BACKGROUND
the consumption of a low-calorie, but nutritionally sufficient, diet has been found to improve indices of disease risk in humans, and has been studied as an intervention that increases lifespan in mice of most genetic backgrounds.
these effects of calorie restriction  are hypothesized to reflect not only prevention of particular age-related diseases, but an actual interference with the process of aging  <cit> .
the physiological effects of cr have therefore been of great interest from the standpoint of human health  <cit> , and much effort has been devoted to development of drug compounds that "mimic" beneficial aspects of low-calorie diets, without requiring a reduction in overall caloric intake  <cit> .
despite decades of work examining rodent responses to cr, however, the key biochemical pathways underlying positive effects of this diet on aging and lifespan remain to be elucidated  <cit> .
the most well-studied genes and pathways have emerged from work in lower invertebrates, including the insulin/insulin-like growth factor  <dig> system, the mammalian target of rapamycin , forkhead transcription factors, and the sirtuin family of histone deacetylase genes  <cit> .
establishing the relative importance of these and other pathways in the mammalian cr effect provides avenues for development of cr mimetic compounds, but also advances a mechanistic understanding that should shed light on unanswered questions.
for instance, it is unclear whether beneficial effects of cr arise from restriction of specific nutrients   <cit> , and for unknown reasons, effects of cr on lifespan differ among genotypes  <cit> .
these issues may be addressed, in part, by identifying pathways perturbed by cr and understanding their functional contribution to increased lifespan.
gene expression analyses using dna microarrays cannot reveal whether genes are causally involved in effects of cr on lifespan, but are nonetheless powerful tools for identifying new, and possibly unexpected, pathways that warrant further experimental investigation  <cit> .
in mice, previous studies have found that cr brings about large shifts in gene expression, including tissue-specific responses to cr, as well as a generalized or common cr response that is shared among tissues  <cit> .
both tissue-specific and common responses to cr are of potential importance, but common responses to cr across multiple tissue types provide especially attractive targets for further study.
swindell   <cit>  analyzed transcriptional responses to cr across  <dig> tissue types in the laboratory mouse, and found that common responses to cr include activation of tumor suppressor genes, as well as anti-oxidant and anti-inflammatory pathways.
for instance, cr was found to increase expression of metallothionein-encoding genes  in several tissues, including liver, heart, muscle, hypothalamus, colon and lung.
additionally, in multiple tissues, cr increased expression of nfkbia, an inhibitor of nf-κb, suggesting a potential anti-inflammatory mechanism of cr diets  <cit> .
more recently, microarrays have been used to evaluate effects of cr in previously unexamined tissues, including thymus, gonads, mammary gland, spinal cord, cerebellum and striatum  <cit> .
these data allow common responses to cr be refined, should lend greater confidence to lists of cr-regulated genes and pathways, and provide an opportunity for applying improved statistical methods.
for instance, previous investigations have focused on how cr affects individual genes, but this may reveal only part of the story.
a more comprehensive and informative approach is to identify regions within the genome-wide transcriptional network that are activated or inhibited by cr  <cit> .
historically, the effects of cr have been viewed as being associated with the aging process  <cit> .
this standpoint argues that effects of cr extend beyond any one disease process , but that cr has multiplex effects on a range of physiological systems, ultimately amounting to an inhibitory effect on the progression of aging.
the association between cr and aging, however, remains poorly understood, largely because the aging process itself remains poorly defined  <cit> .
while an uncontroversial definition of aging may not be developed anytime soon, it should be possible to add rigor to the concept by generating quantitative models of aging that are operationally useful.
in this regard, whole-genome microarray datasets would seem especially valuable  <cit> , and can be used to generate models that test, quantitatively, the assertion that cr acts to oppose the progression of aging  <cit> .
conclusions generated from previous investigations conflict regarding the association between the effects of cr and aging.
on the one hand, an early investigation revealed that age-associated expression patterns in muscle were "either completely or partially prevented by caloric restriction"  <cit> , and this conclusion was supported in subsequent studies  <cit> .
other investigations, however, have yielded different conclusions.
for instance, effects of cr were entirely unrelated to those of aging in muscle tissue from rhesus monkeys  <cit> , and in one aptly designed experiment examining mouse cardiac tissue, only  <dig> of  <dig> age-responsive genes  were significantly altered by cr  <cit> .
clearly, experimental design and statistical methodology are two important considerations for evaluating this diverse set of results.
many studies, for instance, have not evaluated whether the observed overlap between cr and aging effects is larger than expected by chance alone.
this statistical evaluation would not be straight-forward in many cases, since experiments involved a shared control treatment that was used to evaluate the effects of both aging and cr .
given this design, the effect of cr is not estimated independently of the effect of aging, and some correspondence between cr and aging effects would be expected by chance  <cit> .
the present study integrates results from a large number of microarray experiments that have evaluated the effects of cr or aging in the laboratory mouse.
collectively, experiments analyzed in this report have evaluated the effects of cr in  <dig> mouse tissue types, and the effects of aging in  <dig> mouse tissue types.
for many tissues, effects of cr and/or aging have been evaluated in multiple studies, and results from separate studies of the same tissue are combined within a meta-analytic framework.
the overall approach was shaped by three main objectives.
first, it was of interest to identify robust expression patterns associated with cr and aging, particularly common responses occurring within multiple mouse tissues.
secondly, to move beyond single-gene-centric analyses and to take full advantage of the discernable co-expression relationships among genes, regions of the transcriptional network activated or inhibited by cr or aging were identified .
lastly, based on this cross-study comparison of multiple datasets, it was of interest to statistically evaluate whether cr acted to oppose age-associated gene expression patterns.
the overlap between the effects of cr and aging was therefore evaluated using several different methods, including overlap between differential expression signatures  <cit>  and similarity between ordered gene lists  <cit> .
RESULTS
genes regulated by caloric restriction in the laboratory mouse
the cr dataset is a compilation of  <dig> experiments that, taken together, have evaluated the effects of cr in  <dig> tissue types .
for most tissues, data from just 1- <dig> experiments was available, but for well-studied tissues , data was available from as many as  <dig> separate experiments .
additional file  <dig> provides detailed information regarding all experiments incorporated into the analysis, including mouse strains used in each study, sample sizes, array platforms, and whether experimental results from a given study were validated using rt-pcr.
differential expression results from independent studies were combined using fisher's p-value combination method .
the symbol pu is used throughout to denote a p-value generated from a one-sided hypothesis test for up regulation by cr , while pd denotes a p-value generated from a one-sided hypothesis test for down regulation by cr .
the customary p  represents a two-sided p-value, generated by testing whether cr or aging altered gene expression in either direction .
unless otherwise indicated, p-values cited in the text have been adjusted to account for multiple testing of hypotheses among the  <dig>  genes evaluated in the analysis.
the table lists the tissue and cell types for which array data was available and incorporated into the analysis.
each tissue or cell type is referenced by a three-letter symbol listed in the second columns.
the final two columns list, for each tissue or cell type, the number of independent array experiments that have investigated response to cr or aging.
additional file  <dig> provides details regarding each experiment incorporated into the analysis .
there was considerable heterogeneity among tissues in terms of the number of genes for which information was available, and with respect to the percentage of genes significantly influenced by cr.
for heart, liver, cochlea, muscle, cortex, hypothalamus and colon, effects of cr had been evaluated, in one or more experiments, for nearly all  <dig>  genes included in the analysis .
for other tissues, however, no data had been collected for most known genes and information was available for less than  <dig>  genes  .
based upon a nominal type i error rate of  <dig> , it was expected that at least 5% of genes in any one tissue would be significantly altered by cr.
this was the case for  <dig> of  <dig> tissues, in which cr altered more than 5% of the examined genes, with as many as 54% of genes altered in the case of heart .
for cerebellum and striatum, however, cr significantly altered only  <dig> - 3% of the examined genes, which is a smaller number of significant effects than expected on the basis of chance .
the effects of cr were characterized by constructing differential expression signatures associated with each tissue  <cit> , and the overlap among signatures was examined.
differential expression signatures associated with cr in different tissues usually overlapped by less than 40%, but this overlap was often statistically significant .
a total of  <dig> pair-wise comparisons were made among  <dig> signatures, and in  <dig> of these cases , the overlap was stronger than expected based on chance alone .
there was strong similarity between some segments of the central nervous system, such as spinal cord and cerebellum, as well as hypothalamus and hippocampus, but there was no overall tendency for signatures associated with the central nervous system to cluster together .
for instance, effects of cr in the striatum were not significantly similar to those in the cerebellum, spinal cord, hippocampus, cortex and hypothalamus .
for liver, heart and muscle, effects of cr had been evaluated in more than four experiments, so detailed tissue-specific analyses were carried out .
a surprising outcome of these analyses was that, although numerous genes emerged as significant in at least one experiment for each tissue, relatively few genes exhibited a consistent expression response to cr in multiple experiments.
in liver, for instance, after collecting results across  <dig> experiments, only 10- <dig> genes were found to respond to cr in a consistent fashion .
these consistent responses provide liver-specific markers of cr response, and include up regulation of lpin <dig> and lpin <dig> , which were both increased in each of  <dig> experiments for which data was available .
a second interesting aspect of the response to cr in liver was that several expression responses paralleled those resulting from growth hormone/insulin-like growth factor  <dig>  mutations known to increase mouse lifespan  <cit> .
for instance, in liver, cr decreased expression of leukemia inhibitor factor receptor  , insulin-like growth factor acid labile subunit  , kidney expressed gene  <dig>  , and the cytochrome p <dig> gene cyp2f <dig> .
additionally, cr increased expression of isocitrate dehydrogenase  <dig>  , insulin-like growth factor binding protein  <dig>   and, very consistently, elevated expression of flavin-containing monooxygenase  <dig>   .
each of these expression responses was previously identified in association with multiple long-lived dwarf mutants, which have reduced levels of gh and/or igf- <dig> in circulation  <cit> .
common responses to cr in multiple tissues included genes identified previously  <cit> , but also new genes not uncovered in prior analyses.
given the large number of experiments considered, the analysis provided substantial statistical power, and overall,  <dig> % of genes  were significantly up regulated by cr across tissues , while  <dig> % of genes  were down regulated by cr across tissues .
given this large number of significant results, a more discerning criterion was to stratify genes according to the number of tissues in which a gene was significantly up or down regulated by cr.
for instance, less than 1% of genes  were up regulated by cr with respect to  <dig> or more tissue types, or down regulated by cr with respect to  <dig> or more tissues .
