Phosphorylation of p53 by TAF1 inactivates p53-dependent transcription in the DNA damage response.

While p53 activation has long been studied, the mechanisms by which its targets genes are restored to their preactivation state are less clear. We report here that TAF1 phosphorylates p53 at Thr55, leading to dissociation of p53 from the p21 promoter and inactivation of transcription late in the DNA damage response. We further show that cellular ATP level might act as a molecular switch for Thr55 phosphorylation on the p21 promoter, indicating that TAF1 is a cellular ATP sensor. Upon DNA damage, cells undergo PARP-1-dependent ATP depletion, which is correlated with reduced TAF1 kinase activity and Thr55 phosphorylation, resulting in p21 activation. As cellular ATP levels recover, TAF1 is able to phosphorylate p53 on Thr55, which leads to dissociation of p53 from the p21 promoter. ChIP-sequencing analysis reveals p53 dissociates from promoters genome wide as cells recover from DNA damage, suggesting the general nature of this mechanism.

5'-YRNA fragments derived by processing of transcripts from specific YRNA genes and pseudogenes are abundant in human serum and plasma.

Small noncoding RNAs carry out a variety of functions in eukaryotic cells, and in multiple species they can travel between cells, thus serving as signaling molecules. In mammals multiple small RNAs have been found to circulate in the blood, although in most cases the targets of these RNAs, and even their functions, are not well understood. YRNAs are small (84-112 nt) RNAs with poorly characterized functions, best known because they make up part of the Ro ribonucleoprotein autoantigens in connective tissue diseases. In surveying small RNAs present in the serum of healthy adult humans, we have found YRNA fragments of lengths 27 nt and 30-33 nt, derived from the 5'-ends of specific YRNAs and generated by cleavage within a predicted internal loop. Many of the YRNAs from which these fragments are derived were previously annotated only as pseudogenes, or predicted informatically. These 5'-YRNA fragments make up a large proportion of all small RNAs (including miRNAs) present in human serum. They are also present in plasma, are not present in exosomes or microvesicles, and circulate as part of a complex with a mass between 100 and 300 kDa. Mouse serum contains far fewer 5'-YRNA fragments, possibly reflecting the much greater copy number of YRNA genes and pseudogenes in humans. The function of the 5'-YRNA fragments is at present unknown, but the processing and secretion of specific YRNAs to produce 5'-end fragments that circulate in stable complexes are consistent with a signaling function.

5' tRNA halves are present as abundant complexes in serum, concentrated in blood cells, and modulated by aging and calorie restriction.

BACKGROUND: Small RNAs complex with proteins to mediate a variety of functions in animals and plants. Some small RNAs, particularly miRNAs, circulate in mammalian blood and may carry out a signaling function by entering target cells and modulating gene expression. The subject of this study is a set of circulating 30-33 nt RNAs that are processed derivatives of the 5' ends of a small subset of tRNA genes, and closely resemble cellular tRNA derivatives (tRFs, tiRNAs, half-tRNAs, 5' tRNA halves) previously shown to inhibit translation initiation in response to stress in cultured cells. RESULTS: In sequencing small RNAs extracted from mouse serum, we identified abundant 5' tRNA halves derived from a small subset of tRNAs, implying that they are produced by tRNA type-specific biogenesis and/or release. The 5' tRNA halves are not in exosomes or microvesicles, but circulate as particles of 100-300 kDa. The size of these particles suggest that the 5' tRNA halves are a component of a macromolecular complex; this is supported by the loss of 5' tRNA halves from serum or plasma treated with EDTA, a chelating agent, but their retention in plasma anticoagulated with heparin or citrate. A survey of somatic tissues reveals that 5' tRNA halves are concentrated within blood cells and hematopoietic tissues, but scant in other tissues, suggesting that they may be produced by blood cells. Serum levels of specific subtypes of 5' tRNA halves change markedly with age, either up or down, and these changes can be prevented by calorie restriction. CONCLUSIONS: We demonstrate that 5' tRNA halves circulate in the blood in a stable form, most likely as part of a nucleoprotein complex, and their serum levels are subject to regulation by age and calorie restriction. They may be produced by blood cells, but their cellular targets are not yet known. The characteristics of these circulating molecules, and their known function in suppression of translation initiation, suggest that they are a novel form of signaling molecule.

mRNA-Seq reveals complex patterns of gene regulation and expression in the mouse skeletal muscle transcriptome associated with calorie restriction.

