Mice with longer telomeres than those of the natural species

We previously showed that it is possible to generate mouse ES cells with hyper-long telomeres in the absence of any genetic manipulations, and that these ES cells can be used to generate mouse chimeras, which carry cells with longer telomeres than those of the natural species26. In order to address the impact of excessively long telomeres in a given species, here, we have generated a cohort of chimeric mice with a 100% contribution from ES cells with hyper-long telomeres ES cells. To this end, ~100 morulae at the eight-cell state were microinjected with 6–10 GFP-positive female ES cells with hyper-long telomeres at passage 16 by laser-assisted perforation of the “zona pellucida” obtaining chimeric mice which were 100% contributed by the GFP-positive ES cells (Fig. 1a). In particular, adult mice (1.5–2 years of age) showed 100% of their cells positive for eGFP as determined by immunohistochemistry (IHC) with anti GFP antibodies in different mouse tissues (Fig. 1b). The fact that, even at old age, chimeric mice show 100% of the cells that are GFP-positive, and therefore are derived from ES cells with hyper-long telomeres, indicates that these cells with hyper-long telomeres are not negatively selected during aging. Further supporting that cells with hyper-long telomeres are not deleterious, all mouse tissues showed a normal histology even at older ages (Fig. 1b). Strikingly, hyper-long telomere mice were significantly leaner than control mice from the same genetic background (Fig. 1c). To further study this unexpected phenotype, we carried out a longitudinal follow-up of mouse weight throughout their entire lifespan and found that hyper-long telomere mice showed a significant reduction in body weight which started from 40 weeks of age onwards (Fig. 1d).

Fig. 1 Hyper-long telomere mice are leaner than normal mice. a Scheme showing generation of mice with hyper-long telomeres. eGFP ES cells are cultured in 2i medium until passage 16 and they are microinjected into morulae to obtain 100% contributing chimaeras. b Representative images showing 100% eGFP ES cell contribution in different organs and tissues. c, d Longitudinal follow up of weight in the indicated cohorts. Hyper-long telomere mice show a significant reduction in body weight from 40 weeks onwards (c) and this body size reduction is maintained until the last timepoint measured (c, d). Error bars represent standard error. t-test was used for statistical analysis. The number of mice is indicated in each case. *p < 0.05. **p < 0.01. Source data are provided as a Source Data file Full size image

Thus, here we generated viable mice that are 100% contributed by ES cells with hyper-long telomeres. We did not find any overt phenotypes in these mice other than they are leaner compared to normal mice.

Less fat accumulation in hyper-long telomere mice

In order to investigate the leaner phenotype of hyper-long telomere mice, we performed densitometry assays at 75 and 100 weeks of age in both hyper-long telomere mice and normal telomere length controls from the same genetic background. We found that hyper-long telomere mice showed significantly lower fat content than age-matched control mice at two different ages (75 and 100 weeks-old) (Fig. 2a). In contrast, no differences were observed in either bone mineral density (BMD) (Fig. 2b) or in total lean mass between hyper-long telomere mice and the normal telomere length controls (Fig. 2c). In agreement with lower fat accumulation, hyper-long telomere mice also showed a significantly reduced skin subcutaneous fat layer compared to age-matched control mice (100 weeks of age) (Fig. 2d, e). These results indicate that the reduced body size of hyper-long telomere mice is due to a lower accumulation of fat.

Fig. 2 Reduced body size in hyper-long telomere mice is due to lower fat accumulation. a–c Densitometry analysis of hyper-long telomere mice and normal mice at two different ages. a Quantification of percentage of total fat. b Quantification of bone density. c Quantification of total lean mass. d, e Quantification of the skin subcutaneous fat layer in 100 weeks-old hyper-long mice and age-matched controls (d), and representative image showing skin subcutaneous fat layer thickness. Measurements were done by ImageJ software and calculated by the mean value of 30–40 different measurements all over the skin subcutaneous fat layer. Error bars represent standard error. t-test was used for statistical analysis. The number of mice is indicated in each case. *p < 0.05. ***p < 0.001. Source data are provided as a Source Data file Full size image

