the er stress pathway, proteasome degradation was also upregulated, but again only after  <dig> hours of fasting .
a list of these and some additional genes regulated in the er stress and proteasome degradation, with their change level, is shown in table  <dig>
discussion
the major architectural feature of fasting liver is a pronounced decline in cell size  rather than a loss of cell number.
in addition, the liver's metabolic zonation in upstream, periportal and downstream, pericentral regions remains intact.
these findings indicate that the liver can quickly resume its homeostatic functions once feeding resumes.
our microarray data show that the adaptive response of the liver to fasting at the level of gene expression is most pronounced during the early phase, with the upregulation of ammonia detoxification persisting up to  <dig> hours of fasting.
since the technically similar study of bauer et al.
<cit>  reported enhanced expression of lipid-catabolizing and urea-cycle enzymes after  <dig> and  <dig> h of fasting, the collective data show that the response to fasting in the liver starts already at  <dig> hours of fasting and becomes maximal between  <dig> and  <dig> hours.
the hepatic response to total food deprivation, therefore, does not proceed through the global "sugar-fat-protein" sequence that is described for the adaptation on the whole-body level  <cit> .
the expression of genes involved in lipid metabolism and ketone-body synthesis, many under pparα coordination  <cit> , was strongly regulated towards fatty-acid oxidation and ketone-body formation.
interestingly, this adaptive response was seen between  <dig> and  <dig> hours  of fasting only, and then faded out.
in the rat, this response was recently reported to occur between  <dig> and  <dig> days of fasting  <cit> .
fatty-acid oxidation was accommodated by the identical time frame of the upregulation of the expression of tca-cycle enzymes and the proteins of the electron-transport chain in response to fasting.
the associated oxidative stress and mitochondrial radical formation was apparently sufficiently strong to induce the unfolded-protein  response in liver.
thus far, to our knowledge, activation of the unfolded protein response has not been associated with fatty-acid oxidation in the fasting liver, but it is induced by a high-fat diet  <cit> .
similarly, mitochondria in fasting muscle protect themselves against the oxidative stress that results from fat oxidation  <cit>  by accumulating the uncoupling proteins ucp <dig> and ucp <dig>  <cit> .
the role of the liver in gluconeogenesis during fasting is well documented  <cit> .
however, the expression of enzymes associated with gluconeogenesis was upregulated only during the first day of fasting and was mainly confined to the malate-aspartate shuttle and pepck <dig>  in fact, apart from pepck <dig>  the expression of none of the committed steps in gluconeogenesis was regulated.
it is likely that the enhanced expression of tca-cycle and malate-aspartate shuttle enzymes, and the enhanced expression of pepck <dig> enhance the flux towards either glucose-6-phosphate or lactate.
it is, therefore, remarkable that the expression of glucose-6-phosphatase, a periportal enzyme, and pyruvate kinase, a mainly pericentral enzyme, are not regulated, while the expression of lactate dehydrogenase is only upregulated at  <dig> and  <dig> hours.
similarly, the expression and activity of glucose-6-phosphatase in rat liver are upregulated mildly during the first  <dig> hours of fasting only  <cit> .
our unpublished data show a similar response in the kidneys of fasting mice, in which pepck <dig> is 2– <dig> fold upregulated at all time points, whereas glucose-6-phosphatase is not regulated.
the pronounced accumulation of glycogen in pericentral hepatocytes starting after  <dig> hours of fasting, which was also observed in 72- and 96-hour fasted rats  <cit> , indicates that pericentral hepatocytes, which do not express glucose-6-phosphatase  <cit> , channel glucose-6-phosphate towards glycogen.
since all relevant enzymes are also expressed in periportal hepatocytes, which do not accumulate glycogen, we assume that these hepatocytes contain enough glucose-6-phosphatase to produce glucose.
the liver produces ~60% of the newly produced glucose in starvation, while the kidneys account for ~40%  <cit> .
a recent, but controversial series of experiments suggest that, in addition to the liver  <cit>  and kidney  <cit> , the small intestine also has the capacity to produce glucose upon prolonged fasting  <cit> .
it contributes indirectly, by providing lactate and alanine to the liver in short-term fasting  <cit> , and directly by the production of glucose  <cit>  .
the concept is controversial, since other studies were unable to detect glucose formation from glutamine in the isolated small intestine of  <dig> hours fasted rats  <cit> .
