Figure 1.
Impact of long-term calorie restriction on metabolic homeostasis and age-associated pathologies.
(A) Weight given as average ± SEM of WT and TgTERT mice fed with control or CR diet (see Materials and Methods). One way ANOVA was used to assess statistical significance between the four groups (WT Control vs. Wt CR: p<0.0001; TgTERT Control vs. TgTERT CR: p<0.0001; WT Control vs. TgTERT Control: p = 0.72; WT CR vs. TgTERT CR: p = 0.46). (B and C) Total fat mass of the indicated cohorts was measured at 16 months of diet (B) and 24 months of diet (C). Values are given as average ± SEM, and statistical significance was determined by the two-tailed Student’s t-test. (D and E) Glucose tolerance test (GTT) was performed at 12 months of diet. Integrated AUCs (area under the curve; (D)) and curves (E) are shown. Values are given as average ± SEM, and statistical significance was determined by the two-tailed Student’s t-test. (F) Fasting plasma insulin levels, given as mean ± SEM, was measured in the different cohorts at 12 months of diet. Statistical significance was determined by the two-tailed Student’s t-test. (G) Insulin sensitivity, estimated using the homeostatic model assessment score (HOMA-IR), was performed at 12 months of diet. Values are given as average ± SEM, and statistical significance was determined by the two-tailed Student’s t-test. (H) Femur bone mineral density (BMD) variation through lifetime of WT and TgTERT mice under control and CR diets. Values are given as average ± SEM, and statistical significance was determined by the two-tailed Student’s t-test. (I) Representative DEXA image used for BMD and fat mass calculations. (J) Neuromuscular coordination was quantified as the percentage of mice that pass with success the tightrope test. Numbers above the bars represent the number of mice that successfully pass the test over the total number of mice tested. Student’s t-test was used to assess significance between control and CR mice. Values are given as average ± SEM, and statistical significance was determined by the two-tailed Student’s t-test.
Figure 2.
Slower age-dependent telomere shortening in mice under calorie restriction.
(A and C) Mean telomere length (A) and percentage of short telomeres (C) was determined by HT QFISH on white blood cells from the indicated mice under CR and control diet. The number of mice is indicated on the top of each bar (n). Values are given as average ± SEM, and statistical significance was determined by one-tailed Student’s t-test. (B and D) Linear regression lines of the values obtained for mean telomere length (B) and percentage of short telomeres (D) measured in white blood cells. The slope ± SD of each regression line is indicated and represents the rate of telomere loss with time.
Figure 3.
Longitudinal telomere length analyses.
(A, B, C and D) Adjustment of mean telomere length values with aging to a linear model or, alternatively, to a quadratic model in the indicated mouse cohorts. Linear regression analysis was used to measure the association between age and mean telomere length. The slope of the regression line is indicated and represents the rate of telomere shortening per year (Kb). Second order polynomial adjustment (quadratic) was used for the non-linear fit model. The R2 indicates the goodness of the data adjustment to each model. The number of mice per group is shown (n). (E, F, G and H) Adjustment of the percentage of short telomeres (<15kb) to a linear model or alternatively to a quadratic model in the indicated mouse cohorts. Linear regression analysis was used to measure the association between age and the percentage of short telomeres. The slope of the regression line is indicated and represents the percentage of short telomeres enrichment per year. Second order polynomial adjustment (quadratic) was used for the non-linear fit model. The R2 indicates the goodness of the data adjustment to each model. The number of mice per group is shown (n). (I and J) Linear regression lines of the association between age and mean telomere length are shown for mice under CR (red lines) or a control diet (black lines), in both WT (I) and TgTERT (J) backgrounds. Multiple regression analysis was used to evaluate the statistical differences between the slopes of the different linear regression lines. The number of mice in each group is indicated (n). (K and L) Linear regression lines of the association between age and the percentage of short telomeres (<15 kb) are shown for mice under CR (red lines) or a control diet (black lines), in both WT (K) and TgTERT (L) backgrounds. Multiple regression analysis was used to assess the statistical differences between the different linear regression lines. The number of mice in each group is indicated (n).