the most widespread effect of cr was up regulation of serum/glucocorticoid regulated kinase  <dig>  in  <dig> tissues, including colon, cochlea, kidney, lung, heart, liver and white adipose tissue, as well as four regions of the central nervous system   .
additionally, and consistent with previous analyses  <cit> , cr increased expression of metallothionein  <dig>  in  <dig> tissues , and down regulated the collagen-specific molecular chaperone serpinh <dig> in  <dig> tissues, including regions within the central nervous system   .
a previous study has confirmed, by rt-pcr, that serpinh <dig> is down regulated by cr in heart, liver and hypothalamus  <cit> .
in figure 2a and 2b, genes are ranked according to the total number of tissues in which cr increased or decreased expression significantly.
additional file  <dig> provides a comprehensive list using an alternative ranking scheme .
this alternative approach highlighted some strong cr effects not shown in figure  <dig>  including increased expression of tsc22d <dig>  zbtb <dig>  bc <dig>  sult1a <dig>  per <dig>  timp <dig>  s3- <dig>  pnpla <dig>  ppargc1a and cp, as well as decreased expression of ifitm <dig>  lasp <dig>  tia <dig>  5830428h23rik, hsp90b <dig>  hist1h2bc, cxcl <dig>  col6a <dig> and dock <dig> .
additional file  <dig> also provides analyses of gene ontology terms, kegg pathways, associated micrornas, and chromosomal locations.
overall, genes most strongly increased by cr across tissues were associated with fatty acid metabolism, the citrate cycle, ppar signaling, oxidative phophorylation, amino acid degradation and metabolism, circadian rhythm, renal cell carcinoma, fatty acid elongation in mitochondria and the insulin signaling pathway  .
genes commonly down regulated by cr were associated with focal adhesion, antigen processing and presentation, ecm-receptor interaction, dna replication, mapk signaling, cell communication, vegf signaling and natural killer cell mediated cytotoxicity  .
genes listed in figures 2a and 2b have not been well-studied in relation to the effect of cr in mouse.
for instance, a series of pubmed searches was conducted , where each gene name was searched in conjunction with the phrase "caloric restriction".
this search yielded one record associated with sgk <dig> and hspb <dig>  respectively, but no pubmed records were retrieved for any other gene shown in figures 2a and 2b.
figure 2c shows differential expression patterns of genes receiving greater research attention, such as sirtuin  <dig> , for which the above-mentioned search procedure retrieved  <dig> pubmed records.
sirt <dig> expression was significantly increased by cr in the cochlea, and significantly decreased in muscle and white adipose tissue, but overall, was not significantly regulated by cr across tissues  .
of the  <dig> sirtuin gene family members, cr had the strongest effect on the expression of sirtuin  <dig> , which was significantly increased by cr in colon, cochlea and cortex, and significantly decreased by cr in heart, liver and muscle  .
interestingly, the igf <dig> transcript was significantly regulated by cr, but there was heterogeneity among tissues in the direction of this effect.
in colon, lung, muscle, white adipose tissue and hypothalamus, igf <dig> expression was decreased by cr, but the opposite trend was found in cochlea, heart, cortex and mammary gland .
overall, across all tissues, there was significant evidence for decreased expression of igf <dig> by cr .
gene expression modules regulated by caloric restriction
the above analyses consider cr-responsiveness of individual genes, taken one at a time, but do not consider the organization among genes based upon their patterns of co-expression.
for each gene, therefore, a "nearest-neighbor" co-expression module was generated by finding "neighbor" genes with correlated patterns of expression, based upon a reference set of  <dig>  affymetrix oligonucleotide arrays .
nearest-neighbor modules of varying sizes were constructed for each gene included in the analysis .
each module was then scored based upon an m statistic designed to quantify responsiveness of modules to cr, and the significance of observed m values was evaluated by simulations in which modules of a given size were formed at random .
a total of  <dig>   <dig>   <dig>   <dig> and  <dig> significant cr-responsive modules with  <dig>   <dig>   <dig>   <dig> and  <dig> genes, respectively, were identified.
table  <dig> lists the most significant cr-regulated co-expression modules of smaller size, and all significant modules of each size are shown in additional file  <dig>
the table lists the most significant cr-regulated co-expression modules containing  <dig>   <dig>  or  <dig> genes.
the m statistic is an indicator of responsiveness to cr, with larger values suggesting stronger effects of cr on the expression of genes contained in a given module .
p-values associated with m statistics were generated by simulation, and reflect the likelihood of obtaining an equal or larger m value by randomly selecting genes to form a module of the same size .
the final column provides an indication of the degree to which module members are co-expressed with one another.
this value is an element of the  <cit>  interval, and is obtained by calculating all possible pair-wise co-expression relationships among members of the module , and identifying the weakest co-expression relationship between any two module members .
smaller minimal |r| values indicate more "diffuse" co-expression modules with weaker co-expression relationships, and larger minimal |r| values indicate "tighter" co-expression modules with stronger co-expression relationships.
additional file  <dig> provides a more detailed description of these and other co-expression modules that emerged as significantly associated with cr.
the approach identified 2-gene co-expression modules that were responsive to cr, some of which included functionally related gene pairs, such as {hist2h3c <dig>  hist2h3c2} and {per <dig>  per2}  .
of the 2-gene modules, only {fmo <dig>  1200016e24rik} was marginally significant , although others were often components of larger modules that were statistically significant .
for instance, significant 3-gene modules included {hist2h3c <dig>  hist2h3c <dig>  hist1h2bc} and {per <dig>  per <dig>  zbtb16} .
the composition of cr-responsive modules often revealed unexpected associations between genes.
for instance, the most significant 10-gene module included six genes that encode histone cluster subunits, along with genes encoding an rna binding protein  and a peroxisomal beta-oxidation system enzyme  .
the second-most significant 10-gene module included three period homologue genes , a tsc <dig> domain family gene with anti-inflammatory effects   <cit> , and a gene known to negatively regulate tumorigenesis   <cit>  .
it was often the case that genes belonging to the same module exhibited similar responses to cr within the same tissues .
the most cr-responsive 40-gene module included genes associated with mrna processing and transcriptional regulation .
the "center" of this module was the zinc finger protein  <dig> , with the remaining  <dig> genes being nearest neighbors of zfp <dig>  genes belonging to this module were highly responsive to cr, but the direction of response differed among tissue types .
in cochlea, hypothalamus and colon, nearly all member genes were significantly up regulated by cr.
however, in hippocampus, mammary gland and liver, genes in this module were usually down regulated by cr .
gene ontology analysis revealed that member genes were involved in transcriptional processes, particularly splicing, metabolism, processing and biosynthesis of rna .
along these lines, a disproportionately large number of module genes were associated with the nucleus, chromosome and spliceosome .
genes regulated by aging in the laboratory mouse
the effects of aging on gene expression had been evaluated in  <dig> experiments and a total of  <dig> tissue types .
on average, aging altered  <dig> % of genes, with the strongest effects found in the cortex  .
among those tissues with the strongest age effects , more genes increased with age than decreased with age .
the weakest effects of aging were associated with cochlea and striatum, in which aging altered only 2-4% of genes, which is fewer than expected by chance at the nominal type i error rate of  <dig>  .
for some tissues, aging had a larger impact on gene expression than observed in prior studies.
in cerebellum, for example, at a type i error rate of  <dig> , aging altered the expression of  <dig> of  <dig> genes .
this percentage is considerably higher than that observed in the agemap project  <cit> , in which aging altered the expression of only  <dig> of  <dig> genes  .
this apparent difference in effect size may reflect increased statistical power associated with a meta-analytic approach, or differences in the relative amount of noise influencing expression measurements generated using different array platforms.
differential expression signatures were constructed to characterize the effects of aging in each tissue, and comparisons of these signatures suggested that, relative to cr, effects of aging were more correspondent among different tissue types.
for instance, among  <dig> pairwise comparisons between signatures, significant overlap was found in  <dig> cases  , and a cluster analysis indicated that  <dig> of the  <dig> tissues formed a single group with similar patterns of differential expression .
a side-by-side comparison of results from multiple experiments examining the same tissue highlighted robust tissue-specific markers of aging .
for instance, in each of seven experiments evaluating the effects of aging in liver, expression of immunoglobulin joining chain  increased , while expression of kidney expressed gene  <dig>  decreased  .
the effects of aging in cardiac tissue had been examined in an especially large number of experiments .
comparison of these experiments indicated that aging increased cardiac expression of phenylalanine hydroxylase  in all  <dig> experiments for which data was available .
likewise, aging increased cardiac expression of complement component 4b  in  <dig> experiments for which data was available .
these results confirm and extend observations from a recent expression profiling study of age-associated gene expression patterns in cardiac tissue from multiple mouse strains, which identified both pah and c4b as robust biomarkers of cardiac aging, and confirmed these age-related expression patterns using rt-pcr  <cit> .
in muscle, an age-related increase of heat shock protein  <dig>  expression was observed in four separate experiments , as well as an age-associated decrease of col1a <dig> expression .
genes with age-related expression patterns in multiple tissue types were identified using fisher's method.
this revealed that  <dig> genes  were significantly up regulated with age across all tissues , while  <dig> genes  were significantly down regulated with age across all tissues .
there were strong functional differences between genes that increased with age in multiple tissues and genes that decreased with age in multiple tissues .
genes up regulated with aging were frequently associated with immune response, and over represented gene ontology terms included antigen processing and presentation, lymphocyte mediated immunity, immunoglobulin mediated immune response, humoral immune response, lysosome organization/biogenesis, and regulation of adaptive immune response .
however, this was not generally true of genes decreased with age.
genes down regulated with aging in multiple tissues were more commonly associated with metabolic processes, chromosome organization/biogenesis, cell division and cycle, mrna processing and splicing, and protein folding  .
the age-related expression patterns of identified genes were more widespread than those of some genes previously associated mammalian aging and cellular senescence .
based on previous investigations  <cit> , for instance, it was expected that aging would increase expression of cyclin-dependent kinase inhibitor 2a  and ets homologous factor , and that aging would decrease expression of senescence marker protein  <dig> .
for these genes, however, significant effects of aging were observed only in a few tissues, and often, the effect was inconsistent with prior expectations.
for instance, cdkn2a/p16ink4a is a regulator of the cell cycle, and it has been proposed that expression of cdkn2a is a biomarker of aging in multiple mouse tissues  <cit> .
but there was little indication that cdkn2a expression increased with age in most tissues, and overall, the effect of age on cdkn2a expression was nonsignificant among all tissue types .