furthermore, the expression of the key gluconeogenic enzyme phosphoenolpyruvate kinase  in the mouse small intestine was reported to amount to only  <dig> –1% of that in the liver after  <dig> h hours of fasting  <cit> , also arguing against intestinal gluconeogenesis.
we, therefore, compared pepck mrna levels in these two organs by qpcr in the fed and 3-days fasting condition .
while pepck <dig> expression in the gut at  <dig> hours of fasting only amounted to ~5% of that in liver, its expression increased to  <dig> and 53% of that in the liver after  <dig> and  <dig> hours of fasting, respectively.
this finding demonstrates that the issue of intestinal gluconeogenesis during prolonged fasting deserves additional study.
the plasma concentrations of both glucose and lactate remained unchanged during the first  <dig> hours of fasting, declined temporarily by 35–40% at  <dig> hours, and returned to control values between  <dig> and  <dig> hours.
the maintenance of normal concentrations of glucose and lactate during the first  <dig> hours of fasting is most likely the result of gluconeogenesis in the liver and kidney.
since the expression of enzymes that are shared by the glycolytic and gluconeogenic pathways, declines after  <dig> hours of fasting, the observed decline at  <dig> hours may represent a declining contribution of the liver to gluconeogenesis.
as we argued earlier, the accumulation of glycogen in the pericentral hepatocytes between  <dig> and  <dig> hours of fasting indicates that gluconeogenic intermediates flow towards glucose-6-phosphate and accumulate as glycogen due to the low expression of glucose-6-phosphatase in these hepatocytes.
most likely, a similar or higher flow of gluconeogenic intermediates is present in the periportal hepatocytes, but is exported as glucose due to the high concentration of glucose-6-phosphatese in these cells.
furthermore, the putative production of glucose in the 72-hour fasted intestine can also contribute to the circulating glucose level.
the urea-cycle enzymes distinguish themselves from most other genes in the liver in that they were upregulated in expression throughout the period of fasting that was studied.
furthermore, cytosolic glutamate-oxaloacetate transaminase, which mediates the availability of aspartate to the urea-cycle enzyme argininosuccinate synthetase, was also strongly upregulated at  <dig> hours.
similarly, oat and prodh, which supply glutamate to glutamine synthetase for glutamine synthesis in pericentral hepatocytes, were strongly upregulated at all time points studied, but glutamine synthetase itself was not regulated .
since few amino-acid catabolizing enzymes were upregulated , most amino-groups were probably carried to the liver as alanine or glutamine, although neither glutamate-pyruvate transaminase nor liver glutaminase was upregulated.
the coordinate control of ammonia detoxification and the source of ammonia during prolonged fasting therefore deserve attention.
an important question is to what extent we can extrapolate the observations in a small mammal like the mouse to larger animals like humans.
the ability to tolerate the absence of food does indeed decline with body size: in the mouse the maximum duration of fasting is  <dig> days  <cit> , in rat 12– <dig> days  <cit> , in children  <dig> weeks and in adult humans 8– <dig> weeks  <cit> .
qualitatively, however, the response to fasting is probably comparable between these mammals, as long as the time scale is adjusted to the size of the animal.
rather than questioning the comparability of small and large animals, our data question whether the implicit extrapolation of the "sugars-fats-proteins" succession of energy substrates during fasting that is based on whole-body energy expenditure  <cit>  to individual organs is valid.
microarray studies in rodents that have prospected the adaptive response to fasting of the small intestine  <cit> , liver , muscle  <cit> , and a more limited study in kidney focusing on circadian differences in gene expression  <cit> , reveal a different scenario.
muscle and kidney respond to fasting with a progressive change over time in mrna concentrations of enzymes involved in protein, carbohydrate and fat metabolism.
the response in liver peaked at 24– <dig> hours of fasting in mouse, while most adaptive changes had abated by  <dig> hours.
the intestine, finally, showed an early, but temporary peak of adaptive changes in amino-acid, carbohydrate and fat metabolism at  <dig> hours of fasting, while a late response, existing almost exclusively of amino-acid catabolizing and gluconeogenic enzymes, gradually developed towards  <dig> hours of fasting.
these differences in pattern and amplitude of gene expression change in different organs can be used to look for circulating biomarkers that reflect the functions of organs during adaptive responses.
