Patent Publication Number: US-2012040855-A1

Title: Tissue-specific aging biomarkers

Description:
CROSS REFERENCE TO RELATED APPLICATIONS 
     This application is a national stage application under 35 U.S.C. §371 of PCT/US2010/000721 filed Mar. 10, 2010, which claims priority to U.S. Provisional Application Ser. No. 61/209854 filed Mar. 11, 2009, the disclosures of which are incorporated herein by this reference. 
    
    
     BACKGROUND OF THE INVENTION 
     1. Field of the Invention 
     The invention relates generally to the field of nutritional support of health and longevity in animals. In particular, the invention provides methods of developing tissue-specific universal biomarkers of aging in animals, as well as sets of robust biomarkers identified by those methods, and use of the tissue-specific universal aging biomarkers to identify nutrients and other functional ingredients or agents having anti-aging properties in animals. 
     2. Description of Related Art 
     Restriction of caloric intake well below ad libitum levels has been shown to increase lifespan, reduce or delay the onset of many age-related conditions, improve stress resistance and decelerate functional decline in numerous animal species, including mammals such as rodents and primates (See, e.g., D. K. Ingram et al. (2004) Ann. N.Y. Acad. Sci. 1019: 412-423). Indeed, clinical trials have been initiated to evaluate the longevity-promoting effect of caloric restriction (CR) in humans. But in humans and animals alike, it seems unlikely that CR is a viable strategy for increasing longevity in most individuals, due to the degree and length of restriction required. For this reason, research has focused on the identification of substances, e.g., pharmaceutical agents, nutritional substances and the like, capable of mimicking the effect of CR without a substantive change in dietary intake. 
     Efforts have been directed toward identifying agents that can mimic one or more of the physiological or biochemical effects of CR (See, e.g., Ingram et al., 2004, supra), or that can mimic the gene expression profile associated with CR in certain tissues and organs (e.g., Spindler, U.S. Pat. No. 6,406,853; U.S. Patent Pub. 2003/0124540). In connection with the latter, methods to analyze genes associated with CR and to screen for CR mimetics based on gene expression profiling have been disclosed (Spindler et al., U.S. Patent Pubs. 2004/0180003, 2004/0191775 and 2005/0013776; Pan et al., U.S. Patent Pub. 2007/0231371). 
     Despite the availability of the approaches summarized above, there remains a need for more robust, faster and less costly methods to screen for agents that can retard or reverse the aging process, to promote healthy aging and increase longevity. The present invention satisfies this need. 
     SUMMARY OF THE INVENTION 
     It is, therefore, an object of the present invention to provide methods of identifying robust and universally applicable gene expression markers of aging in selected tissues, and to provide sets of robust and universally applicable gene expression markers identified by those methods. 
     It is another object of the invention to provide to provide one or more genes or gene segments that are differentially expressed in selected tissues of old subjects as compared with young subjects. 
     It is a further object of the invention to provide a combination comprising a plurality of polynucleotides that are differentially expressed in selected tissues of old subjects as compared with young subjects. 
     It is another object of the invention to provide compositions of two or more polynucleotide or polypeptide probes suitable for detecting the expression of genes differentially expressed in selected tissues of old subjects as compared with young subjects, and devices such as substrate arrays containing the probes. 
     It is a further object of the invention to provide methods for detecting differential expression of one or more genes differentially expressed in selected tissues of old subjects, as compared with young subjects or a standard reference. 
     It is another object of the invention to provide a method for measuring the effect of a test substance on the expression profile of one or more genes differentially expressed in selected tissues of old subjects, as compared with young subjects or a standard reference. 
     One or more of these other objects are achieved using novel methods of identifying tissue-specific aging biomarkers, and novel combinations of polynucleotides or polypeptides representing genes and gene segments that are differentially expressed in selected tissues of old subjects as compared with young subjects. The polynucleotides are used to produce compositions, probes, devices based on the probes, and methods for determining the status of polynucleotides differentially expressed in selected tissues of old subjects, as compared with young subjects or a standard reference, which are useful for achieving the above-identified objects, e.g., prognosing and diagnosing age-related conditions in selected tissues and for screening substances to determine if they are likely to have an anti-aging effect in a particular tissue. Various kits comprising combinations of probes, devices utilizing the probes, and substances are also provided, as are various computer programs for manipulating information, and communication media for communicating information pertaining to the differentially expressed genes and methods of their use. 
     Other and further objects, features, and advantages of the invention will be readily apparent to those skilled in the art. 
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     Definitions 
     As used throughout, ranges are used herein as shorthand, so as to avoid having to set out at length and describe each and every value within the range. Any appropriate value within the range can be selected, where appropriate, as the upper value, lower value, or the terminus of the range. It is understood that any and all whole or partial integers between any ranges or intervals set forth herein are included herein. 
     As used herein and in the appended claims, the singular form of a word includes the plural, and vice versa, unless the context clearly dictates otherwise. Thus, the references “a,” “an,” and “the” are generally inclusive of the plurals of the respective terms. For example, reference to “an animal”, “a method”, or “a substance” includes a plurality of such “animals”, “methods”, or “substances”. Similarly, the words “comprise”, “comprises”, and “comprising” are to be interpreted inclusively rather than exclusively. 
     The term “animal” means a human or other animal, including avian, bovine, canine, equine, feline, hicrine, murine, ovine, and porcine animals. When the term is used in the context of comparing test subjects, the animals that are compared are animals of the same species and possibly of the same race or breed. A “companion animal” is any domesticated animal, and includes, without limitation, cats, dogs, rabbits, guinea pigs, ferrets, hamsters, mice, gerbils, horses, cows, goats, sheep, donkeys, pigs, and the like. Preferably, the animal is a human or a companion animal such as a canine or feline. 
     The term “antibody” means any immunoglobulin that binds to a specific antigen, including IgG, IgM, IgA, IgD, and IgE antibodies. The term includes polyclonal, monoclonal, monovalent, humanized, heteroconjugate, antibody compositions with polyepitopic specificity, chimeric, bispecific antibodies, diabodies, single-chain antibodies, and antibody fragments such as Fab, Fab′, F(ab′) 2 , and Fv, or other antigen-binding fragments. 
     The term “array” means an ordered arrangement of at least two probes on a substrate. At least one of the probes is a control or standard and at least one of the probes is a diagnostic probe. The arrangement of from about two to about 40,000 probes on a substrate assures that the size and signal intensity of each labeled complex formed between a probe and a sample polynucleotide or polypeptide is individually distinguishable. 
     The term “binding complex” refers to a complex formed when a polypeptide in a sample specifically binds (as defined herein) to a binding partner, such as an antibody or functional fragment thereof. 
     The term “calorie restriction” or “caloric restriction” refers to any diet regimen low in calories without undernutrition. In general, the limitation is of total calories derived from of carbohydrates, fats, and proteins. The limitation is typically, although not limited to, about 25% to about 40% of the caloric intake relative to ad libitum consumption. 
     The term “dietary supplement” means a product that is intended to be ingested in addition to the normal diet of an animal. Dietary supplements may be in any form—e.g. solid, liquid, gel, tablets, capsules, powder, and the like. Preferably they are provided in convenient dosage forms. In some embodiments they are provided in bulk consumer packages such as bulk powders or liquids. In other embodiments, supplements are provided in bulk quantities to be included in other food items such as snacks, treats, supplement bars, beverages and the like. 
     The term “differential expression” or “differentially expressed” means increased or unregulated gene expression or means decreased or downregulated gene expression as detected by the absence, presence, or at least two-fold change in the amount of transcribed messenger RNA or translated protein in a sample. 
     The term “effective amount” means an amount of a compound, material, composition, medicament, or other material that is effective to achieve a particular biological result, such as reversing or delaying aging in a selected tissue, as described herein. 
     The term “food” or “food composition” means a composition that is intended for consumption by an animal, including a human, and provides nutrition thereto. As used herein, a “food product formulated for human consumption” is any composition specifically intended for ingestion by a human being. “Pet foods” are compositions intended for consumption by pets, preferably by companion animals. A “complete and nutritionally balanced pet food,” is one that contains all known required nutrients for the intended recipient or consumer of the food, in appropriate amounts and proportions, based for example on recommendations of recognized authorities in the field of companion animal nutrition. Such foods are therefore capable of serving as a sole source of dietary intake to maintain life or promote production, without the addition of supplemental nutritional sources. Nutritionally balanced pet food compositions are widely known and widely used in the art. 
     The term “fragment” means (1) an oligonucleotide or polynucleotide sequence that is a portion of a complete sequence and that has the same or similar activity for a particular use as the complete polynucleotide sequence or (2) a peptide or polypeptide sequence that is a portion of a complete sequence and that has the same or similar activity for a particular use as the complete polypeptide sequence. Such fragments can comprise any number of nucleotides or amino acids deemed suitable for a particular use. Generally, oligonucleotide or polynucleotide fragments contain at least about 10, 50, 100, or 1000 nucleotides and polypeptide fragments contain at least about 4, 10, 20, or 50 consecutive amino acids from the complete sequence. The term encompasses polynucleotides and polypeptides variants of the fragments. 
     The term “gene” or “genes” means a complete or partial segment of DNA involved in producing a polypeptide, including regions preceding and following the coding region (leader and trailer) and intervening sequences (introns) between individual coding segments (exons). The term encompasses any DNA sequence that hybridizes to the complement of gene coding sequences. 
     The term “gene product” means the product of transcription of a gene, such as mRNA or derivatives thereof (e.g., cDNA), or translation of a gene transcript. The term “gene product” generally refers to the translation product, which is a protein. The term “gene product” may be used interchangeably with the term “protein” herein. 
     The term “homolog” means (1) a polynucleotide, including polynucleotides from the same or different animal species, having greater than 30%, 50%, 70%, or 90% sequence similarity to a reference polynucleotide, and having the same or substantially the same properties and performing the same or substantially the same function as the reference polynucleotide, or having the capability of specifically hybridizing to a reference polynucleotide under stringent conditions or (2) a polypeptide, including polypeptides from the same or different animal species, having greater than 30%, 50%, 70%, or 90% sequence similarity to a reference polypeptide and having the same or substantially the same properties and performing the same or substantially the same function as the reference polypeptide, or having the capability of specifically binding to a reference polypeptide. When referring to fragments of full length coding sequences, the function of those fragments may simply be to encode a selected portion of a polypeptide of a certain sequence, or to be of suitably similar sequence to hybridize to another polynucleotide fragment encoding that polypeptide. When referring to fragments of polypeptides, the function of those fragments may simply be to form an epitope suitable for generation of an antibody. Sequence similarity of two polypeptide sequences or of two polynucleotide sequences is determined using methods known to skilled artisans, e.g., the algorithm of Karlin and Altschul (Proc. Natl. Acad. Sci. USA 87:2264-2268 (1990)). Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al. (J. Mol. Biol. 215:403-410 (1990)). To obtain gapped alignments for comparison purposes, Gapped Blast can be utilized as described in Altschul et al. (Nucl. Acids Res. 25: 3389-3402 (1997)). When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) are used. See http://ww.ncbi.nlm.nih.gov. 
     The term “hybridization complex” means a complex that is formed between sample polynucleotides when the purines of one polynucleotide hydrogen bond with the pyrimidines of the complementary polynucleotide, e.g., 5′-A-G-T-C-3′ base pairs with 3′-T-C-A-G-5′. The degree of complementarily and the use of nucleotide analogs affect the efficiency and stringency of hybridization reactions. 
     The term “in conjunction” means that a drug, food, or other substance is administered to an animal (1) together in a composition, particularly food composition, or (2) separately at the same or different frequency using the same or different administration routes at about the same time or periodically. “Periodically” means that the substance is administered on a dosage schedule acceptable for a specific substance. “About the same time” generally means that the substance (food or drug) is administered at the same time or within about 72 hours of each other. “In conjunction” specifically includes administration schemes wherein substances such as drugs are administered for a prescribed period and compositions of the invention are administered indefinitely. 
     The term “individual” when referring to an animal means an individual animal of any species or kind. This term may be used interchangeably with the term “subject.” 
     The term “Longevity” refers generally to the duration of life beyond the average life expectancy for a particular species, or for a particular strain, breed or ethnic group within that species when distinctions within species exist. “Enhanced longevity” or “increased longevity” refers to any significant extension of the life span of a particular animal beyond the average life expectancy for the species to which the animal belongs. 
     The term “polynucleotide” or “oligonucleotide” means a polymer of nucleotides. The term encompasses DNA and RNA (including cDNA and mRNA) molecules, either single or double stranded and, if single stranded, its complementary sequence in either linear or circular form. The term also encompasses fragments, variants, homologs, and alleles, as appropriate for the sequences, which have the same or substantially the same properties and perform the same or substantially the same function as the original sequence. In particular, the term encompasses homologs from different species, e.g., a mouse and a dog or cat. The sequences may be fully complementary (no mismatches) when aligned or may have up to about a 30% sequence mismatch. Preferably, for polynucleotides, the chain contains from about 50 to 10,000 nucleotides, more preferably from about 150 to 3,500 nucleotides. Preferably, for oligonucleotides, the chain contains from about 2 to 100 nucleotides, more preferably from about 6 to 30 nucleotides. The exact size of a polynucleotide or oligonucleotide will depend on various factors and on the particular application and use of the polynucleotide or oligonucleotide. The term includes nucleotide polymers that are synthesized and that are isolated and purified from natural sources. The term “polynucleotide” is inclusive of “oligonucleotide.” 
     The term “polypeptide,” “peptide,” or “protein” means a polymer of amino acids. The term encompasses naturally occurring and non-naturally occurring (synthetic) polymers and polymers in which artificial chemical mimetics are substituted for one or more amino acids. The term also encompasses fragments, variants, and homologs that have the same or substantially the same properties and perform the same or substantially the same function as the original sequence. The term encompass polymers of any length, preferably polymers containing from about 2 to 1000 amino acids, more preferably from about 5 to 500 amino acids. The term includes amino acid polymers that are synthesized and that are isolated and purified from natural sources. 
     The term “probe” means (1) an oligonucleotide or polynucleotide, either RNA or DNA, whether occurring naturally as in a purified restriction enzyme digest or produced synthetically, that is capable of annealing with or specifically hybridizing to a polynucleotide with sequences complementary to the probe or (2) a compound or substance, including a peptide or polypeptide, capable of specifically binding a particular protein or protein fragment to the substantial exclusion of other proteins or protein fragments. An oligonucleotide or polynucleotide probe may be either single or double stranded. The exact length of the probe will depend upon many factors, including temperature, source, and use. For example, for diagnostic applications, depending on the complexity of the target sequence, an oligonucleotide probe typically contains about 10 to 100, 15 to 50, or 15 to 25 nucleotides. In certain diagnostic applications, a polynucleotide probe contains about 100-1000, 300-600, nucleotides, preferably about 300 nucleotides. The probes herein are selected to be “substantially” complementary to different strands of a particular target sequence. This means that the probes must be sufficiently complementary to specifically hybridize or anneal with their respective target sequences under a set of predetermined conditions. Therefore, the probe sequence need not reflect the exact complementary sequence of the target. For example, a noncomplementary nucleotide fragment may be attached to the 5′ or 3′ end of the probe, with the remainder of the probe sequence being complementary to the target sequence. Alternatively, noncomplementary bases or longer sequences can be interspersed into the probe if the probe sequence has sufficient complementarity with the sequence of the target polynucleotide to specifically anneal to the target polynucleotide. A peptide or polypeptide probe may be any molecule to which the protein or peptide specifically binds, including DNA (for DNA binding proteins), antibodies, cell membrane receptors, peptides, cofactors, lectins, sugars, polysaccharides, cells, cell membranes, organelles and organellar membranes. 
     The term “sample” means any animal tissue or fluid containing, e.g., polynucleotides, polypeptides, antibodies, metabolites, and the like, including cells and other tissue containing DNA and RNA. Examples include adipose, blood, cartilage, connective, epithelial, lymphoid, muscle, nervous, sputum, and the like. A sample may be solid or liquid and may be DNA, RNA, cDNA, bodily fluids such as blood or urine, cells, cell preparations or soluble fractions or media aliquots thereof, chromosomes, organelles, and the like. 
     The term “single package” means that the components of a kit are physically associated in or with one or more containers and considered a unit for manufacture, distribution, sale, or use. Containers include, but are not limited to, bags, boxes, bottles, shrink wrap packages, stapled or otherwise affixed components, or combinations thereof. A single package may be containers of individual food compositions physically associated such that they are considered a unit for manufacture, distribution, sale, or use. 
     The term “specifically bind” means a special and precise interaction between two molecules which is dependent upon their structure, particularly their molecular side groups. For example, the intercalation of a regulatory protein into the major groove of a DNA molecule, the hydrogen bonding along the backbone between two single stranded nucleic acids, or the binding between an epitope of a protein and an agonist, antagonist, or antibody. 
     The term “specifically hybridize” means an association between two single stranded polynucleotides of sufficiently complementary sequence to permit such hybridization under predetermined conditions generally used in the art (sometimes termed “substantially complementary”). For example, the term may refer to hybridization of a polynucleotide probe with a substantially complementary sequence contained within a single stranded DNA or RNA molecule according to an aspect of the invention, to the substantial exclusion of hybridization of the polynucleotide probe with single stranded polynucleotides of non-complementary sequence. 
     The term “standard” means (1) a control sample that contains tissue from a subject administered a control or reference substance, or no substance, as compared with a sample that contains tissue from a subject administered a test substance, for example, to determine if the test substance causes differential gene expression, as appropriate for the context of its use. 
     The term “stringent conditions” means (1) hybridization in 50% (vol/vol) formamide with 0.1% bovine serum albumin, 0.1% Ficoll, 0.1% polyvinylpyrrolidone, 50 mM sodium phosphate buffer at pH 6.5 with 750 mM NaCl, 75 mM sodium citrate at 42° C., (2) hybridization in 50% formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5× Denhardt&#39;s solution, sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS, and 10% dextran sulfate at 42° C.; with washes at 42° C. in 0.2×SSC and 0.1% SDS or washes with 0.015 M NaCl, 0.0015 M sodium citrate, 0.1% Na2SO4 at 50° C. or similar procedures employing similar low ionic strength and high temperature washing agents and similar denaturing agents. 
     The term “tissue-specific marker” or “tissue-specific biomarker” as used herein refers to genes and their expression products that are differentially expressed in a selected tissue of an old, as compared with young, subject. The term “tissue-specific” is intended to encompass tissues and organs. For example, the selected tissue may be smooth muscle tissue from the heart, and the tissue specific markers may be referred to as “heart-specific.” As another example, the selected tissue may be adipose tissue, which may not be associated with any particular organ. The skilled artisan will understand these terms as they are used in context throughout the specification. 
     The term “variant” means (1) a polynucleotide sequence containing any substitution, variation, modification, replacement, deletion, or addition of one or more nucleotides from or to a polynucleotide sequence and that has the same or substantially the same properties and performs the same or substantially the same function as the original sequence and (2) a polypeptide sequence containing any substitution, variation, modification, replacement, deletion, or addition of one or more amino acids from or to a polypeptide sequence and that has the same or substantially the same properties and performs the same or substantially the same function as the original sequence. The term therefore includes single nucleotide polymorphisms (SNPs) and allelic variants and includes conservative and non-conservative amino acid substitutions in polypeptides. The term also encompasses chemical derivatization of a polynucleotide or polypeptide and substitution of nucleotides or amino acids with nucleotides or amino acids that do not occur naturally, as appropriate. 
     The term “virtual package” means that the components of a kit are associated by directions on one or more physical or virtual kit components instructing the user how to obtain the other components, e.g., in a bag containing one component and directions instructing the user to go to a website, contact a recorded message, view a visual message, or contact a caregiver or instructor to obtain instructions on how to use the kit. 
     “Young” refers generally to an individual in young adulthood, i.e., matured past puberty or adolescence, as would be defined by species, or by strain, breed or ethnic group within a species, in accordance with known parameters. “Aged” or “old,” as used herein, refers to an individual who is physically or chronologically within the last 30% of its average life expectancy, as determined by species, or by strain, breed or ethnic group within a species, in accordance with known parameters. 
     The methods and compositions and other advances disclosed here are not limited to particular methodology, protocols, and reagents described herein because, as the skilled artisan will appreciate, they may vary. Further, the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to and does not limit the scope of that which is disclosed or claimed. 
     Unless defined otherwise, all technical and scientific terms, terms of art, and acronyms used herein have the meanings commonly understood by one of ordinary skill in the art in the field(s) of the invention, or in the field(s) where the term is used. Although any compositions, methods, articles of manufacture, or other means or materials similar or equivalent to those described herein can be used in the practice of the invention, the preferred compositions, methods, articles of manufacture, or other means or materials are described herein. 
     All patents, patent applications, publications, and other references cited or referred to herein are incorporated herein by reference to the extent allowed by controlling law. The discussion of those references is intended merely to summarize the assertions made therein. No admission is made that any such patents, patent applications, publications or references, or any portion thereof, is relevant, material, or prior art. The right to challenge the accuracy and pertinence of any assertion of such patents, patent applications, publications, and other references as relevant, material, or prior art is specifically reserved. 
     The Invention 
     The present invention arises in part from the inventors&#39; development of a method for identifying robust gene expression markers of aging in selected tissues. The method involves the step of screening for differential gene expression in selected tissues in a plurality of strains, breeds or ethnic groups in a species, and employs a criterion that a candidate gene expression marker must be differentially expressed in a majority of the strains, breeds or ethnic groups that are screened. Using this and, optionally, one or more secondary screening criteria, robust sets of gene expression markers of aging in several selected tissues have been identified. 
     In certain embodiments of the invention, the markers are used to measure expression of at least one differentially expressed gene. In preferred embodiments, the markers are used to measure expression of two or more differentially expressed genes. Measuring two or more differentially expressed genes provides a gene expression pattern or gene expression profile for the selected tissue. More preferably, measurement of a multiplicity of differentially expressed genes in several selected tissues may be performed, providing additional information for a gene expression pattern or profile. 
     In various embodiments of the invention, changes in gene expression may be measured in one or both of two ways: (1) measuring transcription through detection of mRNA produced by a particular gene; and (2) measuring translation through detection of protein produced by a particular transcript. 
     Decreased or increased expression can be measured at the RNA level using any of the methods well known in the art for the quantitation of polynucleotides, such as, for example, PCR (including, without limitation, RT-PCR and qPCR), RNase protection, Northern blotting, microarray, macroarray, and other hybridization methods. The genes that are assayed or interrogated according to the invention are typically in the form of mRNA or reverse transcribed mRNA. The genes may be cloned and/or amplified. The cloning itself does not appear to bias the representation of genes within a population. However, it may be preferable to use polyA+ RNA as a source, as it can be used with fewer processing steps. 
     Thus, in one aspect, the invention provides methods for identifying gene expression markers of aging in a selected tissue. The methods comprise selecting one or more genes differentially expressed in the tissue in old subjects as compared with young subjects, using a criterion that the gene is differentially expressed in the selected tissues in a multiplicity of strains, breeds or ethnic groups of a species, preferably at a pre-determined significance level (e.g., p&lt;0.10, p&lt;0.05, or p&lt;0.01). In certain embodiments, the gene is differentially expressed in, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more strains, breeds or ethnic groups. In other embodiments, the criterion is set that the gene is differentially expressed in a majority of the strains, breeds or ethnic groups tested, and can be elevated such that the gene should be differentially expressed in at least 50%, 60%, 70%, 80%, 90% or 100% of the strains, breeds or ethnic groups tested. 
     The method can be practiced on strains, breeds or ethnic groups of any species. In particular embodiments, the species is a mammal, and in particular a human or a companion animal, such as a canine or feline, or other companion animals as defined above. 
     The tissue selected for practice of the method may be any tissue or organ, including but not limited to adipose, bladder, blood, bone, bone marrow, bowel, brain and central nervous system, breast, bronchus, cartilage, colo-rectal, connective tissue, endocrine system, eye, female reproductive organs, glands, heart, intestine, kidney, liver, lung and nasal/bronchial system, lymph node and lymphoid organs, male reproductive organs, mouth and tongue, neural tissue other than brain/CNS, pancreas, peritoneum, spleen, and stomach, to name a few. In exemplary embodiments, the tissue is selected from heart, muscle, brain or adipose tissue. 
     The method described above can include further criteria for identifying robust markers of aging in selected tissues. For example, the method can further comprise a criterion that differential expression of the gene differentially expressed in old subjects as compared with young subjects is at least partially reversed by caloric restriction. The method can also further comprise a criterion that the gene differentially expressed in old subjects as compared with young subjects is known or suspected to be associated with one or more aging-related physiological functions. The functionality of a gene product can be determined from experimentation or from the literature available to the skilled artisan. 