the expression of cdkn2a increased significantly only in cardiac tissue with age, and in cortex, cdkn2a expression decreased with age .
gene expression modules regulated by aging
nearest-neighbor co-expression modules ranging in size from  <dig> to  <dig> genes were formed and the collective response of each module to aging across tissues was evaluated.
the results demonstrate strong modularity of age-related expression patterns, with certain sets of co-expressed genes exhibiting correspondent differential expression patterns with age.
a total of  <dig>   <dig>   <dig>   <dig> and  <dig> significant age-responsive modules with  <dig>   <dig>   <dig>   <dig> and  <dig> genes, respectively, were identified .
the most significant 3-gene module included two proteasome subunit genes , along with the mhc antigen h2-k <dig> .
the three genes contained in this module exhibited highly correspondent patterns of differential expression, with decreased expression occurring in spleen with age, and an age-related up regulation of expression across  <dig> tissues .
a similar pattern was present with respect to other 3-gene co-expression modules, such as {tyrobp, mpeg <dig>  ctss} and {sfi <dig>  pisd, 4933439c20rik}, and with significant co-expression modules of larger size .
in each of these cases, genes belonging to the same module exhibited similar differential expression patterns in the same tissues, indicating that patterns of co-expression had considerable explanatory power in terms of age-related transcriptional effects.
the table lists the most significant age-regulated co-expression modules containing  <dig>   <dig>  or  <dig> genes.
values in this table are interpreted in the same way as those listed in table  <dig>  except here the m statistic reflects responsiveness of a module to aging .
additional file  <dig> provides a more detailed description of these and other co-expression modules that emerged as significantly associated with aging.
the most significant 40-gene module was centered around cathepsin s  .
the rest of the module consisted of the  <dig> nearest neighbors of ctss, which included genes encoding complement components  and antigens .
strikingly, nearly every gene in this module exhibited an age-related expression increase in liver, kidney, cerebellum, cortex, eye, aorta, lung, heart, hippocampus and whole brain .
among genes belonging to the module, a gene ontology analysis revealed numerous over-represented biological processes, including activation of immune response, positive regulation of b cell mediated immunity, phagocytosis, inflammatory response and neutrophil chemotaxis .
half of module genes were associated with membrane , particularly the plasma membrane .
the second-most significant 40-gene module  was not associated with inflammation or immune response, but interestingly, included genes with roles in mrna processing, rna transport and rna localization.
most of the genes in this module were associated with the nucleus  and several were among the most age-responsive genes identified in the analysis, such as gspt <dig>  ints <dig>  syncrip and creb <dig> .
in contrast to the module shown in figure  <dig>  most member genes were predominantly decreased with aging, particularly in aorta, gametes and cardiac tissue .
relationship between caloric restriction and aging
cr is thought to inhibit the progression of aging and to oppose age-related gene expression patterns.
it was therefore expected that a negative association would exist between transcriptional effects of cr and aging, and several approaches were taken to evaluate whether such an association was present.
first, genes most strongly regulated by cr or aging across multiple tissue types were considered .
there was no general tendency for genes increased by cr across tissues to be decreased by aging, and likewise, genes decreased by cr across tissues were not disproportionately increased by aging .
individual genes can be identified that either support or contradict the expected pattern.
for instance, cr decreased expression of igh- <dig> and marcks, and this effect does indeed oppose increased expression of these genes with age across tissues.
on the other hand, cr decreased expression of col3a <dig>  and this effect would seem to mimic, rather than oppose, age-related decreases in col3a <dig> expression .
the association between cr and aging was next examined at the global scale, among all genes, and also with respect to each of the four most well-studied tissue types  .
in liver, there was a slight, positive association between the effects of cr and aging  .
this association was significant , although given the large number of genes involved in the comparison, this significance test was not too informative.
in the heart, muscle and central nervous system, the expected negative association between cr and aging did emerge, albeit weakly, with the estimated correlation coefficient less than or equal to - <dig>  in each case.
the strongest association was found in heart , in which age-related expression patterns were weakly opposed by cr .
in muscle and central nervous system , the association between cr and aging was again weak , and non-significant in the case of muscle , despite the large number of genes upon which the association was based.
with respect to central nervous system, a large fraction of genes  were both increased by cr and decreased with age , although very few genes  were decreased by cr and increased with age .
the global effects of cr and aging were then compared by evaluating the association between differential expression signatures.
in this analysis, it was expected that genes significantly increased by cr would tend to be decreased significantly with age, and conversely, that genes significantly decreased by cr would increase significantly with age.
associations between differential expression patterns were found , and were moderately strong in liver , weaker in heart , weakest in muscle , and strongest in the central nervous system .
the pattern of association, however, varied among tissues, and was often not consistent with the expectation.
in liver and central nervous system, cr did oppose age-related expression patterns, but these trends were complicated by the fact that, in each tissue type, there was a significant tendency for cr to actually reinforce the effects of aging, which is in complete contrast to the expectation stated above .
in muscle, the relationship between cr and age-related differential expression patterns was effectively random, with little or no evidence of any association .
the strongest evidence for the expected cr-aging relationship was again found with respect to heart.
in cardiac tissue, there was a significant abundance of genes increased by cr and decreased by age, as well as an abundance of genes decreased by cr and increased by age .
these conclusions were reinforced by results from an alternative analytical method, based upon correspondence between ranked gene lists  <cit> , which also showed conflicting associations between cr and aging in liver, muscle and central nervous system , with a stronger and more clear-cut relationship in heart .
the above results suggest that, among all genes, the transcriptional association between effects of cr and aging is limited, with a stronger relationship in heart relative to other tissue types.
one possibility is that the transcriptional association between cr and aging is stronger among genes associated with some biological processes, and weaker among certain genes associated with other processes.
the cr-aging association was thus examined with respect to gene ontology biological process terms that were associated with genes increased or decreased by aging across all mouse tissues .
this analysis revealed definite differences among gene categorizations with respect to the transcriptional relationship between cr and aging.
in particular, among genes involved in immune response, innate immune response and immune system processes, the effects of cr were negatively associated with those of aging .
these results are in agreement with the rt-pcr investigation of park et al.
<cit> , which found that, in heart and cerebellum, cr opposed the effects of aging for certain genes associated with immunity and defense response .
figures  <dig> and  <dig> also show that cr opposes age-associated expression patterns among other classes of genes as well, such as groups of genes with roles in apoptosis, protein amino acid dephosphorylation, lipid transport, chemotaxis, biopolymer metabolic process and inflammation.
at the same time, for some processes, the opposite pattern of association was found , and for many gene groups, the association between cr and aging was non-significant and weakly inverse, in agreement with analyses based upon global transcriptional patterns among all genes.
discussion
caloric restriction  and aging have been found to influence the expression of numerous genes in a variety of mouse tissues, but there has been considerable heterogeneity in results generated from different laboratories.
this study has identified genes and gene expression modules that, based upon currently available data, are most robustly associated with cr and/or aging in the laboratory mouse.
these transcriptional effects provide a well-supported basis for the generation of new research hypotheses, which can serve as starting points for experimental studies into mechanisms of cr and aging.
the identified expression patterns influence a wide range of tissue and cell types, and thus represent the most systemic transcriptional effects of cr and aging, which may be associated with pathways required for positive effects of cr on survivorship, or linked to mechanisms that drive increased disease and mortality risk with age.
these transcriptional effects were often modular, with groups of co-expressed genes responding to cr or aging in coordination across multiple tissues.
for instance, the analysis uncovered a cr-responsive group of co-expressed genes encoding histone clusters , as well as a large module that contains genes involved in mrna processing .
age-related expression patterns affecting multiple tissues were, to a considerable degree, explained by the modularity of the transcriptome.
this was best demonstrated by a large module containing  <dig> inflammation and immune response genes , nearly all of which increased expression with age in ten mouse tissues.
it was expected that effects of cr and aging would be associated, with cr acting of oppose age-related changes in gene expression.
this was the case among genes associated with certain biological processes , but at the genome-wide level among all genes, the transcriptional association between cr and aging was limited.
common responses to cr represent systemic effects of low-calorie diets that influence multiple organ systems  <cit> .
a previous study found that cr increased expression of genes encoding the anti-oxidant metallothioneins , the nf-κb inhibitor iκbα , as well as the tumor suppressor p <dig>   <cit> .
the present investigation has revealed that these effects are even more widespread than previously thought, each occurring in at least seven mouse tissues .
additionally, widespread transcriptional effects of cr were identified that could influence the predisposition of mice to develop cancers, which is thought to be a main factor underlying pro-longevity effects of the cr diet  <cit> .
these expression patterns were associated with a broad range of tissue types, and include increased expression of thioredoxin interacting protein , zinc finger and btb domain containing  <dig>  and tissue inhibitor of metalloproteinase  <dig>  .
furthermore, cr reduced expression of thymosin , secreted acidic cysteine rich glycoprotein  and heat shock protein  <dig>  , and each of these effects could also inhibit transformation or cellular proliferation and migration  <cit> .
increased expression of txnip and zbtb <dig> expression by cr could reflect an anti-proliferative effect of low calorie diets on a common pathway, since txnip encodes a protein known to induce expression of zbtb <dig>  <cit> , and both genes encode proteins that can promote apoptosis or limit the invasive capacity of tumor cells  <cit> .
widespread effects of cr on txnip expression are especially noteworthy, since txnip-deficient mice exhibit several abnormal responses to fasting  <cit> , and because txnip can regulate activation of the pten tumor suppressor protein   <cit> .
like txnip and zbtb <dig>  timp <dig> has tumor-suppressive properties  <cit> , and its expression limits tumor invasiveness and has been correlated with survivorship in cancer patients  <cit> .
additionally, expression of timp <dig> negatively controls tumor necrosis factor alpha  levels, leading to reduced nf-κb activation and inflammation  <cit> .
taken together, these results begin to outline a generalized response of the mammalian cell to cr.
this response involves increased expression of genes with tumor-suppressive properties , decreased expression of genes associated with cellular transformation and proliferation , as well as the induction of genes with anti-inflammatory  or anti-oxidative effects .
the beneficial effects of cr on survivorship may be related to loss of insulin-like growth factor  <dig>  signaling  <cit> .
this type of mechanism has been shown to increase lifespan and decrease cancer incidence in mutant dwarf mice, in which serum igf- <dig> levels are greatly diminished  <cit> , or in which the localized bioavailability of igf- <dig> within tissues is reduced  <cit> .