CONCLUSIONS
based on whole-body energy expenditure, the "sugars-fats-proteins" sequence of energy substrates during fasting was suggested.
in our extensive microarray studies of the response to fasting in the gut  <cit>  and liver , we found no support for this intuitively attractive model at the individual organ level.
the liver markedly differed from the biphasic response pattern in the small intestine  in that its adaptive response peaked at 24– <dig> hours of fasting, while most adaptive changes had abated by  <dig> hours.
expression profiling and pathway analysis revealed that genes involved in amino-acid, lipid, carbohydrate and energy metabolism responded most significantly to fasting, with no temporal separation between them.
even though the liver lost 50% of its initial weight during  <dig> days of fasting, its basic morphology remained preserved, showing that the liver can quickly resume its homeostatic functions when feeding resumes.

 abstinence or absence of food requires the body to recruit metabolites from pre-existing stores. based on the rate of weight loss, nitrogen excretion, concentration of plasma metabolites and resting metabolic rate, the body is thought to pass through three successive adaptive phases during fasting that have been associated with the primary fuel that is putatively available to the tissues. during the brief postabsorptive period, the rate of weight loss is relatively high. the decreasing insulin levels induce glycogenolysis and lipolysis to support circulating glucose, triglyceride and cholesterol levels. during the subsequent "coping" phase, the loss of body mass is slower. this state, which can be maintained for several weeks in humans, for almost a week in rats, and for 2– days in mice, is thought to depend, at least in humans, on lipids as the main fuel source. however, amino-acid oxidation and, hence, protein catabolism remains necessary for continuous anaplerosis of the tca cycle. it is widely accepted that muscle is a main source of amino acids from protein catabolism, that protein catabolism is maintained by an increased in the circulating levels of glucocorticosteroids, and that glutamine and alanine are the main carriers of this energy source to the intestine, liver and kidney. as a result, total splanchnic glucose production amounts to approximately grams daily in humans after several weeks of starvation. despite this enhanced glucose production, but reflecting the enhanced fatty-acid oxidation and ketone-body synthesis in muscle and splanchnic region, the brain gradually switches to ketone-body oxidation after several weeks of starvation. during the preterminal phase, finally, the rate of loss of body weight may increase again. because the fat stores are depleted, proteolysis remains the sole, nonsustainable source of fuel. the maintenance of the fuel supply during fasting requires an extensive exchange of metabolites from organs that break down the stores of fats or proteins to organs that consume these metabolites. this exchange mainly occurs as glucose, lactate, amino acids, triglycerides and ketone bodies. the question that arises from these global findings concerns the contribution of individual organs to the whole-body adaptive response to fasting. our previous study of the effects of fasting on the small intestine suggested, in comparison with that of liver and muscle, an organ-specific response to fasting. our study included both shorter and longer periods of fasting than earlier published studies. the aim of the current study was, therefore, to determine the characteristics of gene-expression profile of mouse liver between and hours of fasting, using a genome-wide transcriptomics approach. our findings show that the adaptive response of the liver peaks around hours after food withdrawal and, unexpectedly, declines thereafter. the major components of the response were fatty-acid β-oxidation and ketone-body synthesis, and oxidative and energy metabolism during the first hours of fasting, and glycogen synthesis and the urea cycle throughout the entire fasting period.  effects of food withdrawal on liver structure during the first hours of fasting, mice lost ~12% of their body weight. thereafter, weight loss remained steady at a rate of ~7% per day, so that mice had lost ~30% of their initial weight after hours of fasting. note that we expressed daily differences in the rate of weight loss on a per-day basis to define a common denominator for the h- and h-fasted animals. liver wet weight declined more than body weight, especially during the first hours of fasting, and amounted to ~ ~ ~ and ~10% per day after and hours of fasting, respectively. after hours, the liver had, therefore, lost almost 50% of its initial weight. the basic architecture of the liver lobules and the zonation of gene expression as studied by the expression of glutamine synthetase and carbamoylphosphate synthetase remained unaffected. staining for the appearance of active-caspase revealed no changes in the number of apoptotic cells upon fasting, not even after hours. in agreement, the apoptotic genes that were represented on the microarrays showed no significant change in expression in fasted compared to fed mice. since there was no reduction in the number of liver