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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online September 8, 2000 as doi:10.1096/fj.00-0242fje. |
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Department of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center, New Orleans, Louisiana 70112, USA
2Correspondence: Department of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center, 1901 Perdido St., Box P7–2, New Orleans, LA 70112, USA. E-mail: sjazwi{at}lsuhsc.edu
SPECIFIC AIMS
We addressed the hypothesis that caloric restriction acts at the cellular level to extend longevity and postpone senescence in eukaryotes, and provides one of multiple mechanisms of metabolic control in aging. The effect of progressive reduction in the glucose or amino acids concentration of the growth medium on the life span and aging of individual Saccharomyces cerevisiae cells has been examined, and the interaction of this caloric restriction effect with the retrograde response pathway, which signals the functional status of the mitochondrion and determines longevity, has been investigated.
PRINCIPAL FINDINGS
1. Lowering the glucose concentration increases mean and maximum
life span of yeast cells in both broth and synthetic medium
Reduction of the glucose concentration in a modified broth
routinely used for culturing yeast resulted in an increase in the life
span of individual cells, measured by the number of daughters they
produced, which was the more extensive the greater the reduction in
nutrient levels. Corresponding increases in the mean and maximum life
span were observed—up to 75% in some experiments. Similar results
have been obtained with three different yeast strains. The effect on
life span of reducing glucose levels was duplicated in a standard,
chemically defined growth medium. Extension of life span by as much as
81% was found. However, the increased longevity was abrogated when
glucose was reduced beyond a certain concentration, indicating that
below this point glucose becomes limiting and malnutrition sets in.
2. Reduction of glucose levels postpones the development of a
senescent phenotype during the yeast life span
One of the manifestations of aging in yeast is an increase in
generation time, which is the interval between consecutive buddings of
individual cells. The appearance of this age-related phenotype was
delayed when life span was extended by lowering glucose levels in the
medium, as measured by the rate of bud production. After a brief lag,
there appeared to be a small increase in this rate at the lower glucose
concentrations.
3. Extension of life span by adjustment of glucose levels does not
depend on the retrograde response
Metabolic control plays a role in determining yeast life
span, as evidenced by the increased longevity afforded by the induction
of the retrograde response, a form of interorganelle communication that
signals the functional status of the mitochondrion to the nucleus
resulting in changes in the expression of nuclear genes that encode a
variety of mitochondrial, cytoplasmic, and peroxisomal proteins. To
ascertain whether the effect on life span of lowering glucose levels is
mediated by the retrograde response, the expression of the
CIT2 gene, a diagnostic of the retrograde response, was
assessed. Not only were CIT2 transcript levels not increased
under conditions that extend life span, they were reduced by 49%.
The RTG genes are required for the retrograde response.
Rtg2p promotes the formation of an active heterodimeric Rtg1p-Rtg3p
transcription factor by transducing mitochondrial signals that affect
the phosphorylation state and subcellular localization of Rtg3p.
Deletion of the RTG2 gene did not suppress the life span
extension caused by reduction of glucose levels (Fig. 1A
), suggesting the lack of a requirement for the retrograde
response for life span extension. In fact, the small decrease in life
span at high glucose concentrations that has been often observed on
various growth media on deletion of this gene was eliminated at low
glucose levels (Fig. 2A
).
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It is possible that the prolonged life span resulting from
reduced glucose levels by-passed the requirement for Rtg2p but was
still dependent on the Rtg1p-Rtg3p transcription factor. To examine
this possibility, we deleted RTG3. This deletion did not
prevent the extension of life span upon reduction of glucose
concentration in the growth medium (Fig. 2B
). There was a
55% increase in mean life span of the rtg3
strain even
without a reduction in glucose levels. Thus, the expression of genes
under the control of the Rtg1p-Rtg3p transcription factor suppresses
longevity to an extent under these growth conditions, in contrast to
the requirement of the Rtg pathway for optimal longevity when the
retrograde response signal is present in yeast whose mitochondria are
not fully functional. The increase in mean life span seen upon
reduction of glucose levels (80%) was additive (123%) to that
obtained upon deletion of RTG3 (Fig. 2B
),
generating the largest increase in longevity described thus far in
yeast. These results have been observed in two different yeast strains,
where the details of the induction of the retrograde response differ
but its effect on life span is the same.
4. Decreasing the amino acids concentration of the growth medium
promotes an increase in the mean and maximum life span of yeast
The extension of life span detected on reduction of glucose
concentration in broth was effective only when the levels of other
nutrients were decreased, suggesting that manipulation of these
nutrients, especially amino acids, might result in life span extension.