     The methods described above are used to identify biomarkers of aging in selected tissues. Accordingly, in another aspect, the invention provides combinations comprising a plurality of polynucleotides or proteins expressed therefrom that are differentially expressed in selected tissues of old subjects as compared with young subjects, wherein the polynucleotides are selected from genes encoding proteins listed in Table 2, Table 5, Table 8 or Table 10, or fragments thereof. These tables list gene designations, gene names and “Entrez” numbers, which enable access to the full description of the genes and gene products in the National Institutes of Health National Center for Biotechnology Information (NCBI) database. 
     In one embodiment, the selected tissue is heart and the polynucleotides are selected from genes encoding two or more of Amy1, Apod, Bdh1, C3, Casq1, Ce18, Kcnd2, Lcn2, Mt2, Myot, Pah, Prkeq, Serpina3n, Skap2, Tmeml6k, and Vllg2. Of this group the differential expression is reversed by caloric restriction in C3, Cc18, Lcn2, Mt2, Pah, Prkcq, Serpina3n, Tmeml6k, and Vgll2. 
     In another embodiment, the selected tissue is adipose and the polynucleotides are selected from genes encoding two or more of Aspn, Clec4n, Col6a2, Coll8a1, Cox8b, Crip2, Ear11, Emilin2, Otop1, Pla2g2d, Rhbd13, Slc6a13, and Sycp3. Of this group, the differential expression is reversed by caloric restriction in Aspn, Col6a2, Crip2, Emilin2, Otop1, Pla2g2d, Rhbd13, and Slc6a13. 
     In another embodiment, the selected tissue is brain and the polynucleotides are selected from genes encoding two or more of Apod, B2m, Clqa, Clqb, Cd68, Clec7a, Cst7, Ctsd, Gfap, Il33, Lgals3, Lyzs, and Spp1. Of this group, the differential expression is reversed by caloric restriction in Apod, B2m, Clqa, Clqb, Ctsd, Gfap, Il33, Lyzs, and Spp1. 
     In another embodiment, the selected tissue is muscle and the polynucleotides are selected from genes encoding two or more of C4, Cdkn2c, Cds1, Colla1, Col1a2, Col3a1, Dusp26, Edg2, Igh-6, Mt2, Plk2, Rhpn2, and Syt9. Of this group, the differential expression is reversed by caloric restriction in C4, Cdkn2c, Cds1, Colla1, Col1a2, Col3a1, Edg2, Igh-6, Mt2, Plk2, and Syt9. 
     In one embodiment, the combination comprises two or more polynucleotides or proteins expressed from the polynucleotides. Preferably, the combination comprises a plurality of polynucleotides or proteins expressed from polynucleotides, generally about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100 or more polynucleotides or proteins, or fragments thereof, as appropriate for a particular species, tissue and use. When the combination comprises one or more fragments, the fragments can be of any size that retains the properties and function of the original polynucleotide or protein, preferably from about 30%, 60%, or 90% of the original. 
     The polynucleotides and proteins can be from any animal including humans, particularly canines and felines, most particularly canines. Homologs of the polynucleotides and proteins from different animal species are obtainable by standard information mining and molecular methods well known to the skilled artisan. For example, the name, public database accession number, or description of function of a gene or protein may be entered into one of several publicly available databases, which will generate a list of sources providing information about that gene from different species, including sequence information. One such database is the “Information Hyperlinked over Proteins (iHOP) database, which is accessible on the interne via the url: ihop-net.org. Alternatively, a public database accession number of a known gene or protein may be utilized to access sequence information for that gene or protein and to search for homologs or orthologs in other species using a sequence comparison search. For example, the GenBank accession number of a gene or protein from mouse may be entered into the National Institutes of Health&#39;s National Center for Biotechnology Information (NCBI) database, thereby accessing DNA or polypeptide sequences for that mouse gene. Using the same database, a BLAST search may be performed on the mouse DNA or protein sequence, or fragments thereof of sufficient length to define the gene or protein, to identify sequences of sufficient homology from other species, e.g., a canine. Accession numbers of the sequences from the other species of interest may then be entered into the database to obtain information pertaining to those full-length nucleotide or protein sequences, as well as other descriptive information. 
     In another aspect, the invention provides compositions comprising two or more probes for detecting differential gene expression in a selected tissue in old subjects as compared with young subjects. In certain embodiments, the selected tissue is heart, adipose, brain or muscle tissue, and the probes comprise: (a) polynucleotides that specifically hybridize to two or more genes encoding proteins listed in Table 2, Table 5, Table 8 or Table 10, or fragments thereof; or (b) polypeptide binding agents that specifically bind to two or more polypeptides selected from proteins listed in Table 2, Table 5, Table 8 or Table 10, or fragments thereof. 
     In one embodiment, the selected tissue is heart, and the proteins encoded by the differentially expressed genes are Amy1, Apod, Bdh1, C3, Casq1, Cc18, Kcnd2, Lcn2, Mt2, Myot, Pah, Prkcq, Serpina3n, Skap2, Tmeml6k, and Vgll2. In another embodiment, the selected tissue is adipose and the proteins encoded by the differentially expressed genes are Aspn, Clec4n, Col6a2, Coll8a1, Cox8b, Crip2, Emilin2, Otop1, Pla2g2d, Rhbdl3, Slc6a13, and Sycp3. In another embodiment, the selected tissue i sbrain and the proteins encoded by the differentially expressed genes are Apod, B2m, Clqa, Clqb, Cd68, Clec7a, Cst7, Ctsd, Gfap, Il33, Lgals3, Lyzs, and Spp1. In still another embodiment, the selected tissue is muscle and the proteins encoded by the differentially expressed genes are C4, Cdkn2c, Cds1, Colla1, Colla2, Col3a1, Dusp26, Edg2, Igh-6, Mt2, Plk2, Rhpn2, and Syt9. 
     In particular embodiments, the differential expression is reversed by caloric restriction and the proteins encoded by the differentially expressed genes are: (a) C3, Cc18, Lcn2, Mt2, Pah, Prkcq, Serpina3n, Tmeml6k, and Vgll2 in the heart; (b) Aspn, Col6a2, Crip2, Emilin2, Otop1, Pla2g2d, Rhbd13, and Slc6a13 in adipose tissue; (c) Apod, B2m, Clqa, Clqb, Ctsd, Gfap, Il33, Lyzs, and Spp 1 in the brain; or (d) C4, Cdkn2c, Cds1, Colla1, Colla2, Col3a1, Edg2, Igh-6, Mt2, Plk2, and Syt9 in muscle. 
     Preferably, the composition comprises a plurality of probes, generally about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200, 500 or more probes for detecting the polynucleotides or proteins, or fragments thereof, as appropriate for a particular species, tissue and use. It Will be understood by the skilled artisan that multiple different probes for a single target gene or protein may be utilized, to refine the sensitivity or accuracy of an assay utilizing the probes. For example, several oligonucleotide probes, specifically hybridizing to different sequences on a target polynucleotide, may be employed. Likewise, several antibodies, immunologically specific for different epitopes on a target protein, may be utilized. 
     One or more oligonucleotide or polynucleotide probes for interrogating a sample may be prepared using the sequence information for any of the genes listed herein, from any species, preferably canine or feline. The probes should be of sufficient length to specifically hybridize substantially exclusively with appropriate complementary genes or transcripts. In certain embodiments, the oligonucleotide probes will be at least about 10, 12, 14, 16, 18, 20 or 25 nucleotides in length. In some embodiments, longer probes of at least about 30, 40, 50, 60, 70, 80, 90 or 100 nucleotides are desirable, and probes longer than about 100 nucleotides may be suitable in some embodiments. The probes may comprise full length sequences encoding functional proteins. The nucleic acid probes are made or obtained using methods known to skilled artisans, e.g., in vitro synthesis from nucleotides, isolation and purification from natural sources, or enzymatic cleavage of the polynucleotides of the invention. 
     Hybridization complexes comprising nucleic acid probes hybridized to a polynucleotide of the invention may be detected by a variety of methods known in the art. In certain embodiments of the invention, immobilized nucleic acid probes may be used for the rapid and specific detection of polynucleotides and their expression patterns. Typically, a nucleic acid probe is linked to a solid support and a target polynucleotide (e.g., a gene, a transcription product, an amplicon, or, most commonly, an amplified mixture) is hybridized to the probe. Either the probe, or the target, or both, can be labeled, typically with a fluorophore or other tag, such as streptavidin. Where the target is labeled, hybridization may be detected by detecting bound fluorescence. Where the probe is labeled, hybridization is typically detected by quenching of the label. Where both the probe and the target are labeled, detection of hybridization is typically performed by monitoring a color shift resulting from proximity of the two bound labels. A variety of labeling strategies, labels, and the like, particularly for fluorescent based applications, are known in the art. 
     In another embodiment, the probes comprise polypeptide binding agents that specifically bind to polypeptides produced by expression of one or more of the polypeptides listed herein, or fragments thereof. Such protein binding probes may be prepared using the sequence information available for any of the proteins identified in Table 2, Table 5, Table 8 and Table 10, or fragments thereof. 
     Assay techniques that can be used to determine levels of a protein in a sample are also well known to those of skill in the art. Such assay methods include radioimmunoassays, competitive-binding assays, Western blot analysis and ELISA assays. In the assay methods utilizing antibodies, both polyclonal and monoclonal antibodies are suitable for use in the invention. Such antibodies may be immunologically specific for a particular protein, or an epitope of the protein, or a protein fragment, as would be well understood by those of skill in the art. Methods of making polyclonal and monoclonal antibodies immunologically specific for a protein or peptide are also well known in the art. 
     Preferred embodiments of the invention may utilize antibodies for the detection and quantification of proteins produced by expression of the genes described herein. Though proteins may be detected by immunoprecipitation, affinity separation, Western blot analysis and the like, a preferred method utilizes ELISA-type methodology wherein the antibody is immobilized on a solid support and a target protein or peptide is exposed to the immobilized antibody. Either the probe, or the target, or both, can be labeled. A variety of labeling strategies, labels, and the like, are known in the art. 
     In another aspect, the invention provides devices comprising a solid support to which is affixed an array comprising a plurality of probes for detecting differential gene expression in a selected tissue in old subjects as compared with young subjects. In certain embodiments, the selected tissue is heart, adipose, brain or muscle tissue, and the probes comprise: (a) polynucleotides that specifically hybridize to two or more genes encoding proteins listed in Table 2, Table 5, Table 8 or Table 10, or fragments thereof; or (b) polypeptide binding agents that specifically bind to two or more polypeptides selected from proteins listed in Table 2, Table 5, Table 8 or Table 10, or fragments thereof. In a preferred embodiment, the device is uses to detect differential expression of genes from canines or felines. 
     In one embodiment, the selected tissue is heart, and the proteins encoded by the differentially expressed genes are Amy1, Apod, Bdh1, C3, Casq1, Cc18, Kcnd2, Lcn2, Mt2, Myot, Pah, Prkcq, Serpina3n, Skap2, Tmeml6k, and Vgll2. In another embodiment, the selected tissue is adipose and the proteins encoded by the differentially expressed genes are Aspn, Clec4n, Col6a2, Coll8a1, Cox8b, Crip2, Ear11, Emilin2, Otop1, Pla2g2d, Rhbdl3, Slc6a13, and Sycp3. In another embodiment, the selected tissue is brain and the proteins encoded by the differentially expressed genes are Apod, B2m, Clqa, Clqb, Cd68, Clec7a, Cst7, Ctsd, Gfap, Il33, Lgals3, Lyzs, and Spp1. In still another embodiment, the selected tissue is muscle and the proteins encoded by the differentially expressed genes are C4, Cdkn2c, Cds1, Colla1, Colla2, Col3a1, Dusp26, Edg2, Igh-6, Mt2, Plk2, Rhpn2, and Syt9. 
     In particular embodiments, the differential expression is reversed by caloric restriction and the proteins encoded by the differentially expressed genes are: (a) C3, Cc18, Lcn2, Mt2, Pah, Prkcq, Serpina3n, Tmeml6k, and Vgll2 in the heart; (b) Aspn, Col6a2, Crip2, Emilin2, Otop1, Pla2g2d, Rhbd13, in adipose tissue; (c) Apod, B2m, Clqa, Clqb, Ctsd, Gfap, Il33, Lyzs, and Spp1 in the brain; or (d) C4, Cdkn2c, Cds1, Colla1, Colla2, Col3a1, Edg2, Igh-6, Mt2, Plk2, and Syt9 in muscle. 
     In one embodiment, arrays of oligonucleotide or polynucleotide probes may be utilized, whereas another embodiment may utilize arrays of antibodies or other proteins that bind specifically to the differentially expressed gene products. Such arrays may be custom made according to known methods, such as, for example, in-situ synthesis on a solid support or attachment of pre-synthesized probes to a solid support via micro-printing techniques. In preferred embodiments, arrays of nucleic acid or protein-binding probes are custom made to specifically detect transcripts or proteins produced by two or more of the differentially expressed genes or gene fragments described herein. 
     In another aspect, the invention provides methods for detecting differential expression of one or more genes differentially expression in a selected tissue in old subjects as compared with a standard or with young subjects. In particular embodiments, the tissue is heart, adipose, brain or muscle, and the methods generally comprise: (a) providing probes comprising (i) polynucleotides that specifically hybridize to two or more genes encoding proteins listed in Table 2, Table 5, Table 8 or Table 10, or fragments thereof; or (ii) polypeptide binding agents that specifically bind to two or more polypeptides selected from proteins listed in Table 2, Table 5, Table 8 or Table 10, or fragments thereof; (b) adding the probes to a sample comprising mRNA or proteins from an old subject, in a manner enabling hybridization or binding of the probes to the mRNA or proteins in the sample, thereby forming hybridization or binding complexes in the sample; (c) optionally, adding the probes to another sample comprising mRNA or proteins from a young subject, in a manner enabling hybridization or binding of the probes to the mRNA or proteins in the second sample, thereby forming hybridization or binding complexes in the other sample; (d) detecting the hybridization complexes in the sample or samples; and (e) comparing the hybridization or binding complexes from the first sample with the hybridization or binding complexes from a standard or, optionally, from the other sample, wherein at least one difference between the amount of hybridization or binding in the sample as compared with the standard or the optional other sample indicates differential expression of the one or more genes differentially expressed in the old subjects. 
     The method may be used to detect differential expression of genes encoding the gene products set forth in Tables 2, 5, 8 or 10, or in subsets thereof. Thus, in one embodiment, the selected tissue is heart, and the proteins encoded by the differentially expressed genes are Amy1, Apod, Bdh1, C3, Casq1, Cc18, Kcnd2, Lcn2, Mt2, Myot, Pah, Prkcq, Serpina3n, Skap2, Tmeml6k, and Vgll2. In another embodiment, the selected tissue is adipose and the proteins encoded by the differentially expressed genes are Aspn, Clec4n, Col6a2, Coll8a1, Cox8b, Crip2, Ear11, Emilin2, Otop1, Pla2g2d, Rhbdl3, Slc6a13, and Sycp3. In another embodiment, the selected tissue is brain and the proteins encoded by the differentially expressed genes are Apod, B2m, Clqa, Clqb, Cd68, Clec7a, Cst7, Ctsd, Gfap, Il33, Lgals3, Lyzs, and Spp1. In still another embodiment, the selected tissue is muscle and the proteins encoded by the differentially expressed genes are C4, Cdkn2c, Cds1, Colla1, Colla2, Col3a1, Dusp26, Edg2, Igh-6, Mt2, Plk2, Rhpn2, and Syt9. 
     In particular embodiments, the differential expression is reversed by caloric restriction and the proteins encoded by the differentially expressed genes are: (a) C3, Cc18, Lcn2, Mt2, Pah, Prkcq, Serpina3n, Tmeml6k, and Vgll2 in the heart; (b) Aspn, Col6a2, Crip2, Emilin2, Otop1, Pla2g2d, Rhbd13, and Slc6a13 in adipose tissue; (c) Apod, B2m, Clqa, Clqb, Ctsd, Gfap, Il33, Lyzs, and Spp 1 in the brain; or (d) C4, Cdkn2c, Cds1, Colla1, Colla2, Col3a1, Edg2, Igh-6, Mt2, Plk2, and Syt9 in muscle. 
     In a preferred embodiment, the method is uses to detect differential expression of genes from canines or felines. In particular embodiments, the probes are bound to a substrate, preferably in an array. 
     Step (c) and part of steps (d) and (e) are optional and are used if a relatively contemporaneous comparison of two or more test systems (i.e., tissues from old and young subjects) is to be conducted. However, in another embodiment, the standard used for comparison is based upon data previously obtained using the method. In this embodiment, the probes are exposed to a sample to form hybridization or binding complexes that are detected and compared with those of a standard. The differences between the hybridization or binding complexes from the sample and standard indicate differential expression of polynucleotides and therefore genes differentially expressed in tissue of the old subject versus the standard, which can comprise mRNA previously isolated from a young subject or another type of reference subject. In a preferred embodiment, probes are made to specifically detect polynucleotides or fragments thereof produced by one or more of the genes or gene fragments identified by the invention. Methods for detecting hybridization complexes are known to skilled artisans. 
     The assays described herein utilizing tissue-specific biomarkers for the detection of aging related transcription and translation products are useful in methods for determining the physiological age of a tissue in a subject. Such methods may be useful for implementing, facilitating, or guiding an anti-aging regimen, such as caloric restriction and/or a nutritional regimen. Such methods comprise obtaining a sample of the selected tissue from a subject undergoing such a regimen. The tissue sample is then analyzed for modulated expression of one or more genes associated with a young versus old phenotype, using a gene- or protein-array or other detection method as described herein. The results of the analysis will reveal whether the regimen is effective in delaying or reversing the aging process in the tissue. 
     In another aspect, the invention provides methods of determining if a test substance is likely to be useful in reversing or delaying the aging process in at least one selected tissue when administered to an animal. The methods comprise (a) determining a first gene expression profile by measuring the transcription or translation products of two or more polynucleotides selected from genes encoding proteins listed in Table 2, Table 5, Table 8 or Table 10, or fragments thereof, in a test system in the absence of the test substance; (b) determining a second gene expression profile by measuring the transcription or translation products of two or more polynucleotides selected from genes encoding proteins listed in Table 2, Table 5, Table 8 or Table 10, or fragments thereof, in a test system in the presence of the test substance; and (c) comparing the first gene expression profile with the second gene expression profile, wherein a change in the second gene expression profile as compared with the first gene expression profile indicates that the test substance is likely to be useful in reversing or delaying the aging process when administered to an animal. When comparing the first gene expression profile with the second gene expression profile, the comparison can be either at the individual transcription or translation product level or as an average of the aging changes for all transcription or translation products. This method is useful for generating an aging retardation index. 
     In one embodiment, the selected tissue is heart, and the proteins encoded by the differentially expressed genes are Amy1, Apod, Bdh1, C3, Casq1, Ccl8, Kcnd2, Lcn2, Mt2, Myot, Pah, Prkcq, Serpina3n, Skap2, Tmem16k, and Vgll2. In another embodiment, the selected tissue is adipose and the proteins encoded by the differentially expressed genes are Aspn, Clec4n, Col6a2, Col18a1, Cox8b, Crip2, Ear11, Emilin2, Otop1, Pla2g2d, Rhbdl3, Slc6a13, and Sycp3. In another embodiment, the selected tissue is brain and the proteins encoded by the differentially expressed genes are Apod, B2m, Clqa, Clqb, Cd68, Clec7a, Cst7, Ctsd, Gfap, Il33, Lgals3, Lyzs, and Spp1. In still another embodiment, the selected tissue is muscle and the proteins encoded by the differentially expressed genes are C4, Cdkn2c, Cds1, Colla1, Colla2, Col3a1, Dusp26, Edg2, Igh-6, Mt2, Plk2, Rhpn2, and Syt9. 
     In particular embodiments, the differential expression is reversed by caloric restriction and the proteins encoded by the differentially expressed genes are: (a) C3, Cc18, Lcn2, Mt2, Pah, Prkcq, Serpina3n, Tmeml6k, and Vgll2 in the heart; (b) Aspn, Col6a2, Crip2, Emilin2, Otop1, Pla2g2d, Rhbd13, and Slc6a13, in adipose tissue; (c) Apod, B2m, Clqa, Clqb, Ctsd, Gfap, Il33, Lyzs, and Spp 1 in the brain; or (d) C4, Cdkn2c, Cds1, Colla1, Colla1, Col3a1, Edg2, Igh-6, Mt2, Plk2, and Syt9 in muscle. 
     In certain embodiments, the method may further include the step of comparing at least the second gene expression profile with a reference or standard gene expression profile obtained by measuring the transcription or translation products of two or more polynucleotides selected from genes encoding proteins listed in Tables 2, 5, 8 or 10, or fragments thereof, in a test system in the presence of a reference substance or composition known reverse or delay aging in a particular tissue or tissues when administered to animals. 
     In one embodiment, the test system comprises a population of cultured cells. A nucleic acid construct comprising an aging-related gene according to the invention is introduced into cultured host cells. The host cells can be mammalian cell lines, such as but are not limited, to NIH3T3, CHO, HELA, and COS, although non-mammalian cells such as yeast, bacteria and insect cells can also be used. The coding sequences of the genes are operably linked to appropriate regulatory expression elements suitable for the particular host cell to be utilized. The nucleic acid constructs can be introduced into the host cells according to any acceptable means in the art, including but not limited to, transfection, transformation, calcium phosphate precipitation, electroporation and lipofection. Such techniques are well known and routine in the art. Transformed cells can be also used to identify compounds that modulate expression of the aging-related genes. 
     Gene expression assays can be carried out using a gene construct comprising the promoter of a selected aging-related gene operably linked to a reporter gene. The reporter construct may be introduced into a suitable cultured cell, including, without limitation, the standard host cell lines described above, or cells freshly isolated from a subject, such as adipose or muscle cells. The assay is performed by monitoring expression of the reporter gene in the presence or absence of a test compound. 
     In a preferred embodiment, the test system comprises animals. Typically, a test compound is administered to a subject and the gene expression profile in a selected tissue of the subject is analyzed to determine the effect of the test compound on transcription or the translation of the aging-related genes or gene products of the invention. Gene expression can be analyzed in situ or ex vivo to determine the effect of the test compound. In another embodiment, a test compound is administered to a subject and the activity of a protein expressed from a gene is analyzed in situ or ex vivo according to any means suitable in the art to determine the effect of the test compound on the activity of the proteins of interest. In addition, where a test compound is administered to a subject, the physiological, systemic, and physical effects of the compound, as well as potential toxicity of the compound can also be evaluated. 
     Test substances can be any substance and combination of substances that may have an effect on polynucleotides or genes differentially expressed in selected tissues of old versus young subjects. Suitable test substances include, but are not limited to, amino acids; proteins, peptides, polypeptides, nucleic acids, oligonucleotides, polynucleotides, small molecules, macromolecules, vitamins, minerals, simple sugars; complex sugars; polysaccharides; carbohydrates; medium-chain triglycerides (MCTs); triacylglycerides (TAGs); n-3 (omega-3) fatty acids including DHA, EPA, ALA; n-6 (omega-6) fatty acids including LA, γ-linolenic acid (GLA) and ARA; SA, conjugated linoleic acid (CLA); choline sources such as lecithin; fat-soluble vitamins including vitamin A and precursors thereof such as carotenoids (e.g., (n-carotene), vitamin D sources such as vitamin D2 (ergocalciferol) and vitamin D3 (cholecalciferol), vitamin E sources such as tocopherols (e.g., a-tocopherol) and tocotrienols, and vitamin K sources such as vitamin K1 (phylloquinone) and vitamin K2 (menadione); water-soluble vitamins including B vitamins such as riboflavin, niacin (including nicotinamide and nicotinic acid), pyridoxine, pantothenic acid, folic acid, biotin and cobalamin; and vitamin C (ascorbic acid); antioxidants, including some of the vitamins listed above, especially vitamins E and C; also bioflavonoids such as catechin, quercetin and theaflavin; quinones such as ubiquinone; carotenoids such as lycopene and lycoxanthin; resveratrol; and a-lipoic acid; L-carnitine; D-limonene; glucosamine; S-adenosylmethionine; and chitosan. In a preferred embodiment, test substances are nutrients that may be added to food or consumed as a supplement. Substances identified by the foregoing method are also contemplated as part of the invention. 
     In another aspect, the invention provides kits comprising, in separate containers in a single package, or in separate containers in a virtual package, two or more probes for detecting differential gene expression in a selected tissue in old subjects as compared with young subjects. In certain embodiments, the tissue is heart, adipose, brain or muscle tissue and the probes comprise (a) polynucleotides that specifically hybridize to two or more genes encoding proteins listed in Table 2, Table 5, Table 8 or Table 10, or fragments thereof; or (b) polypeptide binding agents that specifically bind to two or more polypeptides selected from proteins listed in Table 2, Table 5, Table 8 or Table 10, or fragments thereof; wherein the kit further comprises at least one of (1) instructions for how to use the probes in a gene expression assay for detecting differential gene expression in selected tissues of subjects, (2) reagents and equipment for using the probes, and (3) a composition known to reverse or delay the aging process in the selected tissue when administered to the subject. 
     When the kit comprises a virtual package, the kit is limited to instructions in a virtual environment in combination with one or more physical kit components. In one embodiment, the kit contains probes and/or other physical components and the instructions for using the probes and other components are available via the interne. The kit may contain additional items such as a device for mixing samples, probes, and reagents and device for using the kit, e.g., test tubes or mixing utensils. 
     In another aspect, the invention provides computer systems comprising a database containing information about polynucleotides that are differentially expressed in a selected tissue of old subjects as compared with young subjects. The database can contain information identifying the expression level of one or more polynucleotides selected from genes encoding proteins listed in Tables 2, 5, 8 or 10, and/or polypeptides that specifically bind to the proteins listed in Tables 2, 5, 8 or 10, and a user interface to interact with the database, particularly to input, manipulate, and review the information for different animals or categories of animals. In one embodiment, the database further contains information identifying the activity level of one or more polypeptides listed in Tables 2, 5, 8 or 10. In another, the database further comprises sequence information for one or more of the polynucleotides or polypeptides as listed in Tables 2, 5, 8 or 10, preferably from a variety of species. In other embodiments, the database contains additional information pertaining to the description of the genes in one or more animal species. The computer system is any electronic device capable of containing and manipulating the data and interacting with a user, e.g., a typical computer or an analytical instrument designed to facilitate using the invention and outputting the results relating to the status of an animal. 
     In another aspect, the invention provides media for communicating information about or instructions for one or more of compositions and methods described herein. Such media typically comprise documents, digital storage media, optical storage media, audio presentations, visual displays or the like, containing the information or instructions. For example, the communication medium may be a displayed web site, a kiosk, brochure, product label, package insert, advertisement, handout, public announcement audiotape, videotape, DVD, CD, computer-readable chip, computer-readable card, computer-readable disk, computer memory, or any combination thereof. Useful information includes one or more of (1) methods for promoting the health and wellness of animals and (2) contact information for the animal&#39;s caregivers to use if they have a question about the invention and its use. Useful instructions include techniques for using the probes, instructions for performing a gene expression assay, and administration amounts and frequency for the substances. The communication means is useful for instructing on the benefits of using the invention. 
     EXAMPLES 
     Various aspects of the invention can be further illustrated by the following examples. It will be understood that these examples are provided merely for purposes of illustration and do not limit the scope of the invention disclosed herein unless otherwise specifically indicated. 
     Example 1 
     This example briefly describes a study performed to test the ability of certain combinations of substances to mimic the life-extending effects of caloric restriction (CR) without reducing dietary intake. C57BL6 mice were fed a Control diet based on the AIN93M formula (American Institute of Nutrition (AIN) purified diet formula for maintenance of mature rodents) or a diet with similar nutrient composition but representing a 25% calorie restriction (CR) 
     Tissues were collected from mice on the Control diets at five and 25 months of age; tissues from nutrient-supplemented mice were collected at 25 months of age. RNA was isolated from tissues and gene expression changes were determined by qPCR using an Eppendorf “realplex2” instrument. Data for individual genes are set forth in certain of the subsequent examples. 
     Example 2 
     This example describes the identification of biomarkers of aging in heart tissue. 
     The Affymetrix Mouse Genome 430 2.0 array was used to identify gene expression changes in heart of seven strains of mice (129, C57BL6, Balbc, C3H, CBA, DBA and B6C3HF1). A significant change in expression was determined using two-tailed t-tests for young vs. old mice (P&lt;0.05, n=7 mice per strain per age group). Young mice were tested at 5 months of age, old mice were tested at 25 months of age. Table 1 shows the number of genes that were significantly changed in expression with age in each strain (P&lt;0.05), and Table 2 lists the genes that were changed in expression in at least four of the seven strains., 
     
