Animal study

The HCR/LCR contrasting rat model system for intrinsic fitness capacity is the result of an over 15 year two-way artificial selection experiment described previously19. For the current study, we obtained 79 female rats (39 HCR and 40 LCR) and began testing when the rats were ~9 months old. Figure 1a shows the study design. We randomly assigned LCR and HCR rats to control (C) and running (R) subgroups matched within strain for body weight and fitness capacity: 19 HCR-C and 20 LCR-C, maintained in standard cages; and voluntary runners: 20 HCR-R and 20 LCR-R, maintained in cages equipped with a running wheel to permit voluntary running throughout adulthood. Figure 1b,c show details on measured body weight and food intake for all four study groups across 12–30 months of age. As previously shown, LCR rats weigh significantly more than HCR rats (age 3 months, P < 0.001, data not shown) and this difference remained significant throughout adult life (Fig. 1b, HCR-C vs. LCR-C, 12–30 months, P < 0.001). Food intake was significantly greater in runner groups compared to control for both LCR and HCR (Fig. 1c, HCR-C vs. HCR-R, 12–30 months, P < 0.05).

Figure 1 Rat study protocol and measurements. (a) Schematic of study protocol. Rats were bred for high (HCR, blue) or low (LCR, orange) intrinsic fitness, then assigned to control (C) or running (R) subgroups. (b) Body weights from ages 12 to 30 months. Rats per group: 12 months: HCR-C = 18, HCR-R = 15, LCR-C = 20 and LCR-R = 20; 21 months: HCR-C = 19, HCR-R = 17, LCR-C = 18 and LCR-R = 13; 30 months: HCR-C = 12, HCR-R = 8, LCR-C = 7 and LCR-R = 3. (c) Food intakes from ages 12 to 30 months. Rats per group: same as in b), except at 12 months: HCR-C = 16. (d) Average daily spontaneous activity measured over 3 days between 13 and 15 months of age (activity index). Rats per group: 13 months: HCR-C = 18, HCR-R = 12, LCR-C = 20 and LCR-R = 11; 15 months: HCR-C = 19, HCR-R = 13, LCR-C = 19 and LCR-R = 10. (e) Average running distance per day. Rats per group: 12 months: HCR-R = 15 and LCR-R = 15; 21 months: HCR-R = 16 and LCR-R = 14; 30 months: HCR-R = 4 and LCR-R = 2. Error bars represent SEMs. Figure was drawn by Sira Karvinen. Full size image

We measured the natural total spontaneous physical activity (horizontal and vertical movements) every three months throughout each rat’s lifespan using a custom designed force-plate system20. The activity index was clearly higher among the runners than among the corresponding controls (Fig. 1d). Consistent with previous reports, the HCR-C group exhibited higher spontaneous activity than the LCR-C group by 23% (Fig. 1d, P < 0.01). In addition, for cages equipped with running wheels, the average wheel distance per day was longer in HCR-R than in the LCR-R group at time points from 12 to 29 months (Fig. 1e, P < 0.05).

Because physical activity is known to have positive effects on glucose metabolism, we measured insulin sensitivity at baseline (age 9 months) and after 1 year of intervention (age 21 months). At baseline, all four rat groups had similar insulin sensitivity, based on the HOMA-IR index21. After one year of voluntary running, the LCR-R group had a significantly lower HOMA-IR index than the LCR-C group (3.5 ± 3.0 vs. 5.7 ± 2.8, P < 0.05). In HCR groups, there was no difference in the HOMA index between groups (4.3 ± 3.1 vs. 4.6±3.0, respectively), which is consistent with the fact that HCRs represent a healthier phenotype compared to LCRs, having lower risk of metabolic diseases even without physical training.

As expected, the Cox proportional hazards model showed that LCR rats had a higher risk of death than HCR rats, the hazard ratio (HR) for LCR compared to HCR rats adjusted for running group and age at randomization being 1.7 (95% CI: 1.1–2.6, P = 0.028). Interestingly, when combined, the pool of LCR and HCR runner rats had an increased risk of death compared to control groups, with a HR of 2.1 (95% CI: 1.3–3.4; P < 0.001). This finding persisted after adjustments for strain, age at randomization and body weight at 9 months of age (multivariate-adjusted HR = 2.3, 95% CI: 1.4–3.6; P < 0.001). The decreased survivability for runners vs. controls was similar for both HCR and LCR strains. The survival curves in Fig. 2 shows the mean lifespan among runners was consistently 16% shorter than among controls in both the HCR (mean 26.4 vs. 31.5 months, P < 0.05) and LCR (23.8 vs. 28.4 months, P < 0.01) groups. The deaths caused by development of spontaneous tumours with aging did not explain the group differences in lifespan (21–45% of deaths within group).