Indeed, decreasing amino acids concentration while maintaining glucose
levels resulted in an increased longevity (up to 95%) that was larger
the greater the reduction in the nonessential amino acids. This life
span extension does not operate through the retrograde response
pathway, because it did not entail the induction of CIT2 and
it was not suppressed by deletion of RTG2. Thus, it is not
the decreased availability of a specific nutrient but rather the
restriction of the caloric content of the growth medium that plays a
role in life span extension.
CONCLUSIONS
This study demonstrates that yeast life span can be modulated physiologically. The life span extension observed upon manipulation of nutritional status possesses many of the hallmarks of caloric restriction in mammals. First, this effect is continuous, operating like a rheostat—the greater the reduction in nutrient concentrations, the larger the life extension (up to a point). This evokes a mechanism involving changes in flux through a pathway rather than a threshold effect that triggers an all-or-none response. Second, the increased longevity is associated with a postponement in the appearance of an aging phenotype. Third, the effect is not due to limitation of a specific nutrient. On the basis of these similar features, we tentatively call this mechanism of life span extension in yeast caloric restriction.
The increase in life span upon reduction of glucose levels does not appear to be simply due to release of the cells from glucose repression, because the life span extension seen is continuous as glucose concentration is lowered progressively. Furthermore, the glucose concentrations at which the maximal effect on longevity was observed were quite different in broth and in chemically defined medium. Finally, life span extension by limitation of amino acids is clearly not the result of release from glucose repression.
A reduction in blood glucose levels is an inevitable, early response to caloric restriction in mammals. The fact that lowering the concentration of glucose available to individual yeast cells has such a marked effect on their longevity and aging suggests that a key component of the anti-aging effect of caloric restriction in mammals may be changes elicited by glucose at the cellular level. The reduction of glucose levels delayed the decline in budding rate normally seen during aging. The calorie-restricted yeast displayed a higher level of metabolic activity than the controls, all other things being equal, as indicated by an increased budding rate. This supports the notion that a change in the characteristics of fuel use underlies the caloric restriction effect.
Caloric restriction did not induce the retrograde response; in fact, a
substantial reduction in the expression of the diagnostic gene for this
response, CIT2, was found. This suggests that caloric
restriction operates by down-regulating at least part of the retrograde
response. Significantly, the extension of life span by caloric
restriction did not require RTG2. Caloric restriction
removed any need of this gene for a normal life span, suggestive of a
positive relationship with certain features of the retrograde response.
Deletion of RTG3 also did not suppress the caloric
restriction effect; instead, it potentiated a maximal increase in
longevity that was additive to caloric restriction. Thus, the
retrograde response and caloric restriction represent two distinct
metabolic mechanisms of life extension (Fig. 2
). The results suggest some separation of functions of the
RTG2 and RTG3 mediators of the retrograde
response. They are consistent with the hypothesis that caloric
restriction diminishes or prevents certain aspects of the retrograde
response and that certain aspects of the retrograde response have a
negative effect on the increased longevity provided by caloric
restriction, in effect preventing the full benefits of caloric
restriction (Fig. 2)
. This does not rule out some overlap between the
longevity effectors induced by the retrograde response and by caloric
restriction.
There are similarities between the metabolic effects of the retrograde
response in yeast and of the daf mutants of Caenorhabditis
elegans that display extended adult longevities. These metabolic
effects are reminiscent of some of the features of the respective
dispersal forms of yeast and worms, the spore and the dauer larva,
which represent evolutionarily conserved responses to environmentally
unfavorable conditions. The retrograde response, however, appears to be
a compensatory adaptation to the deleterious effects of the internal
stress of mitochondrial dysfunction that can accumulate with age (Fig. 2)
. In contrast, caloric restriction may constitute an adjustment that
prevents the deficits associated with aging. Although clues of its
operation exist, evidence for the presence of the retrograde response
in organisms other than yeast is currently lacking. A fundamental
question concerning caloric restriction is whether the reduction in
calories triggers responses that enhance survival, or whether it is the
elimination of excess nutrients that have a negative effect on life
span that is responsible.
The present study makes apparent the utility of yeast to elucidate the molecular mechanisms underlying caloric restriction, and it may bridge the gap between studies of aging across wide phylogenetic divisions. Our results provide evidence for multiple mechanisms of metabolic control in aging. Inasmuch as caloric restriction lowers blood glucose levels, this study raises the possibility that reduced glucose alters aging at the cellular level in mammals.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0242fje To cite this article, use (September 8, 2000) An intervention resembling caloric restriction prolongs life span and retards aging in yeast. FASEB J. 10.1096/fj.00-0242fje 
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