       
         
           
               
               
               
             
               
                   
                 TABLE 1 
               
               
                   
                   
               
               
                   
                 Strain 
                 Number of Transcripts 
               
               
                   
                   
               
             
            
               
                   
                 129 
                 4,954 
               
               
                   
                 B6 
                 2,190 
               
               
                   
                 Balbc 
                 2,137 
               
               
                   
                 C3H 
                 3,236 
               
               
                   
                 CBA 
                 1,118 
               
               
                   
                 DBA 
                 1,521 
               
               
                   
                 F1 
                 1,287 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
               
               
             
               
                 TABLE 2 
               
               
                   
               
               
                 Gene 
                   
                 Entrez 
               
               
                 Symbol 
                 Gene Title 
                 Gene 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
            
               
                 Adh1 
                 alcohol dehydrogenase 1 (class I) 
                 11522 
               
               
                 Agtrl1 
                 angiotensin receptor-like 1 
                 23796 
               
               
                 Akr1b8 
                 aldo-keto reductase family 1, member B8 
                 14187 
               
               
                 Aldh1a1 
                 aldehyde dehydrogenase family 1, subfamily A1 
                 11668 
               
               
                 Alox5ap 
                 arachidonate 5-lipoxygenase activating protein 
                 11690 
               
               
                 Amy1 
                 amylase 1, salivary 
                 11722 
               
               
                 Angptl2 
                 angiopoietin-like 2 
                 26360 
               
               
                 Ankrd1 
                 ankyrin repeat domain 1 (cardiac muscle) 
                 107765 
               
               
                 Anxa1 
                 annexin A1 
                 16952 
               
               
                 Apod 
                 apolipoprotein D 
                 11815 
               
               
                 Apoe 
                 apolipoprotein E 
                 11816 
               
               
                 Asah3l 
                 N-acylsphingosine amidohydrolase 3-like 
                 230379 
               
               
                 Asph 
                 aspartate-beta-hydroxylase 
                 65973 
               
               
                 Atp6v0e2 
                 ATPase, H+ transporting, lysosomal V0 subunit E2 
                 76252 
               
               
                 Atp6v1c1 
                 ATPase, H+ transporting, lysosomal V1 subunit C1 
                 66335 
               
               
                 Atp9a 
                 ATPase, class II, type 9A 
                 11981 
               
               
                 Atxn10 
                 ataxin 10 
                 54138 
               
               
                 BC023892 
                 cDNA sequence BC023892 
                 212943 
               
               
                 Bckdhb 
                 branched chain ketoacid dehydrogenase E1, beta polypeptide 
                 12040 
               
               
                 Bdh1 
                 3-hydroxybutyrate dehydrogenase, type 1 
                 71911 
               
               
                 Bre 
                 brain and reproductive organ-expressed protein 
                 107976 
               
               
                 Bysl 
                 bystin-like 
                 53414 
               
               
                 C1r 
                 complement component 1, r subcomponent 
                 50909 
               
               
                 C3 
                 complement component 3 
                 12266 
               
               
                 Cacna2d1 
                 calcium channel, voltage-dependent, alpha2/delta subunit 1 
                 12293 
               
               
                 Camk2n1 
                 calcium/calmodulin-dependent protein kinase II inhibitor 1 
                 66259 
               