cells during fasting, we took two approaches to estimate the decrease in average cell size. the summation of hepatocyte diameters in three hours fasted and three control animals, amounted to 25% reduction in cell diameter. based on the liver wet weight, the average cell diameter decreased 20% in the fasted animals. effects of fasting on metabolism ammonia levels had increased -, - and -fold after and hours of fasting, respectively. glucose and lactate concentrations remained stable until h of fasting, but decreased and 43%, respectively in the next hours. the plasma concentration of many amino acids changed at some time point of fasting, but only the changes in the concentration of taurine showed a trend with time. accumulation of taurine helps protect cells from hypertonicity, as may occur during shrinkage of fasting hepatocytes. canonical pathways represent a set of about signalling and metabolic maps in the metacore suit. all maps are drawn from scratch by genego annotators and manually curated. global gene-expression profile in the liver to gain a comprehensive overview of the physiological response of the liver to fasting, whole-genome measurements were made. compared to the fed group, and transcripts, including expressed sequence tags and riken sequences, met our boundary condition for significance after and hours of fasting, respectively. the dendrogram generated by supervised hierarchical clustering shows a clear separation between fed and fasted conditions. among the arrays coming from fasted animals, those from hours stand out, while the branches of the two earlier time points are intertwined, indicating that expression profiles are rather similar after and hours of fasting. this is also reflected in the venn diagrams where the overlap between and hours is larger than the overlap with hours. global analysis reveals a strong early and an abated late response to fasting we used genmapp and, in particular, metacore™ software to deduce the biological processes that change with an increasing duration of fasting from the liver transcriptome data. in metacore, the degree of association of the uploaded datasets with predefined metabolic pathways is defined by p-values, with lower p-values being more relevant. the expression of genes that met our thresholds could be linked to the metacore™ suite. their distribution across time points is shown in figure 4a. the graphs show the numbers of unique, similar and common genes for all three, and for two initial time points separately, showing that the response to fasting at hours was similar to, but more pronounced than that at hours. we performed gene-set enrichment analysis in three different functional ontologies using metacore™: cellular processes, biological processes and canonical pathways. based on the gene ontology categorization of cellular processes, fasting predominantly affected the metabolic processes, in particular the carboxylic-acid metabolizing processes, lipid and glucose metabolism. the enrichment analysis for biological processes showed, more specifically, that genes involved in amino-acid, lipid, carbohydrate and energy metabolism responded most significantly to fasting. the graph presents p-values as parameter of the likelihood that coordinate changes in the pathways shown were indeed present at the different time points of fasting. as statistical parameter, the p-value encompasses no variation. the changes in all processes except amino-acid metabolism showed a response that peaked at hours after food withdrawal and declined thereafter. the response during the late phase of fasting was dominated by amino-acid metabolism, although lipid and carbohydrate metabolism remained significantly regulated. the figure further reveals that the changes in energy metabolism were significant at hours of fasting only. the common denominator of the overall fasting response was, therefore, metabolism of amino acids, carbohydrates, and lipids. regulated pathways since the global analysis does not reveal a direction in the changes and lacks functional detail, we scrutinized the pathways with most pronounced regulation for functional implications. a list of the top-scoring canonical pathways, shown in table points to gluconeogenesis, urea synthesis, and pparα-regulated fatty-acid oxidation as the major characteristics in the response of the liver to fasting. amino-acid catabolism and urea synthesis of all the pathways studied in the liver, the adaptive changes in amino-acid metabolism persisted throughout the fasting period. of the enzymes in this group, those of the urea cycle were upregulated at all three time points. among the genes consistently affected were argininosuccinate synthetase and argininosuccinate lyase, was not represented on the microarrays, but its expression level, as estimated by qpcr, was increased -fold at all time points. urea synthesis occurs in periportal hepatocytes, whereas ammonia detoxification via glutamine synthesis occurs pericentrally. genes for the pericentral enzymes ornithine-aminotransferase and proline dehydrogenase, which provide glutamate for glutamine synthesis, were upregulated -, - and -fold and -, - and - fold at and hours, respectively. glutamine synthetase itself was, however, not regulated. remarkably, the