similarly, it was recently shown that mouse lifespan can be increased by late-life administration of rapamycin, which inhibits mtor, an element downstream of the igf-1/pi3-k pathway  <cit> .
a major question in aging biology, therefore, is whether pro-survival effects of the cr diet are dependent upon inhibition of igf- <dig> signals in a manner that, to some degree, parallels mechanisms underlying increased lifespan in long-lived dwarf mutants and/or mice treated with rapamycin.
along these lines, it is interesting to note that the most widespread effect of cr to emerge from this analysis was increased expression of serum/glucocorticoid regulated kinase  <dig> , which occurred in  <dig> of the  <dig> mouse tissues examined .
the sgk <dig> kinase is activated by igf-1/phosphatidylinositol- <dig> kinase  signals, and data from several microarray analyses indicate that expression of sgk <dig> is up regulated by mutations and treatments that inhibit the igf-1/pi3-k pathway  <cit> .
another widespread effect of cr was increased expression of phosphatase and tensin homolog  , which is a tumor suppressor that inhibits igf- <dig> signals by preventing activation of the pi3k/akt signaling pathway  <cit> .
these effects of cr on sgk <dig> and pten expression may reflect attenuation of igf- <dig> signals across a number of tissue types, possibly in a manner that, to some degree, mimics the influence of dwarf longevity mutations or rapamycin treatment.
such expression patterns thus raise new questions for further work, which can contribute to a developing model of interactions between cr and the igf- <dig> pathway  <cit> .
the metabolism of mrna is a fundamental process that underlies virtually all cellular activities, and perturbation of mrna processing can have important effects on gene regulation, homeostasis and cell cycle control.
this study has found that, in multiple tissues, cr influences the expression of genes involved in the splicing and processing of messenger rna .
these genes are co-expressed and organized into a single large module that includes splicing factors , pre-mrna processing factors , pre-mrna cleavage factor i , an rna/dna binding protein associated with splicing regulation and mrna stability   <cit> , a co-activator of pre-mrna splicing   <cit> , and a yeast homolog of luc7p that associates with the spliceosomal subunit u <dig> snrnp   <cit> .
the possibility that nutrient signals, and in particular cr, can have important effects on mrna processing is consistent with previous investigations.
for example, glucose-6-phosphate dehydrogenase  protein levels and activity are positively regulated by intake of carbohydrates, but without any alteration in transcription rate of the associated g6pdx gene  <cit> .
carbohydrate intake instead appears to increase the efficiency of splicing events within the nucleus, leading to increased abundance of mature mrna transcript and g6pd protein  <cit> .
nutritional regulation of mrna processing has also been reported for thyroid hormone responsive spot <dig> homolog   <cit> , as well as insulin-like growth factor i   <cit> .
these examples suggest that nutritional effects on mrna processing are not altogether unusual, and that some widespread effects of cr could result from an influence on the multi-step process by which mrnas proceed from transcription to translation.
this effect of cr may be consequential for disease processes, since proteins that regulate splicing events can contribute to cell growth and the proliferation of cancer cells  <cit> .
it is interesting to note that expression of genes associated with mrna processing, rna transport or rna localization are decreased by aging in many tissues .
this consistent effect of aging contrasted with that of cr, which variably influenced the expression of functionally similar genes, promoting increased expression in some tissues  but decreased expression in others  .
there is much evidence to suggest that certain mechanisms of aging are conserved across species, with similar pathways regulating life span and the progression of aging in worms, flies and mammals  <cit> .
this notion suggests that there may exist common features of aging that are evolutionarily conserved, which are reflected by age-related patterns of gene expression that are shared among mammalian cell types within diverse tissues.
the present study has identified robust age-related expression patterns that appear to be characteristic of cells within many or most mammalian tissues.
it is not expected that all such age-related expression patterns will be associated with deleterious pathologies, since some modifications of gene expression with age are likely to reflect favorable compensatory mechanisms.
nevertheless, some age-regulated genes identified in this analysis are known to modulate inflammatory processes and contribute to cellular proliferation.
for instance, two widespread effects of aging were up regulation of granulin  and secreted phosphoprotein  <dig>  .
increased expression of grn was in fact the most widespread effect of aging identified, occurring in nearly all of the tissues examined .
grn encodes the granulin protein , which can inhibit tnf-induced inflammatory processes and promote vascularization necessary for wound and tissue repair  <cit> , but can also give rise to cleavage products that stimulate cytokine secretion leading to the exacerbation of inflammatory processes  <cit> .
additionally, granulin has been identified as potent growth factor that stimulates aggressive cell proliferation, and in at least some cell lines, this mitogenic effect depends upon the expression level of grn  <cit> .
increased expression of spp <dig> with age was identified in  <dig> of  <dig> mouse tissue types, with the strongest increase observed in the cerebellum.
spp <dig> encodes osteopontin, a protein involved in localized recruitment of macrophages and in the production of cytokines within certain immune cells, which can contribute to a state of chronic inflammation  <cit> .
osteopontin is also a regulator of tumor progression and its abundance within tumors has emerged as an impressive biomarker for numerous types of cancer  <cit> .
expression of osteopontin within tissues and in circulation has also been associated with a wide range of other conditions, including obesity, diabetes and cardiovascular disease  <cit> .
an important component of future work will be rt-pcr investigation of grn and spp <dig> expression patterns with age, both across mouse tissues and among different mouse strains  <cit> .
a widespread effect of aging was increased expression of inflammation and immune response genes, which involved class i mhc genes , immunoglobulin chain components , and a large module of  <dig> co-expressed genes known to participate in inflammation and immune processes .
these observations are consistent with those from previous investigations that have evaluated genome-wide expression patterns in aging mice  <cit> , and thus provide further evidence that heightened expression of inflammation and immune response genes is a systemic feature of aging that influences a broad range of organ systems.
indeed, exceptions to this pattern may represent the most interesting cases.
for example, only  <dig> of  <dig> tissues did not show an age-related increase in the expression of either h2-d <dig> or h2-k <dig> .
it is likely that, at least in part, increased expression of inflammation and immune response genes reflects age-related increases in immune cell populations within aging tissues.
in liver, a recent histological analysis demonstrated that several immune cell types  infiltrate perivascular regions with aging to establish proliferating cell clusters  <cit> .
this type of mechanism is likely to explain, for example, the increased expression of igk-v <dig> and igh- <dig> described in this analysis, which could be driven by b-cell lymphocyte infiltration of aging tissues.
such immune cell infiltration could be driven by chronic inflammation, possibly in a manner that parallels tertiary lymphoid neogenesis  <cit> , and it is further possible that such inflammation is exacerbated by an age-associated accumulation of fat cells  <cit> .
an important direction for future studies will be to evaluate mechanisms by which immune cells, such as lymphocytes, are drawn to aging tissues and whether such mechanisms are comparable in various organ systems.
an inspection of current data, for example, revealed that the b-cell attractants ccl <dig> and cxcl <dig> increase with age in some tissues  <cit> .
in particular, expression of ccl <dig> increased with age in  <dig> tissues , while an age-related increase in cxcl <dig> expression was observed in  <dig> tissues .
the identification of robust age-related gene expression patterns may lead to the development of useful "biomarkers", which could provide tools for tracking the progression of the aging in experimental studies  <cit> .
development of such indicators is requisite for making objective statements regarding the effects of interventions, or of particular mutations, on the deceleration  of aging.
the age-related expression patterns highlighted in this analysis represent systemic or near-systemic effects, which have repeatedly emerged in multiple experimental settings.
such transcriptional effects may thus be suitable for further evaluation as aging biomarkers, or as components of a quantitative model whose output serves as an aging biomarker  <cit> .
the current data, for example, suggest that increased expression of immune response genes  is a reliable feature of normal aging, perhaps representing a widespread outcome of aging that compares with the prominent but localized effects of aging in the thymus .
localized expression of such genes may be either a cause of consequence of multiple age-related pathologies  <cit> , particularly if increased expression arises from sustained inflammation in certain tissues  <cit> .
consequently, such age-related effects may provide informative endpoints that, in combination with other indicators of aging and healthspan, will eventually lead to surrogates for lifespan in experimental studies of anti-aging interventions using the laboratory mouse  <cit> .
in this sense, other aspects of the aging immune system have also proven informative, such as t-cell subsets, which have been found to predict mouse lifespan within experimental cohorts  <cit> .
along these lines, extended lifespan of mutant mice lacking the pregnancy-associated plasma protein a  gene is reflected by a delayed rate of thymic atrophy  <cit> .
a conceptual association between cr and aging has been maintained for decades, and numerous investigations have been designed around the expectation that cr interferes with the progression of aging.
the alternative view is that cr and aging are in fact disparate phenomena, with cr having many favorable phenotypic effects , but not having effects broad enough to be characterized in terms of the actual process of "aging".
gene expression data is not, by itself, sufficient to discriminate between these two views, and it is clear that any sufficient definition of aging must include measurements from multiple physiological domains  <cit> .
moreover, it is certainly possible that deleterious aspects of aging are prevented by cr in ways that are not reflected by gene expression patterns, and many age-associated expression patterns may indeed reflect favorable compensatory mechanisms rather than pathological processes.
nevertheless, gene expression data reflect the behavior of numerous biochemical pathways, and such data are also well-suited to quantitative investigation.
genome-wide expression data can, in this regard, provide at least some insight into the conceptual links between cr and aging.
there are elements of this analysis that support an association between cr and aging, and elements that do not.
on the one hand, individual genes can be identified for which the effects of cr oppose those of aging in multiple tissues , and this also appears to be true among genes associated with certain types of biological processes .
at a global scale, however, among all genes, this association is fairly weak.
this is most clearly shown by the scatterplots in figure  <dig>  which indicate that genome-wide correlations between cr and aging effects within particular tissues do not exceed  <dig>  in magnitude, and this basic conclusion is supported by analyses shown in figures  <dig> and  <dig>  among different tissues, the genome-wide association was stronger in cardiac tissue, but weaker in liver, muscle and central nervous system.
taken together, therefore, these analyses show that cr counters certain types of age-associated expression patterns, but not others, with a fairly weak global association among all genes, and some degree of variability in the strength of the association among mouse tissue types.