               
                 Casq1 
                 calsequestrin 1 
                 12372 
               
               
                 Ccdc72 
                 coiled-coil domain containing 72 
                 66167 
               
               
                 Ccl6 
                 chemokine (C-C motif) ligand 6 
                 20305 
               
               
                 Ccl8 
                 chemokine (C-C motif) ligand 8 
                 20307 
               
               
                 Ccnd1 
                 cyclin D1 
                 12443 
               
               
                 Cd163 
                 CD163 antigen 
                 93671 
               
               
                 Cdh22 
                 cadherin 22 
                 104010 
               
               
                 Cebpd 
                 CCAAT/enhancer binding protein (C/EBP), delta 
                 12609 
               
               
                 Cfb 
                 complement factor B 
                 14962 
               
               
                 Chek2 
                 CHK2 checkpoint homolog ( S. pombe ) 
                 50883 
               
               
                 Churc1 
                 churchill domain containing 1 
                 211151 
               
               
                 Cilp 
                 cartilage intermediate layer protein, nucleotide pyrophosphohydrolase 
                 214425 
               
               
                 Ckb 
                 creatine kinase, brain 
                 12709 
               
               
                 Clic5 
                 chloride intracellular channel 5 
                 224796 
               
               
                 Col3a1 
                 procollagen, type III, alpha 1 
                 12825 
               
               
                 Col8a1 
                 procollagen, type VIII, alpha 1 
                 12837 
               
               
                 Cp 
                 Ceruloplasmin 
                 12870 
               
               
                 Cpxm2 
                 carboxypeptidase X 2 (M14 family) 
                 55987 
               
               
                 Ctgf 
                 connective tissue growth factor 
                 14219 
               
               
                 Ctss 
                 cathepsin S 
                 13040 
               
               
                 Cxcl14 
                 chemokine (C—X—C motif) ligand 14 
                 57266 
               
               
                 Cyb5r3 
                 cytochrome b5 reductase 3 
                 109754 
               
               
                 Cyp27a1 
                 cytochrome P450, family 27, subfamily a, polypeptide 1 
                 104086 
               
               
                 Dalrd3 
                 DALR anticodon binding domain containing 3 
                 67789 
               
               
                 Dbnl 
                 drebrin-like 
                 13169 
               
               
                 Dhrs1 
                 dehydrogenase/reductase (SDR family) member 1 
                 52585 
               
               
                 Dhrs7c 
                 dehydrogenase/reductase (SDR family) member 7C 
                 68460 
               
               
                 Dpep1 
                 dipeptidase 1 (renal) 
                 13479 
               
               
                 EG665317 
                 predicted gene, EG665317 
                 665317 
               
               
                 Ehbp1l1 
                 EH domain binding protein 1-like 1 
                 114601 
               
               
                 Ehmt2 
                 euchromatic histone lysine N-methyltransferase 2 
                 110147 
               
               
                 Enpp2 
                 ectonucleotide pyrophosphatase/phosphodiesterase 2 
                 18606 
               
               
                 Fads1 
                 fatty acid desaturase 1 
                 76267 
               
               
                 Fbln2 
                 fibulin 2 
                 14115 
               
               
                 Fcgr3 
                 Fc receptor, IgG, low affinity III 
                 14131 
               
               
                 Fez2 
                 fasciculation and elongation protein zeta 2 (zygin II) 
                 225020 
               
               
                 Fgfr1op2 
                 FGFR1 oncogene partner 2 
                 67529 
               
               
                 Fkbp5 
                 FK506 binding protein 5 
                 14229 
               
               
                 Fmo2 
                 flavin containing monooxygenase 2 
                 55990 
               
               
                 Ftl2 
                 ferritin light chain 2 
                 14337 
               
               
                 Fxyd6 
                 FXYD domain-containing ion transport regulator 6 
                 59095 
               
               
                 Fzr1 
                 fizzy/cell division cycle 20 related 1 ( Drosophila ) 
                 56371 
               
               
                 Gcdh 
                 glutaryl-Coenzyme A dehydrogenase 
                 270076 
               
               
                 Gda 
                 guanine deaminase 
                 14544 
               
               
                 Gpm6b 
                 glycoprotein m6b 
                 14758 
               
               
                 Hlx1 
                 H2.0-like homeo box 1 ( Drosophila ) 
                 15284 
               
               
                 Hod 
                 homeobox only domain 
                 74318 
               
               
                 Hp1bp3 
                 heterochromatin protein 1, binding protein 3 
                 15441 
               
               
                 Icam1 
                 intercellular adhesion molecule 
                 15894 
               
               
                 Ier3 
                 immediate early response 3 
                 15937 
               
               
                 Ifit1 
                 interferon-induced protein with tetratricopeptide repeats 1 
                 15957 
               
               
                 Il4ra 
                 interleukin 4 receptor, alpha 
                 16190 
               
               
                 Isoc1 
                 Isochorismatase domain containing 1 
                 66307 
               
               
                 Itm2a 
                 integral membrane protein 2A 
                 16431 
               
               
                 Jph2 
                 junctophilin 2 
                 59091 
               
               
                 Kbtbd2 
                 kelch repeat and BTB (POZ) domain containing 2 
                 210973 
               
               
                 Kcnd2 
                 potassium voltage-gated channel, Shal-related family, member 2 
                 16508 
               
               
                 Kcne1 
                 potassium voltage-gated channel, Isk-related subfamily, member 1 
                 16509 
               
               
                 Klhdc1 
                 kelch domain containing 1 
                 271005 
               
               
                 Lcn2 
                 lipocalin 2 
                 16819 
               
               
                 Lect1 
                 leukocyte cell derived chemotaxin 1 
                 16840 
               
               
                 Letm1 
                 leucine zipper-EF-hand containing transmembrane protein 1 
                 56384 
               
               
                 Lgals3bp 
                 lectin, galactoside-binding, soluble, 3 binding protein 
                 19039 
               
               
                 Lrp1 
                 low density lipoprotein receptor-related protein 1 
                 16971 
               
               
                 Lrp11 
                 low density lipoprotein receptor-related protein 11 
                 237253 
               
               
                 Ly6a 
                 lymphocyte antigen 6 complex, locus A 
                 110454 
               
               
                 Man2a1 
                 mannosidase 2, alpha 1 
                 17158 
               
               
                 Mef2a 
                 myocyte enhancer factor 2A 
                 17258 
               
               
                 Mfge8 
                 milk fat globule-EGF factor 8 protein 
                 17304 
               
               
                 Mgp 
                 matrix Gla protein 
                 17313 
               
               
                 Mier3 
                 mesoderm induction early response 1, family member 3 
                 218613 
               
               
                 Mlf1 
                 myeloid leukemia factor 1 
                 17349 
               
               
                 Mrc1 
                 mannose receptor, C type 1 
                 17533 
               
               
                 Mt2 
                 metallothionein 2 
                 17750 
               
               
                 Mybpc3 
                 myosin binding protein C, cardiac 
                 17868 
               
               
                 Myom2 
                 myomesin 2 
                 17930 
               
               
                 Myot 
                 Myotilin 
                 58916 
               
               
                 Ndrg4 
                 N-myc downstream regulated gene 4 
                 234593 
               
               
                 Nfkbia 
                 nuclear factor of kappa light chain gene enhancer in B-cells inhibitor, 
                 18035 
               
               
                   
                 alpha 
               
               
                 Npr3 
                 natriuretic peptide receptor 3 
                 18162 
               
               
                 Nt5c2 
                 5′-nucleotidase, cytosolic II 
                 76952 
               
               
                 Oas2 
                 2′-5′ oligoadenylate synthetase 2 
                 246728 
               
               
                 Osmr 
                 oncostatin M receptor 
                 18414 
               
               
                 Pah 
                 phenylalanine hydroxylase 
                 18478 
               
               
                 Pbxip1 
                 pre-B-cell leukemia transcription factor interacting protein 1 
                 229534 
               
               
                 Pde1c 
                 Phosphodiesterase 1C 
                 18575 
               
               
                 Pdlim4 
                 PDZ and LIM domain 4 
                 30794 
               
               
                 Pgm5 
                 phosphoglucomutase 5 
                 226041 
               
               
                 Phlda1 
                 pleckstrin homology-like domain, family A, member 1 
                 21664 
               
               
                 Pkn2 
                 protein kinase N2 
                 109333 
               
               
                 Pld3 
                 phospholipase D family, member 3 
                 18807 
               
               
                 Plp2 
                 proteolipid protein 2 
                 18824 
               
               
                 Postn 
                 periostin, osteoblast specific factor 
                 50706 
               
               
                 Ppp1r3b 
                 protein phosphatase 1, regulatory (inhibitor) subunit 3B 
                 244416 
               
               
                 Prg4 
                 proteoglycan 4 (megakaryocyte stimulating factor, articular 
                 96875 
               
               
                   
                 superficial zone protein) 
               
               
                 Prkar1a 
                 protein kinase, cAMP dependent regulatory, type I, alpha 
                 19084 
               
               
                 Prkcq 
                 protein kinase C, theta 
                 18761 
               
               
                 Ranbp5 
                 RAN binding protein 5 
                 70572 
               
               
                 Rnf5 
                 ring finger protein 5 
                 54197 
               
               
                 Rpl3l 
                 ribosomal protein L3-like 
                 66211 
               
               
                 Rras 
                 Harvey rat sarcoma oncogene, subgroup R 
                 20130 
               
               
                 Rtn2 
                 reticulon 2 (Z-band associated protein) 
                 20167 
               
               
                 Rtn4 
                 reticulon 4 
                 68585 
               
               
                 Scn1b 
                 sodium channel, voltage-gated, type I, beta 
                 20266 
               
               
                 Scn4b 
                 sodium channel, type IV, beta 
                 399548 
               
               
                 Serpina3n 
                 serine (or cysteine) peptidase inhibitor, clade A, member 3N 
                 20716 
               
               
                 Serpine2 
                 serine (or cysteine) peptidase inhibitor, clade E, member 2 
                 20720 
               
               
                 Skap2 
                 src family associated phosphoprotein 2 
                 54353 
               
               
                 Slc6a6 
                 solute carrier family 6 (neurotransmitter transporter, taurine), member 6 
                 21366 
               
               
                 Snx10 
                 sorting nexin 10 
                 71982 
               
               
                 Socs3 
                 suppressor of cytokine signaling 3 
                 12702 
               
               
                 Srf 
                 serum response factor 
                 20807 
               
               
                 Svep1 
                 sushi von Willebrand factor type A, EGF and pentraxin domain 
                 64817 
               
               
                   
                 containing 1 
               
               
                 Tbc1d10c 
                 TBC1 domain family, member 10c 
                 108995 
               
               
                 Tfpi 
                 tissue factor pathway inhibitor 
                 21788 
               
               
                 Tgfb2 
                 transforming growth factor, beta 2 
                 21808 
               
               
                 Tgm2 
                 transglutaminase 2, C polypeptide 
                 21817 
               
               
                 Thbs2 
                 thrombospondin 2 
                 21826 
               
               
                 Thbs4 
                 thrombospondin 4 
                 21828 
               
               
                 Timp2 
                 tissue inhibitor of metalloproteinase 2 
                 21858 
               
               
                 Tln1 
                 talin 1 
                 21894 
               
               
                 Tmem16k 
                 transmembrane protein 16K 
                 102566 
               
               
                 Tmem176a 
                 transmembrane protein 176A 
                 66058 
               
               
                 Tmem43 
                 transmembrane protein 43 
                 74122 
               
               
                 Tnfaip8 
                 tumor necrosis factor, alpha-induced protein 8 
                 106869 
               
               
                 Tomm40 
                 translocase of outer mitochondrial membrane 40 homolog (yeast) 
                 53333 
               
               
                 Tpte2 
                 transmembrane phosphoinositide 3-phosphatase and tensin homolog 2 
                 57914 
               
               
                 Trim47 
                 tripartite motif protein 47 
                 217333 
               
               
                 Tspan13 
                 tetraspanin 13 
                 66109 
               
               
                 Tspan17 
                 tetraspanin 17 
                 74257 
               
               
                 Uap1l1 
                 UDP-N-acteylglucosamine pyrophosphorylase 1-like 1 
                 227620 
               
               
                 Ube2z 
                 ubiquitin-conjugating enzyme E2Z (putative) 
                 268470 
               
               
                 Uchl1 
                 ubiquitin carboxy-terminal hydrolase L1 
                 22223 
               
               
                 Vgll2 
                 vestigial like 2 homolog ( Drosophila ) 
                 215031 
               
               
                 Vwf 
                 Von Willebrand factor homolog 
                 22371 
               
               
                 Wdr13 
                 WD repeat domain 13 
                 73447 
               
               
                 Wisp2 
                 WNT1 inducible signaling pathway protein 2 
                 22403 
               
               
                 Wtap 
                 Wilms&#39; tumour 1-associating protein 
                 60532 
               
               
                 Yipf7 
                 Yip1 domain family, member 7 
                 75581 
               
               
                 Zadh2 
                 zinc binding alcohol dehydrogenase, domain containing 2 
                 225791 
               
               
                 Zfp697 
                 zinc finger protein 697 
                 242109 
               
               
                   
               
            
           
         
       
     
     Sixteen potential markers of heart aging were selected for confirmation of array data by qPCR. Genes were selected based on multiple factors including (but not limited to): abundant expression in the microarray experiment, robust change in expression in the B6 strain, previous reports of gene associated with cardiac aging. Using the RNA samples from B6 used in the array study, qPCR analysis revealed that all 16 genes showed a change in expression with age. These genes are shown in Table 3. Of the 16 qPCR verified markers of heart aging, qPCR further revealed that the age-related expression pattern was reversed by CR in nine markers by at least about 32%. These nine markers are C3, Ccl8, Lcn2, Mt2, Pah, Prkcq, Serpina3n, Tmeml6k, and Vgll2. 
     
       
         
           
               
               
               
             
               
                 TABLE 3 
               
               
                   
               
               
                 Gene 
                   
                   
               
               
                 Symbol 
                 Gene Title 
                 Entrez Gene 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
            
               
                 Amy1 
                 amylase 1, salivary 
                 11722 
               
               
                 Apod 
                 apolipoprotein D 
                 11815 
               
               
                 Bdh1 
                 3-hydroxybutyrate dehydrogenase, type 1 
                 71911 
               
               
                 C3 
                 complement component 3 
                 12266 
               
               
                 Casq1 
                 calsequestrin 1 
                 12372 
               
               
                 Ccl8 
                 chemokine (C-C motif) ligand 8 
                 20307 
               
               
                 Kcnd2 
                 potassium voltage-gated channel, 
                 16508 
               
               
                   
                 Shal-related family, member 2 
               
               
                 Lcn2 
                 lipocalin 2 
                 16819 
               
               
                 Mt2 
                 metallothionein 2 
                 17750 
               
               
                 Myot 
                 Myotilin 
                 58916 
               
               
                 Pah 
                 phenylalanine hydroxylase 
                 18478 
               
               
                 Prkcq 
                 protein kinase C, theta 
                 18761 
               
               
                 Serpina3n 
                 serine (or cysteine) peptidase inhibitor, 
                 20716 
               
               
                   
                 clade A, member 3N 
               
               
                 Skap2 
                 src family associated phosphoprotein 2 
                 54353 
               
               
                 Tmem 16k 
                 transmembrane protein 16K 
                 102566 
               
               
                 Vgll2 
                 vestigial like 2 homolog ( Drosophila ) 
                 215031 
               
               
                   
               
            
           
         
       
     
