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.

Organ reserve, excess metabolic capacity, and aging.

"Organ reserve" refers to the ability of an organ to successfully return to its original physiological state following repeated episodes of stress. Clinical evidence shows that organ reserve correlates with the ability of older adults to cope with an added workload or stress, suggesting a role in the process of aging. Although organ reserve is well documented clinically, it is not clearly defined at the molecular level. Interestingly, several metabolic pathways exhibit excess metabolic capacities (e.g., bioenergetics pathway, antioxidants system, plasticity). These pathways comprise molecular components that have an excess of quantity and/or activity than that required for basic physiological demand in vivo (e.g., mitochondrial complex IV or glycolytic enzymes). We propose that the excess in mtDNA copy number and tandem DNA repeats of telomeres are additional examples of intrinsically embedded structural components that could comprise excess capacity. These excess capacities may grant intermediary metabolism the ability to instantly cope with, or manage, added workload or stress. Therefore, excess metabolic capacities could be viewed as an innate mechanism of adaptability that substantiates organ reserve and contributes to the cellular defense systems. If metabolic excess capacities or organ reserves are impaired or exhausted, the ability of the cell to cope with stress is reduced. Under these circumstances cell senescence, transformation, or death occurs. In this review, we discuss excess metabolic and structural capacities as integrated metabolic pathways in relation to organ reserve and cellular aging.

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.

Plasma miRNA Profile of Crohn's Disease and Rheumatoid Arthritis Patients.

Crohn's disease (CD) and rheumatoid arthritis (RA) are immune mediated inflammatory diseases. Several studies indicate a role for microRNAs (miRNAs) in the pathogenesis of a variety of autoimmune diseases, including CD and RA. Our study's goal was to investigate circulating miRNAs in CD and RA patients to identify potential new biomarkers for early detection and personalized therapeutic approaches for autoimmune diseases. For this study, subjects with CD (n = 7), RA (n = 8) and healthy controls (n = 7) were recruited, and plasma was collected for miRNA sequencing. Comparison of the expression patterns of miRNAs between CD and healthy patients identified 99 differentially expressed miRNAs. Out of these miRNAs, 4 were down regulated, while 95 were up regulated. Comparison of miRNAs between RA and healthy patients identified 57 differentially expressed miRNAs. Out of those, 12 were down regulated, while 45 were up regulated. For all the miRNAs down regulated in CD and RA patients, 420 GO terms for biological processes were similarly regulated between both groups. Therefore, the identification of new plasma miRNAs allows the emergence of new biomarkers that can assist in the diagnosis and treatment of CD and RA.

Specific PIWI-Interacting RNAs and Related Small Noncoding RNAs Are Associated With Ovarian Aging in Ames Dwarf (df/df) Mice.

The Ames dwarf (df/df) mouse is a well-established model for delayed aging. MicroRNAs (miRNAs), the most studied small noncoding RNAs (sncRNAs), may regulate ovarian aging to maintain a younger ovarian phenotype in df/df mice. In this study, we profile other types of ovarian sncRNAs, PIWI-interacting RNAs (piRNAs) and piRNA-like RNAs (piLRNAs), in young and aged df/df and normal mice. Half of the piRNAs derive from transfer RNA fragments (tRF-piRNAs). Aging and dwarfism alter the ovarian expression of these novel sncRNAs. Specific tRF-piRNAs that increased with age might target and decrease the expression of the breast cancer antiestrogen resistance protein 3 (BCAR3) gene in the ovaries of old df/df mice. A set of piLRNAs that decreased with age and map to D10Wsu102e mRNA may have trans-regulatory functions. Other piLRNAs that decreased with age potentially target and may de-repress transposable elements, leading to a beneficial impact on ovarian aging in df/df mice. These results identify unique responses in ovarian tissues with regard to aging and dwarfism. Overall, our findings highlight the complexity of the aging effects on gene expression and suggest that, in addition to miRNAs, piRNAs, piLRNAs, tRF-piRNAs, and their potential targets can be central players in the maintenance of a younger ovarian phenotype in df/df mice.

Data Mining of Small RNA-Seq Suggests an Association Between Prostate Cancer and Altered Abundance of 5' Transfer RNA Halves in Seminal Fluid and Prostatic Tissues.

Extracellular RNAs are gaining clinical interest as biofluid-based noninvasive markers for diseases, especially cancer. In particular, derivatives of transfer RNA (tRNA) are emerging as a new class of small-noncoding RNAs with high biomarker potential. We and others previously reported alterations in serum levels of specific tRNA halves in disease states including cancer. Here, we explored seminal fluid for tRNA halves as potential markers of prostate cancer. We found that 5' tRNA halves are abundant in seminal fluid and are elevated in prostate cancer relative to noncancer patients. Importantly, most of these tRNA halves are also detectable in prostatic tissues, and a subset were increased in malignant relative to adjacent normal tissue. These findings emphasize the potential of 5' tRNA halves as noninvasive markers for prostate cancer screening and diagnosis and provide leads for future work to elucidate a putative role of the 5' tRNA halves in carcinogenesis.