Sarcopenia is an age-associated loss of skeletal muscle mass and strength that increases the risk of disability. Calorie restriction (CR), the consumption of fewer calories while maintaining adequate nutrition, mitigates sarcopenia and many other age-related diseases. To identify potential mechanisms by which CR preserves skeletal muscle integrity during aging, we used mRNA-Seq for deep characterization of gene regulation and mRNA abundance in skeletal muscle of old mice compared with old mice subjected to CR. mRNA-Seq revealed complex CR-associated changes in expression of mRNA isoforms, many of which occur without a change in total message abundance and thus would not be detected by methods other than mRNA-Seq. Functional annotation of differentially expressed genes reveals CR-associated upregulation of pathways involved in energy metabolism and lipid biosynthesis, and downregulation of pathways mediating protein breakdown and oxidative stress, consistent with earlier microarray-based studies. CR-associated changes not noted in previous studies involved downregulation of genes controlling actin cytoskeletal structures and muscle development. These CR-associated changes reflect generally healthier muscle, consistent with CR's mitigation of sarcopenia. mRNA-Seq generates a rich picture of the changes in gene expression associated with CR, and may facilitate identification of genes that are primary mediators of CR's effects.

Delayed and accelerated aging share common longevity assurance mechanisms.

Mutant dwarf and calorie-restricted mice benefit from healthy aging and unusually long lifespan. In contrast, mouse models for DNA repair-deficient progeroid syndromes age and die prematurely. To identify mechanisms that regulate mammalian longevity, we quantified the parallels between the genome-wide liver expression profiles of mice with those two extremes of lifespan. Contrary to expectation, we find significant, genome-wide expression associations between the progeroid and long-lived mice. Subsequent analysis of significantly over-represented biological processes revealed suppression of the endocrine and energy pathways with increased stress responses in both delayed and premature aging. To test the relevance of these processes in natural aging, we compared the transcriptomes of liver, lung, kidney, and spleen over the entire murine adult lifespan and subsequently confirmed these findings on an independent aging cohort. The majority of genes showed similar expression changes in all four organs, indicating a systemic transcriptional response with aging. This systemic response included the same biological processes that are triggered in progeroid and long-lived mice. However, on a genome-wide scale, transcriptomes of naturally aged mice showed a strong association to progeroid but not to long-lived mice. Thus, endocrine and metabolic changes are indicative of "survival" responses to genotoxic stress or starvation, whereas genome-wide associations in gene expression with natural aging are indicative of biological age, which may thus delineate pro- and anti-aging effects of treatments aimed at health-span extension.

Development of a bioassay to screen for chemicals mimicking the anti-aging effects of calorie restriction.