Improved metabolic parameters in hyper-long telomere mice

As mice with hyper-long telomeres showed reduced fat accumulation, we next set to determine different metabolic parameters. First, we measured the levels of albumin, creatinine, bilirubin, urea, alanine aminotransferase (ALT), cholesterol, the low-density lipoprotein (LDL), and the high-density lipoprotein (HDL) in blood serum from both hyper-long telomere mice and age-matched controls at 50, 75, and 100 weeks of age. We found no significant differences in the levels of creatinine, bilirubin, albumin, urea and, HDL at any of the time points between hyper-long telomere mice and control mice (Fig. 3a–e). Interestingly, hyper-long telomere mice showed significantly lower levels of the “bad cholesterol”, LDL, compared to control mice at the three time points studied (Fig. 3f). Hyper-long telomere mice also showed significantly lower levels of cholesterol and of the hepatic damage marker ALT compared with control mice (Fig. 3g, h). Thus, we found lower cholesterol levels, including LDL levels, and decreased liver damage in hyper-long telomere mice compared to control mice.

Fig. 3 Hyper-long telomere mice show less metabolic aging. a–h Pentra quantification of different serum metabolites at 50, 75, and 100 weeks-old. Metabolites were measured in serum obtained from blood extracts using ABX Pentra, a quantification of creatinine levels, b quantification of total bilirubin levels, c quantification of albumin levels, d quantification of urea levels, e quantification of HDL (high density lipoprotein) levels, f quantification of LDL (low density lipoprotein) levels, g quantification of ALT (alanine aminotransferase) levels and h quantification of cholesterol levels. i–k Glucose tolerance test (GTT). GTT was performed in hyper-long telomere mice and age-matched controls at 50 (i), 75 (j) and 100 (k) weeks of age by intraperitoneal glucose injection after 16 h fasting. l–n Insulin tolerance test (ITT). ITT was performed in hyper-long telomere mice and age-matched controls at 50 (l), 75 (m) and 100 (n) weeks of age by intraperitoneal insulin injection. Error bars represent standard error. t-test was used for statistical analysis. The number of mice is indicated in each case. *p < 0.05. **p < 0.01. ***p < 0.001. Source data are provided as a Source Data file Full size image

To further asses the metabolic effects of hyper-long telomeres, we next studied glucose metabolism by performing a glucose tolerance test (GTT). To this end, glucose was injected intraperitoneally in age-matched hyper-long telomere mice and normal telomere mice at 50, 75, and 100 weeks of age previously fasted for 16 h. At all timepoints, hyper-long telomere mice showed an increased glucose sensitivity (Fig. 3i–k). We obtained similar results when we performed an insulin tolerance test (ITT). In this case, hyper-long telomere mice showed a better insulin tolerance at both 75 and 100 weeks of age compared to the normal telomere length controls (Fig. 3l–n).

In summary, these results indicate that hyper-long telomere mice have an improved metabolic fitness compared to normal telomere length mice. In particular, they show lower levels of LDL, cholesterol, and ALT in blood, as well as are more sensitive to glucose and insulin even at old age (100-weeks old), suggesting less metabolic aging compared to age-matched control mice.

Hyper-long telomere mice live longer and have less spontaneous tumour incidence

Critically short telomeres in humans and mice can lead to premature aging and shorter lifespans by limiting the ability of stem cells to regenerate tissues11,12,27. In turn, we previously showed that longer telomeres owing to telomerase over-expression in adult mice can increase mouse longevity17,28. Thus, here we set to address whether longer telomeres than normal in the adult organism, in the absence of telomerase over-expression, were sufficient to increase longevity. Strikingly, we found that hyper-long telomere mice showed a significant increase of 12.75% in median longevity as well as an increase in maximum longevity of 8.40% compared to normal telomere length controls (Fig. 4a). These findings indicate that long telomeres per se, even in the absence of telomerase overexpression, are sufficient to increase mouse longevity.