expression of amino-acid catabolizing enzymes themselves was barely affected by fasting. only the degradation of branched-chain keto-acids was upregulated, as shown by the upregulation of acetyl-coenzyme a dehydrogenase, medium chain, enoyl-coenzyme a, hydratase/3-hydroxyacyl-coenzyme, hydroxyacyl-coenzyme a dehydrogenase, short chain, acetyl-coenzyme a acyltransferase, and 3-hydroxy-3-methylglutaryl-coenzyme a lyase – all within first the hours. this finding suggests that the adaptations in amino-acid catabolism during fasting mainly occur outside the liver. since neither glutamate-pyruvate transaminase nor ammonia-inducible liver glutaminase were upregulated, the capacity of the liver to deaminate the amino-carriers alanine and glutamine must have been sufficient. tca cycle and electron-transport chain the strong induction of the urea cycle suggests a strong stimulation of amino-acid oxidation or gluconeogenesis. in agreement with this hypothesis, both the expression of enzymes of the tricarboxylic-acid cycle and oxidative phosphorylation were induced in fasted liver, again mainly at hours. aconitase, isocitrate dehydrogenase 3β, oxoglutarate dehydrogenase, dihydrolipoamide s-succinyltransferase, fumarate hydratase and malate dehydrogenase were all upregulated at hours of fasting, indicating an increased capacity of the cycle. dlst and fh were and times induced at hours of fasting, while aco expression was also -fold increased at hours. in agreement with an increased capacity of the tca cycle, the expression of the genes of the electron-transport chain was strongly stimulated. four genes belonging to nadh-ubiquinone oxidoreductase complex: nadh dehydrogenase 1α subcomplex subunit, nadh dehydrogenase 1α subcomplex subunit, nadh dehydrogenase flavoprotein and nadh dehydrogenase flavoprotein, were all approximately -fold upregulated. expression of the genes of the atp synthase complex, atp synthase subunit α, atp synthase δ chain and atp synthase lipid-binding protein, was – -fold induced at hours. ubiquinol-cytochrome-c reductase complex core protein was -fold upregulated after hours, whereas the energy-dissipating uncoupling protein was -fold downregulated at this time point. taken together, these data indicate that the capacity for atp synthesis in the liver is strongly upregulated during the first day of food deprivation. gluconeogenesis phosphoenolpyruvate carboxykinase, a key enzyme in the gluconeogenic route, was upregulated -, - and -fold on the microarrays and -,, and -fold in the qpcr measurements at and hours of fasting, respectively. cytosolic glutamate oxaloacetate transaminase was also strongly upregulated at all three time points. in addition, malate dehydrogenase and mitochondrial glutamate oxaloacetate transaminase were induced. together, these data suggest an increased capacity of the malate-aspartate shuttle across the mitochondrial membrane, which would accommodate an enhanced carbon flux from the mitochondria. all other steps that were affected by fasting were shared by the glycolytic and gluconeogenic pathways and were regulated during the first day of fasting only. phosphoglycerate mutase was -fold upregulated at hours of fasting, while glucosephosphate isomerase, aldolase 1a isoform, triosephosphate isomerase, glyceraldehyde-3-phosphate dehydrogenase and enolase 1α were -, -, -, -, - fold upregulated at hours of fasting, respectively. these data indicate that the enhanced capacity of the gluconeogenic pathway would largely depend on enhanced tca and malate-aspartate cycling, and that this adaptive response in gene expression might be restricted to a single day in the mouse. liver glycogen accumulation upon prolonged fasting the near total return to "normalcy" of gene expression at hours was striking. because glucose-6-phosphatase expression was not upregulated, we explored the possibility that glucose precursors were channelled into glycogen. as expected, periodic acid-schiff staining showed the complete disappearance of glycogen from the liver after hours of fasting, but some staining had returned at hours and intense staining was seen in 72-hours fasted liver. whereas glycogen was localized around the portal veins in fed liver, it was deposited exclusively around the central veins after hours of fasting, with sharp borders towards the empty cells. fatty-acid catabolism and ketone-body synthesis the enhanced expression of fatty-acid catabolizing enzymes was also limited to the initial phase of fasting. the expression of the transcription factor pparα, a major regulator of fatty-acid oxidation, was -fold upregulated at hours of fasting. furthermore, the mitochondrial carnitine/acylcarnitine fatty-acid translocase was -fold upregulated at hours, while carnitine palmitoyltransferase on the inner mitochondrial membrane was -fold upregulated at both and hours of fasting. however, the expression of cpt which is present on the outer mitochondrial membrane and is sensitive to malonyl-coa inhibition, remained unchanged. the acyl-coenzyme a dehydrogenases, involved in oxidation of very long-, long-, medium- and short-chain fatty acids, were all upregulated in the first hours of fasting. the β-subunit of the trifunctional protein was 2- and -fold upregulated at and