Suppression of the growth hormone/insulin-like growth factor-I pathway in Ames dwarf (DF) mice, and caloric restriction (CR) in normal mice extends lifespan and delays the onset of age-related disorders. In combination, these interventions have an additive effect on lifespan in Ames DF mice. Therefore, common signaling pathways regulated by DF and CR could have additive effects on longevity. In this study, we tried to identity the signaling mechanism and develop a system to assess pro-longevity status in cells and mice. We previously identified genes up-regulated in the liver of DF and CR mice by DNA microarray analysis. Motif analysis of the upstream sequences of those genes revealed four major consensus sequence motifs, which have been named dwarfism and calorie restriction-responsive elements (DFCR-REs). One of the synthesized sequences bound to hepatocyte nuclear factor-4α (HNF-4α), an important transcription factor involved in liver metabolism. Furthermore, using this sequence information, we developed a highly sensitive bioassay to identify chemicals mimicking the anti-aging effects of CR. When the reporter construct, containing an element upstream of a secreted alkaline phosphatase (SEAP) gene, was co-transfected with HNF-4α and its regulator peroxisome proliferator-activated receptor (PPAR) γ coactivator-1α (PGC-1α), SEAP activity was increased compared with untransfected controls. Moreover, transient transgenic mice established using this construct showed increased SEAP activity in CR mice compared with ad libitum-fed mice. These data suggest that because of its rapidity, ease of use, and specificity, our bioassay will be more useful than the systems currently employed to screen for CR mimetics, which mimic the beneficial effects of CR. Our system will be particularly useful for high-throughput screening of natural and synthetic candidate molecules.

Use of microarray biomarkers to identify longevity therapeutics.

A number of lines of evidence, including nonhuman primate and human studies, suggest that regulatory pathways similar to those invoked by caloric restriction (CR) may be involved in determining human longevity. Thus, pharmaceuticals capable of mimicking the molecular mechanisms of life- and health-span extension by CR (CR mimetics) may have application to human health. CR acts rapidly, even in late adulthood, to begin to extend life- and health-span in mice. We have linked these effects with rapid changes in the levels of specific gene transcripts in the liver and the heart. Our results are consistent with the rapid effects of caloric intake on the lifespan and/or biochemistry and physiology of Drosophila, rodents, rhesus macaques and humans. To test the hypothesis that existing pharmaceuticals can mimic the physiologic effects of CR, we evaluated the effectiveness of glucoregulatory drugs and putative cancer chemo-preventatives in reproducing the hepatic gene-expression profiles produced by long-term CR (LTCR). We found that 8 weeks of metformin treatment was superior to 8 weeks of CR at reproducing the specific changes in transcript levels produced by LTCR. Consistent with these results, metformin reduces cancer incidence in diabetic humans and ameliorates the onset and severity of metabolic syndrome. Metformin extends the mean and maximum lifespans of female transgenic HER-2/neu mice by 8% and 13.1% in comparison with control mice. Phenformin, a close chemical relative of metformin, extends lifespan and reduces tumor incidence in C3H mice. These results indicate that gene-expression biomarkers can be used to identify promising candidate CR mimetics.

Screening candidate longevity therapeutics using gene-expression arrays.

BACKGROUND: We review studies showing that CR acts rapidly, even in late adulthood, to extend health- and lifespan in mice. These rapid physiological effects are closely linked to patterns of gene expression in liver and heart. Non-human primate and human studies suggest that the signal transduction pathways responsible for the lifespan and health effects of caloric restriction (CR) may also be involved in human longevity. Thus, pharmaceuticals capable of mimicking the effects of CR (and other methods of lifespan extension) may have application to human health. OBJECTIVE: We show that lifespan studies are an inefficient and theoretically problematic way of screening for longevity therapeutics. We review studies suggesting that rapid changes in patterns of gene expression can be used to identify pharmaceuticals capable of mimicking some positive effects of caloric restriction. RESULTS: We present a traditional study of the effects of melatonin, melatonin and pregnenolone, aminoguanidine, aminoguanidine and alpha-lipoic acid, aminoguanidine, alpha-lipoic acid, pregnenolone, and coenzyme-Q(10) on the lifespan of mice. No treatment extended lifespan. However, because the mice die mostly of cancer, only chemopreventives active against specific cancers can be identified by such studies. The studies were also time-consuming and expensive. We discuss high-density microarray studies of the effectiveness of glucoregulatory drugs and putative cancer chemopreventatives at reproducing the hepatic gene-expression profiles of long-term and short-term CR. We describe the identification of one compound, metformin, which reproduces a subset of the gene-expression and physiological effects of CR. CONCLUSION: Taken together, our results suggest that gene-expression biomarkers may be superior to lifespan studies for initial screening of candidate longevity therapeutics.