     The effects of the dietary interventions set forth in Example 1 on specific markers of heart aging are described below, along with reported function(s) of each marker. 
     Stress Response 
     Metallothionein 2 (Mt2): Metallothionein genes are known to be induced in response to oxidative stress, and transgenic mice overexpressing human metallothionein 2A from a cardiac-specific promoter were protected from doxorubicin cardiotoxicity. Reports indicate that Mt2 can protect the heart from oxidative injury only if it is present before induction of oxidative stress. It should be noted that this gene was identified as a possible supermarker of skeletal muscle aging from the microarray data analysis, but a change in the expression of this gene was not confirmed by qPCR. In the heart, we observed an increase in the expression of this gene with age which was partially opposed by CR. 
     Apolipoprotein D (Apod): Apolipoprotein D is a member of the lipocalin family of genes and is involved in the immune and stress responses. Apod is induced in response to stress in the brain, and we previously identified this gene as a supermarker of mouse neocortex aging. In mouse heart, this gene was increased ˜2.5-fold with age, but the increase was not prevented by CR. 
     Lipocalin 2 (Lcn2). Numerous reports indicate that lipocalin 2 is induced by inflammatory and oxidative stress, and this increase is opposed in the presence of the reactive oxygen species scavengers cysteamine and DMSO. Therefore, Lcn2 could be a useful biomarker to identify oxidative stress both in vitro and in vivo. In mouse heart, this gene was increased nearly threefold with age, and the increase was entirely blunted by CR. 
     Immune Response/Inflammation 
     Complement component 3 (C3): Complement component 3 plays a central role in the activation of complement system. Its activation is required for both classical and alternative complement activation pathways. We previously identified a closely related gene (C4) as a supermarker of aging in mouse skeletal muscle; these data are in agreement with many reports of increased immune activation with age. In mouse heart, C3 was nearly doubled in expression with age, and this increase was prevented by CR. 
     Chemokine (C-C motif) ligand 8 (Cc18): This cytokine displays chemotactic activity for monocytes, lymphocytes, basophils and eosinophils, and by recruiting leukocytes to sites of inflammation, this cytokine may contribute to tumor-associated leukocyte infiltration and to the antiviral state against HIV infection. In mouse heart, this gene was robustly increased (nearly sixfold) with age, and this increase was partially prevented by CR. 
     Protein kinase c theta (Prkcq): Protein kinase C (PKC) family members phosphorylate a wide variety of protein targets and are known to be involved in diverse cellular signaling pathways. Each member of the PKC family has a specific expression profile and is believed to play a distinct role. Prkcq is required for activation of T-lymphocytes; interestingly, a closely related gene (Prkcz) was increased in expression in skeletal muscle of multiple strains of mice, though this was not confirmed by qPCR. In heart, expression was increased nearly threefold with age and was slightly opposed by CR. 
     Serine (or cysteine) peptidase inhibitor, Glade A, member 3N (Serpina3n): This gene has been shown to suppress granzyme B-mediated apoptosis in cytotoxic T-lymphocytes, and the net effect of an increase in expression of this gene would be an inhibition of apoptosis of immune cells. In mouse heart, this gene was increased fourfold with age, and this increase was almost entirely prevented by CR. src family associated phosphoprotein 2 (Skap2): The protein encoded by this gene facilitates immune cell adhesion to sites of inflammation. In mouse heart, this gene was increased nearly twofold with age, and this increase was not affected by CR. 
     Metabolism 
     3-hydroxybutyrate dehydrogenase, type 1 (Bdh1): This gene encodes a member of the short-chain dehydrogenase/reductase gene family and is involved in ketone body production by catalyzing the interconversion of acetoacetate and 3-hydroxybutyrate, the two major ketone bodies produced during fatty acid catabolism. In mouse heart, we observed a slight increase in the expression of this gene with age, which was further elevated by CR. 
     Phenylalanine hydroxylase (Pah): Pah encodes the enzyme phenylalanine hydroxylase that is the rate-limiting step in phenylalanine catabolism to tyrosine. Deficiency of this enzyme activity results in the autosomal recessive disorder phenylketonuria. In mouse heart, the expression of this gene was increased in expression nearly tenfold with age, and CR reduced the expression of this gene to about half of that seen in old controls. 
     Heart Function 
     Amylase 1 (Amy1). Amylases are secreted proteins that hydrolyze 1,4-alpha-glucoside bonds in oligosaccharides and polysaccharides, and thus catalyze the first step in digestion of dietary starch and glycogen. The protein encoded by this gene may be related to heart function, as increased plasma levels of this protein have been reported in humans with chronic heart failure. We previously observed an increase in the expression of this gene in skeletal muscle of multiple strains of mice, though this gene was not identified as a supermarker in muscle. In mouse heart, the expression of this gene was increased ˜2.5-fold with age and was not markedly affected by CR. 
     Vestigial like 2 homolog ( Drosophila ) (Vgll2): This gene encodes a transcriptional cofactor that promotes skeletal muscle differentiation, so its expression in the heart is probably related to general cardiac muscle maintenance. In mouse heart, the expression of this gene was increased ˜7.5-fold with age, and CR prevented about half of the age-related increase in expression. 
     Myotilin (Myot): This gene encodes a protein found in the z-disc region of striated muscle and is involved in maintaining muscle structure and sarcomere organization. In humans, a mutation in this gene is associated with a form of muscular dystrophy. The expression of this gene was modestly increased with age in mouse heart, and the increase was not opposed by CR. 
     Calsequestrin (Casq1): Calsequestrin is the major calcium binding protein in the sarcoplasmic reticulum, and release of calcium ions bound to Casq1 results in muscle contraction. The expression of this gene was reduced in expression with age, possibly reflecting a general decline in muscle contraction with age. The expression of this gene was not changed by CR 
     Potassium voltage-gated channel, Sha1-related family, member 2 (Kcnd2): The protein encoded by this gene is responsible for outward potassium transport in the heart and expression of this gene is regulated by other genes sensitive to calcium status. The expression of this gene was decreased ˜25% with age, but was not changed by CR. 
     Transmembrane protein 16K (Tmem 16k): No functional data are available for this gene, however, the Gene Ontology consortium indicates that it is an integral membrane protein as inferred from electronic annotation. The expression of this gene was increased by ˜50% with age, and CR reduced the expression of this gene to below that seen in young control mice. 
     To gauge the overall effectiveness of an intervention designed to oppose age-related changes in gene expression, it was useful to generate an index which enables comparison of the effectiveness of an intervention in opposing the expression of the markers of heart aging. Below are describes two indices, though other analyses may also be devised. Each index is an average of the effect of a dietary intervention to oppose age-related changes in gene expression; the first index considers all 16 universal markers of cardiac aging described in this report; the second index considers only nine universal markers that were changed with both age and CR. For each gene, a “percent prevention” was calculated as the percent of the aging change that was opposed by an intervention. For example, a value of “100%” would indicate that the dietary intervention maintained the expression of a gene to the same level as seen in young controls. A prevention estimate greater than 100% would indicate the expression of a gene was shifted to a level that is “younger” than observed in young controls; conversely, a negative percent prevention would indicate that the expression of a gene was exacerbated beyond that seen in the old control group. The values for each gene are then averaged across a treatment, and the resulting index reveals the extent to which an intervention can oppose age-related changes in the expression of the markers of cardiac aging. For both indices, mild CR had the greatest ability to oppose age-related changes in the expression of the markers of heart aging. 
     Example 3 
     This example describes the identification of transcriptional markers of aging in adipose tissue. 
     The Affymetrix Mouse Genome 430 2.0 array was used to identify gene expression changes in epididymal adipose tissue of seven strains of mice (129, C57BL6, Balbc, C3H, CBA, DBA and B6C3HF1). 
     A significant change in expression was determined using two-tailed t-tests for young vs. old mice (P&lt;0.05, n=7 mice per strain per age group). Young mice were tested at five months of age, old mice were tested at 25 months of age. Table 4 shows the number of genes that were significantly changed with age in each strain, and Table 5 lists all genes that were changed in expression in at least five of the seven strains. 
     
       
         
           
               
               
               
             
               
                   
                 TABLE 4 
               
               
                   
                   
               
               
                   
                 Strain 
                 Number of Transcripts 
               
               
                   
                   
               
             
            
               
                   
                 129 
                 2,420 
               
               
                   
                 B6 
                 1,980 
               
               
                   
                 Balbc 
                 1,646 
               
               
                   
                 C3H 
                 5,391 
               
               
                   
                 CBA 
                 5,993 
               
               
                   
                 DBA 
                 3,788 
               
               
                   
                 F1 
                 4,187 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
               
               
             
               
                 TABLE 5 
               
               
                   
               
               
                 Gene Symbol 
                 Gene Title 
                 Entrez Gene 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
            
               
                 Amotl1 
                 angiomotin-like 1 
                 75723 
               
               
                 Cdc42ep1 
                 CDC42 effector protein (Rho GTPase binding) 1 
                 104445 
               
               
                 Chkb 
                 choline kinase beta 
                 12651 
               
               
                 AI597468 
                 expressed sequence AI597468 
                 103266 
               
               
                 Flna 
                 filamin, alpha 
                 192176 
               
               
                 Mst1r 
                 macrophage stimulating 1 receptor (c-met-related tyr 
                 19882 
               
               
                   
                 kinase) 
               
               
                 Nmb 
                 neuromedin B 
                 68039 
               
               
                 Nsd1 
                 nuclear receptor-binding SET-domain protein 1 
                 18193 
               
               
                 Nap1l5 
                 nucleosome assembly protein 1-like 5 
                 58243 
               
               
                 Otop1 
                 otopetrin 1 
                 21906 
               
               
                 Osbpl8 
                 oxysterol binding protein-like 8 
                 237542 
               
               
                 Pla2g2d 
                 phospholipase A2, group IID 
                 18782 
               
               
                 Polr2d 
                 polymerase (RNA) II (DNA directed) polypeptide D 
                 69241 
               
               
                 Pcdhb22 
                 protocadherin beta 22 
                 93893 
               
               
                 4921524J17Rik 
                 RIKEN cDNA 4921524J17 gene 
                 66714 
               
               
                 Rc3h2 
                 ring finger and CCCH-type zinc finger domains 2 
                 319817 
               
               
                 Rbms3 
                 RNA binding motif, single stranded interacting protein 
                 207181 
               
               
                 Akap2 
                 A kinase (PRKA) anchor protein 2 
                 11641 
               
               
                 Aco2 
                 aconitase 2, mitochondrial 
                 11429 
               
               
                 Acsf3 
                 acyl-CoA synthetase family member 3 
                 257633 
               
               
                 Acadl 
                 acyl-Coenzyme A dehydrogenase, long-chain 
                 11363 
               
               
                 Acadvl 
                 acyl-Coenzyme A dehydrogenase, very long chain 
                 11370 
               
               
                 Arf2 
                 ADP-ribosylation factor 2 
                 11841 
               
               
                 Arl5b 
                 ADP-ribosylation factor-like 5B 
                 75869 
               
               
                 Ankib1 
                 ankyrin repeat and IBR domain containing 1 
                 70797 
               
               
                 Anxa2 
                 annexin A2 
                 12306 
               
               
                 Aspn 
                 Asporin 
                 66695 
               
               
                 Abcd4 
                 ATP-binding cassette, sub-family D (ALD), member 4 
                 19300 
               
               
                 Bmp1 
                 bone morphogenetic protein 1 
                 12153 
               
               
                 C1galt1c1 
                 C1GALT1-specific chaperone 1 
                 59048 
               
               
                 Cidea 
                 cell death-inducing DNA fragmentation factor, alpha 
                 12683 
               
               
                   
                 subunit-like effector A 
               
               
                 Coq9 
                 coenzyme Q9 homolog (yeast) 
                 67914 
               
               
                 Col5a2 
                 collagen, type V, alpha 2 
                 12832 
               
               
                 Col6a1 
                 collagen, type VI, alpha 1 
                 12833 
               
               
                 Col6a2 
                 collagen, type VI, alpha 2 
                 12834 
               
               
                 Col18a1 
                 collagen, type XVIII, alpha 1 
                 12822 
               
               
                 Clec4n 
                 C-type lectin domain family 4, member n 
                 56620 
               
               
                 Crip2 
                 cysteine rich protein 2 
                 68337 
               
               
                 Cyb5r1 
                 cytochrome b5 reductase 1 
                 72017 
               
               
                 Cox4i1 
                 cytochrome c oxidase subunit IV isoform 1 
                 12857 
               
               
                 Cox6c 
                 cytochrome c oxidase, subunit VIc 
                 12864 
               
               
                 Dnmt3a 
                 DNA methyltransferase 3A 
                 13435 
               
               
                 Dnaja3 
                 DnaJ (Hsp40) homolog, subfamily A, member 3 
                 83945 
               
               
                 Emilin2 
                 elastin microfibril interfacer 2 
                 246707 
               
               
                 Ear11 
                 eosinophil-associated, ribonuclease A family, member 11 
                 93726 
               
               
                 Efemp1 
                 epidermal growth factor-containing fibulin-like 
                 216616 
               
               
                   
                 extracellular matrix protein 1 
               
               
                 Fbxo6 
                 F-box protein 6 
                 50762 
               
               
                 Fndc3b 
                 fibronectin type III domain containing 3B 
                 72007 
               
               
                 Fyttd1 
                 forty-two-three domain containing 1 
                 69823 
               
               
                 Gcnt2 
                 glucosaminyl (N-acetyl) transferase 2, I-branching 
                 14538 
               
               
                   
                 enzyme 
               
               
                 Gpd2 
                 glycerol phosphate dehydrogenase 2, mitochondrial 
                 14571 
               
               
                 Gnb4 
                 guanine nucleotide binding protein (G protein), beta 4 
                 14696 
               
               
                 Hddc3 
                 HD domain containing 3 
                 68695 
               
               
                 Hfe 
                 Hemochromatosis 
                 15216 
               
               
                 Hadha 
                 hydroxyacyl-Coenzyme A dehydrogenase/3-ketoacyl- 
                 97212 
               
               
                   
                 Coenzyme A thiolase/enoyl-Coenzyme A hydratase 
               
               
                   
                 (trifunctional protein), alpha subunit 
               
               
                 C130006E23 
                 Hypothetical protein C130006E23 
                 331563 
               
               
                 Hif1a 
                 hypoxia inducible factor 1, alpha subunit 
                 15251 
               
               
                 Itga6 
                 integrin alpha 6 
                 16403 
               
               
                 Irf7 
                 interferon regulatory factor 7 
                 54123 
               
               
                 Ldhb 
                 lactate dehydrogenase B 
                 16832 
               
               
                 Lace1 
                 lactation elevated 1 
                 215951 
               
               
                 Lxn 
                 Latexin 
                 17035 
               
               
                 Lenep 
                 lens epithelial protein 
                 57275 
               
               
                 Lims1 
                 LIM and senescent cell antigen-like domains 1 
                 110829 
               
               
                 Macrod1 
                 MACRO domain containing 1 
                 107227 
               
               
                 Msln 
                 Mesothelin 
                 56047 
               
               
                 Myadm 
                 myeloid-associated differentiation marker 
                 50918 
               
               
                 Gnptg 
                 N-acetylglucosamine-1-phosphotransferase, gamma 
                 214505 
               
               
                   
                 subunit 
               
               
                 Ndufa10 
                 NADH dehydrogenase (ubiquinone) 1 alpha subcomplex 
                 67273 
               
               
                   
                 10 
               
               
                 Ndufa5 
                 NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 5 
                 68202 
               
               
                 Ntn4 
                 netrin 4 
                 57764 
               
               
                 Ntrk3 
                 neurotrophic tyrosine kinase, receptor, type 3 
                 18213 
               
               
                 Nmnat3 
                 nicotinamide nucleotide adenylyltransferase 3 
                 74080 
               
               
                 Nid1 
                 nidogen 1 
                 18073 
               
               
                 Ogdh 
                 oxoglutarate dehydrogenase (lipoamide) 
                 18293 
               
               
                 Ptcd3 
                 pentatricopeptide repeat domain 3 
                 69956 
               
               
                 Ppic 
                 peptidylprolyl isomerase C 
                 19038 
               
               
                 Pxdn 
                 peroxidasin homolog ( Drosophila ) 
                 69675 
               
               
                 Kcnj15 
                 potassium inwardly-rectifying channel, subfamily J, 
                 16516 
               
               
                   
                 member 15 
               
               
                 EG665378 
                 predicted gene, EG665378 
                 665378 
               
               
                 Prss23 
                 protease, serine, 23 
                 76453 
               
               
                 Pim1 
                 proviral integration site 1 
                 18712 
               
               
                 Rmnd1 
                 required for meiotic nuclear division 1 homolog ( S. cerevisiae ) 
                 66084 
               
               
                 Rp2h 
                 retinitis pigmentosa 2 homolog (human) 
                 19889 
               
               
                 Arhgef10 
                 Rho guanine nucleotide exchange factor (GEF) 10 
                 234094 
               
               
                 Rhbdl3 
                 rhomboid, veinlet-like 3 ( Drosophila ) 
                 246104 
               
               
                 1810013D10Rik 
                 RIKEN cDNA 1810013D10 gene 
                 66278 
               
               
                 2010005J08Rik 
                 RIKEN cDNA 2010005J08 gene 
                 72046 
               
               
                 2810482I07Rik 
                 RIKEN cDNA 2810482I07 gene 
                 67243 
               
               
                 4933439C20Rik 
                 RIKEN cDNA 4933439C20 gene 
                 66776 
               
               
                 9530058B02Rik 
                 RIKEN cDNA 9530058B02 gene 
                 68241 
               
               
                 D830012I24Rik 
                 RIKEN cDNA D830012I24 gene 
                 320070 
               
               
                 Sec23a 
                 SEC23A ( S. cerevisiae ) 
                 20334 
               
               
                 Sema4a 
                 sema domain, immunoglobulin domain (Ig), 
                 20351 
               
               
                   
                 transmembrane domain (TM) and short cytoplasmic 
               
               
                   
                 domain, (semaphorin) 4A 
               
               
                 Serpina3n 
                 serine (or cysteine) peptidase inhibitor, clade A, member 
                 20716 
               
               
                   
                 3N 
               
               
                 Serpinf1 
                 serine (or cysteine) peptidase inhibitor, clade F, member 1 
                 20317 
               
               
                 Serhl 
                 serine hydrolase-like 
                 68607 
               
               
                 LOC100047339 
                 similar to lysyl oxidase-like 2 
                 100047339 
               
               
                 LOC100046998 
                 similar to optic atrophy 1 (autosomal dominant) 
                 100046998 
               
               
                 LOC100046740 
                 similar to Secreted acidic cysteine rich glycoprotein 
                 100046740 
               
               
                 Smcr7 
                 Smith-Magenis syndrome chromosome region, candidate 
                 237781 
               
               
                   
                 7 homolog (human) 
               
               
                 Slc46a3 
                 solute carrier family 46, member 3 
                 71706 
               
               
                 Slc6a13 
                 solute carrier family 6 (neurotransmitter transporter, 
                 14412 
               
               
                   
                 GABA), member 13 
               
               
                 Stard3nl 
                 STARD3 N-terminal like 
                 76205 
               
               
                 Sdhb 
                 succinate dehydrogenase complex, subunit B, iron sulfur 
                 67680 
               
               
                   
                 (Ip) 
               
               
                 Suclg1 
                 succinate-CoA ligase, GDP-forming, alpha subunit 
                 56451 
               
               
                 Sucla2 
                 succinate-Coenzyme A ligase, ADP-forming, beta subunit 
                 20916 
               
               
                 Supv3l1 
                 suppressor of var1, 3-like 1 ( S. cerevisiae ) 
                 338359 
               
               
                 Syngr1 
                 synaptogyrin 1 
                 20972 
               
               
                 Sycp3 
                 synaptonemal complex protein 3 
                 20962 
               
               
                 Tspan2 
                 tetraspanin 2 
                 70747 
               
               
                 Tanc1 
                 tetratricopeptide repeat, ankyrin repeat and coiled-coil 
                 66860 
               
               
                   
                 containing 1 
               
               
                 Timm44 
                 translocase of inner mitochondrial membrane 44 
                 21856 
               
               
                 Usp47 
                 ubiquitin specific peptidase 47 
                 74996 
               
               
                 Uckl1 
                 uridine-cytidine kinase 1-like 1 
                 68556 
               
               
                 Vps13a 
                 vacuolar protein sorting 13A (yeast) 
                 271564 
               
               
                 Vash1 
                 vasohibin 1 
                 238328 
               
               
                 Vcan 
                 Versican 
                 13003 
               
               
                 Wwtr1 
                 WW domain containing transcription regulator 1 
                 97064 
               
               
                 Zkscan17 
                 zinc finger with KRAB and SCAN domains 17 
                 268417 
               
               
                   
               
            
           
         
       
     
     Thirty-one potential markers of adipose tissue aging were selected for confirmation of array data by qPCR. In selecting candidate genes for validation by RT PCR, genes changed in all strains were given highest priority for further testing. Other considerations included avoiding genes with low expression (as judged by the average signal intensity from the microarray experiment) and avoiding genes that did not show change in expression by at least 50% (&lt;1.5 or &gt;1.5 fold change). Four genes that changed in all seven strains did not have a commercially available primer, and thus these genes could not be screened by qPCR. Beta actin (Actb) was not changed in any strain and served as the housekeeping gene for qPCR analyses. 
     For the remaining 27 genes identified as candidate markers of adipose tissue aging, qPCR analysis was used to test samples from the nutrient feeding study of Example 1 for validation of an aging change and opposition of the aging change by mild CR. A statistically significant change in expression with age was observed for 13 of the 27 genes when analyzed by qPCR. These are shown in Table 6. Eight of these 13 genes were further determined to be changed by age, and at least about 33% of the aging change was prevented by CR; these eight genes are: Aspn, Col6a2, Crip2, Emilin2, Otop1, Pla2g2d, Rhbd3and Slc6a13. 
     
