Report

Extended Life-Span Conferred by Cotransporter Gene Mutations in Drosophila

See allHide authors and affiliations

Science  15 Dec 2000:
Vol. 290, Issue 5499, pp. 2137-2140
DOI: 10.1126/science.290.5499.2137

Abstract

Aging is genetically determined and environmentally modulated. In a study of longevity in the adult fruit fly,Drosophila melanogaster, we found that five independent P-element insertional mutations in a single gene resulted in a near doubling of the average adult life-span without a decline in fertility or physical activity. Sequence analysis revealed that the product of this gene, named Indy (for I'm not dead yet), is most closely related to a mammalian sodium dicarboxylate cotransporter—a membrane protein that transports Krebs cycle intermediates. Indy was most abundantly expressed in the fat body, midgut, and oenocytes: the principal sites of intermediary metabolism in the fly. Excision of the P element resulted in a reversion to normal life-span. These mutations may create a metabolic state that mimics caloric restriction, which has been shown to extend life-span.

Single gene mutations can greatly enhance our understanding of complex biological processes such as aging. Mutations in Caenorhabditis elegans and mice have highlighted the importance of hormone signal transduction, mitochondrial function, food intake, and the growth hormone–prolactin–thyroid-stimulating hormone system in life-span extension (1–9). To date, only one mutation that extends life-span in Drosophila has been reported. A partial loss-of-function mutation in the methuselah (mth) gene extends the average life-span of Drosophila by 35%, but neither the function of the methuselah gene product nor its tissue localization is known (10). In mammals, the only intervention that extends life-span is caloric restriction, and it has been postulated that the mechanism by which some of the mutations inC. elegans (for example, daf) extend life-span may be through a similar alteration in energy use (5–7).

In studies of Drosophila enhancer-trap lines (11), we noticed that male and female flies of two lines, 206 and 302, showed a doubling of mean life-span (from ∼37 to ∼70 days) and a 50% increase in maximal life-span. This occurred when only one copy of the enhancer-trap chromosome was present (in heterozygotes) (Fig. 1). Chromosomal in situ hybridization revealed that the P element in both 206 and 302 was inserted at the same cytological location (12). Genomic DNA flanking the site of insertion in the two enhancer-trap lines (206 and 302) was obtained by plasmid rescue (13) and sequenced. The insertion sites in the 206 and 302 enhancer-trap lines were 5753 base pairs (bp) from each other and were in the same gene, which we have namedIndy (for I'm not dead yet).

Figure 1

Life-span extension in Indymutants. Survival curves of males heterozygous for three differentIndy mutations, a precise excision of the P -element from Indy 302 (revertant), and an enhancer-trap control are shown. All flies were tested as heterozygotes over a wild-type Canton-S strain. TheIndy mutants are Indy302 (open green circles),Indy206 (solid blue circles), and Indy159 (open orange circles) (see Fig. 2 for mutation map). The excision line (open red squares) is one of four exact excisions (sequence confirmed) of the P element obtained by mobilizing the P element from either theIndy302 or Indy206 line, using delta 2-3 transposase (17). The control (solid black squares) is one of four other enhancer-trap control lines from the same mutagenesis that generated Indy302 and Indy206(11), tested as a heterozygote over Canton-S (28). A similar control survival curve was found for a control from the mutagenesis that gave rise to Indy159(12, 13). The mean 25°C life-spans of controls were 37 days, whereas the mean life-spans for Indy206,Indy302, and Indy159 were 71, 69, and 69 days, respectively. Indy206, Indy302, andIndy159 extended mean life-span by 92, 87, and 87% respectively. Extension of 1% maximal life-span of theseIndy mutants was greater than 45%. At 18°C, the increase in mean life-span conferred by Indy mutations approached 100%, whereas the increase in 1% maximum life-span approached 50% (12). Flies were maintained in a humidified, temperature-controlled environmental chamber at 25°C and were transferred to fresh food vials and scored for survival every 2 to 3 days as in (20). Each survivorship curve represents data from over 300 male flies. A total of 5430 male and femaleIndy heterozygote flies were tested.