Gene expression and physiologic responses of the heart to the initiation and withdrawal of caloric restriction.

Aging increases and caloric restriction (CR) decreases morbidity and mortality associated with the cardiovascular system. Using Affymetrix microarrays, we identified changes in heart gene expression induced by aging and CR in male mice. Eight weeks of CR (CR8) reproduced 19% of the long-term CR (LTCR)-related expression changes. Because CR8 begins to extend the life span of these mice, these genes may be keys to its cardioprotective effects. CR8 and LTCR changed gene expression in a manner consistent with reduced remodeling and fibrosis, and enhanced contractility and energy production via lipid beta-oxidation. Molecular and histochemical studies indicated that CR reduced natriuretic peptide precursor type B and collagen expression, and reduced perivascular collagen deposition. We found smaller cardiomyocytes in the left ventricle of old-LTCR mice, suggesting reduced age-related cell death. Eight weeks of control feeding returned 97% of the LTCR-responsive genes to control expression levels. Thus, key CR-induced effects are rapidly responsive to diet, suggesting reduced caloric intake has rapid, positive effects on the heart.

Combined statin and angiotensin-converting enzyme (ACE) inhibitor treatment increases the lifespan of long-lived F1 male mice.

Statins, such as simvastatin, and ACE inhibitors (ACEis), such as ramipril, are standard therapies for the prevention and treatment of cardiovascular diseases. These types of drugs are commonly administered together. More recently, angiotensin II type 1 receptor (AT1R) antagonists, such as candesartan cilexetil (candesartan), have been used in the place of, or in combination with, ACEis. Here, we investigated the effects of simvastatin and ramipril single and combination therapy, and candesartan treatment on the lifespan of isocalorically fed, long-lived, B6C3F1 mice. Males were used for their relative endocrine simplicity and to minimize animal usage. The drugs were administered daily in food. The simvastatin and ramipril combination therapy significantly increased the mean and median lifespan by 9 %. In contrast, simvastatin, ramipril, or candesartan monotherapy was ineffective. All groups consumed the same number of calories. Simvastatin, alone or administered with ramipril, decreased body weight without changing caloric consumption, suggesting it may alter energy utilization in mice. Combination therapy elevated serum triglyceride and glucose levels, consistent with altered energy homeostasis. Few significant or consistent differences were found in mortality-associated pathologies among the groups. Simvastatin treatment did not reduce normal serum cholesterol or lipid levels in these mice, suggesting that the longevity effects may stem from the pleiotropic, non-cholesterol-related, effects of statins. Together, the results suggest that statins and ACEis together may enhance mouse longevity. Statins and ACE inhibitors are generally well-tolerated, and in combination, they have been shown to increase the lifespan of normotensive, normocholesterolemic humans.

MicroRNAs Circulate in the Hemolymph of Drosophila and Accumulate Relative to Tissue microRNAs in an Age-Dependent Manner.

In mammals, extracellular miRNAs circulate in biofluids as stable entities that are secreted by normal and diseased tissues, and can enter cells and regulate gene expression. Drosophila melanogaster is a proven system for the study of human diseases. They have an open circulatory system in which hemolymph (HL) circulates in direct contact with all internal organs, in a manner analogous to vertebrate blood plasma. Here, we show using deep sequencing that Drosophila HL contains RNase-resistant circulating miRNAs (HL-miRNAs). Limited subsets of body tissue miRNAs (BT-miRNAs) accumulated in HL, suggesting that they may be specifically released from cells or particularly stable in HL. Alternatively, they might arise from specific cells, such as hemocytes, that are in intimate contact with HL. Young and old flies accumulated unique populations of HL-miRNAs, suggesting that their accumulation is responsive to the physiological status of the fly. These HL-miRNAs in flies may function similar to the miRNAs circulating in mammalian biofluids. The discovery of these HL-miRNAs will provide a new venue for health and disease-related research in Drosophila.