hours, respectively, while another subunit, hydroxyacyl-coenzyme a dehydrogenase, short chain showed increased expression after hours of fasting only, indicating altogether a strong stimulation of fatty-acid oxidation at the gene-expression level during the first day of fasting. the expression of hmgcoa synthase was also strongly stimulated during the first day of fasting, indicating an increased capacity of the synthesis of ketone bodies from acetyl-coa. this process is further facilitated by increased expression of genes involved in branched-chain keto-acid degradation at and hours. interestingly, neither the cytoplasmic hmgcoa synthase nor hmgcoa reductase, the key enzyme in de novo cholesterol synthesis pathway, have changed the expression levels in fasted liver. among the genes involved in fatty-acid synthesis, enoyl coenzyme a hydratase domain containing was and -fold downregulated at and hours, while stearoyl-coenzyme a desaturase showed a -fold decrease in expression at hours of fasting. these data underscore the importance of enhanced lipid catabolism in the liver, which, in the mouse, apparently occurs during the first day of fasting only. oxidative stress and unfolded protein response the enhanced expression of tca cycle and oxidative-phosphorylation enzymes often causes oxidative stress. indeed, cytosolic superoxide dismutase was -fold upregulated after hours, and the early growth response protein, its transcriptional regulator, -fold. furthermore, catalase and stress-regulated mitogen-activated protein kinase were both -fold upregulated at this time point. in addition, metallothionein gene, known to be involved in protection against oxidative stress and metal toxicity, was intensely upregulated. interestingly, the top-scoring processes obtained from a biological-process enrichment analysis all belonged to the unfolded-protein response stress). to present the relevant data in a single figure, we created a network using the shortest-path algorithm. the resulting network provides links based on the known interaction data between the nodes from the query data set, and also between the nodes that regulate the given genes or metabolites. it shows heat-shock and other proteins, all upregulated – fold, indicating upregulation of this stress-response pathway in fasted liver. downstream of the er stress pathway, proteasome degradation was also upregulated, but again only after hours of fasting. a list of these and some additional genes regulated in the er stress and proteasome degradation, with their change level, is shown in table discussion the major architectural feature of fasting liver is a pronounced decline in cell size rather than a loss of cell number. in addition, the liver's metabolic zonation in upstream, periportal and downstream, pericentral regions remains intact. these findings indicate that the liver can quickly resume its homeostatic functions once feeding resumes. our microarray data show that the adaptive response of the liver to fasting at the level of gene expression is most pronounced during the early phase, with the upregulation of ammonia detoxification persisting up to hours of fasting. since the technically similar study of bauer et al. reported enhanced expression of lipid-catabolizing and urea-cycle enzymes after and h of fasting, the collective data show that the response to fasting in the liver starts already at hours of fasting and becomes maximal between and hours. the hepatic response to total food deprivation, therefore, does not proceed through the global "sugar-fat-protein" sequence that is described for the adaptation on the whole-body level. the expression of genes involved in lipid metabolism and ketone-body synthesis, many under pparα coordination, was strongly regulated towards fatty-acid oxidation and ketone-body formation. interestingly, this adaptive response was seen between and hours of fasting only, and then faded out. in the rat, this response was recently reported to occur between and days of fasting. fatty-acid oxidation was accommodated by the identical time frame of the upregulation of the expression of tca-cycle enzymes and the proteins of the electron-transport chain in response to fasting. the associated oxidative stress and mitochondrial radical formation was apparently sufficiently strong to induce the unfolded-protein response in liver. thus far, to our knowledge, activation of the unfolded protein response has not been associated with fatty-acid oxidation in the fasting liver, but it is induced by a high-fat diet. similarly, mitochondria in fasting muscle protect themselves against the oxidative stress that results from fat oxidation by accumulating the uncoupling proteins ucp and ucp. the role of the liver in gluconeogenesis during fasting is well documented. however, the expression of enzymes associated with gluconeogenesis was upregulated only during the first day of fasting and was mainly confined to the malate-aspartate shuttle and pepck in fact, apart from pepck the expression of none of the committed steps in gluconeogenesis was regulated. it is likely that the enhanced expression of tca-cycle and malate-aspartate shuttle enzymes, and the enhanced expression of pepck enhance the flux towards either glucose-6-phosphate or