We report the outcomes of a quantitative genetic study on aging and longevity in the mouse. We studied an extant series of recombinant inbred strains (ILSXISS) that have been used both in DR aging studies as well as to study alcohol sensitivity (Williams et al., 2004
). The major advantage of using RI strains is that their genotypes have been extensively assessed, thus minimizing costs, and detailed characterizations of numerous traits have already been conducted. Subsequent testing for significant relationships among traits in a stable set of RIs is another key advantage of using a large genetic panel for studying complex mechanisms (Chesler et al., 2008
). These strains were recently imported by the Jackson Laboratory and 65 strains should eventually be available.
This study indicated a large amount of genetic variation for mouse longevity; heritability was 34% for AL and 36% for DR (60% of AL food intake). There was no significant correlation between mean longevity under these two conditions, although maximum lifespans of the AL and DR mice were significantly correlated. Similar observations were made at the UTHSCSA on the ILSXISS RI mice (Liao et al., 2010a
; Mattson 2010
), where they also observed similar heritability (28% AL males, 36% AL females, 55% DR males, 53% DR females). These two sets of observations were generated under different conditions (e.g., SPF status, age at which DR was initiated, one or both sexes tested, additional tests performed at each site, etc).
A very unexpected observation is that in about half of the strains, longevity was shortened by DR compared with the longevity under AL feeding. Similar results were also obtained at the UTHSCSA (Liao et al. 2010a
). They also saw differential effects of DR on longevity in which some strains showed a positive response but about one-third showed a negative response and about one-third showed no significant response at all. Thus the observations of differential effects are replicable, providing a critical validation of this unexpected result. These findings are not completely novel but no previous study has examined more than two or three genotypes simultaneously. Forster et al. (2003)
and Turturro et al. (1999)
previously suggested that DBA mice get less life extension under DR than C57BL/6 mice, but no studies have found anything close to the range of variation observed here and no genetic studies have been carried out.
The lifespan shortening under DR was unexpected based on the wide efficacy of DR in many species. This and other issues have led some to question the relevance of these studies and suggest several possibilities that could make this observation of less interest. Nutrient deficiency might explain some of the life-shortening, but there were no obvious signs of malnutrition. First, all strains were able to establish a stable BW on the DR diet. Second, few animals died in the first three months and those that died early did not appear to be slowly wasting away, but usually died without being noticeably moribund or cachectic; the diet composition provided multiples of all micronutrients except selenium and choline (Liao et al., 2010
). Third, the shortened lifespans did not cluster as might be expected if there were one overriding cause of death. Because most of the mice in our lifespan study were cannibalized before they were found, we did not conduct pathology studies, nor did we have sufficient funds to perform detailed autopsies.
It’s also important to note that our lifespan data correlated significantly with female fertility, post DR (R
= 0.44, P
= 0.006, N
= 33 strains). This observation suggests genetic segregation of a common anti-aging component, which we called Aging Measure 1. Several previous studies of female reproductive capabilities under DR (Weindruch and Walford, 1988
; Merry and Holehan, 1991
; Johnston et al., 2006
) found that females returned to an AL diet, after as long as 16 months on the DR diet, were, in general, able to regain fertility. This fertility data can also be examined to ask if there were negative effects of DR on female fertility, as might be expected if it were harmful to the mice: We found few negative effects on fertility caused by DR; early deaths and SPF status were not an issue. In addition, we collected fertility data on three of the four strains with significantly shorter DR lifespan and found that fertility was improved for two and only slightly decreased in the third (Supplementary Table 4
). This also argues against a malnutrition effect of the DR regimen.
A related question is whether the observed lifespan shortening was genetically distinct from the observed lifespan extension. Under that model we would expect very different correlations with female fertility—which was almost uniformly affected positively by DR. Of the 33 strains tested for both fertility and lifespan, 19 strains had life extension under DR, and the correlation between DR fertility and DR mean and maximum lifespan was 0.36 and 0.24 (P = 0.06, 0.16, respectively, 1-tailed). In the 14 remaining strains, which showed lifespan shortening under DR, the correlation between DR fertility and mean and maximum lifespan was essentially the same: 0.32 and 0.47 (P = 0.13 and 0.04, respectively, 1-tailed). The significant QTL on chromosome 7 modulating both fertility and longevity also suggests common uniform action. The simplest interpretation is that lifespan shortening and lengthening under DR were determined by common genes.