       
         
           
               
               
               
             
               
                 TABLE 6 
               
               
                   
               
               
                 Gene Symbol 
                 Gene Title 
                 Entrez Gene 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
            
               
                 Aspn 
                 Asporin 
                 66695 
               
               
                 Clec4n 
                 C-type lectin domain family 4, member n 
                 56620 
               
               
                 Col6a2 
                 Collagen, type VI, alpha 2 
                 12834 
               
               
                 Col18a1 
                 Collagen, type XVIII, alpha 1 
                 12822 
               
               
                 Cox8b 
                 Cytochrome c oxidase, subunit VIIIb 
                 12869 
               
               
                 Crip2 
                 Cysteine rich protein 2 
                 68337 
               
               
                 Ear11 
                 Eosinophil-associated, ribonuclease A 
                 93726 
               
               
                   
                 family, member 11 
               
               
                 Emilin2 
                 Elastin microfibril interfacer 2 
                 246707 
               
               
                 Otop1 
                 Otopetrin 1 
                 21906 
               
               
                 Pla2g2d 
                 Phospholipase A2, group IID 
                 18782 
               
               
                 Rhbdl3 
                 Rhomboid, veinlet-like 3 ( Drosophila ) 
                 246104 
               
               
                 Slc6a13 
                 Solute carrier family 6 (neurotransmitter 
                 14412 
               
               
                   
                 transporter, GABA), member 13 
               
               
                 Sycp3 
                 Synaptonemal complex protein 3 
                 20962 
               
               
                   
               
            
           
         
       
     
     The functional significance of the markers of adipose tissue aging is discussed below. 
     Growth Factor-Responsive Genes 
     Asporin (Aspn): Transforming growth factor beta (TGF beta) is a secreted protein that plays a role in maintenance of the extracellularmatrix (ECM) by regulating the expression of genes involved in cytoskeletal maintenance (1). Asporin is a component of the ECM, and expression of asporin has been shown to be induced by TGF beta (2) in articular cartilage; we observed a decrease in asporin expression (2.0 fold) in adipose tissue with age. Taken together, these data suggest a decline in the maintenance of the ECM with age in adipose tissue. This decline may be prevented by CR, as the age related decline in Aspn expression was almost completely prevented by CR (1.8 fold increase in Old CR vs. Old Control mice). 
     Cysteine rich protein 2 (Crip2): Although little is known about the function of Crip2, it appears to belong to a family of proteins involved in cytoskeletal remodeling (3), and as with asporin (above), Crip2 expression has been shown to be induced by TGF beta (4). The pattern of Crip2 expression was very similar to that observed with asporin, including a significant change in expression with age (2.3 fold) that was prevented by CR (2.0 fold). 
     Rhomboid, veinlet-like 3 (Rhbdl3): The protein encoded by the Rhbdl3 gene has been characterized as the most evolutionarily conserved cDNA of the  Drosophila  gene rho which is modulate by epidermal growth factor signaling, and it plays a role in neural development in mice. This gene was robustly decreased in expression with age and CR completely prevented the age related change in the expression of this gene (6.2 fold change with age and 7.2 fold increase with CR). 
     In summary, the above three genes represent universal markers of adipose tissue aging that are regulated by growth factors, the expression of which can be modulated by diet. 
     Decrease in ECM Microfiber Assembly with Age 
     Collagen, type VI, alpha 2 (Col6a2): Collagen VI is a component of the extracellular matrix and mutations in the Col6a2 gene in humans are associated with several congenital myopathies because of disrupted microfiber formation (6, 7). We have previously shown in our study of universal markers of mouse skeletal muscle aging that several collagen genes (Col1a1, Col1a2 and Col3a1) are decreased in expression in with age, and that this decline is opposed by CR. A significant reduction (2.1 fold decrease) in Col6a2 in adipose tissue suggesting a decreased maintenance of the ECM with age due to decreased microfiber assembly. 
     Elastin microfibril interfacer 2 (Emilin2). Little is known about the function of Emilin2, although it is reported to be synthesized and located in the ECM. Emilin2 may play a role in cell death via the extrinsic apoptotic pathway, as binding of apoptotic factors to Emilin2 results in caspase activation. Alternatively, others have suggested that elevated serum levels of the protein encoded by this gene may be a biomarker of ovarian cancer. In any case, a significant reduction in Emilin2 expression was observed with age in adipose tissue (-2.1 fold change with age), and CR opposed ˜35% of the aging change. 
     Increased Inflammation with Age 
     Phospholipase A2, group IID (Pla2g2d): Phospholipases A2 (PLA2) are well-known for their ability to mobilize fatty acids from phospholipids which are subsequently converted to proinflammatory prostaglandins and leukotrienes (11). Interestingly, elevated levels of PLA2 may have consequences beyond lipid mobilization, as increased extracellular PLA2 is associated with increased levels of the proinflammatory cytokines TNFa and interleukin 1 (12). Expression of the Pla2g2d gene increased 3.1 fold with age and was partially (but not significantly) prevented by CR (1.4 fold change in expression). 
     Less Characterized Genes 
     Otopetrin 1 (Otop1): The only report regarding the function of Otop1 shows that it is important for the formation of otoconia, structures of the inner ear that are responsible for perception of gravity and acceleration (13). Because these structures are formed by mineralization of calcium carbonate, it is likely that the function of this gene in adipose tissue is related to calcium homeostasis. Otop1 expression was increased in expression 3.0 fold with age and was partially (but not significantly) opposed by CR,(1.5 fold change in expression). 
     Solute carrier family 6, member 13 (Slc6a13): There are no reports regarding the function of this gene in mice or humans. However, it can be inferred from a single study in rats (14) that the protein encoded by the Slc6a13 gene is involved in transport of the neurotransmitter gamma aminobutyric acid (GABA). Slc6a13 was decreased in expression (1.8 fold change) with age, and this was partially (but not significantly) opposed by CR (1.3 fold change relative to Old Controls). 
     To gauge the overall effectiveness of an intervention designed to oppose age-related changes in the universal markers of aging, it was useful to generate an index which allows one to compare how an intervention opposes the expression of the universal markers of aging. Accordingly, an “aging prevention index” was calculated to describe the average effect of an intervention on age related changes in the expression of the eight universal markers of adipose tissue aging. 
     For each of the eight universal markers of adipose tissue aging, a “percent prevention” was calculated as the percent of the aging change that was opposed by an intervention. For example, a value of “100%” would indicate that the dietary intervention maintained the expression of a gene at the same level as seen in young controls. A prevention estimate greater than 100% would indicate the expression of a gene was shifted to a level that is “younger” than observed in young controls; conversely, a negative percent prevention would indicate that the expression of a gene was exacerbated beyond that seen in the Old Control group. The values for each gene are then averaged across a treatment, and the resulting index reveals the overall extent to which an intervention can oppose age-related changes at the transcriptional level. 
     Example 4 
     This example describes the identification of transcriptional markers of aging in brain tissue. 
     The Affymetrix Mouse Genome 430 2.0 array was used to identify gene expression changes in neocortex of six strains of mice (C57BL6, Balbc, C3H, CBA, DBA and B6C3HF1). A significant change in expression was determined using two-tailed t-tests for young versus old mice (P&lt;0.05, n=7 mice per strain per age group). Young mice were tested at five months of age, old mice were tested at 25 months of age. 
     Table 7 shows the number of genes that were significantly changed with age in each strain. Table 8 lists all genes that were changed in expression in at least three of the six strains. 
     
       
         
           
               
               
               
             
               
                   
                 TABLE 7 
               
               
                   
                   
               
               
                   
                 Strain 
                 Number of Transcripts 
               
               
                   
                   
               
             
            
               
                   
               
            
           
           
               
               
               
            
               
                   
                 B6 
                 1,029 
               
               
                   
                 Balbc 
                 415 
               
               
                   
                 C3H 
                 2,151 
               
               
                   
                 CBA 
                 1,542 
               
               
                   
                 DBA 
                 1,604 
               
               
                   
                 F1 
                 1,957 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
               
               
             
               
                 TABLE 8 
               
               
                   
               
               
                 Gene Symbol 
                 Gene Title 
                 Entrez Gene 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
            
               
                 Abca8a 
                 ATP-binding cassette, sub-family A (ABC1), member 8a 
                 217258 
               
               
                 Adamtsl4 
                 ADAMTS-like 4 
                 229595 
               
               
                 Agxt2l1 
                 alanine-glyoxylate aminotransferase 2-like 1 
                 71760 
               
               
                 AI465270 
                 expressed sequence AI465270 
                 102097 
               
               
                 AI838057 
                 expressed sequence AI838057 
                 101160 
               
               
                 Ak3l1 
                 adenylate kinase 3 alpha-like 1 
                 11639 
               
               
                 Alad 
                 aminolevulinate, delta-, dehydratase 
                 17025 
               
               
                 Alkbh3 
                 alkB, alkylation repair homolog 3 (E. coli) 
                 69113 
               
               
                 Ankrd17 
                 ankyrin repeat domain 17 
                 81702 
               
               
                 Ankrd39 
                 ankyrin repeat domain 39 
                 109346 
               
               
                 Anln 
                 anillin, actin binding protein (scraps homolog,  Drosophila ) 
                 68743 
               
               
                 Anxa4 
                 annexin A4 
                 11746 
               
               
                 Anxa5 
                 annexin A5 
                 11747 
               
               
                 Apod 
                 apolipoprotein D 
                 11815 
               
               
                 Arid4a 
                 AT rich interactive domain 4A (Rbp1 like) 
                 238247 
               
               
                 Arl2 
                 ADP-ribosylation factor-like 2 
                 56327 
               
               
                 Arpc1b 
                 actin related protein 2/3 complex, subunit 1B 
                 11867 
               
               
                 Aspa 
                 aspartoacylase (aminoacylase) 2 
                 11484 
               
               
                 Atp2b1 
                 ATPase, Ca++ transporting, plasma membrane 1 
                 67972 
               
               
                 B2m 
                 beta-2 microglobulin 
                 12010 
               
               
                 BC035295 
                 cDNA sequence BC035295 
                 207785 
               
               
                 BC061194 
                 cDNA sequence BC061194 
                 381350 
               
               
                 Bcor 
                 Bcl6 interacting corepressor 
                 71458 
               
               
                 Bid 
                 BH3 interacting domain death agonist 
                 12122 
               
               
                 Bok 
                 Bcl-2-related ovarian killer protein 
                 51800 
               
               
                 C030017B01Rik 
                 RIKEN cDNA C030017B01 gene 
                 77524 
               
               
                 C130006E23 
                 Hypothetical protein C130006E23 
                 331563 
               
               
                 C1qa 
                 complement component 1, q subcomponent, alpha polypeptide 
                 12259 
               
               
                 C1qb 
                 complement component 1, q subcomponent, beta polypeptide 
                 12260 
               
               
                 C1qc 
                 complement component 1, q subcomponent, C chain 
                 12262 
               
               
                 C330006P03Rik 
                 RIKEN cDNA C330006P03 gene 
                 320588 
               
               
                 Calb2 
                 calbindin 2 
                 12308 
               
               
                 Camsap1l1 
                 calmodulin regulated spectrin-associated protein 1-like 1 
                 67886 
               
               
                 Capns1 
                 calpain, small subunit 1 
                 12336 
               
               
                 Cast 
                 Calpastatin 
                 12380 
               
               
                 Ccnd2 
                 cyclin D2 
                 12444 
               
               
                 Ccs 
                 copper chaperone for superoxide dismutase 
                 12460 
               
               
                 Cd14 
                 CD14 antigen 
                 12475 
               
               
                 Cd52 
                 CD52 antigen 
                 23833 
               
               
                 Cd59a 
                 CD59a antigen 
                 12509 
               
               
                 Cd68 
                 CD68 antigen 
                 12514 
               
               
                 Cd74 
                 CD74 antigen (invariant polypeptide of major histocompatibility 
                 16149 
               
               
                   
                 complex, class II antigen-associated) 
               
               
                 Cd81 
                 CD 81 antigen 
                 12520 
               
               
                 Cd9 
                 CD9 antigen 
                 12527 
               
               
                 Cdh11 
                 cadherin 11 
                 12552 
               
               
                 Cdh8 
                 cadherin 8 
                 12564 
               
               
                 Cdkl2 
                 cyclin-dependent kinase-like 2 (CDC2-related kinase) 
                 53886 
               
               
                 Cebpd 
                 CCAAT/enhancer binding protein (C/EBP), delta 
                 12609 
               
               
                 Cend1 
                 cell cycle exit and neuronal differentiation 1 
                 57754 
               
               
                 Chkb 
                 choline kinase beta 
                 12651 
               
               
                 Cited1 
                 Cbp/p300-interacting transactivator with Glu/Asp-rich carboxy- 
                 12705 
               
               
                   
                 terminal domain 1 
               
               
                 Clec7a 
                 C-type lectin domain family 7, member a 
                 56644 
               
               
                 Cmtm3 
                 CKLF-like MARVEL transmembrane domain containing 3 
                 68119 
               
               
                 Cmtm5 
                 CKLF-like MARVEL transmembrane domain containing 5 
                 67272 
               
               
                 Cnksr2 
                 connector enhancer of kinase suppressor of Ras 2 
                 245684 
               
               
                 Cnn3 
                 calponin 3, acidic 
                 71994 
               
               
                 Cntn6 
                 contactin 6 
                 53870 
               
               
                 Commd9 
                 COMM domain containing 9 
                 76501 
               
               
                 Comtd1 
                 catechol-O-methyltransferase domain containing 1 
                 69156 
               
               
                 Cope 
                 coatomer protein complex, subunit epsilon 
                 59042 
               
               
                 Coro1b 
                 coronin, actin binding protein 1B 
                 23789 
               
               
                 Crim1 
                 cysteine rich transmembrane BMP regulator 1 (chordin like) 
                 50766 
               
               
                 Cst7 
                 cystatin F (leukocystatin) 
                 13011 
               
               
                 Ctsd 
                 cathepsin D 
                 13033 
               
               
                 Ctss 
                 cathepsin S 
                 13040 
               
               
                 Ctsz 
                 cathepsin Z 
                 64138 
               
               
                 Cyb5r1 
                 cytochrome b5 reductase 1 
                 72017 
               
               
                 Cyba 
                 cytochrome b-245, alpha polypeptide 
                 13057 
               
               
                 Cyp20a1 
                 cytochrome P450, family 20, subfamily A, polypeptide 1 
                 77951 
               
               
                 Cyp27a1 
                 cytochrome P450, family 27, subfamily a, polypeptide 1 
                 104086 
               
               
                 D12Ertd647e 
                 DNA segment, Chr 12, ERATO Doi 647, expressed 
                 52668 
               
               
                 D19Wsu12e 
                 DNA segment, Chr 19, Wayne State University 12, expressed 
                 226090 
               
               
                 D1Ertd471e 
                 DNA segment, Chr 1, ERATO Doi 471, expressed 
                 27877 
               
               
                 Ddc 
                 dopa decarboxylase 
                 13195 
               
               
                 Ddt 
                 D-dopachrome tautomerase 
                 13202 
               
               
                 Dhrs1 
                 dehydrogenase/reductase (SDR family) member 1 
                 52585 
               
               
                 Diap2 
                 diaphanous homolog 2 ( Drosophila ) 
                 54004 
               
               
                 Dnalc1 
                 dynein, axonemal, light chain 1 
                 105000 
               
               
                 Dusp6 
                 dual specificity phosphatase 6 
                 67603 
               
               
                 E330009J07Rik 
                 RIKEN cDNA E330009J07 gene 
                 243780 
               
               
                 Efha1 
                 EF hand domain family A1 
                 68514 
               
               
                 EG434128 
                 predicted gene, EG434128 
                 434128 
               
               
                 Egr1 
                 early growth response 1 
                 13653 
               
               
                 Egr3 
                 early growth response 3 
                 13655 
               
               
                 Epha4 
                 Eph receptor A4 
                 13838 
               
               
                 Ephx1 
                 epoxide hydrolase 1, microsomal 
                 13849 
               
               
                 Ero1lb 
                 ERO1-like beta ( S. cerevisiae ) 
                 67475 
               
               
                 Eya4 
                 eyes absent 4 homolog ( Drosophila ) 
                 14051 
               
               
                 Fand2a 
                 fumarylacetoacetate hydrolase domain containing 2A 
                 68126 
               
               
                 Fbxo39 
                 F-box protein 39 
                 327959 
               
               
                 Fcer1g 
                 Fc receptor, IgE, high affinity I, gamma polypeptide 
                 14127 
               
               
                 Fcgr3 
                 Fc receptor, IgG, low affinity III 
                 14131 
               
               
                 Fnbp1l 
                 formin binding protein 1-like 
                 214459 
               
               
                 Fndc3b 
                 fibronectin type III domain containing 3B 
                 72007 
               
               
                 Foxg1 
                 forkhead box G1 
                 15228 
               
               
                 Fxyd1 
                 FXYD domain-containing ion transport regulator 1 
                 56188 
               
               
                 Gabrb3 
                 gamma-aminobutyric acid (GABA-A) receptor, subunit beta 3 
                 14402 
               
               
                 Gal 
                 Galanin 
                 14419 
               
               
                 Gatm 
                 glycine amidinotransferase (L-arginine: glycine 
                 67092 
               
               
                   
                 amidinotransferase) 
               
               
                 Gda 
                 guanine deaminase 
                 14544 
               
               
                 Gfap 
                 glial fibrillary acidic protein 
                 14580 
               
               
                 Golph2 
                 golgi phosphoprotein 2 
                 105348 
               
               
                 Gprin3 
                 GPRIN family member 3 
                 243385 
               
               
                 Gpx3 
                 glutathione peroxidase 3 
                 14778 
               
               
                 Grhpr 
                 glyoxylate reductase/hydroxypyruvate reductase 
                 76238 
               
               
                 Gria3 
                 glutamate receptor, ionotropic, AMPA3 (alpha 3) 
                 53623 
               
               
                 Gstm1 
                 glutathione S-transferase, mu 1 
                 14862 
               
               
                 Gstm6 
                 glutathione S-transferase, mu 6 
                 14867 
               
               
                 Gstm7 
                 glutathione S-transferase, mu 7 
                 68312 
               
               
                 Gsto1 
                 glutathione S-transferase omega 1 
                 14873 
               
               
                 Gstt3 
                 glutathione S-transferase, theta 3 
                 103140 
               
               
                 Gtf2f1 
                 general transcription factor IIF, polypeptide 1 
                 98053 
               
               
                 H2-D1 
                 histocompatibility 2, D region locus 1 
                 14964 
               
               
                 H2-M3 
                 histocompatibility 2, M region locus 3 
                 14991 
               
               
                 Hapln2 
                 hyaluronan and proteoglycan link protein 2 
                 73940 
               
               
                 Hebp2 
                 heme binding protein 2 
                 56016 
               
               
                 Hectd2 
                 HECT domain containing 2 
                 226098 
               
               
                 Helz 
                 helicase with zinc finger domain 
                 78455 
               
               
                 Homer1 
                 homer homolog 1 ( Drosophila ) 
                 26556 
               
               
                 Hsd17b11 
                 hydroxysteroid (17-beta) dehydrogenase 11 
                 114664 
               
               
                 Hsd3b7 
                 hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta- 
                 101502 
               
               
                   
                 isomerase 7 
               
               
                 Idh2 
                 isocitrate dehydrogenase 2 (NADP+), mitochondrial 
                 269951 
               
