The in vivo gene expression signature of oxidative stress.

How higher organisms respond to elevated oxidative stress in vivo is poorly understood. Therefore, we measured oxidative stress parameters and gene expression alterations (Affymetrix arrays) in the liver caused by elevated reactive oxygen species induced in vivo by diquat or by genetic ablation of the major antioxidant enzymes CuZn-superoxide dismutase (Sod1) and glutathione peroxidase-1 (Gpx1). Diquat (50 mg/kg) treatment resulted in a significant increase in oxidative damage within 3-6 h in wild-type mice without any lethality. In contrast, treatment of Sod1(-/-) or Gpx1(-/-) mice with a similar concentration of diquat resulted in a significant increase in oxidative damage within an hour of treatment and was lethal, i.e., these mice are extremely sensitive to the oxidative stress generated by diquat. The expression response to elevated oxidative stress in vivo does not involve an upregulation of classic antioxidant genes, although long-term oxidative stress in Sod1(-/-) mice leads to a significant upregulation of thiol antioxidants (e.g., Mt1, Srxn1, Gclc, Txnrd1), which appears to be mediated by the redox-sensitive transcription factor Nrf2. The main finding of our study is that the common response to elevated oxidative stress with diquat treatment in wild-type, Gpx1(-/-), and Sod1(-/-) mice and in untreated Sod1(-/-) mice is an upregulation of p53 target genes (p21, Gdf15, Plk3, Atf3, Trp53inp1, Ddit4, Gadd45a, Btg2, Ndrg1). A retrospective comparison with previous studies shows that induction of these p53 target genes is a conserved expression response to oxidative stress, in vivo and in vitro, in different species and different cells/organs.

Tissue specific and non-specific changes in gene expression by aging and by early stage CR.

Aging alters the expression of a variety of genes. Calorie restriction (CR), which extends life span in laboratory rodents, also changes gene expression. This study investigated changes in gene expression across three different tissues from the same mouse to examine how aging and early stage CR influence gene expression in different tissues of an organism. Expression profiling of heart, liver, and hypothalamus tissues was done in young (4-6 months) ad libitum fed (AL), young CR (2.5-4.5 months of CR), and old (26-28 months) AL male C57BL/6 mice. Aging significantly altered the expressions of 309, 1819, and 1085 genes in heart, liver, and hypothalamus tissues, respectively. In nine genes, aging altered expression across all three tissues although the regulation directions did not agree across all three tissues for some genes. Early stage CR in young mice significantly changed the expressions of 192, 839, and 100 genes in heart, liver, and hypothalamus tissues, respectively, and seven genes altered expression across all three tissues; three were up regulated and four were down regulated. The results of Gene Ontology (GO) Biological Process analysis indicated up regulation of antigen processing/presentation genes by aging and down regulation of stress response genes by early stage CR in all three tissues. The comparison of the results of aging and short term CR studies showed there were 389 genes, 18 GO biological processes, and 20 GO molecular functions in common.

Early hypothalamic response to age-dependent gene expression by calorie restriction.

Molecular events linking the initial detection of calorie restriction (CR) to changes in gene expression throughout the organism that ultimately retard aging in CR animals are unknown. This study measured changes in gene expression induced by CR and by aging in the hypothalamus, which likely plays a central role in the initial perception of and response to CR. Hypothalamic expression profiling was done in young (4-6 months) ad libitum fed (AL), young CR (2.5-4.5 months of CR), and old (26-28 months) AL male C57BL/6 mice. CR altered the expression of 137 genes and aging altered 1222. Only 8 age-related genes were oppositely regulated by CR. To test whether reduced plasma glucose is a signal in altering hypothalamic gene expression, we examined GLUT4 transgenic mice (C57BL/6 background; 4-6 months), which have reduced plasma glucose similar to that of CR mice. Twenty-seven genes differed between transgenic and non-transgenic mice; nine of these were only altered by CR. The decreased plasma glucose had a limited role in CR mediated hypothalamic gene expression.

Microarray evaluation of dietary restriction.