CONCLUSIONS
cr and aging have widespread biochemical effects and systems-oriented approaches are needed to develop comprehensive in silico models that link these effects conceptually.
microarray analyses provide a data-driven approach for judging which pathways serve as "hubs" that connect the diverse processes modulated by cr and aging, and this can lead to the construction of large gene interaction networks, with scope substantially greater than those generated by standard methods.
this study has identified sub-networks, embedded within the genome-wide transcriptional network, that are responsive to either dietary intake or aging across a variety of mouse tissues.
cr influenced small co-expression modules containing genes that encode histone clusters , period homologues , as well as a large module containing genes involved in the splicing and processing of mrna .
aging increased expression of granulin  and secreted phosphoprotein  <dig>  in more than  <dig> tissue types, and led to elevated expression of genes and modules associated with inflammation and immune response .
in general, many age-associated differential expression patterns were explained by the underlying modularity of the transcriptome.
it was expected that the transcriptional effects of cr and aging would be negatively associated, and this was true for genes associated with certain biological processes .
at the genome-wide level, however, cr and aging were found to have weak association, which was stronger in heart relative to liver, muscle and central nervous system.

 the consumption of a low-calorie, but nutritionally sufficient, diet has been found to improve indices of disease risk in humans, and has been studied as an intervention that increases lifespan in mice of most genetic backgrounds. these effects of calorie restriction are hypothesized to reflect not only prevention of particular age-related diseases, but an actual interference with the process of aging. the physiological effects of cr have therefore been of great interest from the standpoint of human health, and much effort has been devoted to development of drug compounds that "mimic" beneficial aspects of low-calorie diets, without requiring a reduction in overall caloric intake. despite decades of work examining rodent responses to cr, however, the key biochemical pathways underlying positive effects of this diet on aging and lifespan remain to be elucidated. the most well-studied genes and pathways have emerged from work in lower invertebrates, including the insulin/insulin-like growth factor system, the mammalian target of rapamycin, forkhead transcription factors, and the sirtuin family of histone deacetylase genes. establishing the relative importance of these and other pathways in the mammalian cr effect provides avenues for development of cr mimetic compounds, but also advances a mechanistic understanding that should shed light on unanswered questions. for instance, it is unclear whether beneficial effects of cr arise from restriction of specific nutrients, and for unknown reasons, effects of cr on lifespan differ among genotypes. these issues may be addressed, in part, by identifying pathways perturbed by cr and understanding their functional contribution to increased lifespan. gene expression analyses using dna microarrays cannot reveal whether genes are causally involved in effects of cr on lifespan, but are nonetheless powerful tools for identifying new, and possibly unexpected, pathways that warrant further experimental investigation. in mice, previous studies have found that cr brings about large shifts in gene expression, including tissue-specific responses to cr, as well as a generalized or common cr response that is shared among tissues. both tissue-specific and common responses to cr are of potential importance, but common responses to cr across multiple tissue types provide especially attractive targets for further study. swindell analyzed transcriptional responses to cr across tissue types in the laboratory mouse, and found that common responses to cr include activation of tumor suppressor genes, as well as anti-oxidant and anti-inflammatory pathways. for instance, cr was found to increase expression of metallothionein-encoding genes in several tissues, including liver, heart, muscle, hypothalamus, colon and lung. additionally, in multiple tissues, cr increased expression of nfkbia, an inhibitor of nf-κb, suggesting a potential anti-inflammatory mechanism of cr diets. more recently, microarrays have been used to evaluate effects of cr in previously unexamined tissues, including thymus, gonads, mammary gland, spinal cord, cerebellum and striatum. these data allow common responses to cr be refined, should lend greater confidence to lists of cr-regulated genes and pathways, and provide an opportunity for applying improved statistical methods. for instance, previous investigations have focused on how cr affects individual genes, but this may reveal only part of the story. a more comprehensive and informative approach is to identify regions within the genome-wide transcriptional network that are activated or inhibited by cr. historically, the effects of cr have been viewed as being associated with the aging process. this standpoint argues that effects of cr extend beyond any one disease process, but that cr has multiplex effects on a range of physiological systems, ultimately amounting to an inhibitory effect on the progression of aging. the association between cr and aging, however, remains poorly understood, largely because the aging process itself remains poorly defined. while an uncontroversial definition of aging may not be developed anytime soon, it should be possible to add rigor to the concept by generating quantitative models of aging that are operationally useful. in this regard, whole-genome microarray datasets would seem especially valuable, and can be used to generate models that test, quantitatively, the assertion that cr acts to oppose the progression of aging. conclusions generated from previous investigations conflict regarding the association between the effects of cr and aging. on the one hand, an early investigation revealed that age-associated expression patterns in muscle were "either completely or partially prevented by caloric restriction", and this conclusion was supported in subsequent studies. other investigations, however, have yielded different conclusions. for instance, effects of cr were entirely unrelated to those of aging in muscle tissue from rhesus monkeys, and in one aptly designed experiment examining mouse cardiac tissue, only of age-responsive genes were significantly altered by cr. clearly, experimental design and statistical methodology are two important considerations for evaluating this diverse set of results. many studies, for instance, have not evaluated whether the observed overlap between cr and aging effects is larger than expected by chance alone. this statistical evaluation would not be straight-forward in many cases, since experiments involved a shared control treatment that was used to evaluate the effects of both aging and cr. given this design, the effect of cr is not estimated independently of the effect of aging, and some correspondence between cr and aging effects would be expected by chance. the present study integrates results from a large number of microarray experiments that have evaluated the effects of cr or aging in the laboratory mouse. collectively, experiments analyzed in this report have evaluated the effects of cr in mouse tissue types, and the effects of aging in mouse tissue types. for many tissues, effects of cr and/or aging have been evaluated in multiple studies, and results from separate studies of the same tissue are combined within a meta-analytic framework. the overall approach was shaped by three main objectives. first, it was of interest to identify robust expression patterns associated with cr and aging, particularly common responses occurring within multiple mouse tissues. secondly, to move beyond single-gene-centric analyses and to take full advantage of the discernable co-expression relationships among genes, regions of the transcriptional network activated or inhibited by cr or aging were identified. lastly, based on this cross-study comparison of multiple datasets, it was of interest to statistically evaluate whether cr acted to oppose age-associated gene expression patterns. the overlap between the effects of cr and aging was therefore evaluated using several different methods, including overlap between differential expression signatures and similarity between ordered gene lists.  genes regulated by caloric restriction in the laboratory mouse the cr dataset is a compilation of experiments that, taken together, have evaluated the effects of cr in tissue types. for most tissues, data from just 1- experiments was available, but for well-studied tissues, data was available from as many as separate experiments. additional file provides detailed information regarding all experiments incorporated into the analysis, including mouse strains used in each study, sample sizes, array platforms, and whether experimental results from a given study were validated using rt-pcr. differential expression results from independent studies were combined using fisher's p-value combination method. the symbol pu is used throughout to denote a p-value generated from a one-sided hypothesis test for up regulation by cr, while pd denotes a p-value generated from a one-sided hypothesis test for down regulation by cr. the customary p represents a two-sided p-value, generated by testing whether cr or aging altered gene expression in either direction. unless otherwise indicated, p-values cited in the text have been adjusted to account for multiple testing of hypotheses among the genes evaluated in the analysis. the table lists the tissue and cell types for which array data was available and incorporated into the analysis. each tissue or cell type is referenced by a three-letter symbol listed in the second columns. the final two columns list, for each tissue or cell type, the number of independent array experiments that have investigated response to cr or aging. additional file provides details regarding each experiment incorporated into the analysis. there was considerable heterogeneity among tissues in terms of the number of genes for which information was available, and with respect to the percentage of genes significantly influenced by cr. for heart, liver, cochlea, muscle, cortex, hypothalamus and colon, effects of cr had been evaluated, in one or more experiments, for nearly all genes included in the analysis. for other tissues, however, no data had been collected for most known genes and information was available for less than genes. based upon a nominal type i error rate of, it was expected that at least 5% of genes in any one tissue would be significantly altered by cr. this was the case for of tissues, in which cr altered more than 5% of the examined genes, with as many as 54% of genes altered in the case of heart. for cerebellum and striatum, however, cr significantly altered only - 3% of the examined genes, which is a smaller number of significant effects than expected on the basis of chance. the effects of cr were characterized by constructing differential expression signatures associated with each tissue, and the overlap among signatures was examined. differential expression signatures associated with cr in different tissues usually overlapped by less than 40%, but this overlap was often statistically significant. a total of pair-wise comparisons were made among signatures, and in of these cases, the overlap was stronger than expected based on chance alone. there was strong similarity between some segments of the central nervous system, such as spinal cord and cerebellum, as well as hypothalamus and hippocampus, but there was no overall tendency for signatures associated with the central nervous system to cluster together. for instance, effects of cr in the striatum were not significantly similar to those in the cerebellum, spinal cord, hippocampus, cortex and hypothalamus. for liver, heart and muscle, effects of cr had been evaluated in more than four experiments, so detailed tissue-specific analyses were carried out. a surprising outcome of these analyses was that, although numerous genes emerged as significant in at least one experiment for each tissue, relatively few genes exhibited a consistent expression response to cr in multiple experiments. in liver, for instance, after collecting results across experiments, only 10- genes were found to respond to cr in a consistent fashion. these consistent responses provide liver-specific markers of cr response, and include up regulation of lpin and lpin, which were both increased in each of experiments for which data was available. a second interesting aspect of the response to cr in liver was that several expression responses paralleled those resulting from growth hormone/insulin-like growth factor mutations known to increase mouse lifespan. for instance, in liver, cr decreased expression of leukemia inhibitor factor receptor, insulin-like growth factor acid labile subunit, kidney expressed gene, and the cytochrome p gene cyp2f. additionally, cr increased expression of isocitrate dehydrogenase, insulin-like growth factor binding protein and, very consistently, elevated expression of flavin-containing monooxygenase. each of these expression responses was previously identified in association with multiple long-lived dwarf mutants, which have reduced levels of gh and/or igf- in circulation. common responses to cr in multiple tissues included genes identified previously, but also new genes not uncovered in prior analyses. given the large number of experiments considered, the analysis provided substantial statistical power, and overall, % of genes were significantly up regulated by cr across tissues, while % of genes were down regulated by cr across tissues. given this large number of significant results, a more discerning criterion was to stratify genes according to the number of tissues in which a gene was significantly up or down regulated by cr. for instance, less than 1% of genes were up regulated by cr with respect to or more tissue types, or