Identification of potential caloric restriction mimetics by microarray profiling.

To facilitate the development of assays for the discovery of pharmaceuticals capable of mimicking the effects of caloric restriction (CR) on life- and healthspan (CR mimetics), we evaluated the effectiveness of glucoregulatory and putative cancer chemopreventatives in reproducing the hepatic gene expression profile produced by long-term CR (LTCR), using Affymetrix microarrays. We have shown that CR initiated late in life begins to extend lifespan, reduce cancer as a cause of death, and reproduce approximately three-quarters of the genomic effects of LTCR in 8 wk (CR8). Eight weeks of metformin treatment was superior to CR8 at reproducing LTCR-like gene expression changes, maintaining a superior number of such changes over a broad range of statistical stringencies, and producing more Gene Ontology terms overlapping those produced by LTCR. Consistent with these results, metformin has been shown to reduce cancer incidence in mice and humans. Phenformin, a chemical cousin of metformin, extends lifespan and reduces tumor incidence in mice. Taken together, these results indicate that gene expression biomarkers can be used to identify promising candidate CR mimetics.

Rapid and reversible induction of the longevity, anticancer and genomic effects of caloric restriction.

It is widely held that caloric restriction (CR) extends lifespan by preventing or reducing the age-related accumulation of irreversible molecular damage. In contrast, our results suggest that CR can act rapidly to begin life and health span extension, and that its rapid genomic effects are closely linked to its health effects. We found that CR begins to extend lifespan and reduce cancer as a cause of death within 8 weeks in older mice, apparently by reducing the rate of tumor growth. Further, 8 weeks of CR progressively reproduces nearly three quarters of the genomic effects of long-term CR (LTCR) in liver. Fewer of the genomic effects of LTCR are rapidly reproduced by the initiation of CR in the heart, but the changes produced are keys to cardiovascular health. Thus, the genomic effects of CR may be established more rapidly in mitotic than in postmitotic tissues. Most of the genomic effects of LTCR dissipate 8 weeks after switching to a control diet. Consistent with these results, others have shown that acute CR rapidly and reversibly reduces the short-term risk of death in Drosophila to that of LTCR treated flies. Further, in late adulthood, acute CR partially or completely reverses age-related alterations of liver, brain and heart proteins. CR also rapidly and reversibly mitigates biomarkers of aging in adult rhesus macaques and humans. These data argue that highly conserved mechanisms for the rapid and reversible enhancement of life- and health-span exist for mitotic and postmitotic tissues.

Nordihydroguaiaretic Acid Extends the Lifespan of Drosophila and Mice, Increases Mortality-Related Tumors and Hemorrhagic Diathesis, and Alters Energy Homeostasis in Mice.

Mesonordihydroguaiaretic acid (NDGA) extends murine lifespan. The studies reported here describe its dose dependence, effects on body weight, toxicity-related clinical chemistries, and mortality-related pathologies. In flies, we characterized its effects on lifespan, food consumption, body weight, and locomotion. B6C3F1 mice were fed AIN-93M diet supplemented with 1.5, 2.5, 3.5, or 4.5 g NDGA/kg diet (1.59, 2.65, 3.71 and 4.77 mg/kg body weight/day) beginning at 12 months of age. Only the 3.5 mg/kg diet produced a highly significant increase in lifespan, as judged by either the Mantel-Cox log-rank test (p = .008) or the Gehan-Breslow-Wilcoxon test (p = .009). NDGA did not alter food intake, but dose-responsively reduced weight, suggesting it decreased the absorption or increased the utilization of calories. NDGA significantly increased the incidence of liver, lung, and thymus tumors, and peritoneal hemorrhagic diathesis found at necropsy. However, clinical chemistries found little evidence for overt toxicity. While NDGA was not overtly toxic at its therapeutic dosage, its association with severe end of life pathologies does not support the idea that NDGA consumption will increase human lifespan or health-span. The less toxic derivatives of NDGA which are under development should be explored as anti-aging therapeutics.