Figure 2 Effects of genetic background and environment on lifespan. Control rats (C) had longer lifespans than rats in the runner groups (R) of the same strain (HCR-C vs. HCR-R, P < 0.05 and LCR-C vs. LCR-R, P < 0.01). Mean lifespans were also significantly different between rat strains (HCR-C vs. LCR-C, P < 0.05). Values in the table show means±SDs. Full size image

Human study

The prospective Finnish Twin Cohort22 includes all same-sex twin pairs born in Finland before 1958. Physical activity was measured with a structured questionnaire. We used persistence and changes in vigorous physical activity during the years 1975, 1981 and 1990 as baseline predictors of mortality. Altogether, 11 325 twin individuals (4190 complete twin pairs) answered the required physical activity questions for all three baseline time points (for more details of the cohort, see Table S1). A Cox proportional hazards model was used to analyse mortality, starting from the 1990 response date and ending July 31, 2013. Altogether, 458 deaths were observed among individuals that had performed no vigorous activity at baseline (in 1975, 1981 and 1990) and 201 deaths among individuals that persistently performed vigorous activity. An individual-based analysis showed that, compared to individuals with no vigorous activity, individuals with persistent vigorous physical activity showed decreased mortality (in 1975, 1981 and 1990); the age- and sex-adjusted HR of death was 0.55 (95% CI: 0.46–0.64) and the HR adjusted for sex, age, education level, smoking status in 1990, alcohol use (grams per day) in 1990, BMI in 1990, work activity and health status in 1990 was 0.73 (0.61–0.88) (Fig. 3a and Table S2).

Figure 3 Kaplan-Meier survival curves of mortality in the human study. Follow-up started from the date of the 1990 questionnaire response to the end of July 2013. Groups comprised individuals with no vigorous activity (orange) vs. those with persistent vigorous activity (blue) at baseline (start of follow-up). (a) Survival of 2428 individuals with no vigorous activity and 2145 individuals with persistent vigorous activity; (b) survival of 134 discordant DZ twin pairs; (c) survival of 34 discordant MZ twin pairs. Full size image

Of the 4190 same-sex twin pairs (MZ = 1388, DZ = 2547, 255 unknown zygosity), we identified 179 (4.3%) persistently discordant for participation in vigorous physical activity. These activity-discordant twin pairs comprised 2.4% (34 of 1388) of all MZ pairs and 5.3% (134 of 2547) of all DZ pairs (P < 0.001; Fisher’s exact test for a difference in persistent discordances between MZ and DZ pairs). A pairwise analysis of these 179 twin pairs showed that the mortality HR for persistent vs. non-persistent vigorous activity was 0.65 (0.46–0.91) and after adjusting for all covariates including health status, the HR was 0.72 (0.48–1.07). Consistent with our previous twin analysis, which was based on a shorter period of physical activity discordances23, the activity-discordant DZ pairs showed a difference in mortality (Fig. 3b, HR = 0.58, 95% CI: 0.39–0.88). However, no difference was observed in the pairwise analysis of the smaller group of activity-discordant MZ pairs (Fig. 3c, HR = 1.00, 95% CI: 0.52–1.94). The heritability of physical activity (see below) contributed to the statistical power of the analysis among MZ pairs.

To describe the total volume of physical activity performed during leisure time, we calculated a metabolic equivalent (MET) index expressed as the sum of leisure MET-hours per day at each time-point (1975, 1981 and 1990). The means of the three MET index values showed that MZ pairs (0.54) had a higher Intraclass Correlation Coefficients (ICC) than DZ pairs (0.26). This result indicates that the differences in the genetic component of the total variance influenced the long-term levels of physical activity compared to the environmental component. Using standard techniques for genetic modelling24 (AE model), we estimate the narrow-sense heritability for physical activity to be 53% (95% CI: 46–59%).

Due to the low number of deaths among MZ twin pairs we repeated the pairwise analyses among DZ and MZ pairs separately using cohort members who were discordant for vigorous physical activity in 1975 and 1981 (similar criteria as in our primary analysis but shorter PA discordance at baseline). This material also included older cohort members than in our primary analysis. This dataset included 778 DZ and 231 MZ vigorous activity discordant twin pairs. Among these DZ pairs there were 204 deaths and among MZ pairs 55 deaths during follow-up (between 1981 and 31st August 2013). Unadjusted pairwise HR for death among active compared to inactive members for DZ pairs was lower (HR 0.58 [95% CI 0.46–0.74]) than among MZ pairs (0.85 [0.56–1.30]. When this secondary analysis was repeated in the baseline-healthy subgroup the HRs were 0.64 (0.45–0.89) for DZ pairs and 1.05 (0.58–1.88) for MZ pairs, respectively.

We also performed an individual-based analysis of how work-related (non-voluntary) physical activity affected mortality. We found that individuals with persistently non-sedentary work had a higher risk of death than those with persistently sedentary work (age- and sex-adjusted HR = 1.15, 95% CI: 1.00–1.32; P = 0.046). Additional analyses that adjusted for other covariates (e.g. sex, age, education, smoking status, alcohol consumption and BMI) attenuated the HR. Also, multivariate models that analysed the persistence or change in work-related physical activity during 1975–1990 did not show any statistically significant associations with subsequent mortality. Moreover, no differences for the risk of death by work-related physical activity were observed in the pairwise analyses of all twin pairs.