Caloric restriction impacts plasma microRNAs in rhesus monkeys.

Caloric restriction (CR) is one of the most robust interventions shown to delay aging in diverse species, including rhesus monkeys (Macaca mulatta). Identification of factors involved in CR brings a promise of translatability to human health and aging. Here, we show that CR induced a profound change in abundance of circulating microRNAs (miRNAs) linked to growth and insulin signaling pathway, suggesting that miRNAs are involved in CR's mechanisms of action in primates. Deep sequencing of plasma RNA extracts enriched for short species revealed a total of 243 unique species of miRNAs including 47 novel species. Approximately 70% of the plasma miRNAs detected were conserved between rhesus monkeys and humans. CR induced or repressed 24 known and 10 novel miRNA species. Regression analysis revealed correlations between bodyweight, adiposity, and insulin sensitivity for 10 of the CR-regulated known miRNAs. Sequence alignment and target identification for these 10 miRNAs identify a role in signaling downstream of the insulin receptor. The highly abundant miR-125a-5p correlated positively with adiposity and negatively with insulin sensitivity and was negatively regulated by CR. Putative target pathways of CR-associated miRNAs were highly enriched for growth and insulin signaling that have previously been implicated in delayed aging. Clustering analysis further pointed to CR-induced miRNA regulation of ribosomal, mitochondrial, and spliceosomal pathways. These data are consistent with a model where CR recruits miRNA-based homeostatic mechanisms to coordinate a program of delayed aging.

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.

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.

Combined activation of the energy and cellular-defense pathways may explain the potent anti-senescence activity of methylene blue.

Methylene blue (MB) delays cellular senescence, induces complex-IV, and activates Keap1/Nrf2; however, the molecular link of these effects to MB is unclear. Since MB is redox-active, we investigated its effect on the NAD/NADH ratio in IMR90 cells. The transient increase in NAD/NADH observed in MB-treated cells triggered an investigation of the energy regulator AMPK. MB induced AMPK phosphorylation in a transient pattern, which was followed by the induction of PGC1α and SURF1: both are inducers of mitochondrial and complex-IV biogenesis. Subsequently MB-treated cells exhibited >100% increase in complex-IV activity and a 28% decline in cellular oxidants. The telomeres erosion rate was also significantly lower in MB-treated cells. A previous research suggested that the pattern of AMPK activation (i.e., chronic or transient) determines the AMPK effect on cell senescence. We identified that the anti-senescence activity of MB (transient activator) was 8-times higher than that of AICAR (chronic activator). Since MB lacked an effect on cell cycle, an MB-dependent change to cell cycle is unlikely to contribute to the anti-senescence activity. The current findings in conjunction with the activation of Keap1/Nrf2 suggest a synchronized activation of the energy and cellular defense pathways as a possible key factor in MB's potent anti-senescence activity.

ApoHRP-based assay to measure intracellular regulatory heme.

The majority of the heme-binding proteins possess a "heme-pocket" that stably binds to heme. Usually known as housekeeping heme-proteins, they participate in a variety of metabolic reactions (e.g., catalase). Heme also binds with lower affinity to the "Heme-Regulatory Motifs" (HRM) in specific regulatory proteins. This type of heme binding is known as exchangeable or regulatory heme (RH). Heme binding to HRM proteins regulates their function (e.g., Bach1). Although there are well-established methods for assaying total cellular heme (e.g., heme-proteins plus RH), currently there is no method available for measuring RH independent of the total heme (TH). The current study describes and validates a new method to measure intracellular RH. This method is based on the reconstitution of apo-horseradish peroxidase (apoHRP) with heme to form holoHRP. The resulting holoHRP activity is then measured with a colorimetric substrate. The results show that apoHRP specifically binds RH but not with heme from housekeeping heme-proteins. The RH assay detects intracellular RH. Furthermore, using conditions that create positive (hemin) or negative (N-methyl protoporphyrin IX) controls for heme in normal human fibroblasts (IMR90), the RH assay shows that RH is dynamic and independent of TH. We also demonstrated that short-term exposure to subcytotoxic concentrations of lead (Pb), mercury (Hg), or amyloid-ß (Aß) significantly alters intracellular RH with little effect on TH. In conclusion the RH assay is an effective assay to investigate intracellular RH concentration and demonstrates that RH represents ∼6% of total heme in IMR90 cells.

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.

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.

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.

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.