Health Effects of Long-Term Rapamycin Treatment: The Impact on Mouse Health of Enteric Rapamycin Treatment from Four Months of Age throughout Life

Abstract

Rapamycin, an mTOR inhibitor, has been shown to extend lifespan in a range of model organisms. It has been reported to extend lifespan in multiple strains of mice, administered chronically or acutely early or late in life. The ability of rapamycin to extend health (healthspan) as opposed to life is less well documented. To assess the effects chronic rapamycin treatment on healthspan, enteric rapamycin was given to male and female C57BL/6J mice starting at 4 months of age and continued throughout life. Repeated, longitudinal assessments of health in individual animals were made starting at 16 months of age (=12 months of treatment) until death. A number of health parameters were improved (female grip strength, female body mass and reduced sleep fragmentation in both sexes), others showed no significant difference, while at least one (male rotarod performance) was negatively affected. Rapamycin treatment affected many measures of health in a highly sex-specific manner. While sex-specific phenotypic effects of rapamycin treatment have been widely reported, in this study we document sex differences in the direction of phenotypic change. Rapamycin-fed males and females were both significantly different from controls; however the differences were in the opposite direction in measures of body mass, percent fat and resting metabolic rate, a pattern not previously reported.

Fig 1. Rapamycin concentration in whole blood at 10 months of age (after 6 months of rapamycin feeding).

Blood concentrations of rapamycin were significantly higher in females than males after 6 months of rapamycin feeding (n = 18 and 14 respectively).

Fig 2. Body mass and composition in rapamycin-fed mice (filled circles) versus controls (hollow circles).

P-values shown on individual panels only if there is a significant treatment effect independent of age. Sample sizes varied, depending on age, control females, n = 32–2; rapamycin females, n = 30–4; control males, n = 33–2; rapamycin males, n = 36–3. A, B: Total body mass. Highly significant differences (p << 0.001) exist in treatment x age effects in body for both sexes. Although they weighed less than controls by 16 months of age, rapamycin-fed females retained body mass longer, whereas rapamycin-fed males were similar to controls at 16 months but lost body mass earlier and remained lighter as they aged. C, D: Percent body fat. Highly significant differences (p << 0.001) in treatment x age effects exist for both sexes. As in with body mass, aging rapamycin-fed females retained body fat longer and lost body fat more slowly than age-matched controls. In contrast, rapamycin-fed males initially had a higher percentage of body fat, but lost fat mass earlier than controls. E, F: Fat-free mass, sometimes referred to as lean mass. Although obscured by the scaling, rapamycin-fed females had lower fat-free mass than controls at all ages measured. Fat-free mass declined more slowly with age in rapamycin-fed females than males.

Fig 3. Metabolic activity in rapamycin-fed mice (filled circles) compared to controls (hollow circles).

P-values shown on individual panels only if there is a significant treatment effect independent of age. Sample sizes varied, depending on age, control females, n = 8–4; rapamycin females, n = 11–3; control males, n = 9–5; rapamycin males, n = 16–2. A, B: Mass-specific metabolic rate during the light (= inactive) phase. Males and females showed no effects of rapamycin treatment on mass-specific metabolic rate during the inactive phase of their daily 24-hour cycle, although both sexes showed highly significant (p << 0.001) sex x age treatment effects. C, D: Mass-specific metabolic rate during the dark (= active) phase. Aging rapamycin-fed females, but not males, maintained significantly higher metabolic rates between measures taken at 24 and 28 months of age compared to controls during the dark (= active) phase of the 24-hour light cycle. Both males and females showed highly significant (p <<0.001) decline dark-phase metabolic rate with age irrespective of treatment. E, F: Resting mass-specific metabolic rate. Resting metabolic rate declined with age in females, but aging rapamycin-fed females had higher resting metabolic rates compared to age-matched controls. Resting metabolic rate declined significantly in aging rapamycin-fed males but not in age-matched controls (treatment x age, p << 0.001).

Fig 4. Spontaneous activity and sleep in rapamycin-fed mice (filled circles) compared to controls (hollow circles).

P-values shown on individual panels only if there is a significant treatment effect independent of age. Sample sizes varied, depending on age, control females, n = 14–2; rapamycin females, n = 18–5; control males, n = 13–4; rapamycin males, n = 22–7. A, B: Spontaneous 24-hour activity was greater in females than males (p<< 0.001). Activity increased with age (p = 0.008) in females and decreased with age in males (p = 0.003), regardless of treatment. C, D: Total sleep Rapamycin treatment marginally increased total sleep in both sexes (p = 0.05) when taken together, but aging affected sleep patterns in the two sexes differently (age x sex, p << 0.001). E, F: Sleep fragmentation increased with age in all animals (p << 0.001); however, rapamycin treatment reduced sleep fragmentation in males and showed a trend to reduce it in females.

Fig 5. Strength, coordination and movement in rapamycin-fed mice (filled circles) compared to controls (hollow circles).

P-values shown on individual panels only if there is a significant treatment effect independent of age. A, B: Grip strength declined significantly with age in all animals, regardless of treatment (p << 0.001); however rapamycin treatment affected males and females differently (treatment x sex, p = 0.003). Rapamycin-fed females had greater grip strength than controls at all ages measured; whereas grip strength in control and rapamycin treated males did not differ. Sample sizes varied, depending on age, control females, n = 17–7; rapamycin females, n = 27–4; control males, n = 22–4; rapamycin males, n = 31–3. C, D: Stride length increased in males and females until 27 months of age and then declined with increasing age (p << 0.001). Rapamycin treatment had no effect on stride length in either sex. Sample sizes varied, depending on age, control females, n = 15–6; rapamycin females, n = 21–5; control males, n = 19–4; rapamycin males, n = 26–9. E, F: Rotarod performance, measured as maximum latency to fall, was significantly affected by body mass (p << 0.001) and so body mass was included as a covariate in the analysis. With the effects of body size removed, females showed no effects of rapamycin treatment and males showed a marginally significant negative effect of rapamycin treatment on rotarod performance. The y-axis shows the residuals of rotarod performance (latency to fall) regressed against body mass. Sample sizes varied, depending on age, control females, n = 11–6; rapamycin females, n = 19–6; control males, n = 13–7; rapamycin males, n = 21–8.

Fig 6. Age-related changes in inner ear histology was not altered by rapamycin treatment.

A, B: The number of cochlear neurons in male and female mice were not statistically different between control and rapamycin-fed animals. C,D: The number of outer hair cells in male and female mice were not statistically different between control and rapamycin-fed animals. E,F: The number of inner hair cells in male and female mice were not statistically different between control and rapamycin-fed animals.