Circulating microRNA signature of genotype-by-age interactions in the long-lived Ames dwarf mouse.

Recent evidence demonstrates that serum levels of specific miRNAs significantly change with age. The ability of circulating sncRNAs to act as signaling molecules and regulate a broad spectrum of cellular functions implicates them as key players in the aging process. To discover circulating sncRNAs that impact aging in the long-lived Ames dwarf mice, we conducted deep sequencing of small RNAs extracted from serum of young and old mice. Our analysis showed genotype-specific changes in the circulating levels of 21 miRNAs during aging [genotype-by-age interaction (GbA)]. Genotype-by-age miRNAs showed four distinct expression patterns and significant overtargeting of transcripts involved in age-related processes. Functional enrichment analysis of putative and validated miRNA targets highlighted cellular processes such as tumor suppression, anti-inflammatory response, and modulation of Wnt, insulin, mTOR, and MAPK signaling pathways, among others. The comparative analysis of circulating GbA miRNAs in Ames mice with circulating miRNAs modulated by calorie restriction (CR) in another long-lived mouse suggests CR-like and CR-independent mechanisms contributing to longevity in the Ames mouse. In conclusion, we showed for the first time a signature of circulating miRNAs modulated by age in the long-lived Ames mouse.

Differential Effects of Hepatocyte Nuclear Factor 4α Isoforms on Tumor Growth and T-Cell Factor 4/AP-1 Interactions in Human Colorectal Cancer Cells.

The nuclear receptor hepatocyte nuclear factor 4α (HNF4α) is tumor suppressive in the liver but amplified in colon cancer, suggesting that it also might be oncogenic. To investigate whether this discrepancy is due to different HNF4α isoforms derived from its two promoters (P1 and P2), we generated Tet-On-inducible human colon cancer (HCT116) cell lines that express either the P1-driven (HNF4α2) or P2-driven (HNF4α8) isoform and analyzed them for tumor growth and global changes in gene expression (transcriptome sequencing [RNA-seq] and chromatin immunoprecipitation sequencing [ChIP-seq]). The results show that while HNF4α2 acts as a tumor suppressor in the HCT116 tumor xenograft model, HNF4α8 does not. Each isoform regulates the expression of distinct sets of genes and recruits, colocalizes, and competes in a distinct fashion with the Wnt/ß-catenin mediator T-cell factor 4 (TCF4) at CTTTG motifs as well as at AP-1 motifs (TGAXTCA). Protein binding microarrays (PBMs) show that HNF4α and TCF4 share some but not all binding motifs and that single nucleotide polymorphisms (SNPs) in sites bound by both HNF4α and TCF4 can alter binding affinity in vitro, suggesting that they could play a role in cancer susceptibility in vivo. Thus, the HNF4α isoforms play distinct roles in colon cancer, which could be due to differential interactions with the Wnt/ß-catenin/TCF4 and AP-1 pathways.

Circulating small non-coding RNA signature in head and neck squamous cell carcinoma.

The Head and Neck Squamous Cell Carcinoma (HNSCC) is the sixth most common human cancer, causing 350,000 individuals die worldwide each year. The overall prognosis in HNSCC patients has not significantly changed for the last decade. Complete understanding of the molecular mechanisms in HNSCC carcinogenesis could allow an earlier diagnosis and the use of more specific and effective therapies. In the present study we used deep sequencing to characterize small non-coding RNAs (sncRNAs) in serum from HNSCC patients and healthy donors. We identified, for the first time, a multi-marker signature of 3 major classes of circulating sncRNAs in HNSCC, revealing the presence of circulating novel and known miRNAs, and tRNA- and YRNA-derived small RNAs that were significantly deregulated in the sera of HNSCC patients compared to healthy controls. By implementing a triple-filtering approach we identified a subset of highly biologically relevant miRNA-mRNA interactions and we demonstrated that the same genes/pathways affected by somatic mutations in cancer are affected by changes in the abundance of miRNAs. Therefore, one important conclusion from our work is that during cancer development, there seems to be a convergence of oncogenic processes driven by somatic mutations and/or miRNA regulation affecting key cellular pathways.