down regulated by cr with respect to or more tissues. the most widespread effect of cr was up regulation of serum/glucocorticoid regulated kinase in tissues, including colon, cochlea, kidney, lung, heart, liver and white adipose tissue, as well as four regions of the central nervous system. additionally, and consistent with previous analyses, cr increased expression of metallothionein in tissues, and down regulated the collagen-specific molecular chaperone serpinh in tissues, including regions within the central nervous system. a previous study has confirmed, by rt-pcr, that serpinh is down regulated by cr in heart, liver and hypothalamus. in figure 2a and 2b, genes are ranked according to the total number of tissues in which cr increased or decreased expression significantly. additional file provides a comprehensive list using an alternative ranking scheme. this alternative approach highlighted some strong cr effects not shown in figure including increased expression of tsc22d zbtb bc sult1a per timp s3- pnpla ppargc1a and cp, as well as decreased expression of ifitm lasp tia 5830428h23rik, hsp90b hist1h2bc, cxcl col6a and dock. additional file also provides analyses of gene ontology terms, kegg pathways, associated micrornas, and chromosomal locations. overall, genes most strongly increased by cr across tissues were associated with fatty acid metabolism, the citrate cycle, ppar signaling, oxidative phophorylation, amino acid degradation and metabolism, circadian rhythm, renal cell carcinoma, fatty acid elongation in mitochondria and the insulin signaling pathway. genes commonly down regulated by cr were associated with focal adhesion, antigen processing and presentation, ecm-receptor interaction, dna replication, mapk signaling, cell communication, vegf signaling and natural killer cell mediated cytotoxicity. genes listed in figures 2a and 2b have not been well-studied in relation to the effect of cr in mouse. for instance, a series of pubmed searches was conducted, where each gene name was searched in conjunction with the phrase "caloric restriction". this search yielded one record associated with sgk and hspb respectively, but no pubmed records were retrieved for any other gene shown in figures 2a and 2b. figure 2c shows differential expression patterns of genes receiving greater research attention, such as sirtuin, for which the above-mentioned search procedure retrieved pubmed records. sirt expression was significantly increased by cr in the cochlea, and significantly decreased in muscle and white adipose tissue, but overall, was not significantly regulated by cr across tissues. of the sirtuin gene family members, cr had the strongest effect on the expression of sirtuin, which was significantly increased by cr in colon, cochlea and cortex, and significantly decreased by cr in heart, liver and muscle. interestingly, the igf transcript was significantly regulated by cr, but there was heterogeneity among tissues in the direction of this effect. in colon, lung, muscle, white adipose tissue and hypothalamus, igf expression was decreased by cr, but the opposite trend was found in cochlea, heart, cortex and mammary gland. overall, across all tissues, there was significant evidence for decreased expression of igf by cr. gene expression modules regulated by caloric restriction the above analyses consider cr-responsiveness of individual genes, taken one at a time, but do not consider the organization among genes based upon their patterns of co-expression. for each gene, therefore, a "nearest-neighbor" co-expression module was generated by finding "neighbor" genes with correlated patterns of expression, based upon a reference set of affymetrix oligonucleotide arrays. nearest-neighbor modules of varying sizes were constructed for each gene included in the analysis. each module was then scored based upon an m statistic designed to quantify responsiveness of modules to cr, and the significance of observed m values was evaluated by simulations in which modules of a given size were formed at random. a total of and significant cr-responsive modules with and genes, respectively, were identified. table lists the most significant cr-regulated co-expression modules of smaller size, and all significant modules of each size are shown in additional file the table lists the most significant cr-regulated co-expression modules containing or genes. the m statistic is an indicator of responsiveness to cr, with larger values suggesting stronger effects of cr on the expression of genes contained in a given module. p-values associated with m statistics were generated by simulation, and reflect the likelihood of obtaining an equal or larger m value by randomly selecting genes to form a module of the same size. the final column provides an indication of the degree to which module members are co-expressed with one another. this value is an element of the interval, and is obtained by calculating all possible pair-wise co-expression relationships among members of the module, and identifying the weakest co-expression relationship between any two module members. smaller minimal |r| values indicate more "diffuse" co-expression modules with weaker co-expression relationships, and larger minimal |r| values indicate "tighter" co-expression modules with stronger co-expression relationships. additional file provides a more detailed description of these and other co-expression modules that emerged as significantly associated with cr. the approach identified 2-gene co-expression modules that were responsive to cr, some of which included functionally related gene pairs, such as {hist2h3c hist2h3c2} and {per per2}. of the 2-gene modules, only {fmo 1200016e24rik} was marginally significant, although others were often components of larger modules that were statistically significant. for instance, significant 3-gene modules included {hist2h3c hist2h3c hist1h2bc} and {per per zbtb16}. the composition of cr-responsive modules often revealed unexpected associations between genes. for instance, the most significant 10-gene module included six genes that encode histone cluster subunits, along with genes encoding an rna binding protein and a peroxisomal beta-oxidation system enzyme. the second-most significant 10-gene module included three period homologue genes, a tsc domain family gene with anti-inflammatory effects, and a gene known to negatively regulate tumorigenesis. it was often the case that genes belonging to the same module exhibited similar responses to cr within the same tissues. the most cr-responsive 40-gene module included genes associated with mrna processing and transcriptional regulation. the "center" of this module was the zinc finger protein, with the remaining genes being nearest neighbors of zfp genes belonging to this module were highly responsive to cr, but the direction of response differed among tissue types. in cochlea, hypothalamus and colon, nearly all member genes were significantly up regulated by cr. however, in hippocampus, mammary gland and liver, genes in this module were usually down regulated by cr. gene ontology analysis revealed that member genes were involved in transcriptional processes, particularly splicing, metabolism, processing and biosynthesis of rna. along these lines, a disproportionately large number of module genes were associated with the nucleus, chromosome and spliceosome. genes regulated by aging in the laboratory mouse the effects of aging on gene expression had been evaluated in experiments and a total of tissue types. on average, aging altered % of genes, with the strongest effects found in the cortex. among those tissues with the strongest age effects, more genes increased with age than decreased with age. the weakest effects of aging were associated with cochlea and striatum, in which aging altered only 2-4% of genes, which is fewer than expected by chance at the nominal type i error rate of. for some tissues, aging had a larger impact on gene expression than observed in prior studies. in cerebellum, for example, at a type i error rate of, aging altered the expression of of genes. this percentage is considerably higher than that observed in the agemap project, in which aging altered the expression of only of genes. this apparent difference in effect size may reflect increased statistical power associated with a meta-analytic approach, or differences in the relative amount of noise influencing expression measurements generated using different array platforms. differential expression signatures were constructed to characterize the effects of aging in each tissue, and comparisons of these signatures suggested that, relative to cr, effects of aging were more correspondent among different tissue types. for instance, among pairwise comparisons between signatures, significant overlap was found in cases, and a cluster analysis indicated that of the tissues formed a single group with similar patterns of differential expression. a side-by-side comparison of results from multiple experiments examining the same tissue highlighted robust tissue-specific markers of aging. for instance, in each of seven experiments evaluating the effects of aging in liver, expression of immunoglobulin joining chain increased, while expression of kidney expressed gene decreased. the effects of aging in cardiac tissue had been examined in an especially large number of experiments. comparison of these experiments indicated that aging increased cardiac expression of phenylalanine hydroxylase in all experiments for which data was available. likewise, aging increased cardiac expression of complement component 4b in experiments for which data was available. these results confirm and extend observations from a recent expression profiling study of age-associated gene expression patterns in cardiac tissue from multiple mouse strains, which identified both pah and c4b as robust biomarkers of cardiac aging, and confirmed these age-related expression patterns using rt-pcr. in muscle, an age-related increase of heat shock protein expression was observed in four separate experiments, as well as an age-associated decrease of col1a expression. genes with age-related expression patterns in multiple tissue types were identified using fisher's method. this revealed that genes were significantly up regulated with age across all tissues, while genes were significantly down regulated with age across all tissues. there were strong functional differences between genes that increased with age in multiple tissues and genes that decreased with age in multiple tissues. genes up regulated with aging were frequently associated with immune response, and over represented gene ontology terms included antigen processing and presentation, lymphocyte mediated immunity, immunoglobulin mediated immune response, humoral immune response, lysosome organization/biogenesis, and regulation of adaptive immune response. however, this was not generally true of genes decreased with age. genes down regulated with aging in multiple tissues were more commonly associated with metabolic processes, chromosome organization/biogenesis, cell division and cycle, mrna processing and splicing, and protein folding. the age-related expression patterns of identified genes were more widespread than those of some genes previously associated mammalian aging and cellular senescence. based on previous investigations, for instance, it was expected that aging would increase expression of cyclin-dependent kinase inhibitor 2a and ets homologous factor, and that aging would decrease expression of senescence marker protein. for these genes, however, significant effects of aging were observed only in a few tissues, and often, the effect was inconsistent with prior expectations. for instance, cdkn2a/p16ink4a is a regulator of the cell cycle, and it has been proposed that expression of cdkn2a is a biomarker of aging in multiple mouse tissues. but there was little indication that cdkn2a expression increased with age in most tissues, and overall, the effect of age on cdkn2a expression was nonsignificant among all tissue types. the expression of cdkn2a increased significantly only in cardiac tissue with age, and in cortex, cdkn2a expression decreased with age. gene expression modules regulated by aging nearest-neighbor co-expression modules ranging in size from to genes were formed and the collective response of each module to aging across tissues was evaluated. the results demonstrate strong modularity of age-related expression patterns, with certain sets of co-expressed genes exhibiting correspondent differential expression patterns with age. a total of and significant age-responsive modules with and genes, respectively, were identified. the most significant 3-gene module included two proteasome subunit genes, along with the mhc antigen h2-k. the three genes contained in this module exhibited highly correspondent patterns of differential expression, with decreased expression occurring in spleen with age, and an age-related up regulation of expression across tissues. a similar pattern was present with respect to other 3-gene co-expression modules, such as {tyrobp, mpeg ctss} and {sfi pisd, 4933439c20rik}, and with significant co-expression modules of larger size. in each of these cases, genes belonging to the same module exhibited similar differential expression patterns in the same tissues, indicating that patterns of co-expression had considerable explanatory power in terms of age-related transcriptional effects. the table lists the most significant age-regulated co-expression modules containing or genes. values in this table are interpreted in the same way as those listed in table except here the m statistic reflects responsiveness of a module to aging. additional file provides a more detailed description of these and other co-expression modules that emerged as significantly associated with aging. the most significant 40-gene module was centered around cathepsin s. the rest of the module consisted of the nearest neighbors of ctss, which included genes encoding complement components and antigens. strikingly, nearly every gene in this module exhibited an age-related expression increase in liver, kidney, cerebellum, cortex, eye, aorta, lung, heart, hippocampus and whole brain. among genes belonging to the