Fig. 4 Hyper-long telomere mice live longer and have less spontaneous cancer. a Survival proportions of 100% hyper-long chimeric mice compared to control mice from the same background. Hyper-long telomere mice show a 12.75% increase in median lifespan and an 8.4% increase in maximum lifespan. b Quantification of spontaneous tumor incidence in hyper-long telomere mice and controls. Hyper-long telomere mice show a decrease in almost 20% in tumor incidence compared to control mice from the same background. Error bars represent standard error. Mantel–Cox test was used for statistical analysis in survival curves and Chi-square test for spontaneous tumor incidence. The number of mice is indicated in each case. *p < 0.05. **p < 0.01. ***p < 0.001. Source data are provided as a Source Data file Full size image

The fact that hyper-long telomere mice had an increased longevity also suggested that they were unlikely to have any deleterious effects promoting cancer. This question is of particular importance because previous works have correlated presence of long telomeres in humans with increased cancer incidence29,30,31,32,33. To address this, we studied the spontaneous tumor incidence of hyper-long telomere mice and control mice. We found that hyper-long telomere mice showed a reduction of almost 50% in the number of mice that developed tumors compared to the normal telomere length control mice, although this difference did not reach significance (Fig. 4b). Of notice, the cause of death of tumor free mice was associated to general body degeneration associated to the aging process, as well as uterine infection in some female mice.

These findings clearly demonstrate that long telomeres per se do not lead to increased cancer, at least in mice. Instead, longer telomeres are clearly correlated with an increased mouse longevity.

Hyper-long telomere mice have normal cognitive capabilities

Next, we wondered whether having longer telomeres than normal could affect cognitive capabilities in these mice. To address this, we performed different cognitive tests at 50, 75, and 100 weeks of age. To evaluate neuromuscular endurance, we performed a rotarod test, which measures the time that mice are able to stay on a rotating platform with accelerated movement without falling (Supplementary Fig. 1a). We observed no differences between the hyper-long telomere mice and the control mice at the different time points (Supplementary Fig. 1b). In order to study mouse coordination, we performed a tightrope test, which evaluates the capability of the mice to stay on a rope without falling during at least 1 min (Supplementary Fig. 1c). Again, we did not see any significance difference in performance between the hyper-long telomere mice and the controls (Supplementary Fig. 1d). Finally, to measure the sensory perception of mice we used a buried food test, in which we measured the ability of mice to find a buried food pellet after 16 h fasting (Supplementary Fig. 1e). Again, we found no differences between hyper-long telomere mice and controls (Supplementary Fig. 1f).

Together, these results suggest that hyper-long telomere mice show normal cognitive capabilities, such as normal coordination, balance and smell.

Hyper-long telomere mice retain longer telomeres at old ages

In order to determine whether hyper-long telomere mice retained longer telomeres even at old age, we measured telomere length in different tissues at 100 weeks of age. Telomere length was determined in intestine and skin, as examples of proliferative tissues, and in brain as a non-proliferative tissue. We found that in all cases, 100-weeks old hyper-long telomere mice showed longer telomeres on average, than the normal age-matched controls (Fig. 5a–f). In agreement with longer telomeres, hyper-long telomere mice also showed a significantly lower accumulation of short telomeres at old age compared with the age-matched control mice (Fig. 5a–f).

Fig. 5 Hyper-long telomere mice show longer telomeres throughout their lifespan in both proliferative and non-proliferative tissues. a–f Mean telomere fluorescence and percentage of short telomeres in back skin (a, b), intestine (c, d), brain (e, f) in 100 weeks-old hyper-long telomere mice and age-matched controls. Error bars represent standard error. t-test was used for statistical analysis. The number of mice is indicated in each case. *p < 0.05. **p < 0.01. ***p < 0.001. Source data are provided as a Source Data file Full size image

Importantly, as one of the main phenotypes in the hyper-long telomere mice is an improved metabolic profile, we measured telomere length and percentage of short telomeres in metabolically relevant tissues, such as the liver, white adipose tissue (WAT), and brown adipose tissue (BAT) at 100–110 weeks old. Interestingly, we found a very pronounced increase in telomere length in the hyper-long telomere mice in all three metabolic tissues compared to control mice from the same background (Fig. 6a–f). Accordingly, we observed a significant decrease in the percentage of short telomeres in the hyper-long telomere mice compared to controls in all three tissues (Fig. 6a–f).