               
                 Ier3 
                 immediate early response 3 
                 15937 
               
               
                 Ifit3 
                 interferon-induced protein with tetratricopeptide repeats 3 
                 15959 
               
               
                 Ifitm3 
                 interferon induced transmembrane protein 3 
                 66141 
               
               
                 Ikbkg 
                 inhibitor of kappaB kinase gamma 
                 16151 
               
               
                 Il33 
                 interleukin 33 
                 77125 
               
               
                 Imp4 
                 IMP4, U3 small nucleolar ribonucleoprotein, homolog (yeast) 
                 27993 
               
               
                 Itih3 
                 inter-alpha trypsin inhibitor, heavy chain 3 
                 16426 
               
               
                 Jmjd1c 
                 jumonji domain containing 1C 
                 108829 
               
               
                 Kcnf1 
                 potassium voltage-gated channel, subfamily F, member 1 
                 382571 
               
               
                 Kcnj16 
                 potassium inwardly-rectifying channel, subfamily J, member 16 
                 16517 
               
               
                 Kcnv1 
                 potassium channel, subfamily V, member 1 
                 67498 
               
               
                 Klf10 
                 Kruppel-like factor 10 
                 21847 
               
               
                 Klf9 
                 Kruppel-like factor 9 
                 16601 
               
               
                 Klk6 
                 kallikrein related-peptidase 6 
                 19144 
               
               
                 Lcat 
                 lecithin cholesterol acyltransferase 
                 16816 
               
               
                 Lcp1 
                 lymphocyte cytosolic protein 1 
                 18826 
               
               
                 Lefty1 
                 left right determination factor 1 
                 13590 
               
               
                 Lgals3 
                 lectin, galactose binding, soluble 3 
                 16854 
               
               
                 Lgals8 
                 lectin, galactose binding, soluble 8 
                 56048 
               
               
                 Lhfp 
                 lipoma HMGIC fusion partner 
                 108927 
               
               
                 LOC629605 
                 hypothetical protein LOC629605 
                 629605 
               
               
                 LOC672274 
                 similar to Transcription factor SOX-4 
                 672274 
               
               
                 Loh11cr2a 
                 loss of heterozygosity, 11, chromosomal region 2, gene A 
                 67776 
               
               
                   
                 homolog (human) 
               
               
                 Lyzs 
                 Lysozyme 
                 17105 
               
               
                 M6prbp1 
                 mannose-6-phosphate receptor binding protein 1 
                 66905 
               
               
                 Malat1 
                 Metastasis associated lung adenocarcinoma transcript 1 (non- 
                 72289 
               
               
                   
                 coding RNA) 
               
               
                 Marcks 
                 myristoylated alanine rich protein kinase C substrate 
                 17118 
               
               
                 Mccc2 
                 methylcrotonoyl-Coenzyme A carboxylase 2 (beta) 
                 78038 
               
               
                 Mdk 
                 Midkine 
                 17242 
               
               
                 Mfhas1 
                 malignant fibrous histiocytoma amplified sequence 1 
                 52065 
               
               
                 Mrpl10 
                 mitochondrial ribosomal protein L10 
                 107732 
               
               
                 Mysm1 
                 myb-like, SWIRM and MPN domains 1 
                 320713 
               
               
                 Nfyb 
                 nuclear transcription factor-Y beta 
                 18045 
               
               
                 Nol4 
                 nucleolar protein 4 
                 319211 
               
               
                 Ntsr2 
                 neurotensin receptor 2 
                 18217 
               
               
                 Optn 
                 Optineurin 
                 71648 
               
               
                 Pank3 
                 pantothenate kinase 3 
                 211347 
               
               
                 Pcdh17 
                 protocadherin 17 
                 219228 
               
               
                 Pcdh8 
                 protocadherin 8 
                 18530 
               
               
                 Pcdhb2 
                 protocadherin beta 2 
                 93873 
               
               
                 Pcdhb21 
                 protocadherin beta 21 
                 93892 
               
               
                 Pcdhb9 
                 protocadherin beta 9 
                 93880 
               
               
                 Pcolce2 
                 procollagen C-endopeptidase enhancer 2 
                 76477 
               
               
                 Peci 
                 peroxisomal delta3, delta2-enoyl-Coenzyme A isomerase 
                 23986 
               
               
                 Phlda1 
                 pleckstrin homology-like domain, family A, member 1 
                 21664 
               
               
                 Plek 
                 Pleckstrin 
                 56193 
               
               
                 Plekhf1 
                 pleckstrin homology domain containing, family F (with FYVE 
                 72287 
               
               
                   
                 domain) member 1 
               
               
                 Plxna2 
                 plexin A2 
                 18845 
               
               
                 Pmp22 
                 peripheral myelin protein 
                 18858 
               
               
                 Ppap2c 
                 phosphatidic acid phosphatase type 2c 
                 50784 
               
               
                 Ppm1d 
                 protein phosphatase 1D magnesium-dependent, delta isoform 
                 53892 
               
               
                 Ppp5c 
                 protein phosphatase 5, catalytic subunit 
                 19060 
               
               
                 Prdm5 
                 PR domain containing 5 
                 70779 
               
               
                 Psmb8 
                 proteosome (prosome, macropain) subunit, beta type 8 (large 
                 16913 
               
               
                   
                 multifunctional peptidase 7) 
               
               
                 Psmb9 
                 proteosome (prosome, macropain) subunit, beta type 9 (large 
                 16912 
               
               
                   
                 multifunctional peptidase 2) 
               
               
                 Ptpn12 
                 protein tyrosine phosphatase, non-receptor type 12 
                 19248 
               
               
                 Pvrl3 
                 poliovirus receptor-related 3 
                 58998 
               
               
                 Rab11fip2 
                 RAB11 family interacting protein 2 (class I) 
                 74998 
               
               
                 Rabl3 
                 RAB, member of RAS oncogene family-like 3 
                 67657 
               
               
                 Rcbtb2 
                 regulator of chromosome condensation (RCC1) and BTB (POZ) 
                 105670 
               
               
                   
                 domain containing protein 2 
               
               
                 Sbno1 
                 sno, strawberry notch homolog 1 ( Drosophila ) 
                 243272 
               
               
                 Shox2 
                 short stature homeobox 2 
                 20429 
               
               
                 Slc14a1 
                 solute carrier family 14 (urea transporter), member 1 
                 108052 
               
               
                 Slc15a4 
                 solute carrier family 15, member 4 
                 100561 
               
               
                 Slit2 
                 slit homolog 2 ( Drosophila ) 
                 20563 
               
               
                 Slit3 
                 slit homolog 3 ( Drosophila ) 
                 20564 
               
               
                 Sord 
                 sorbitol dehydrogenase 
                 20322 
               
               
                 Sos2 
                 Son of sevenless homolog 2 ( Drosophila ) 
                 20663 
               
               
                 Sparc 
                 secreted acidic cysteine rich glycoprotein 
                 20692 
               
               
                 Spp1 
                 secreted phosphoprotein 1 
                 20750 
               
               
                 Spred2 
                 sprouty-related, EVH1 domain containing 2 
                 114716 
               
               
                 Ssbp3 
                 single-stranded DNA binding protein 3 
                 72475 
               
               
                 Sstr4 
                 somatostatin receptor 4 
                 20608 
               
               
                 St18 
                 suppression of tumorigenicity 18 
                 240690 
               
               
                 Suhw4 
                 suppressor of hairy wing homolog 4 ( Drosophila ) 
                 235469 
               
               
                 Tcea2 
                 transcription elongation factor A (SII), 2 
                 21400 
               
               
                 Th 
                 tyrosine hydroxylase 
                 21823 
               
               
                 Tmbim1 
                 transmembrane BAX inhibitor motif containing 1 
                 69660 
               
               
                 Tmem144 
                 transmembrane protein 144 
                 70652 
               
               
                 Tmem176a 
                 transmembrane protein 176A 
                 66058 
               
               
                 Tmem176b 
                 transmembrane protein 176B 
                 65963 
               
               
                 Tnnt1 
                 troponin T1, skeletal, slow 
                 21955 
               
               
                 Tnnt2 
                 troponin T2, cardiac 
                 21956 
               
               
                 Trf 
                 Transferring 
                 22041 
               
               
                 Tspan4 
                 tetraspanin 4 
                 64540 
               
               
                 Tspo 
                 translocator protein 
                 12257 
               
               
                 Ube2j1 
                 ubiquitin-conjugating enzyme E2, J1 
                 56228 
               
               
                 Ubtd2 
                 ubiquitin domain containing 2 
                 327900 
               
               
                 Vim 
                 Vimentin 
                 22352 
               
               
                 Wwox 
                 WW domain-containing oxidoreductase 
                 80707 
               
               
                 Zfp292 
                 zinc finger protein 292 
                 30046 
               
               
                 Zmat5 
                 zinc finger, matrin type 5 
                 67178 
               
               
                 Zswim6 
                 zinc finger, SWIM domain containing 6 
                 67263 
               
               
                 Zwilch 
                 Zwilch, kinetochore associated, homolog ( Drosophila ) 
                 68014 
               
               
                   
               
            
           
         
       
     
     Eighteen potential markers of brain aging were selected for confirmation of array data by qPCR. Genes were selected based on multiple factors including but not limited to: abundant expression in the microarray experiment, robust change in expression in the B6 strain, previous reports of genes associated with brain aging. Using the RNA samples from B6 mice used in the array study, qPCR analysis revealed that 13/18 genes showed a change in expression with age. These 13 genes are shown in Table 9. Eight of the 13 genes were further determined to be changed by age and at least about 33% of the aging change was prevented by CR; these eight genes are: Apod, B2m, Clqa, Clqb, Ctsd, Gfap, Il33, Lyzs, and Spp1. 
     
       
         
           
               
               
               
             
               
                 TABLE 9 
               
               
                   
               
               
                 Gene 
                   
                   
               
               
                 Symbol 
                 Gene Title 
                 Entrez Gene 
               
               
                   
               
             
            
               
                 Apod 
                 apolipoprotein D 
                 11815 
               
               
                 B2m 
                 beta-2 microglobulin 
                 12010 
               
               
                 C1qa 
                 complement component 1, q subcomponent, alpha 
                 12259 
               
               
                   
                 polypeptide 
               
               
                 C1qb 
                 complement component 1, q subcomponent, beta 
                 12260 
               
               
                   
                 polypeptide 
               
               
                 Cd68 
                 CD68 antigen 
                 12514 
               
               
                 Clec7a 
                 C-type lectin domain family 7, member a 
                 56644 
               
               
                 Cst7 
                 cystatin F (leukocystatin) 
                 13011 
               
               
                 Ctsd 
                 cathepsin D 
                 13033 
               
               
                 Gfap 
                 glial fibrillary acidic protein 
                 14580 
               
               
                 Il33 
                 interleukin 33 
                 77125 
               
               
                 Lgals3 
                 lectin, galactose binding, soluble 3 
                 16854 
               
               
                 Lyzs 
                 lysozyme 
                 17105 
               
               
                 Spp1 
                 secreted phosphoprotein 1 
                 20750 
               
               
                   
               
            
           
         
       
     
     The markers of brain aging identified above were used to asses the efficacy of CR to oppose age-related changes in brain aging. The extent to which the change in expression with age was opposed by CR (“percent prevention”) was determined using the indices described in the previous examples. 
     Many genes previously reported to be biomarkers of aging were identified in the array study and confirmed by qPCR, including Cd68, Ctsd and Gfap. CR opposed the change in expression for Ctsd, but was only moderately effective for Cd68 and Gfap. 
     Several genes were identified that have a neuroprotective action when expressed acutely, but have a detrimental effect when overexpressed on a chronic timescale (e.g., aging). Some of those genes include Apod, Cblqa and Clqb. A mild CR diet opposed the changes in Apod. The increase in the expression of Clqb seen with age was opposed by CR and in all supplemented mice. 
     There was a marked increase in the expression of genes involved in the immune and inflammatory responses (e.g., B2m, Clec71, Cst7, 1133, Lgals3). This is in strong agreement with previous reports of increased neuroinflammation with age. CR has been reported to oppose age-related changes in the expression of neuroinflammatory genes, though CR appeared to only oppose the age-related increase in B2m expression. Overall, the changes in the expression of inflammatory supermarkers was large (especially Clec7a and Cst7) and resistant to dietary intervention in this study. However, a larger restriction of caloric intake (˜40%) has been show to oppose the age-related changes in neuroinflammatory genes, thus alternative interventions have the potential to oppose age-related changes in these supermarkers. 
     Finally, many of the markers have been associated with neurodegenerative disorders such as Alzheimer&#39;s diseases (e.g., Apod, Clqa, Clqb, Ctsd, Lyzs, Spp1). Calorie restriction has been proposed to oppose the progression of Alzheimer&#39;s disease in humans and in mouse disease models. Accordingly, the mild CR used in this study opposed age-related changes in many of these genes. 
     Background information for reported or proposed functions of genes represented by the markers is set forth below. 
     Apod: Increased Apolipoprotein D expression has been reported in various neurological disorders, including Alzheimer&#39;s disease, schizophrenia, and stroke, and in the aging brain. Apod may be a stress response protein, since overexpression of this gene in  Drosophila  has been reported to lead to neuroprotection and lifespan extension. Thus, the level of Apod correlates with the level of endogenous stress. 
     B2M: beta 2-microglobulin (part of the class I major histocompatibility complex molecules) is also a reported marker of inflammation, and has previously been shown to increase with age in the cerebro-spinal fluid of aged humans and in the Parkinsonian brain. 
     Clqa and Clqb: These genes are reportedly involved in innate immunity and are markers of inflammation that we have previously shown to be activated with age in the mouse brain, and have also been shown to be activated in human neurodegenerative diseases such as Alzheimer&#39;s disease. 
     Cd68: Also known as macrosialin, Cd68 is a macrophage-specific protein, is reportedly increased by aging in selected brain regions of male C57BL/6NNia mice, and is thought to be expressed in microglia. 
     Clec7a: Also know as Dectin-1, this receptor reportedly can induce a variety of cellular responses in macrophages, including phagocytosis, the respiratory burst and cytokine production. This gene encodes a member of the C-type lectin/C-type lectin-like domain (CTL/CTLD) superfamily. The encoded glycoprotein is a small type II membrane receptor with an extracellular C-type lectin-like domain fold and a cytoplasmic domain with an immunoreceptor tyrosine-based activation motif. It reportedly functions as a pattern-recognition receptor that recognizes a variety of beta-1,3-linked and beta-1,6-linked glucans from fungi and plants, and in this way plays a role in innate immune response. Alternate transcriptional splice variants, encoding different isoforms, have been characterized. This gene is closely linked to other CTL/CTLD superfamily members on chromosome 12p13 in the natural killer gene complex region. 
     Cst7: Cystatin F is a glycosylated human low molecular weight cysteine proteinase inhibitor. Cystatins are important natural cysteine protease inhibitors targeting primarily papain-like cysteine proteases, including cathepsins and parasitic proteases like cruzipain, but also mammalian asparaginyl endopeptidase. Mammalian cystatin F, which is expressed almost exclusively in hematopoietic cells and accumulates in lysosome-like organelles, has been implicated in the regulation of antigen presentation and other immune processes. It is an unusual cystatin superfamily member with a reported redox-regulated activation mechanism and a restricted specificity profile. 
     Ctsd: Cathepsin D is a major lysosomal protease, and mutations in Ctsd that render it enzymatically defective have been reported recently in subsets of neuronal ceroid lipofuscinosces/Batten disease. The disease phenotype does not require Bax-mediated apoptosis, and instead appears to be mediated through autophagy. Cathepsin D-mediated proteolysis of apolipoprotein E may have a role in Alzheimer&#39;s disease. Up-regulation of the lysosomal system in experimental models of neuronal injury has also been reported. 
     Gfap: Glial fibrillary acidic protein is a classical marker of astrocyte activation, and probably the most well established brain aging marker. This gene encodes one of the major intermediate filament proteins of mature astrocytes. It is used as a marker to distinguish astrocytes from other glial cells during development. Mutations in this gene cause Alexander disease, a rare disorder of astrocytes in the central nervous system. 
     Il33: A poorly characterized cytokine, IL-33 is reported to be a dual function protein that may function as both a proinflammatory cytokine and an intracellular nuclear factor with transcriptional regulatory properties. 
     Lgals3: Galectin-3 is a multi-functional protein and reportedly participates in mediating inflammatory reactions. Galectin-3 is reportedly upregulated in microglial cells. Interestingly, Galectin-3 also reportedly promotes neural cell adhesion and neurite growth. 
     Lyzs: Also known as lysozyme, it is a poorly characterized and presumably lysosomal protein, which is abundant in leukocytes and is involved in inflammatory reactions. 
     Spp1: Secreted phosphoprotein-1, also known as osteopontin, is a secreted arginine-glycine-aspartate (RGD)-containing phosphoprotein. Spp1 appears to be overexpressed in Parkinson&#39;s disease. Osteopontin (OPN) has been implicated in inflammatory and wound-healing processes, including autoimmune uveitis. 
     Example 5 
     This example describes the identification of transcriptional markers of aging in muscle tissue. To generate a panel of genes likely to be robust markers of age in mouse skeletal muscle, transcriptional profiling was performed using the Affymetrix Mouse Genome 430 2.0 Array on gastrocnemius muscle from seven mouse strains: 129/J, C57BL/6, CBA/J, DBA2J, C3H/HeJ, Balb/c, and B6C3HF1. Profiling was performed in seven young (five months of age) and seven old (28-30 months) mice from each strain. Two-tailed t-tests were performed to test for statistical significance of the change in gene expression with age. Of the ˜21,000 transcripts that were represented on the array, 172 transcripts were significantly (P&lt;0.05) changed with age in at least six of the seven strains (Table 10). 
     