module, a gene ontology analysis revealed numerous over-represented biological processes, including activation of immune response, positive regulation of b cell mediated immunity, phagocytosis, inflammatory response and neutrophil chemotaxis. half of module genes were associated with membrane, particularly the plasma membrane. the second-most significant 40-gene module was not associated with inflammation or immune response, but interestingly, included genes with roles in mrna processing, rna transport and rna localization. most of the genes in this module were associated with the nucleus and several were among the most age-responsive genes identified in the analysis, such as gspt ints syncrip and creb. in contrast to the module shown in figure most member genes were predominantly decreased with aging, particularly in aorta, gametes and cardiac tissue. relationship between caloric restriction and aging cr is thought to inhibit the progression of aging and to oppose age-related gene expression patterns. it was therefore expected that a negative association would exist between transcriptional effects of cr and aging, and several approaches were taken to evaluate whether such an association was present. first, genes most strongly regulated by cr or aging across multiple tissue types were considered. there was no general tendency for genes increased by cr across tissues to be decreased by aging, and likewise, genes decreased by cr across tissues were not disproportionately increased by aging. individual genes can be identified that either support or contradict the expected pattern. for instance, cr decreased expression of igh- and marcks, and this effect does indeed oppose increased expression of these genes with age across tissues. on the other hand, cr decreased expression of col3a and this effect would seem to mimic, rather than oppose, age-related decreases in col3a expression. the association between cr and aging was next examined at the global scale, among all genes, and also with respect to each of the four most well-studied tissue types. in liver, there was a slight, positive association between the effects of cr and aging. this association was significant, although given the large number of genes involved in the comparison, this significance test was not too informative. in the heart, muscle and central nervous system, the expected negative association between cr and aging did emerge, albeit weakly, with the estimated correlation coefficient less than or equal to - in each case. the strongest association was found in heart, in which age-related expression patterns were weakly opposed by cr. in muscle and central nervous system, the association between cr and aging was again weak, and non-significant in the case of muscle, despite the large number of genes upon which the association was based. with respect to central nervous system, a large fraction of genes were both increased by cr and decreased with age, although very few genes were decreased by cr and increased with age. the global effects of cr and aging were then compared by evaluating the association between differential expression signatures. in this analysis, it was expected that genes significantly increased by cr would tend to be decreased significantly with age, and conversely, that genes significantly decreased by cr would increase significantly with age. associations between differential expression patterns were found, and were moderately strong in liver, weaker in heart, weakest in muscle, and strongest in the central nervous system. the pattern of association, however, varied among tissues, and was often not consistent with the expectation. in liver and central nervous system, cr did oppose age-related expression patterns, but these trends were complicated by the fact that, in each tissue type, there was a significant tendency for cr to actually reinforce the effects of aging, which is in complete contrast to the expectation stated above. in muscle, the relationship between cr and age-related differential expression patterns was effectively random, with little or no evidence of any association. the strongest evidence for the expected cr-aging relationship was again found with respect to heart. in cardiac tissue, there was a significant abundance of genes increased by cr and decreased by age, as well as an abundance of genes decreased by cr and increased by age. these conclusions were reinforced by results from an alternative analytical method, based upon correspondence between ranked gene lists, which also showed conflicting associations between cr and aging in liver, muscle and central nervous system, with a stronger and more clear-cut relationship in heart. the above results suggest that, among all genes, the transcriptional association between effects of cr and aging is limited, with a stronger relationship in heart relative to other tissue types. one possibility is that the transcriptional association between cr and aging is stronger among genes associated with some biological processes, and weaker among certain genes associated with other processes. the cr-aging association was thus examined with respect to gene ontology biological process terms that were associated with genes increased or decreased by aging across all mouse tissues. this analysis revealed definite differences among gene categorizations with respect to the transcriptional relationship between cr and aging. in particular, among genes involved in immune response, innate immune response and immune system processes, the effects of cr were negatively associated with those of aging. these results are in agreement with the rt-pcr investigation of park et al., which found that, in heart and cerebellum, cr opposed the effects of aging for certain genes associated with immunity and defense response. figures and also show that cr opposes age-associated expression patterns among other classes of genes as well, such as groups of genes with roles in apoptosis, protein amino acid dephosphorylation, lipid transport, chemotaxis, biopolymer metabolic process and inflammation. at the same time, for some processes, the opposite pattern of association was found, and for many gene groups, the association between cr and aging was non-significant and weakly inverse, in agreement with analyses based upon global transcriptional patterns among all genes. discussion caloric restriction and aging have been found to influence the expression of numerous genes in a variety of mouse tissues, but there has been considerable heterogeneity in results generated from different laboratories. this study has identified genes and gene expression modules that, based upon currently available data, are most robustly associated with cr and/or aging in the laboratory mouse. these transcriptional effects provide a well-supported basis for the generation of new research hypotheses, which can serve as starting points for experimental studies into mechanisms of cr and aging. the identified expression patterns influence a wide range of tissue and cell types, and thus represent the most systemic transcriptional effects of cr and aging, which may be associated with pathways required for positive effects of cr on survivorship, or linked to mechanisms that drive increased disease and mortality risk with age. these transcriptional effects were often modular, with groups of co-expressed genes responding to cr or aging in coordination across multiple tissues. for instance, the analysis uncovered a cr-responsive group of co-expressed genes encoding histone clusters, as well as a large module that contains genes involved in mrna processing. age-related expression patterns affecting multiple tissues were, to a considerable degree, explained by the modularity of the transcriptome. this was best demonstrated by a large module containing inflammation and immune response genes, nearly all of which increased expression with age in ten mouse tissues. it was expected that effects of cr and aging would be associated, with cr acting of oppose age-related changes in gene expression. this was the case among genes associated with certain biological processes, but at the genome-wide level among all genes, the transcriptional association between cr and aging was limited. common responses to cr represent systemic effects of low-calorie diets that influence multiple organ systems. a previous study found that cr increased expression of genes encoding the anti-oxidant metallothioneins, the nf-κb inhibitor iκbα, as well as the tumor suppressor p. the present investigation has revealed that these effects are even more widespread than previously thought, each occurring in at least seven mouse tissues. additionally, widespread transcriptional effects of cr were identified that could influence the predisposition of mice to develop cancers, which is thought to be a main factor underlying pro-longevity effects of the cr diet. these expression patterns were associated with a broad range of tissue types, and include increased expression of thioredoxin interacting protein, zinc finger and btb domain containing and tissue inhibitor of metalloproteinase. furthermore, cr reduced expression of thymosin, secreted acidic cysteine rich glycoprotein and heat shock protein, and each of these effects could also inhibit transformation or cellular proliferation and migration. increased expression of txnip and zbtb expression by cr could reflect an anti-proliferative effect of low calorie diets on a common pathway, since txnip encodes a protein known to induce expression of zbtb, and both genes encode proteins that can promote apoptosis or limit the invasive capacity of tumor cells. widespread effects of cr on txnip expression are especially noteworthy, since txnip-deficient mice exhibit several abnormal responses to fasting, and because txnip can regulate activation of the pten tumor suppressor protein. like txnip and zbtb timp has tumor-suppressive properties, and its expression limits tumor invasiveness and has been correlated with survivorship in cancer patients. additionally, expression of timp negatively controls tumor necrosis factor alpha levels, leading to reduced nf-κb activation and inflammation. taken together, these results begin to outline a generalized response of the mammalian cell to cr. this response involves increased expression of genes with tumor-suppressive properties, decreased expression of genes associated with cellular transformation and proliferation, as well as the induction of genes with anti-inflammatory or anti-oxidative effects. the beneficial effects of cr on survivorship may be related to loss of insulin-like growth factor signaling. this type of mechanism has been shown to increase lifespan and decrease cancer incidence in mutant dwarf mice, in which serum igf- levels are greatly diminished, or in which the localized bioavailability of igf- within tissues is reduced. similarly, it was recently shown that mouse lifespan can be increased by late-life administration of rapamycin, which inhibits mtor, an element downstream of the igf-1/pi3-k pathway. a major question in aging biology, therefore, is whether pro-survival effects of the cr diet are dependent upon inhibition of igf- signals in a manner that, to some degree, parallels mechanisms underlying increased lifespan in long-lived dwarf mutants and/or mice treated with rapamycin. along these lines, it is interesting to note that the most widespread effect of cr to emerge from this analysis was increased expression of serum/glucocorticoid regulated kinase, which occurred in of the mouse tissues examined. the sgk kinase is activated by igf-1/phosphatidylinositol- kinase signals, and data from several microarray analyses indicate that expression of sgk is up regulated by mutations and treatments that inhibit the igf-1/pi3-k pathway. another widespread effect of cr was increased expression of phosphatase and tensin homolog, which is a tumor suppressor that inhibits igf- signals by preventing activation of the pi3k/akt signaling pathway. these effects of cr on sgk and pten expression may reflect attenuation of igf- signals across a number of tissue types, possibly in a manner that, to some degree, mimics the influence of dwarf longevity mutations or rapamycin treatment. such expression patterns thus raise new questions for further work, which can contribute to a developing model of interactions between cr and the igf- pathway. the metabolism of mrna is a fundamental process that underlies virtually all cellular activities, and perturbation of mrna processing can have important effects on gene regulation, homeostasis and cell cycle control. this study has found that, in multiple tissues, cr influences the expression of genes involved in the splicing and processing of messenger rna. these genes are co-expressed and organized into a single large module that includes splicing factors, pre-mrna processing factors, pre-mrna cleavage factor i, an rna/dna binding protein associated with splicing regulation and mrna stability, a co-activator of pre-mrna splicing, and a yeast homolog of luc7p that associates with the spliceosomal subunit u snrnp. the possibility that nutrient signals, and in particular cr, can have important effects on mrna processing is consistent with previous investigations. for example, glucose-6-phosphate dehydrogenase protein levels and activity are positively regulated by intake of carbohydrates, but without any alteration in transcription rate of the associated g6pdx gene. carbohydrate intake instead appears to increase the efficiency of splicing events within the nucleus, leading to increased abundance of mature mrna transcript and g6pd protein. nutritional regulation of mrna processing has also been reported for thyroid hormone responsive spot homolog, as well as insulin-like growth factor i. these examples suggest that nutritional effects on mrna processing are not altogether unusual, and that some widespread effects of cr could result from an influence on the multi-step process by which mrnas proceed from transcription to translation. this effect of cr may be consequential for disease processes, since proteins that regulate splicing events can contribute to cell growth and the proliferation of cancer cells. it is interesting to note that expression of genes associated