Dietary restriction (DR) extends the life span and retards many age-related cellular and molecular changes in laboratory rodents. However, neither its underlying mechanism nor the limits of its action are fully understood. In this review, we assessed the effect of DR on gene expression in vertebrate and invertebrate animals using data generated by microarrays. Altered genes in DR mice reported in 15 articles published since 1999 were compared. A comparison of altered genes by DR in mice, rats, pigs, monkeys, yeast, and flies showed no common gene altered by DR among different species. It seems that individual genes altered in the expression by DR were constrained within species. When we compared the functions of altered genes across all species, we found that certain functions such as metabolism, energy metabolism, stress and immune response, cell growth, and transcription regulation were shared among species. Although individual genes seem to be affected by DR differently among species, the overall physiologic influence of DR may be similar.

SAS programs for real-time RT-PCR having multiple independent samples.

Relative real-time reverse transcription PCR (RT-PCR) has become an important tool for quantifying changes in messenger RNA (mRNA) populations following differential development or stimulation of tissues or cells. However, the best methods for conducting such experiments and analyzing the resultant data remain an issue of discussion. In this report we describe an appropriate experimental methodology and the computer programs necessary to generate a meaningful statistical analysis of the combined biological and experimental variability in such experiments. Specifically, logarithmic transformations of raw fluorescence data from the log-linear portion of real-time PCR growth curves for both target and reference genes are analyzed using a SAS/STAT Mixed Procedure program specifically designed to give a point estimate of the relative expression ratio of the target gene with associated 95% confidence interval. The program code is open-source and is printed in the text.

Hepatic genes altered in expression by food restriction are not influenced by the low plasma glucose level in young male GLUT4 transgenic mice.

Because food restriction (FR) has a profound effect on most tissues, it is plausible that the modulation of aging by FR occurs through cellular processes such as gene expression. The effect of FR in lowering plasma glucose levels has been demonstrated in mice, rats, and nonhuman primates. The consistency of this finding suggests that decreased plasma glucose may be an important consequence of FR. Indeed, lowering plasma glucose in the absence of FR would be expected to change the expression of some of the same genes as seen with FR. GLUT4 transgenic (TG) mice were particularly suited to this examination because they have low plasma glucose levels like FR mice. We investigated altered gene expression by FR and the effect of low plasma glucose levels caused by genetic manipulation by measuring mRNA expression in liver tissues of 4- to 6-mo-old mice with 2.5-4.5 mo of FR using microarrays and 4 groups: GLUT4 TG (C57BL/6 background) consumed food ad libitum (AL), GLUT4 TG FR, wild-type littermates AL, and wild-type littermates FR. The 3 statistical analysis methods commonly indicated that FR altered the expression of 1277 genes; however, none of these genes was altered by additional GLUT4 expression. In fact, the low plasma glucose level in GLUT4 TG mice did not affect gene expression. Some results were confirmed by real-time quantitative RT-PCR. We conclude that a low plasma glucose level does not contribute to or coincide with the effect of FR on gene expression in the liver.

Prospective study of protein-energy supplementation early in life and of growth in the subsequent generation in Guatemala.

BACKGROUND: The secular increase in height is assumed to result from long-term improvements in nutritional intakes and reductions in infectious disease burdens. Nutritional supplementation in early life reduces stunting in chronically undernourished populations. It is unknown whether these improvements can be transmitted to subsequent generations. OBJECTIVE: Our objective was to estimate the intergenerational effect on offspring length of improved nutrition in the mother's childhood. DESIGN: We studied 263 children born in 1996-1999 to 231 women who had received nutritional supplementation, ie, atole (high-protein, moderate-energy drink) or fresco (nonprotein, low-energy drink), prenatally and up to age 7 y as part of a community trial in Guatemala between 1969 and 1977. Child length was measured at different times to age 36 mo. RESULTS: Children born to women who received the enhanced supplement were taller (age-adjusted difference: 0.80 cm; 95% CI: 0.16, 1.44 cm) than were children whose mothers received the low-energy supplement. This increment was independent of the children's birth weight or socioeconomic status but was substantially attenuated and no longer significant after adjustment for maternal height (adjusted difference: 0.43 cm; 95% CI: -0.10, 0.96 cm; P > 0.10). The effect of maternal nutritional supplementation was more pronounced in boys than in girls (P for interaction < 0.10) and in children born to women who received supplements at ages 3-7 y than in children born to women who received supplements at ages 0-3 y (P for interaction < 0.01). CONCLUSION: Nutritional supplementation in childhood has positive effects on both the supplemented persons and on the subsequent generation.