Gene expression profiling of long-lived dwarf mice: longevity-associated genes and relationships with diet, gender and aging

Abstract

Background: Long-lived strains of dwarf mice carry mutations that suppress growth hormone (GH) and insulin-like growth factor I (IGF-I) signaling. The downstream effects of these endocrine abnormalities, however, are not well understood and it is unclear how these processes interact with aging mechanisms. This study presents a comparative analysis of microarray experiments that have measured hepatic gene expression levels in long-lived strains carrying one of four mutations (Prop1(df/df), Pit1(dw/dw), Ghrhr(lit/lit), GHR-KO) and describes how the effects of these mutations relate to one another at the transcriptional level. Points of overlap with the effects of calorie restriction (CR), CR mimetic compounds, low fat diets, gender dimorphism and aging were also examined.

Results: All dwarf mutations had larger and more consistent effects on IGF-I expression than dietary treatments. In comparison to dwarf mutations, however, the transcriptional effects of CR (and some CR mimetics) overlapped more strongly with those of aging. Surprisingly, the Ghrhr(lit/lit) mutation had much larger effects on gene expression than the GHR-KO mutation, even though both mutations affect the same endocrine pathway. Several genes potentially regulated or co-regulated with the IGF-I transcript in liver tissue were identified, including a DNA repair gene (Snm1) that is upregulated in proportion to IGF-I inhibition. A total of 13 genes exhibiting parallel differential expression patterns among all four strains of long-lived dwarf mice were identified, in addition to 30 genes with matching differential expression patterns in multiple long-lived dwarf strains and under CR.

Conclusion: Comparative analysis of microarray datasets can identify patterns and consistencies not discernable from any one dataset individually. This study implements new analytical approaches to provide a detailed comparison among the effects of life-extending mutations, dietary treatments, gender and aging. This comparison provides insight into a broad range of issues relevant to the study of mammalian aging. In this context, 43 longevity-associated genes are identified and individual genes with the highest level of support among all microarray experiments are highlighted. These results provide promising targets for future experimental investigation as well as potential clues for understanding the functional basis of lifespan extension in mammalian systems.

Figure 1

IGF-I expression. The vertical axis corresponds to log-transformed gene expression values. The 31 contrasts listed in Table 1 are ordered along the horizontal axis. Red circles correspond to expression values of replicate observations associated with the A treatment of each contrast, while black diamonds indicate replicate expression values associated with the control B treatment of each contrast (see Table 1).

Figure 2

Potential IGF-I regulated or co-regulated genes. The vertical axis corresponds to log-transformed fold-change. The 31 contrasts listed in Table 1 are ordered along the horizontal axis. The thick black line represents fold-changes associated with IGF-I across the 31 contrasts. Red lines represent five genes for which fold-changes across contrasts are most positively associated with those of IGF-I (Mup3, Es31, Igfals, Keg1, Socs2). Green lines represent five genes for which fold-changes across contrasts are most negatively associated with those of IGF-I (Scd2, Slc16a7, Pcp4l1, Snm1, Igfbp1). Additional files 1 and 2 provide plots for the top 40 genes most positively and negatively associated with the IGF-I expression pattern among all contrasts.

Figure 3

Differential Expression Signatures. Each row corresponds to one of the contrasts listed in Table 1. Each column corresponds to one of 2192 genes differentially expressed with respect to more than one contrast. Rows have been ordered to correspond to the dendrogram shown in Figure 4. Red coloring indicates that a gene is upregulated (P < 0.05), while green coloring indicates that a gene is downregulated (P < 0.05).

Figure 4

Hierarchical cluster analysis. Each branch corresponds to a differential expression signature shown in Figure 3. The horizontal axis indicates the average level of similarity at which two clusters were joined (see Equation 1 in Methods).

Figure 5

Differential expression signature similarity matrix. In the upper-right triangle region, dark red colors indicate high similarity between signatures associated with two contrasts (indicated by row and column labels). This similarity is defined by Equation (1) in the Methods section. The binary coding in the lower-left triangle region indicates whether signatures associated with two contrasts exhibit a significant level of similarity. The statistical procedure used to evaluate similarity is described in the Methods section. Contrast pairs with significant similarity (P < 0.05) are coded dark red, while pairs with non-significant similarity have no coloring.

Figure 6

Longevity-associated genes I. Listed genes are those that are differentially expressed with respect to each of four-long lived dwarf models (Snell, Ames, Little, GHR-KO). Each row corresponds to an individual candidate gene, while each column corresponds to one of the contrasts listed in Table 1. Red squares indicate significant upregulation, while green squares indicate significant downregulation.

Figure 7

Candidate gene expression versus mean lifespan I. The expression level of nine candidate genes was examined among 21 BxD mouse strains. Expression data was generated by Williams et al. [30] (GEO series GSE6621). Lifespans of BxD strains were assayed by Gelman et al. [31]. The dashed horizontal line indicates the average gene expression level for each gene, while the solid line represents the least-squares regression estimate. Individual plots are shown for (A) Hao3, (B) Sult2a2, (C) Spink3, (D) Socs2, (E) Mup4, (F) Igfals, (G) Lifr, (H) Igf1 and (I) Efgr. The spearman rank correlation between expression and mean lifespan is shown in the upper right corner of each plot.

Figure 8

Longevity-associated genes II. Listed genes are those that are differentially expressed with respect to at least three of four long-lived dwarf models (Snell, Ames, Little, GHR-KO), and with respect to at least one of the four caloric restriction contrasts. Each row corresponds to an individual candidate gene, while each column corresponds to one of the contrasts listed in Table 1. Red squares indicate significant upregulation and green squares indicate significant downregulation.

Figure 9

Candidate gene expression versus mean lifespan II. The expression level of six candidate genes was examined among 21 BxD mouse strains (see Fig. 6 caption). The dashed horizontal line indicates the average gene expression level for each gene, while the solid line indicates the estimates least-squares regression estimate. Individual plots are shown for (A) Fmo3, (B) Ero11b, (C) Serpina12, (D) Hes6, (E) Cyp2f2 and (F) Cyp4a14. The spearman rank correlation between expression and mean lifespan is shown in the upper right corner of each plot.