lactate. it is, therefore, remarkable that the expression of glucose-6-phosphatase, a periportal enzyme, and pyruvate kinase, a mainly pericentral enzyme, are not regulated, while the expression of lactate dehydrogenase is only upregulated at and hours. similarly, the expression and activity of glucose-6-phosphatase in rat liver are upregulated mildly during the first hours of fasting only. our unpublished data show a similar response in the kidneys of fasting mice, in which pepck is 2– fold upregulated at all time points, whereas glucose-6-phosphatase is not regulated. the pronounced accumulation of glycogen in pericentral hepatocytes starting after hours of fasting, which was also observed in 72- and 96-hour fasted rats, indicates that pericentral hepatocytes, which do not express glucose-6-phosphatase, channel glucose-6-phosphate towards glycogen. since all relevant enzymes are also expressed in periportal hepatocytes, which do not accumulate glycogen, we assume that these hepatocytes contain enough glucose-6-phosphatase to produce glucose. the liver produces ~60% of the newly produced glucose in starvation, while the kidneys account for ~40%. a recent, but controversial series of experiments suggest that, in addition to the liver and kidney, the small intestine also has the capacity to produce glucose upon prolonged fasting. it contributes indirectly, by providing lactate and alanine to the liver in short-term fasting, and directly by the production of glucose. the concept is controversial, since other studies were unable to detect glucose formation from glutamine in the isolated small intestine of hours fasted rats. furthermore, the expression of the key gluconeogenic enzyme phosphoenolpyruvate kinase in the mouse small intestine was reported to amount to only –1% of that in the liver after h hours of fasting, also arguing against intestinal gluconeogenesis. we, therefore, compared pepck mrna levels in these two organs by qpcr in the fed and 3-days fasting condition. while pepck expression in the gut at hours of fasting only amounted to ~5% of that in liver, its expression increased to and 53% of that in the liver after and hours of fasting, respectively. this finding demonstrates that the issue of intestinal gluconeogenesis during prolonged fasting deserves additional study. the plasma concentrations of both glucose and lactate remained unchanged during the first hours of fasting, declined temporarily by 35–40% at hours, and returned to control values between and hours. the maintenance of normal concentrations of glucose and lactate during the first hours of fasting is most likely the result of gluconeogenesis in the liver and kidney. since the expression of enzymes that are shared by the glycolytic and gluconeogenic pathways, declines after hours of fasting, the observed decline at hours may represent a declining contribution of the liver to gluconeogenesis. as we argued earlier, the accumulation of glycogen in the pericentral hepatocytes between and hours of fasting indicates that gluconeogenic intermediates flow towards glucose-6-phosphate and accumulate as glycogen due to the low expression of glucose-6-phosphatase in these hepatocytes. most likely, a similar or higher flow of gluconeogenic intermediates is present in the periportal hepatocytes, but is exported as glucose due to the high concentration of glucose-6-phosphatese in these cells. furthermore, the putative production of glucose in the 72-hour fasted intestine can also contribute to the circulating glucose level. the urea-cycle enzymes distinguish themselves from most other genes in the liver in that they were upregulated in expression throughout the period of fasting that was studied. furthermore, cytosolic glutamate-oxaloacetate transaminase, which mediates the availability of aspartate to the urea-cycle enzyme argininosuccinate synthetase, was also strongly upregulated at hours. similarly, oat and prodh, which supply glutamate to glutamine synthetase for glutamine synthesis in pericentral hepatocytes, were strongly upregulated at all time points studied, but glutamine synthetase itself was not regulated. since few amino-acid catabolizing enzymes were upregulated, most amino-groups were probably carried to the liver as alanine or glutamine, although neither glutamate-pyruvate transaminase nor liver glutaminase was upregulated. the coordinate control of ammonia detoxification and the source of ammonia during prolonged fasting therefore deserve attention. an important question is to what extent we can extrapolate the observations in a small mammal like the mouse to larger animals like humans. the ability to tolerate the absence of food does indeed decline with body size: in the mouse the maximum duration of fasting is days, in rat 12– days, in children weeks and in adult humans 8– weeks. qualitatively, however, the response to fasting is probably comparable between these mammals, as long as the time scale is adjusted to the size of the animal. rather than questioning the comparability of small and large animals, our data question whether the implicit extrapolation of the "sugars-fats-proteins" succession of energy substrates during fasting that is based on whole-body energy expenditure to individual organs is valid. microarray