A very important insight into the nature of the CR response was provided by an experiment in which flies were initially raised on either a CR or AL dietary regime and were then switched at various ages to the alternative diet (Mair et al., 2003). AL-raised flies normally have a higher age-specific mortality rate than do CR-raised animals. But after a AL ! CR shift, the animals rapidly adopt the age-specific mortality rate characteristic of animals who have been raised on a CR regime for their entire life. A corresponding rapid upward shift in mortality rate is observed following a CR ! AL shift. What this means is that the age-specific mortality rates have no cellular memory; change the physiological state of the cell and you change the resulting mortality rate. An AL ! CR shift, for example, induces the animal to alter its regulatory pathways and shift its gene expression pattern from a pro-growth pattern to a pro-stress resistance pattern. The consequent change in damage patterns affects the mortality rate. The mortality rate is due only to the current levels of damage occurring in the organism. The animal's prior history does not directly play a determining role in its current mortality rate, although certainly the existence of prior unrepaired damage must have some effect. This finding implies that CR/ISP-dependent aging is an environmentally dependent cell-level function, and the presence of the systemic regulatory mechanisms in multicellular eucaryotes does not invalidate this statement.
A cautionary note is in order here: not all flies react to CR in the same manner as described above. Our selected La and Ra strains have a different response pattern (Arking, unpublished data). Mediterranean fruit flies (Ceratitis capitata) are distantly related to Drosophila and might reasonably be expected to react to CR in the same manner if one assumes that the CR mechanism is highly conserved and public. However, a recent study reported that the Mediterranean fruit fly shows a more or less constant longevity at various levels of diet restriction with a sharp decrease in mortality once the diet falls below 50% of the ad libitum level (Carey et al., 2002). There is no evidence for an increase in longevity at some consistent level of dietary restriction. Reproduction occurred across the range of diets tested. The mortality increase occurred in both sexes even though males have no obvious counterpart to the energetic demands of egg production in females. On the other hand, these same flies lived longer when subjected to a ''feast or famine'' dietary regime (Carey et al., 2004). This latter regime likely resembles the normal situation in the wild, and so perhaps that environment selects for animals that can go into a survival mode when food becomes transiently scarce. These data suggest that there are intra- and interspecific differences in the CR response, which is thus a highly conserved but not universal mechanism.
Metabolic control of longevity via insulin-like signaling pathway
The insulin-like signaling pathway (ISP) was initially investigated for its effects on growth and size of the fly, and it was demonstrated that mutants affecting the activity of ISP did affect these parameters (Weinkove and Leevers, 2000). The ISP also affects blood sugar levels. The fly possesses five insulin-like proteins with significant homology to the mouse and human insulin proteins. These proteins are expressed in several tissues but most particularly in small clusters of insulin-producing cells (IPCs) in the brain (Rulifson et al., 2002). These workers showed that ablation of these IPCs caused retarded growth and elevated carbohydrate levels, and that a normal phenotype could be restored by expressing one of the Drosophila insulin-like proteins. Thus there is a remarkable conservation of the insulin-based gluco-regulatory mechanisms in flies and mammals with respect to its effects on growth and blood sugar. Once the experimental data allowed the concept of a conserved longevity regulation mechanism to become clear from the work done with yeast and nematode, then the fly's ISP was investigated to see if it also regulated the fly's longevity. There are some obvious phenotypic differences between homozygous and heterozygous mutants in genes comprising the ISP, but the important point made clear by these investigations is that certain of these mutants can significantly increase longevity.
As you might expect, flies with no ISP activity at all are lethal. But in rare cases, one can construct homozygous flies which contain two different mutations, each with a defect in a different part of the gene. Such unusual animals have a low but detectable level of ISP activity (~25% or less) and can survive through adulthood. These "heteroallelic" homozygotes contain two different mutations in their insulin receptor gene (InR; homologous to the C. elegans daf-2 gene) and gave rise to dwarf adults with different longevity effects on the two sexes (Tatar et al., 2001). Females expressed a delayed onset of senescence, with an 87% increase in their mean life span and a ~45% increase in their maximum life span. These homozygous mutant females are small because the ISP controls cell size, and the low activity levels result in small cells and slow growth. They are sterile because the low ISP activity results in a significant decrease in the juvenile hormone levels which are known to be essential for reproduction (see Reproductive Effects). Male homozygotes are also small and semisterile, for the same reasons. On the other hand, the males show no statistical change in their mean life span even though they have an increased mortality during early to mid-adult life followed by a lower mortality rate thereafter. Incidentally, the smaller effect of the ISP on males seems to be a general phenomenon, and we will touch on it in Reproductive Effects.
Both heterozygous and homozygous InR mutants have an impaired synthesis of the steroid hormone, ecdysone (Tu et al., 2002). This suggests that the increased longevity associated with the heterozygous InR mutants is likely dependent on a decreased level of juvenile hormone synthesis and the consequent reduction of ecdysone synthesis to levels that do not repress the animal's level of stress resistance (see Reproductive Effects for a description of the mechanisms which tie these several observations together).
Flies which are either heterozygous or homozygous for a certain mutation in their IRS proteins (associated with the cytoplasmic side of the InR) also yield long-lived females but not males (Clancy et al., 2001). These flies were also observed to be more resistant to oxidative stress than their controls. Thus disabling the InR and/or IRS genes is sufficient to down-regulate the entire ISP.
