Life extension by diet restriction and N-acetyl-L-cysteine in genetically heterogeneous mice

Abstract

We used a heterogeneous stock of mice-UM-HET3, the first generation offspring of CByB6F1/J and C3D2F1/J parents-to test effects of six antiaging treatments on life span. In the first report of diet restriction in a structured, segregating heterogeneous population, we observed essentially the same increases in mean and maximum life span as found in CByB6F1/J hybrid positive controls. We also report results of treatment with N-acetyl-L-cysteine started at 7 months, and aspirin, nitroflurbiprofen, 4-hydroxy phenyl N-tert-butyl nitrone, and nordihydroguaiaretic acid, all started at 16-18 months. Only male UM-HET3 mice receiving N-acetyl-L-cysteine had significantly increased life span, and this may have been due to treatment-related inadvertent diet restriction. The other agents had no significant effects on life span. The use of UM-HET3 mice helps assure that these results are not the result of unresponsiveness of a single genotype but that they more broadly represent laboratory mice.

Figure 1.

Kaplan–Meier plots of survival curves comparing mice of two stocks—CByB6F1/J and UM-HET3 (HET3), a first-generation four-way cross—treated with diet restriction (DR) or ad lib (AL) fed. At the start of the study, each group consisted of 32 mice, 16 of each sex. We initiated DR at 4–5 weeks of age. Treatment was continued until mice died or became moribund. DR increased life span of both stocks and both sexes (p < .0001, likelihood ratio chi-square tests in a proportional hazards model). The relative response to DR was the same for both stocks, and it did not differ between the sexes (no two-way interactions or three-way interaction among stock, sex, and diet in the proportional hazards model). Data were censored for missing mice (see the Methods section) at the following ages (days): two female HET3 AL mice, both at 478 days; two female HET3 DR mice at 987 and 1,292 days; one male CByB6F1 AL mouse at 460 days; two male CByB6F1 DR mice, both at 1,000 days; two male HET3 AL mice at 92 and 477 days; and one male HET3 DR mouse at 932 days. We censored the life span data for one HET3 AL mouse at 266 days due to bite wounds. No data were censored for female CByB6F1 mice. Overall, we censored data for 6 of the 63 AL mice and 5 of the 64 diet-restricted mice.

Figure 2.

Maximum survivorship in response to diet restriction (DR) is comparable in CByB6F1/J and UM-HET3 (HET3) mice. The figure shows the effect of DR on the proportion of diet-restricted mice in the longest-lived 40% of females and 40% of males for each stock. Statistical analysis employed a nominal logistic model to compare the proportion of diet-restricted mice in the longer-lived 40% survivorship with the proportion of DR mice in the shorter-lived 60% survivorship. Because the effect of DR was sex dependent (interaction of sex and diet, p = .04, likelihood ratio chi-square test), further analysis was separated by sex. Among both males and females, DR increased maximum life span (p < .0001, likelihood ratio chi-square test) comparably in both HET3 and CByB6F1 mice (no significant interaction of stock with diet for either sex). Mice are the same as in Figure 1, except that we dropped all censored mice but one (the “longest-lived” censored mouse) because they could not be unequivocally assigned to a survivorship group. Group sizes were as follows: CByB6F1 females, 32 mice; HET3 females, 29 mice; CByB6F1 males, 29 mice; and HET3 males, 28 mice. Numbers of mice in the lower 60% survivorship were as follows—CByB6F1 females: 16 AL, 3 DR; HET3 females: 13 AL, 4 DR; CByB6F1 males: 13 AL, 4 DR; and HET3 males: 11 AL, 6 DR. AL = ad lib.

Figure 3.

Kaplan–Meier plots of survival curves comparing a control group with N-acetyl-L-cysteine (NAC)–treated groups. Each group consisted of 32 UM-HET3 (HET3) mice, 16 mice of each sex. We treated mice with either 10 g (hi-NAC) or 5 g (lo-NAC) NAC per liter of drinking water starting at 7 months of age. Treatment was continued until mice died or became moribund. The control group was a separate group of HET3 mice from the control group in the diet restriction study. The effect of NAC treatment on life span differed by sex (interaction of sex and treatment in the proportional hazards model, p = .02, likelihood ratio chi-square test); therefore, we analyzed females separately from males. (A) NAC treatment did not affect life span in females. (B) NAC treatment increased life span in males (p = .004, likelihood ratio chi-square test). Each NAC dose had a similar effect (low dose p = .008; high dose p = .0007, likelihood ratio chi-square tests in separate proportional hazards models for each dose). Data were censored for missing mice at the following ages (days): female control, 629 days; female low dose, 791 and 868 days; and female high dose, 292 days. No data were censored for male mice.

Figure 4.

Body weights of UM-HET3 mice on diet restriction (DR) or N-acetyl-L-cysteine (NAC) treatment. Mice were diet restricted from 4 weeks of age or treated with NAC from 7 months of age. Standard errors generally were 1–2 g. AL = ad lib.

Figure 5.

Survival curves for control and treated UM-HET3 (HET3) mice. Starting at 16–18 months of age, groups of about 60 females were given diets containing the following: aspirin, 21 ppm; nitroflurbiprofen (NFP), 200 ppm; 4-hydroxy phenyl N-tert-butyl nitrone (4-OH-PBN), 315 ppm; and nordihydroguaiaretic acid (NDGA), 2,500 ppm. Here, 1 ppm is 1 mg/kg of diet. None of these four treatments affected overall life span or maximum life span (proportion of mice in the longest-lived 40% survivorship). Note: The results from rapamycin-treated mice at The Jackson Laboratory are redrawn from Harrison et al. (11); they are provided here as an illustration of the potential for increased life span of HET3 mice when treatment is started late in life.