Figure 4.
Calorie restriction leads to telomere maintenance and/or elongation with time in a percentage of mice.
(A, C and E) The behavior of mean telomere length was classified in two different profiles (“Decrease” and “Increase or Maintenance”) at different times of diet (1–22 months of diet, 5–22 months of diet and 9–22 months of diet; A, C and E, respectively) in the indicated groups. Numbers above bars indicate the number of mice showing the profile of interest over the total number of mice. Chi-squared test was used to assess the statistical significance of the differences observed. (B, D and F) The behavior in the percentage of short telomeres (<15 kb) was classified in two different profiles (“Increase” and “Decrease or Maintenance”) at different times of diet (1–22 months of diet, 5–22 months of diet and 9–22 months of diet; B, D and F respectively) in the indicated groups. Numbers above bars indicate the number of mice with the profile of interest over the total number of mice. Chi-squared test was used to assess the statistical significance of the differences observed. (G and H) Representative examples of the different assigned profiles for mean telomere length (“Increased”, “Maintained”, and “Shortened”) and percentage of short (<15 kb) telomeres (“Reduced”, “Maintained”, and “Increased”).
Figure 5.
Calorie restriction prevents telomere shortening in different mouse tissues and protects from telomere-mediated chromosomal aberrations in bone marrow cells.
(A,B,C) Telomere fluorescence as determined by QFISH in the indicated tissues from the different mouse cohorts studied here. Histograms represent the frequency (in percentage) of telomere fluorescence per nucleus (in arbitrary units of fluorescence [auf]). Mean telomere length is indicated by a straight line, red for mice under a control diet and yellow for mice under CR. The number of mice (n) and the total number of nuclei analysed is indicated. (D,E,F) Percentage of short telomeres (fraction of telomeres presenting intensity below 50% of the mean intensity) in the indicated tissues from the different mice cohorts studied as determined by QFISH. Student´s t-test was used for statistical analysis. (G) Representative QFISH images for different tissues from the indicated cohorts. Blue colour corresponds to chromosome DNA stained with DAPI; red dots correspond to telomeres (TTAGGG repeats). (H) Telomere length measured in metaphase spreads of BM cells from the different cohorts after hybridization with DAPI and a fluorescent Cy3 labelled PNA-telomeric probe. (I) Frequency of signal-free ends per metaphase in BM cells from the indicated mouse cohorts. (J) Frequency of multitelomeric signals (MTS) per metaphase in BM cells from the indicated mouse cohorts. (K) Representative QFISH images of metaphases from the indicated mouse cohorts. Blue, DNA stained with DAPI; red, telomeres (TTAGGG repeats). (L) Frequency of chromosome fusions per metaphase in BM cells from the indicated mouse cohorts. (M) Representative images of chromosomal fusions. (N) Frequency of other chromosome aberrations (chromosome breaks, fragments and extrachromosomal elements) per metaphase in BM cells from the indicated mouse cohorts. (O) Representative images of the different types of chromosomal aberrations scored. Blue, DAPI staining (DNA); red dots, telomeres (TTAGGG repeats) as detected with a PNA-Cy3 probe.
Figure 6.
Caloric restriction increases median and maximum longevity and protects from cancer.
(A–F) Kaplan-Meyer survival curves of the indicated mouse cohorts. The Log rank test was used for statistical analysis. Mice under CR were more susceptible to unexpected stresses such as the blood extraction procedure carried. 5 mice of the WT CR cohort and 6 mice of the TgTERT cohort died during the blood extraction and were excluded from the survival curves (of note, none of the WT or TgTERT mice show sensibility to the blood extraction procedure). (G–H) Realized lifespan of the 50% shortest (G) and longest (H) lived mice of each cohort. Student´s t-test was used for statistical analysis. (I) Percentage of mice with cancer and cancer-free mice in the different cohorts. All death mice were subjected to full histopathological analysis. (J) Percentage of mice in the different cohorts with the indicated tumours at their time of death. (K) Summary table with the findings regarding telomere length and survival of the different cohorts.