Finally, the dFOXO gene of Drosophila was shown to have sequence homology with the daf-16 gene of C. elegans, and to function in the regulation of growth (Kramer et al., 2003). Flies heterozygous for the dFOXO gene are incapable of expressing the extended longevity otherwise resulting from a mutational down-regulation of the InR gene (Hwangbo et al., 2004). This functional test demonstrates that the dFOXO gene is downstream of InR. Thus the structure of the ISP is the same in yeast and nematode and fly, for effects on the upstream genes such as InR must be mediated through the downstream dFOXO transcription factor. If the latter is mutationally inactivated, then the upstream signals are ignored. This factor is believed to activate various stress-resistance genes and repress various pro-growth genes in a manner similar to that demonstrated in the nematode. Both of these actions will result in a slower accumulation of age-related oxidative damage and in a delayed onset of senescence, as described in Stress Resistance and External Longevity.
The simplest conclusion of these several experiments is that the modulation of longevity via the ISP is highly conserved from yeast to mammals. The fact that longevity regulation in the fly is intertwined in the same system with the regulation of growth, cell size, and fertility suggests that the fly ISP has multiple specific regulatory functions compared to a nematode. This is consistent with the fact that the fly has at least four independent isoloci of the PI3K gene, each of which apparently controls different sets of processes, while humans have at least 16 PI3K genes (Samuels et al., 2004). This increased signaling complexity, which might have arisen as a consequence of the increased size and morphological complexity of the fly, might well account for its apparent greater genetic complexity.
Important though it is, the ISP is not the only signaling pathway involved in assaying nutrient availability.
The TOR signaling pathway is an important regulator of growth and size and is found in organisms from yeast to humans. Experiments with Drosophila show that the TOR pathway senses amino acid availability and uses this information to modulate activity of the S6 kinase regulatory gene so as to enhance growth and repress extended longevity (Kapahl et al., 2004). It also plays an important role in regulating autophagy, or the digestion of the cell's own components for energy (Klionsky, 2004). It was found that either over-expression of the upstream Tscl and Tsc2 genes, or dominant-negative mutations in in the TOR or S6K genes, gave rise to extended longevity. As briefly summarized in Figure 25.3, these data suggest that the two upstream genes act as negative regulators of the TOR or S6K genes, which themselves act as a negative regulator of longevity and a positive regulator of growth. It seems likely that the TOR pathway acts in parallel with, and perhaps even overlaps, the ISP.
Metabolic control of longevity via nuclear-mitochondrial interaction
There exists a large body of information regarding the role of mitochondria in aging. But there is almost no definitive data regarding the role of nuclear-mitochondrial interaction in the Drosophila aging process. This, however, does not mean that nuclear-mitochondrial interaction does not occur in flies, for there are at least three lines of suggestive evidence to the contrary. Taken together, they suggest but do not prove that the observed changes stem from some alteration in nuclear-mitochondrial communication.
First, Ballard and James (2003) found that mitochondria and nuclei taken from animals indigenous to different parts of the species' geographic range yielded low fitness flies when combined. This might come about if the mitochondria had evolved so as to be most effective in certain environments. Combining a (genetically different?) mitochondria from one area with a nucleus which evolved in a different region might result in organelle incompatibilities and hence a decreased life span.
Second, the same laboratory collected strains of Drosophila simulans (a close relative of D. melanogaster) from comparable environments and assayed their longevity as well as aspects of their mitochondrial function. They found that a particular long-lived strain was characterized by a high mitochondrial efficiency. Complex IV of their mitochondria's electron transport chain is more efficient than is the case in normal-lived control strains, as evidenced by their lower than normal oxygen consumption but normal levels of ATP production (Melvin et al., 2005). The nuclear encoded protein subunits of the complex responsible for the increased efficiency turned out to contain several point mutations which resulted in amino acid changes, the presence of which are highly correlated with the altered mitochondrial function. It is reasonable to suspect that the selection of chance mutations in nuclear encoded mitochondrial proteins bespeaks the likely existence of metabolic signals that coordinate nuclear and mitochondrial activities and provide the feedback signals necessary to the selection process.
Third, as a result of the selection experiment summarized in Figure 25.1A, we wound up with a long-lived La strain and a normal-lived Ra control strain. But their extended longevity arises in part from the fact that the La mitochondria produce from ~20 to ~40% less H2O2 than do the normal Ra mitochondria (Ross, 2001). Using the Ra and La strains, Driver and Tawadros (2000) set up various crosses that allowed them to combine mitochondria from different selected strains with the same normal-lived Ra nucleus. They then examined the resulting "cybrid" strains to see if any of the combined genomes had an effect on longevity. They showed that combining mitochondria from either normal-lived strain (Ra or Rb) with the Ra nucleus led to no change in longevity; but combining either of the long-lived mitochondria (La or Lb) with the Ra nucleus led to a significant change. Thus, the Lamt strain achieves about 40% of the extended longevity seen in the La strain solely because it has La type mitochondria. It would seem that mitochondria can make a significant contribution to longevity. Possible mechanisms for this phenomenon have been put forth elsewhere (Arking et al., 2002). The important point here is that different mitochondrial genomes appear to set up different stable metabolic equilibrium settings with the Ra nucleus, and each of these permits different longevities to be expressed.
Whether any of these three cases constitutes nuclear-mitochondrial interaction is still to be determined. The fact that two different labs using different strains and different approaches have observed similar sorts of data
TABLE 25.2 Effect of nonlethal stressors on longevity*
% Response of experimental
Animals over controls
Oxidative Stress Resistance
hsp hsp & ADS
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