Interventions to Slow Aging in Humans: Are We Ready?

The workshop entitled 'Interventions to Slow Aging in Humans: Are We Ready?' was held in Erice, Italy, on October 8-13, 2013, to bring together leading experts in the biology and genetics of aging and obtain a consensus related to the discovery and development of safe interventions to slow aging and increase healthy lifespan in humans. There was consensus that there is sufficient evidence that aging interventions will delay and prevent disease onset for many chronic conditions of adult and old age. Essential pathways have been identified, and behavioral, dietary, and pharmacologic approaches have emerged. Although many gene targets and drugs were discussed and there was not complete consensus about all interventions, the participants selected a subset of the most promising strategies that could be tested in humans for their effects on healthspan. These were: (i) dietary interventions mimicking chronic dietary restriction (periodic fasting mimicking diets, protein restriction, etc.); (ii) drugs that inhibit the growth hormone/IGF-I axis; (iii) drugs that inhibit the mTOR-S6K pathway; or (iv) drugs that activate AMPK or specific sirtuins. These choices were based in part on consistent evidence for the pro-longevity effects and ability of these interventions to prevent or delay multiple age-related diseases and improve healthspan in simple model organisms and rodents and their potential to be safe and effective in extending human healthspan. The authors of this manuscript were speakers and discussants invited to the workshop. The following summary highlights the major points addressed and the conclusions of the meeting.

Additive regulation of hepatic gene expression by dwarfism and caloric restriction.

Disrupted growth hormone/insulin-like growth factor-1 signaling (DF) and caloric restriction (CR) extend life span and delay the onset of age-related diseases in rodents. In combination, these interventions additively extend life span. To investigate the molecular basis for these effects, we performed genome-wide, microarray expression analysis of liver from homozygous and heterozygous Ames dwarf mice fed ad libitum or CR. CR and DF additively affected a group of 95 genes. Individually and together, DF and CR independently affected the expression of 212 and 77 genes, respectively. These results indicate that DF and CR affect overlapping sets of genes and additively affect a subset of genes. Together, the interventions produced changes in gene expression consistent with increased insulin, glucagon and catecholamine sensitivity, gluconeogenesis, protein turnover, lipid beta-oxidation, apoptosis, and xenobiotic and oxidant metabolism; and decreased cell proliferation, lipid and cholesterol synthesis, and chaperone expression. These data suggest that the additive effects of DF and CR on life span develop from their additive effects on the level of expression of some genes and from their independent effects on other genes. These results provide a novel and focused group of genes closely associated with the regulation of life span in mammals.

Hepatic gene expression profiling of streptozotocin-induced diabetes.

Diabetes induces biochemical, morphological, and functional alterations in the liver. The liver is a major target of insulin action, and plays a critical role in maintaining blood glucose homeostasis. We investigated the effects of streptozotocin-induced diabetes (SID) on global hepatic gene expression in mice. We induced SID in mice by intraperitoneal injection of streptozotocin. Affymetrix (Santa Clara, CA) microarrays containing probe sets for approximately 11,000 murine genes and expressed sequence tags were used to assess the effects of SID on hepatic gene expression in mice. SID significantly altered the expression of 87 known genes in the liver. SID increased the expression of genes associated with cytoprotective stress responses, oxidative and reductive xenobiotic metabolism, cell cycle inhibition, growth arrest, apoptosis induction, and protein degradation. SID decreased the expression of genes associated with cell proliferation, growth factor signaling, protein synthesis, and xenobiotic metabolism. The novel results reported here should open new areas of investigation in diabetes research and facilitate the development of novel strategies for gene therapy and drug discovery.