Fig. 6 Hyper-long telomere mice show longer telomeres throughout their lifespan in metabolic-related tissues. a–f Mean telomere fluorescence and percentage of short telomeres in liver (a, b), white adipose tissue (c, d) and brown adipose tissue (e, f), in 100 weeks-old hyper-long telomere mice and age-matched controls. Error bars represent standard error. t-test was used for statistical analysis. The number of mice is indicated in each case. *p < 0.05. **p < 0,01. ***p < 0,001. Source data are provided as a Source Data file Full size image

To rule out that longer telomeres in the hyper-long telomere mice where the result of an altered telomerase expression in the adult organism we determined mRNA levels of the two essential telomerase components Tert and Terc in hyper-long telomeres and normal telomere mice at 100–110 weeks of age in the liver and the WAT. As expected, we found that Tert was not expressed in these tissues in agreement with previous reports showing that Tert expression is undetectable in the majority of adult mouse tissues after birth10,17. Furthermore, we did not see any significant differences in the mRNA expression of Terc between the hyper-long telomere mice and the normal controls in the liver and the WAT (Fig. 7a–d).

Fig. 7 Hyper-long telomere mice does not present altered telomerase expression levels. a–d Tert and Terc mRNA expression levels as measured by Q-PCR in liver (a, b) and in white adipose tissue (c, d) in age-matched (100 weeks old) hyper-long telomere mice and control mice. Error bars represent standard error. t-test was used for statistical analysis. The number of mice is indicated in each case. *p < 0.05. **p < 0,01. ***p < 0,001. Source data are provided as a Source Data file Full size image

Altogether, these results demonstrate that hyper-long telomere mice retain longer telomeres with aging, including metabolic tissues such liver, white adipose tissue and brown adipose tissue, in the absence of Tert overexpression.

Hyper-long telomere mice show less senescence and DNA damage

Next, we addressed whether longer telomeres than normal could be promoting or protecting from age-associated DNA damage. To this end, we performed a telomere Q-FISH to identify telomeres followed by an immunofluorescence against the DNA damage marker 53BP1 in liver of 100 weeks hyper-long telomere chimaeras and controls. To this end, we quantified the number of cells presenting ≥2 53BP1 foci (Fig. 8a, c). Interestingly, hyper-long telomere mice show an 8-fold decrease in 53BP1-positive cells compared to controls (Fig. 8a). Importantly, the percentage of cells presenting ≥1 telomere induced foci (TIF) also show a very significant 6-fold decrease in hyper-long telomere mice compared to controls (Fig. 8b, c).To further investigate whether longer telomeres than normal protect from DNA damage, we quantified the percentage of cells positive for the senescence marker, p21, in the liver of age matched (100 weeks of age) normal length and hyper-long telomere mice. Interestingly, we found a significant 9-fold decrease in the number of p21 positive cells in hyper-long mice compared with age-matched controls (Fig. 8d, e).

Fig. 8 Hyper-long telomere mice show less DNA damage and less senescence. a, b Quantification of DNA damage in age-matched (100 weeks old) hyper-long telomere mice and control mice. Quantification of total damage as indicated by percentage of cells with ≥2 53BP1 foci as determined by immunofluorescence (a) and quantification of cells showing telomere-induced DNA damage as shown by percentage of cells with ≥1 TIF (telomere induced foci) as determined by telomere FISH followed by immunofluorescence with 53BP1 antibody (b). c Representative images of TIFs (yellow arrow). d Quantification of the percentage of p21 positive cells in liver of age matched (100 weeks old) hyper-long telomere mice and control mice. e Representative images of p21 (red arrows) positive cells. Error bars represent standard error. t-test was used for statistical analysis. The number of mice is indicated in each case. **p < 0.01. Source data are provided as a Source Data file Full size image

Together, these results indicate that longer telomeres than normal in hyper-long telomere mice significantly reduce the global DNA damage and the telomeric DNA damage associated with aging in mice.