       
         
           
               
               
               
             
               
                 TABLE 10 
               
               
                   
               
               
                   
                   
                 Entrez 
               
               
                 Gene Symbol 
                 Gene Title 
                 Gene 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
            
               
                 Acsl6 
                 acyl-CoA synthetase long-chain family member 6 
                 216739 
               
               
                 Actr1a 
                 ARP1 actin-related protein 1 homolog A (yeast) 
                 54130 
               
               
                 Adcy2 
                 adenylate cyclase 2 
                 210044 
               
               
                 Agtrl1 
                 angiotensin receptor-like 1 
                 23796 
               
               
                 Akr1b8 
                 aldo-keto reductase family 1, member B8 
                 14187 
               
               
                 Aldh1a1 
                 aldehyde dehydrogenase family 1, subfamily A1 
                 11668 
               
               
                 Amy1 
                 amylase 1, salivary 
                 11722 
               
               
                 Ankrd32 
                 ankyrin repeat domain 32 
                 105377 
               
               
                 Antxr2 
                 anthrax toxin receptor 2 
                 71914 
               
               
                 Apod 
                 apolipoprotein D 
                 11815 
               
               
                 Arrdc4 
                 arrestin domain containing 4 
                 66412 
               
               
                 Atp13a5 
                 ATPase type 13A5 
                 268878 
               
               
                 AU018740 
                 expressed sequence AU018740 
                 98528 
               
               
                 B3gnt1 
                 UDP-GlcNAc:betaGal beta-1,3-N-acetylglucosaminyltransferase 1 
                 53625 
               
               
                 C4 
                 complement component 4 (within H-2S) 
                 12268 
               
               
                 Cd209b 
                 CD209b antigen 
                 69165 
               
               
                 Cdkl2 
                 cyclin-dependent kinase-like 2 (CDC2-related kinase) 
                 53886 
               
               
                 Cdkn2c 
                 cyclin-dependent kinase inhibitor 2C (p18, inhibits CDK4) 
                 12580 
               
               
                 Cds1 
                 CDP-diacylglycerol synthase 1 
                 74596 
               
               
                 Ces2 /// 
                 carboxylesterase 2 /// similar to carboxylesterase 2 
                 234671 /// 
               
               
                 LOC546098 
                   
                 546098 
               
               
                 Chad 
                 chondroadherin 
                 12643 
               
               
                 Chodl 
                 chondrolectin 
                 246048 
               
               
                 Chrna1 
                 cholinergic receptor, nicotinic, alpha polypeptide 1 (muscle) 
                 11435 
               
               
                 Chrnb1 
                 cholinergic receptor, nicotinic, beta polypeptide 1 (muscle) 
                 11443 
               
               
                 Cilp2 
                 cartilage intermediate layer protein 2 
                 68709 
               
               
                 Clybl 
                 citrate lyase beta like 
                 69634 
               
               
                 Col11a1 
                 procollagen, type XI, alpha 1 
                 12814 
               
               
                 Col1a1 
                 procollagen, type I, alpha 1 
                 12842 
               
               
                 Col1a2 
                 procollagen, type I, alpha 2 
                 12843 
               
               
                 Col3a1 
                 procollagen, type III, alpha 1 
                 12825 
               
               
                 Col4a1 
                 procollagen, type IV, alpha 1 
                 12826 
               
               
                 Col5a2 
                 procollagen, type V, alpha 2 
                 12832 
               
               
                 Col6a1 
                 procollagen, type VI, alpha 1 
                 12833 
               
               
                 Cpne2 
                 copine II 
                 234577 
               
               
                 Csprs 
                 component of Sp100-rs 
                 114564 
               
               
                 Ctnnbl1 
                 catenin, beta like 1 
                 66642 
               
               
                 Cuzd1 
                 CUB and zona pellucida-like domains 1 
                 16433 
               
               
                 Cyt1 
                 cytokine like 1 
                 231162 
               
               
                 Ddx6 
                 DEAD (Asp-Glu-Ala-Asp) box polypeptide 6 
                 13209 
               
               
                 Dhx36 
                 DEAH (Asp-Glu-Ala-His) box polypeptide 36 
                 72162 
               
               
                 Dmn 
                 desmuslin 
                 233335 
               
               
                 Dmxl2 
                 Dmx-like 2 
                 235380 
               
               
                 Dnajb14 
                 DnaJ (Hsp40) homolog, subfamily B, member 14 
                 70604 
               
               
                 Dusp26 
                 dual specificity phosphatase 26 (putative) 
                 66959 
               
               
                 E030025D05Rik 
                 RIKEN cDNA E030025D05 gene 
                 216613 
               
               
                 Edg2 
                 endothelial differentiation, lysophosphatidic acid G-protein- 
                 14745 
               
               
                   
                 coupled receptor, 2 
               
               
                 Efha1 
                 EF hand domain family A1 
                 68514 
               
               
                 Egfl6 
                 EGF-like-domain, multiple 6 
                 54156 
               
               
                 Eif2c2 
                 eukaryotic translation initiation factor 2C, 2 
                 239528 
               
               
                 Emb 
                 embigin 
                 13723 
               
               
                 Fbxl4 
                 F-box and leucine-rich repeat protein 4 
                 269514 
               
               
                 Fmo2 
                 flavin containing monooxygenase 2 
                 55990 
               
               
                 Fmod 
                 fibromodulin 
                 14264 
               
               
                 Fscn1 
                 fascin homolog 1, actin bundling protein ( Strongylocentrotus ) 
                 14086 
               
               
                   
                   purpuratus ) 
               
               
                 Fut8 
                 fucosyltransferase 8 
                 53618 
               
               
                 Gadd45a 
                 growth arrest and DNA-damage-inducible 45 alpha 
                 13197 
               
               
                 Gca 
                 grancalcin 
                 227960 
               
               
                 Gdap10 
                 ganglioside-induced differentiation-associated-protein 10 
                 14546 
               
               
                 Gna14 
                 guanine nucleotide binding protein, alpha 14 
                 14675 
               
               
                 Gtl2 /// Lphn1 
                 GTL2, imprinted maternally expressed untranslated mRNA /// 
                 17263///330814 
               
               
                   
                 latrophilin1 
               
               
                 Hist1h3i 
                 Histone 1, H3i (Hist1h3i), mRNA 
                 319153 
               
               
                 Hn1 
                 hematological and neurological expressed sequence 1 
                 15374 
               
               
                 Homer2 
                 homer homolog 2 ( Drosophila ) 
                 26557 
               
               
                 Hook1 
                 hook homolog 1 ( Drosophila ) 
                 77963 
               
               
                 Hook3 
                 hook homolog 3 ( Drosophila ) 
                 320191 
               
               
                 Hsd17b7 
                 hydroxysteroid (17-beta) dehydrogenase 7 
                 15490 
               
               
                 Icam1 
                 intercellular adhesion molecule 
                 15894 
               
               
                 Igh-6 
                 immunoglobulin heavy chain 6 (heavy chain of IgM) 
                 16019 
               
               
                 Itih5 
                 inter-alpha (globulin) inhibitor H5 
                 209378 
               
               
                 Jarid1b 
                 jumonji, AT rich interactive domain 1B (Rbp2 like) 
                 75605 
               
               
                 Jmjd3 
                 Jumonji domain containing 3, mRNA (cDNA clone 
                 216850 
               
               
                   
                 IMAGE: 4037702) 
               
               
                 Kcnab1 
                 potassium voltage-gated channel, shaker-related subfamily, beta 
                 16497 
               
               
                   
                 member 1 
               
               
                 Kera 
                 keratocan 
                 16545 
               
               
                 Krt1-18 
                 keratin complex 1, acidic, gene 18 
                 16668 
               
               
                 Lgals3 
                 lectin, galactose binding, soluble 3 
                 16854 
               
               
                 LOC241944 
                 similar to ZNF43 protein 
                 241944 
               
               
                 LOC545323 
                 similar to neurobeachin-like 1 
                 545323 
               
               
                 Lpgat1 
                 lysophosphatidylglycerol acyltransferase 1 
                 226856 
               
               
                 Lrp11 
                 low density lipoprotein receptor-related protein 11 
                 237253 
               
               
                 Mfap4 
                 microfibrillar-associated protein 4 
                 76293 
               
               
                 Mgst1 
                 microsomal glutathione S-transferase 1 
                 56615 
               
               
                 Mia3 
                 melanoma inhibitory activity 3 
                 338366 
               
               
                 Mll3 
                 myeloid/lymphoid or mixed-lineage leukemia 3 
                 231051 
               
               
                 Mll5 
                 myeloid/lymphoid or mixed-lineage leukemia 5 
                 69188 
               
               
                 Mt2 
                 metallothionein 2 
                 17750 
               
               
                 Mtap7 
                 microtubule-associated protein 7 
                 17761 
               
               
                 Myo5a 
                 myosin Va 
                 17918 
               
               
                 Nipa1 
                 non imprinted in Prader-Willi/Angelman syndrome 1 homolog 
                 233280 
               
               
                   
                 (human) 
               
               
                 Nxn 
                 nucleoredoxin 
                 18230 
               
               
                 Opcml 
                 opioid binding protein/cell adhesion molecule-like 
                 330908 
               
               
                 Pb1 
                 PREDICTED: Mus musculus RIKEN cDNA 2310032M22 gene 
                 76748 
               
               
                   
                 (Pb1) 
               
               
                 Pcgf4 
                 polycomb group ring finger 4 
                 12151 
               
               
                 Pdcd6ip 
                 programmed cell death 6 interacting protein 
                 18571 
               
               
                 Pkp2 
                 plakophilin 2 
                 67451 
               
               
                 Plekhb1 
                 pleckstrin homology domain containing, family B (evectins) 
                 27276 
               
               
                   
                 member 1 
               
               
                 Plk2 
                 polo-like kinase 2 ( Drosophila ) 
                 20620 
               
               
                 Plp2 
                 proteolipid protein 2 
                 18824 
               
               
                 Prkcz 
                 protein kinase C, zeta 
                 18762 
               
               
                 Pura /// 
                 purine rich element binding protein A /// RIKEN cDNA 
                 19290 /// 
               
               
                 6330411E07Rik 
                 6330411E07 gene 
                 70733 
               
               
                 Purb 
                 Purine rich element binding protein B (Purb), mRNA 
                 19291 
               
               
                 Pvrl3 
                 poliovirus receptor-related 3 
                 58998 
               
               
                 Rgs5 
                 regulator of G-protein signaling 5 
                 19737 
               
               
                 Rhpn2 
                 rhophilin, Rho GTPase binding protein 2 
                 52428 
               
               
                 Rnf125 
                 ring finger protein 125 
                 67664 
               
               
                 Sbno1 
                 sno, strawberry notch homolog 1 ( Drosophila ) 
                 243272 
               
               
                 Serpina3n 
                 serine (or cysteine) peptidase inhibitor, clade A, member 3N 
                 20716 
               
               
                 Serpinf1 
                 serine (or cysteine) peptidase inhibitor, clade F, member 1 
                 20317 
               
               
                 Sh3bp5 
                 SH3-domain binding protein 5 (BTK-associated) 
                 24056 
               
               
                 Slc4a10 
                 solute carrier family 4, sodium bicarbonate cotransporter-like, 
                 94229 
               
               
                   
                 member10 
               
               
                 Smc4l1 
                 SMC4 structural maintenance of chromosomes 4-like 1 (yeast) 
                 70099 
               
               
                 Spint2 
                 serine protease inhibitor, Kunitz type 2 
                 20733 
               
               
                 Supt3h 
                 suppressor of Ty 3 homolog ( S. cerevisiae ) 
                 109115 
               
               
                 Syt9 
                 synaptotagmin IX 
                 60510 
               
               
                 Taf9l 
                 TAF9-like RNA polymerase II, TATA box binding protein (TBP)- 
                 407786 
               
               
                   
                 associated factor 
               
               
                 Tekt1 
                 tektin 1 
                 21689 
               
               
                 Tnmd 
                 tenomodulin 
                 64103 
               
               
                 Trim41 
                 tripartite motif-containing 41 
                 211007 
               
               
                 Ttc14 
                 tetratricopeptide repeat domain 14 
                 67120 
               
               
                 Ttc9c 
                 tetratricopeptide repeat domain 9C 
                 70387 
               
               
                 Ubn1 
                 ubinuclein 1 
                 170644 
               
               
                 Vsig4 
                 V-set and immunoglobulin domain containing 4 
                 278180 
               
               
                 Vtn 
                 vitronectin 
                 22370 
               
               
                 Wif1 
                 Wnt inhibitory factor 1 
                 24117 
               
               
                 Zdhhc21 
                 zinc finger, DHHC domain containing 21 
                 68268 
               
               
                   
               
            
           
         
       
     
     Of the 172 transcripts that were identified in the microarray analysis, 21 genes were analyzed by qPCR to determine the change in expression with age and CR. Genes were chosen using several criteria—genes were selected based on having a known biological function, if they had a relatively abundant expression in the array study and/or if studies in peer-reviewed literature suggest a role in muscle aging. RT-PCR analysis was performed using an Applied Biosystems 7000 instrument using off-the-shelf PCR primers designed by Applied Biosystems. TATA box binding protein (Tbp) was used as an internal control for all RT-PCR analyses, as this gene was previously shown not to change with age or CR. Of the 21 genes that were tested, 13 genes showed a change in expression with age in at least six of the seven strains (Table 11). Of the 13 genes, eleven showed reversal by CR. These eleven genes are: C4, Cdkn2c, Cds1, Col1a1, Col1a2, Col3a1, Edg2, Igh-6, Mt2, Plk2, and Syt9. 
     
       
         
           
               
               
               
             
               
                 TABLE 11 
               
               
                   
               
               
                 Gene 
                   
                 Entrez 
               
               
                 Symbol 
                 Gene Title 
                 Gene 
               
               
                   
               
             
            
               
                 C4 
                 complement component 4 (within H-2S) 
                 12268 
               
               
                 Cdkn2c 
                 cyclin-dependent kinase inhibitor 2C 
                 12580 
               
               
                   
                 (p18, inhibits CDK4) 
               
               
                 Cds1 
                 CDP-diacylglycerol synthase 1 
                 74596 
               
               
                 Col1a1 
                 procollagen, type I, alpha 1 
                 12842 
               
               
                 Col1a2 
                 procollagen, type I, alpha 2 
                 12843 
               
               
                 Col3a1 
                 procollagen, type III, alpha 1 
                 12825 
               
               
                 Dusp26 
                 dual specificity phosphatase 26 (putative) 
                 66959 
               
               
                 Edg2 
                 endothelial differentiation, lysophosphatidic acid 
                 14745 
               
               
                   
                 G-protein-coupled receptor, 2 
               
               
                 Igh-6 
                 immunoglobulin heavy chain 6 (heavy chain of IgM) 
                 16019 
               
               
                 Mt2 
                 metallothionein 2 
                 17750 
               
               
                 Plk2 
                 polo-like kinase 2 ( Drosophila ) 
                 20620 
               
               
                 Rhpn2 
                 rhophilin, Rho GTPase binding protein 2 
                 52428 
               
               
                 Syt9 
                 synaptotagmin IX 
                 60510 
               
               
                   
               
            
           
         
       
     
     Of the genes shown in Table 11 two can be broadly classified as being involved in inflammatory response (C4 and Igh6), two can be broadly classified in DNA damage/cell cycle checkpoint (Cdkn2c and Plk2), two can be classified in the stress response (Mt2 and Dusp26), one (Cds1) can be broadly classified as being involved in biosynthesis, one can be classified as involved in calcium metabolism (Syt9) and five genes (Colla1, Colla2, Col3a1, Edg2, Rhpn2) can be classified as being involved in cytoskeletal remodeling. 
     After an appropriate panel of biomarkers had been validated via qPCR in (changed with age and in most instances the change reversed by CR), the expression of those genes was analyzed in mRNA samples from the nutrient study of Example 1. Results pertaining to specific markers, along with a description of the reported functional significance of those markers, are discussed below. 
     Complement C4: The fourth component of the complement cascade is an essential factor in innate immunity. Its activation has been observed during normal brain aging in humans and also in Alzheimer&#39;s disease brain. Different alleles of C4 in humans have been linked in health and survival, suggesting that C4 status impacts health directly. Complement C4 has also been linked to autoimmune disease. 
     Igh-6: Igh6 is a B-cell antigen required for B-cell maturation. Igh6 deficient mice are commonly used as a model of B cell deficiency. Thus, increased Igh-6 in skeletal muscle with age may be secondary to an increase in B-cell infiltration in this tissue, which was completely prevented by CR in these studies. 
     Cdkn2C (p18): CdKn2C, also known as pl8INK4c, is a GI-phase cyclin kinase inhibitor (CKI). It is one of several CKIs involved in cell cycle arrest in response to DNA damage. CKI inhibitors are tumor suppressor genes, since mutations in either p18 or the related p16 lead to tumorigenesis. p16 in particular has been linked to cellular senescence of the adult stem cell population compartment. It is likely that the observed age-related p18 activation reflects accrued DNA damage. 
     Plk2: Polo-kinase 2 is a polo-like kinase expressed at G1 in cultured cells and specific animal tissues that functions in the DNA damage response. Polo, the founding member of this gene family, was identified in  Drosophila  and plays a role in the control of cell division. 
     Mt2: The metallothioneins (I, II and III) are low molecular weight, cysteine rich metal binding proteins found in a wide variety of organisms and known to be induced under a wide variety of stress conditions. Because these proteins control the traffic of intracellular zinc, it is thought that control of zinc levels through metallothioneins is an important aspect of the cellular defense against stress. 
     Dusp26: DUSPs (Dual-specificity tyrosine phosphtases) are similar to tyrosine phosphatases by possessing the tyrosine phosphatase signature domain (I/V)HCXAGXGR(S/T) involved in their catalytic activity. Many members of this class have been identified, including DUSP1-9, DUSP16 and DUSP-26, also known as MKP 8. All of these proteins dephosphorylate serine/threonine and tyrosine residues on different MAP kinase members leading to their inactivation. DUSPs are activated by stimuli that trigger MAP kinase pathways such as heat shock, mitogens and hypoxia. Dusp26 has recently been shown to associate with the heat shock transcriptional factor 4b, establishing a link between this DUSP and the heat shock response. Surprisingly, CR was unable to prevent the age-related induction of Dusp26 
     Cds1: CDP-diacylglycerol synthase is a rate limiting enzyme involved in glycerolipid biosynthesis, which serves as a precursor to both phosphoinositides and phosphatidylglycerol. It is thought that the CDP-diacylglycerol synthases regulate the activity of phospholipids biosynthetic pathways. 
     Edg2: The expression of Edg2 (endothelial differentiation, lysophosphatidic acid G-protein coupled receptor 2) decreased with age, and this decrease was almost entirely prevented by CR. Lysophosphatidic acid induces cytoskeletal rearrangement, and because Edg2 is a receptor for this molecule, these findings recapitulate the general pattern seen in this study that cytoskeletal remodeling is a common feature of aging in mouse skeletal muscle . 
     Colla1, Colla2, Col3a1: Colla and Colla2 encode the chains of type I procollagen. Mutations in these genes are associated with the human disease osteogenesis imperfecta. To our knowledge, this is the first discovery of a coordinated downregulation of collagen genes with aging. Three pro-collagen genes (Colla1, Colla2, Col3a1) showed changes with age and opposition by CR. 
     Rhpn2: Rhophilin 2 is a Rho GTPase binding protein. It has been postulated to play a role in endocytosis, and a two-hybrid yeast screen suggests that components of cytoskeleton are partners of rhophilin 2. 
     Syt9: Calcium influx into presynaptic nerve terminals and neuroendocrine cells triggers exocytosis of synaptic and secretory vesicles. Synaptotagmins, including Syt9 are calcium binding proteins that are thought to be calcium sensors for exocytosis. 
     The specification has disclosed typical preferred embodiments of the invention. Although specific terms are employed, they are used in a generic and descriptive sense only and not for purposes of limitation, the scope of the invention being set forth in the claims. Clearly, many modifications and variations of the invention are possible in light of the above teachings. It is therefore to be understood that within the scope of the appended claims the invention may be practiced otherwise than as specifically described.