studies in rodents that have prospected the adaptive response to fasting of the small intestine, liver, muscle, and a more limited study in kidney focusing on circadian differences in gene expression, reveal a different scenario. muscle and kidney respond to fasting with a progressive change over time in mrna concentrations of enzymes involved in protein, carbohydrate and fat metabolism. the response in liver peaked at 24– hours of fasting in mouse, while most adaptive changes had abated by hours. the intestine, finally, showed an early, but temporary peak of adaptive changes in amino-acid, carbohydrate and fat metabolism at hours of fasting, while a late response, existing almost exclusively of amino-acid catabolizing and gluconeogenic enzymes, gradually developed towards hours of fasting. these differences in pattern and amplitude of gene expression change in different organs can be used to look for circulating biomarkers that reflect the functions of organs during adaptive responses.  based on whole-body energy expenditure, the "sugars-fats-proteins" sequence of energy substrates during fasting was suggested. in our extensive microarray studies of the response to fasting in the gut and liver, we found no support for this intuitively attractive model at the individual organ level. the liver markedly differed from the biphasic response pattern in the small intestine in that its adaptive response peaked at 24– hours of fasting, while most adaptive changes had abated by hours. expression profiling and pathway analysis revealed that genes involved in amino-acid, lipid, carbohydrate and energy metabolism responded most significantly to fasting, with no temporal separation between them. even though the liver lost 50% of its initial weight during days of fasting, its basic morphology remained preserved, showing that the liver can quickly resume its homeostatic functions when feeding resumes. 
the strong induction of the urea cycle, in combination with increased expression of enzymes of the tricarboxylic-acid cycle and oxidative phosphorylation, indicated a strong stimulation of amino-acid oxidation peaking at hours. hence, gene-expression profiling, pathway, network and gene-set enrichment analysis and immunohistochemistry were carried out on mouse liver after and hours of fasting. the continuous high expression of enzymes of the urea cycle, malate-aspartate shuttle, and the gluconeogenic enzyme pepck and the re-appearance of glycogen in the pericentral hepatocytes indicate that amino-acid oxidation yields to glucose and glycogen synthesis during prolonged fasting. liver structure and metabolic zonation were preserved. expression profiling and pathway analysis revealed that genes involved in amino-acid, lipid, carbohydrate and energy metabolism responded most significantly to fasting, that the response peaked at hours, and had largely abated by hours. the induction of genes involved in the unfolded-protein response underscored the cell stress due to enhanced energy metabolism. liver wet weight had declined ~ ~ ~ and ~10% per day after and hours of fasting, respectively. the changes in liver gene expression during fasting indicate that, in the mouse, energy production predominates during early fasting and that glucose production and glycogen synthesis become predominant during prolonged fasting. at this time point, fatty-acid oxidation and ketone-body formation were also induced. the contribution of individual organs to the whole-body adaptive response to fasting has not been established.
 the contribution of individual organs to the whole-body adaptive response to fasting has not been established. hence, gene-expression profiling, pathway, network and gene-set enrichment analysis and immunohistochemistry were carried out on mouse liver after and hours of fasting.  liver wet weight had declined ~ ~ ~ and ~10% per day after and hours of fasting, respectively. liver structure and metabolic zonation were preserved. supervised hierarchical clustering showed separation between the fed, 12– h-fasted and h-fasted conditions. expression profiling and pathway analysis revealed that genes involved in amino-acid, lipid, carbohydrate and energy metabolism responded most significantly to fasting, that the response peaked at hours, and had largely abated by hours. the strong induction of the urea cycle, in combination with increased expression of enzymes of the tricarboxylic-acid cycle and oxidative phosphorylation, indicated a strong stimulation of amino-acid oxidation peaking at hours. at this time point, fatty-acid oxidation and ketone-body formation were also induced. the induction of genes involved in the unfolded-protein response underscored the cell stress due to enhanced energy metabolism. the continuous high expression of enzymes of the urea cycle, malate-aspartate shuttle, and the gluconeogenic enzyme pepck and the re-appearance of glycogen in the pericentral hepatocytes indicate that amino-acid oxidation yields to glucose and glycogen synthesis during prolonged fasting.  the changes in liver gene expression during fasting indicate that, in the mouse, energy production predominates during early fasting and that glucose production and glycogen synthesis become predominant during prolonged fasting. 