Enhanced mitochondrial function in hyper-long telomere mice

Since previous works have reported a relation between telomere and mitochondrial homeostasis34,35, here we next addressed whether hyper-long telomeres could be also affecting mitochondrial function. To this end, we first performed a qPCR-based assay in order to determine the relative mitochondrial DNA (mtDNA) copy number in WAT of 100–110-week old mice using three different mitochondrial genes (Cox1, Cytb, and Nd1). Interestingly, we find that relative mtDNA copy number in WAT of hyper-long telomere mice is double those of control mice from the same background according to the three genes mitochondrial analyzed (Fig. 9a–c).

Fig. 9 Improved mitochondrial function in mice with hyper-long telomeres. a–c. Relative mtDNA content was calculated by the comparative Ct method of the mitochondrial genes Cox1 (a), Cytb1 (b) and Nd1 (c) compared to the nuclear gene H19 in WAT of 100–110 weeks old hyper-long telomere mice and age-matched controls. d–k. mRNA levels from WAT of the OXPHOS genes Cytochrome C (d), ATP synthase (e), Cytochrome C subunit 6 (f) and Cytochrome C subunit 5a (g) as well as mitochondrial regulators PGC-1a (h) and PGC-1b (i) and critical targets, such as ERRa (j) and PPARa (k). Error bars represent standard error. t-test was used for statistical analysis. The number of mice is indicated in each case. *p < 0.05. **p < 0.01. Source data are provided as a Source Data file Full size image

Next, we also measured mRNA expression levels of the different oxidative phosphorylation genes (OXPHOS) Cytochrome C, ATP synthase, Cytochrome C subunit 6 and Cytochrome C subunit 5a as well as mitochondrial regulators PGC-1α and PGC-1β and critical targets such as ERRα and PPARα. Concomitant with our previous result, we found a significant upregulation in all these genes in hyper-long telomere mice compared to the normal length age-matched controls (Fig. 9d–k).

Taken together, these data suggest that mice with hyper-long telomeres have an improved mitochondrial function, which could be also contributing to their delayed aging phenotype and improved metabolic function.

Normal expression of RAP1 in mice with hyper-long telomeres

We previously showed that mice deficient for the RAP1, a component of the shelterin telomere protective complex, showed an obese phenotype as well as signs of metabolic syndrome including abnormal fat accumulation, glucose intolerance and fatty liver36. As we found here that hyper-long telomere mice are protected from age-associated metabolic syndrome, including fat accumulation, high cholesterol levels, liver damage and glucose intolerance, we set to address whether hyper-long telomeres were affecting the levels of the RAP1 protein. To this end, we first determined RAP1 protein levels by Western blot in the white adipose tissue (WAT). We found that RAP1 protein levels were similar in age-matched 100–110 weeks-old hyper-long telomere and normal telomere length controls (Supplementary Fig. 2a, b). Similarly, we detected similar RAP1 protein levels in the liver of hyper-long and normal telomere length mice as determined by immunofluorescence (IF) analysis at 100–110 weeks (Supplementary Fig. 2c, d). These findings suggest that protection from metabolic syndrome in mice with hyper-long telomeres does not seem to be related to altered levels of the RAP1 protein.

Next, we addressed whether hyper-long telomeres resulted in altered levels of the rest of the components of shelterin. To this end, we determined the mRNA expression levels of the different shelterin components in the liver and WAT of 100–110-week old mice. Again, although we observed a tendency to lower expression levels of the different shelterin components in hyper-long telomere mice compared to the controls both in the liver, this difference did not reach statistical significance (Supplementary Fig. 2e–k), similar findings were observed and in the white adipose tissue (Supplementary Fig. 2l–r). Thus, hyper-long telomeres in mice do not alter the mRNA expression of Rap1 or the different shelterin components.