In our analysis of metabolic efficiency, we found significant differences in how much weight the mice could maintain on DR that correlated with maintenance of tail growth rate and the rate of hair regrowth. BW and hair growth rate were correlated at each study site, which led us to define a new parameter that is a composite of BW and growth, a trait which we have called fuel efficiency (FE), which was extracted using Principal Components Analysis. This FE factor was found to be genetically specified and significantly correlated with Aging Measure 1 (R
= 0.34, P
= 0.026). As noted, this factor was defined independent of FE under AL; consequently, whether FE under AL predicts longevity under DR as suggested by Ferguson et al. (2007)
is a separate issue from the analyses conducted in this study (the AL efficiency model will be tested in future studies).
Other studies have also reported that individual mice that maintained the highest BW were likely to be the longest-lived individuals among cohorts of genetically identical mice (Weindruch et al., 1986
; Harper et al., 2006
). This is consistent with a model in which life extension by DR is due, at least in part, to benefits that are a by-product of FE. This might be expected if FE is itself a surrogate measure of metabolic efficiency, which is a survival response in which animals under DR adopt a new metabolic strategy limiting wastage of energy. In brief (), the model suggests that reduced food availability during DR is a signal to reallocate energy by decreasing proton leakage across the mitochondrial membrane (Ramsey et al., 2000
; Lopez-Lluch et al., 2006
). This leads to decreased heat production, increased ATP production, and presumably a concomitant decrease in the production of senescence-promoting reactive oxygen species (ROS) (Ramsey et al., 2000
; Lopez-Lluch et al., 2006
). The higher ATP levels are generally available and help preserve BW, growth, and many other processes, including maintenance and repair functions that might combat aging (Weindruch and Walford, 1988
; Weindruch et al., 1986
). Additional studies will be needed to confirm the molecular details of the ME model.
We found three significant QTLs (genetic regions harboring genes controlling these various aging traits, Supplementary Table 5
). On chromosome 7, we found a QTL affecting lifespan and fertility after DR that we have named Lfdr1
for “longevity and fertility response to dietary restriction, QTL 1; this QTL also has suggestive effects on FE (). Two QTLs having significant effects on FE were identified on chromosomes 9 and 15. These we have named Fedr1
, respectively, for “fuel efficiency response to dietary restriction” QTLs 1 and 2. The QTL on chromosome 9 is also suggestive for an effect on lifespan and fertility in the direction expected (ie, ILS allele associated with increased lifespan and fertility). The QTL on chromosome 15 was not suggestive for an effect on lifespan and fertility, but there is not sufficient statistical power to rule out a QTL of the expected size (only 15% of the genetic variance).
The ME model also aids in predicting what genes are strong candidates for the QTLs identified in this study. Potential candidates include Ppara
(peroxisome proliferator-activated receptor alpha) on chromosome 9 (), which has previously been proposed to modulate not only DR’s health benefits (Corton et al., 2004
; Masternak and Bartke, 2007
) but also ME during DR (Lopez-Lluch et al., 2006
). Candidates on chromosomes 7 and 15 include Cck
(cholecystokinin) and Cckbr
(cholecystokinin receptor B), respectively; the orthologs of these genes have recently been shown to be necessary for DR-induced life extension in C. elegans
(Park and Johnson, 2009
). However, the haplotypes of Ppara, Cck
, and Cckbr
, along with their promoter regions, are not polymorphic between ILS and ISS, illustrating how strong candidate genes can also be immediately ruled out.
Among those genes that are in polymorphic haplotype regions, we also identified several candidates that were particularly interesting. These included the genes Ucp-2
encoding mitochondrial uncoupling proteins, which are obvious candidates for effects on metabolic efficiency; the Ucp-2
gene exhibits a missense polymorphism between ISS and ILS. Another notable candidate gene is Lass5
, an ortholog of the longevity assurance gene lag-1
in yeast (Jazwinski 2002
has an alanine-proline polymorphism between ISS and ILS. We also identified a number of other candidate genes with potential to modulate ATP, NAD, and thyroid hormone signalling consistent with the ME model (Supplementary Table 5
We also looked for polymorphic candidate genes among those identified by Swindell (2009)
as being significantly up or down regulated in a meta-analysis of murine gene expression during DR. One such candidate in the chromosome 7 QTL region was Serpinh1
, serine (or cysteine) peptidase inhibitor, clade H, member 1. This gene is consistently down regulated in variety of tissues (heart, liver, hypothalamus, hippocampus, striatum). According to the UniProt database (http://www.uniprot.org/uniprot/P19324
), the Serpinh1
protein is inducible by heat shock and functions as a possible chaperone that binds specifically to collagen.
For decades, DR has been a prominent and useful tool for retarding aging in rodents. This study demonstrates that applying a systems genetics approach to the analysis of DR (the combination of genetics and detailed physiological analyses) has great potential for elucidating the mechanisms behind DR’s many health and aging benefits.