with mrna processing, rna transport or rna localization are decreased by aging in many tissues. this consistent effect of aging contrasted with that of cr, which variably influenced the expression of functionally similar genes, promoting increased expression in some tissues but decreased expression in others. there is much evidence to suggest that certain mechanisms of aging are conserved across species, with similar pathways regulating life span and the progression of aging in worms, flies and mammals. this notion suggests that there may exist common features of aging that are evolutionarily conserved, which are reflected by age-related patterns of gene expression that are shared among mammalian cell types within diverse tissues. the present study has identified robust age-related expression patterns that appear to be characteristic of cells within many or most mammalian tissues. it is not expected that all such age-related expression patterns will be associated with deleterious pathologies, since some modifications of gene expression with age are likely to reflect favorable compensatory mechanisms. nevertheless, some age-regulated genes identified in this analysis are known to modulate inflammatory processes and contribute to cellular proliferation. for instance, two widespread effects of aging were up regulation of granulin and secreted phosphoprotein. increased expression of grn was in fact the most widespread effect of aging identified, occurring in nearly all of the tissues examined. grn encodes the granulin protein, which can inhibit tnf-induced inflammatory processes and promote vascularization necessary for wound and tissue repair, but can also give rise to cleavage products that stimulate cytokine secretion leading to the exacerbation of inflammatory processes. additionally, granulin has been identified as potent growth factor that stimulates aggressive cell proliferation, and in at least some cell lines, this mitogenic effect depends upon the expression level of grn. increased expression of spp with age was identified in of mouse tissue types, with the strongest increase observed in the cerebellum. spp encodes osteopontin, a protein involved in localized recruitment of macrophages and in the production of cytokines within certain immune cells, which can contribute to a state of chronic inflammation. osteopontin is also a regulator of tumor progression and its abundance within tumors has emerged as an impressive biomarker for numerous types of cancer. expression of osteopontin within tissues and in circulation has also been associated with a wide range of other conditions, including obesity, diabetes and cardiovascular disease. an important component of future work will be rt-pcr investigation of grn and spp expression patterns with age, both across mouse tissues and among different mouse strains. a widespread effect of aging was increased expression of inflammation and immune response genes, which involved class i mhc genes, immunoglobulin chain components, and a large module of co-expressed genes known to participate in inflammation and immune processes. these observations are consistent with those from previous investigations that have evaluated genome-wide expression patterns in aging mice, and thus provide further evidence that heightened expression of inflammation and immune response genes is a systemic feature of aging that influences a broad range of organ systems. indeed, exceptions to this pattern may represent the most interesting cases. for example, only of tissues did not show an age-related increase in the expression of either h2-d or h2-k. it is likely that, at least in part, increased expression of inflammation and immune response genes reflects age-related increases in immune cell populations within aging tissues. in liver, a recent histological analysis demonstrated that several immune cell types infiltrate perivascular regions with aging to establish proliferating cell clusters. this type of mechanism is likely to explain, for example, the increased expression of igk-v and igh- described in this analysis, which could be driven by b-cell lymphocyte infiltration of aging tissues. such immune cell infiltration could be driven by chronic inflammation, possibly in a manner that parallels tertiary lymphoid neogenesis, and it is further possible that such inflammation is exacerbated by an age-associated accumulation of fat cells. an important direction for future studies will be to evaluate mechanisms by which immune cells, such as lymphocytes, are drawn to aging tissues and whether such mechanisms are comparable in various organ systems. an inspection of current data, for example, revealed that the b-cell attractants ccl and cxcl increase with age in some tissues. in particular, expression of ccl increased with age in tissues, while an age-related increase in cxcl expression was observed in tissues. the identification of robust age-related gene expression patterns may lead to the development of useful "biomarkers", which could provide tools for tracking the progression of the aging in experimental studies. development of such indicators is requisite for making objective statements regarding the effects of interventions, or of particular mutations, on the deceleration of aging. the age-related expression patterns highlighted in this analysis represent systemic or near-systemic effects, which have repeatedly emerged in multiple experimental settings. such transcriptional effects may thus be suitable for further evaluation as aging biomarkers, or as components of a quantitative model whose output serves as an aging biomarker. the current data, for example, suggest that increased expression of immune response genes is a reliable feature of normal aging, perhaps representing a widespread outcome of aging that compares with the prominent but localized effects of aging in the thymus. localized expression of such genes may be either a cause of consequence of multiple age-related pathologies, particularly if increased expression arises from sustained inflammation in certain tissues. consequently, such age-related effects may provide informative endpoints that, in combination with other indicators of aging and healthspan, will eventually lead to surrogates for lifespan in experimental studies of anti-aging interventions using the laboratory mouse. in this sense, other aspects of the aging immune system have also proven informative, such as t-cell subsets, which have been found to predict mouse lifespan within experimental cohorts. along these lines, extended lifespan of mutant mice lacking the pregnancy-associated plasma protein a gene is reflected by a delayed rate of thymic atrophy. a conceptual association between cr and aging has been maintained for decades, and numerous investigations have been designed around the expectation that cr interferes with the progression of aging. the alternative view is that cr and aging are in fact disparate phenomena, with cr having many favorable phenotypic effects, but not having effects broad enough to be characterized in terms of the actual process of "aging". gene expression data is not, by itself, sufficient to discriminate between these two views, and it is clear that any sufficient definition of aging must include measurements from multiple physiological domains. moreover, it is certainly possible that deleterious aspects of aging are prevented by cr in ways that are not reflected by gene expression patterns, and many age-associated expression patterns may indeed reflect favorable compensatory mechanisms rather than pathological processes. nevertheless, gene expression data reflect the behavior of numerous biochemical pathways, and such data are also well-suited to quantitative investigation. genome-wide expression data can, in this regard, provide at least some insight into the conceptual links between cr and aging. there are elements of this analysis that support an association between cr and aging, and elements that do not. on the one hand, individual genes can be identified for which the effects of cr oppose those of aging in multiple tissues, and this also appears to be true among genes associated with certain types of biological processes. at a global scale, however, among all genes, this association is fairly weak. this is most clearly shown by the scatterplots in figure which indicate that genome-wide correlations between cr and aging effects within particular tissues do not exceed in magnitude, and this basic conclusion is supported by analyses shown in figures and among different tissues, the genome-wide association was stronger in cardiac tissue, but weaker in liver, muscle and central nervous system. taken together, therefore, these analyses show that cr counters certain types of age-associated expression patterns, but not others, with a fairly weak global association among all genes, and some degree of variability in the strength of the association among mouse tissue types.  cr and aging have widespread biochemical effects and systems-oriented approaches are needed to develop comprehensive in silico models that link these effects conceptually. microarray analyses provide a data-driven approach for judging which pathways serve as "hubs" that connect the diverse processes modulated by cr and aging, and this can lead to the construction of large gene interaction networks, with scope substantially greater than those generated by standard methods. this study has identified sub-networks, embedded within the genome-wide transcriptional network, that are responsive to either dietary intake or aging across a variety of mouse tissues. cr influenced small co-expression modules containing genes that encode histone clusters, period homologues, as well as a large module containing genes involved in the splicing and processing of mrna. aging increased expression of granulin and secreted phosphoprotein in more than tissue types, and led to elevated expression of genes and modules associated with inflammation and immune response. in general, many age-associated differential expression patterns were explained by the underlying modularity of the transcriptome. it was expected that the transcriptional effects of cr and aging would be negatively associated, and this was true for genes associated with certain biological processes. at the genome-wide level, however, cr and aging were found to have weak association, which was stronger in heart relative to liver, muscle and central nervous system. 
the effects of aging were, to a considerable degree, similar among groups of co-expressed genes. with respect to gene subsets associated with certain biological processes, cr opposed age-associated expression patterns. previous analyses of microarray data have identified gene expression responses to cr that are shared among multiple mouse tissues, including the activation of anti-oxidant, tumor suppressor and anti-inflammatory pathways. age-related gene expression patterns characteristic of most mouse tissues were identified, including up regulation of granulin and secreted phosphoprotein. expression patterns identified in this analysis characterize a generalized response of mammalian cells to cr and/or aging. the transcriptional association between cr and aging varied at different levels of analysis. furthermore, cr is thought to oppose age-associated gene expression patterns, but detailed statistical investigations of this hypothesis have not been carried out. the association between cr and aging warrants further study, but most evidence indicates that cr does not induce a genome-wide "reversal" of age-associated gene expression patterns. systemic effects of cr and aging were identified by examining transcriptional responses to cr in mouse tissue types, as well as responses to aging in tissues. these analyses have provided useful research directions, but have been restricted to a limited number of tissues, and have focused on individual genes, rather than whole-genome transcriptional networks.
 caloric restriction counters deleterious effects of aging and, for most mouse genotypes, increases mean and maximum lifespan. previous analyses of microarray data have identified gene expression responses to cr that are shared among multiple mouse tissues, including the activation of anti-oxidant, tumor suppressor and anti-inflammatory pathways. these analyses have provided useful research directions, but have been restricted to a limited number of tissues, and have focused on individual genes, rather than whole-genome transcriptional networks. furthermore, cr is thought to oppose age-associated gene expression patterns, but detailed statistical investigations of this hypothesis have not been carried out.  systemic effects of cr and aging were identified by examining transcriptional responses to cr in mouse tissue types, as well as responses to aging in tissues. cr broadly induced the expression of genes known to inhibit oxidative stress, inflammation and tumorigenesis. additionally, a network-based investigation revealed that cr regulates a large co-expression module containing genes associated with the metabolism and splicing of mrna. the effects of aging were, to a considerable degree, similar among groups of co-expressed genes. age-related gene expression patterns characteristic of most mouse tissues were identified, including up regulation of granulin and secreted phosphoprotein. the transcriptional association between cr and aging varied at different levels of analysis. with respect to gene subsets associated with certain biological processes, cr opposed age-associated expression patterns. however, among all genes, global transcriptional effects of cr were only weakly related to those of aging.  the study of aging, and of interventions thought to combat aging, has much to gain from data-driven and unbiased genomic investigations. expression patterns identified in this analysis characterize a generalized response of mammalian cells to cr and/or aging. these patterns may be of importance in determining effects of cr on overall lifespan, or as factors that underlie age-related disease. the association between cr and aging warrants further study, but most evidence indicates that cr does not induce a genome-wide "reversal" of age-associated gene expression patterns. 