Unknown,Transcriptomics,Genomics,Proteomics

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Calorie Restriction Feminizes Liver Gene Expression and Alters Key Regulators of Conserved Aging Regulatory Pathways

ABSTRACT: Background: Calorie restriction (CR) is the only intervention known to extend lifespan in a wide range of organisms, including mammals. However, the mechanisms by which it regulates mammalian aging remain largely unknown and the involvement of the TOR and Sirtuin pathways (which regulate aging in lower organisms) remain controversial. Femaleness is a second phenotype generally associated with longevity but the relationship between sex-biased and CR-induced gene expression remains undetermined. Methodology/Principal Findings: We generated microarray gene expression data from livers of male mice fed high calorie or CR diets, and we find that CR significantly changes the expression of over 3,000 genes, many between 10- and 50-fold. We compare our data to the GenAge database of known aging-related genes and to prior microarray expression data of genes expressed differently between male and female mice. CR generally feminizes gene expression and many of the most significantly changed individual genes are involved in aging, hormone signaling, and p53-associated regulation of the cell cycle and apoptosis. Among the genes showing the largest and most statistically significant CR-induced expression differences are Ddit4, a key regulator of the TOR pathway, and Nnmt, a regulator of lifespan linked to the Sirtuin pathway. Using Western analyses, we confirmed post-translational inhibition of the TOR pathway. Conclusions: Our data show that CR induces widespread gene expression changes and acts through highly evolutionarily conserved pathways, from microorganisms to mammals, and that its life-extension effects might arise partly from a shift toward a gene expression profile more typical of the longer-lived female sex. Keywords: Two-class gene expression comparison. Calorie restriction (CR) versus HIGH CALORIE feeding. Design of the experiment is a two-class paired design in which the two classes are HIGH CALORIE and CALORIE RESTRICTION (CR) dietary regimens fed to mice, resulting in 15 HIGH CALORIE microarrays and 8 CR microarrays; 23 total microarrays. Four HIGH CALORIE and 2 CR microarrays were produced using pools of 3 RNA samples for each microarray (6 pools of 3, plus 17 individual samples = 35 individual mouse liver samples, 23 microarrays). Total liver RNA was labeled directly. Reference RNA: Stratagene Universal Mouse Reference RNA.

ORGANISM(S): Mus musculus

SUBMITTER: preston estep

PROVIDER: E-GEOD-11244 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Caloric restriction (CR) without malnutrition is one of the most consistent strategies for increasing mean and maximal lifespan and delaying the onset of age-associated diseases. Stress resistance is a common trait of many long-lived mutants and life-extending interventions, including CR. Indeed, better protection against heat shock and other genotoxic insults have helped explain the pro-survival properties of CR. In this study, both in vitro and in vivo responses to heat shock were investigated using two different models of CR. Murine B16F10 melanoma cells treated with serum from CR-fed rats showed lower proliferation, increased tolerance to heat shock and enhanced HSP-70 expression, compared to serum from ad libitum-fed animals. Similar effects were observed in B16F10 cells implanted subcutaneously in male C57BL/6 mice subjected to CR. Microarray analysis identified a number of genes and pathways whose expression profile were similar in both models. These results suggest that the use of an in vitro model could be a good alternative to study the mechanisms by which CR exerts its anti-tumorigenic effects. KEY WORDS: caloric restriction, heat shock, stress response, tumorigenesis, aging In Vivo: Male C57BL/6 mice (3 month old) were single-housed in duplex caging in a room maintained at a constant temperature (20-22 °C) and humidity (30-70%) in a light:dark 12:12-h schedule, according to established animal protocols and NIH guidelines. They were fed either a standard purified mouse diet (NIH-31) ad libitum (AL; n=10) or maintained on a 40% calorie restriction regimen (CR; n=10) for the six week study. B16F10 melanoma cells (ATCC® CRL-6475™) were purchased from American Type Culture Collection (Manassas, VA); they were cultured in Dulbecco's Modified Essential Medium (DMEM) supplemented with 10% fetal bovine serum and penicillin/streptomycin (Gibco, Gaithersburg, MD) under standard cell culture conditions. One month into the study, 5 mice from each diet group were injected with 1x106 B16F10 melanoma cells in the periscapular region. Fourteen days later, animals were euthanized and tumors were excised and frozen for RNA isolation. In Vitro: B16F10 melanoma cells were incubated in media with 10% serum from either AL- or CR-fed rats, serum was obtained from overnight fasted, anesthetized 6-month-old male Fisher 344 rats. Cells were grown for 96 hours before being harvested, washed and collected for RNA isolation. The RNA was extracted using the RNeasy Mini Kit from Qiagen using standard protocols and labeled with Biotin using the standard Illumina protocol. Samples were hybed overnight to Illumina's Sentrix MouseRef-8 v1.1 Expression BeadChips. Arrays were washed, stained with Cy-3 and scanned at 0.8 micron resolution in a 500 GX Illumina Bead Array Reader.

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Calorie Restriction Feminizes Liver Gene Expression and Alters Key Regulators of Conserved Aging Regulatory Pathways

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