Life-span extension in mice by preweaning food restriction and by methionine restriction in middle age

Abstract

Life span can be extended in rodents by restricting food availability (caloric restriction [CR]) or by providing food low in methionine (Meth-R). Here, we show that a period of food restriction limited to the first 20 days of life, via a 50% enlargement of litter size, shows extended median and maximal life span relative to mice from normal sized litters and that a Meth-R diet initiated at 12 months of age also significantly increases longevity. Furthermore, mice exposed to a CR diet show changes in liver messenger RNA patterns, in phosphorylation of Erk, Jnk2, and p38 kinases, and in phosphorylation of mammalian target of rapamycin and its substrate 4EBP1, HE-binding protein 1 that are not observed in liver from age-matched Meth-R mice. These results introduce new protocols that can increase maximal life span and suggest that the spectrum of metabolic changes induced by low-calorie and low-methionine diets may differ in instructive ways.

Figure 1.

Survival curves for mice placed on a methionine-restricted diet at 12 months of age compared with controls. Each symbol represents one mouse. Statistical results are shown for the log-rank test and for a test of the proportion of mice still alive at the 90th percentile of the joint survival distribution.

Figure 2.

Gene expression results for a set of 94 messenger RNAs (mRNAs) evaluated by multiplex reverse transcription–polymerase chain reaction in groups of seven to nine mice. The horizontal axis reports the ratio of expression in liver of methionine-restricted (Meth-R) compared with Meth-C mice. The vertical axis shows the ratio of expression in liver of caloric restriction (CR) compared with ad libitum (AL) control mice. Filled symbols show genes for which a test of false discovery rate showed p < .05 for the comparison of CR with AL mice. Underlined gene names show the three mRNA for which the false discovery statistic reached p < .05 for the comparison between Meth-R and Meth-C mice. Gene names are given for 11 arbitrarily selected genes; a complete listing is included in Supplementary Table 1.

Figure 3.

Evaluation of Erk, JNK, and p38 status in liver of caloric restriction (CR) and methionine-restricted (Meth-R) mice. Each group of immunoblots shows a representative experiment involving four ad libitum and four CR mice (top set) or four Meth-C and four Meth-R mice (bottom set), stained (top to bottom) for phosphorylated Erk1/2, total Erk1/2, phosphorylated JNK2, total JNK2, phosphorylated p38, total p38, and actin. The bar graphics at the right show means and SEs of the mean for eight mice of each type. Asterisks indicate significance at p < .05 by an unpaired t-test for the indicated comparison.

Figure 4.

Evaluation of enzymes in the target of rapamycin pathway in liver of caloric restriction (CR) and methionine-restricted (Meth-R) mice. Each group of immunoblots shows a representative experiment involving four ad libitum and four CR mice (top set) or four Meth-C and four Meth-R mice (bottom set), stained (top to bottom) for phosphorylated Akt, total Akt, phosphorylated mammalian target of rapamycin (mTOR), total mTOR, phosphorylated 4EBP1, and total 4EBP1. The bar graphics at the right show means and SEs of the mean for eight mice of each type. Asterisks indicate significance at p < .05 (*) or p < .01 (**) by an unpaired t-test for the indicated comparison.

Figure 5.

Survival plots for litter enlargement (LE) mice (litters expanded from 8 to 12 pups at birth), PR mice (mothers given a low-protein diet during lactation), and control mice (litters of eight pups, mothers receiving standard diet during lactation). Each symbol represents one mouse. The LE group differs from controls by log-rank test (p = .0007) and by the Wang and Allison test for survival past the 90th percentile (p = .03).

Figure 6.

Body weight curves for male (left) and female (right) mice exposed to differing levels of nutrition during early postnatal development. Each point represents the mean ± SE body weight of 7–26 individuals dependent upon sex and treatment with one exception: only two female control mice were alive at Week 112. For clarity, the inset top panels show body weight curves for mice aged 3–8 weeks.