Sequence analysis identified three expressed sequence tags (ESTs) from the Drosophila genome project (LD13803, LD16220, and HL01773). Genomic and cDNA sequences predicted a 572–amino acid protein with 34% identity and 50% similarity to human and rat renal sodium dicarboxylate cotransporters (14–16) (Fig. 2). Mammalian dicarboxylate cotransporters are membrane proteins responsible for the uptake or reuptake of di- and tricarboxylic acid Krebs cycle intermediates such as succinate, citrate, and alpha-ketoglutarate. They are found in a variety of tissues, including brush border cells of the small intestine, colon, and placenta; the basolateral membrane of perivenous cells in the liver; and epithelial cells of the renal proximal tubule and the brain (14–16).

Figure 2

(A) Genomic organization of theIndy locus, with insertion sites of all five P-element alleles. The organization of the Indy transcription unit is shown. Solid black boxes represent conserved regulatory sequences (AntC and FasI). The red rectangle represents the conserved Hoppel transposable element. PlacW insertion sites in the 206, 302, and 159 insertion lines are shown, as well as the orientation of the insertions. The positions of Birmingham-2 P-element insertions (PBm) in 92 and 265 insertion lines are also shown. The insertions in the 206, 159, and 265 lines are within a Hoppel element in the first intron of the Indy gene just upstream of the putative translational start site. The Hoppel element is present in the same position in wild-type animals, includ- ing P1 clones from the Drosophila Genome Project. The insertion in the 302 line is within 50 bp of the putative transcriptional start site. PlacW (10 kb) is not drawn to scale. (B) Sequence comparisons (29). The proteins most homologous to the Indy protein (GenBank accession no. AE003519) were identified by Blast. Indy-2 is a highly homologous Drosophilagene (accession no. AE003728). hNaDC-1 (accession no. U26209) is a human dicarboxylate cotransporter, and SDCT1 (accession no.AF058714) and SDCT2 (accession no. AF081825) are rat sodium dicarboxylate cotransporters. Red boxes indicate identity across all proteins. Blue indicates identity to Indy. Yellow indicates amino acid similarity to Indy.

Information on the chromosomal location of Indy was used to identify additional mutations in the Indy gene from other laboratories. We examined several candidate lines with P-element insertions in the same cytogenetic region as Indy and found a third enhancer-trap line with a P element inserted 734 bp from the site of the 206 insertion (Fig. 2A). As a heterozygote, this line, 159, showed the same extension in life-span (Fig. 1). Two further P-element insertions in Indy were obtained through site-selected mutagenesis of the Indy locus. In a polymerase chain reaction–based screen of 10,000 mutagenized third chromosomes, we identified two new insertions into the Indy locus (12) (Fig. 2A). Flies heterozygous for either of these new alleles of Indy also showed a large extension in life-span (12).

To confirm that the P-element insertion in Indy caused the life-span extension, we remobilized and excised the P element from theIndy gene in the 302 and 206 lines (17). Four independent lines of flies, shown by sequence analysis to carry exact excisions, reverted to normal life-span (Fig. 1). A nonexcision control line isolated at the same time, which passed through the same genetic background as the excision lines, remained long-lived (12).

The reporter protein β-galactosidase (β-Gal) had an identical pattern of expression in each of the three enhancer-trap lines (206, 302, and 159), despite the P-element insertions being almost 6.5 kb away from each other in the three lines. In adult flies,Indy was expressed in the fat body, midgut, and oenocytes (Fig. 3): organs that are thought to be the primary sites of intermediary metabolism, absorption, and metabolic storage in Drosophila. The fat body is involved in the metabolism and storage of fat, glycogen, and protein and is most often compared to the liver of vertebrates (18, 19).Indy was also expressed at low levels in the halteres; portions of the alimentary canals, including the procardia and restricted regions of the esophagus and hindgut; and the base of the legs. These are regions that have been identified as storage depots for glycogen (18). Finally, Indy was expressed in a subset of cells in the third segment of the antennae (20).

Figure 3

Expression of Indy in adult flies. Whole-mount X-Gal staining showing nuclear localization of β-Gal in cells from lines carrying an enhancer-trap insert in theIndy gene: Indy302, Indy206, andIndy159. Expression is seen in oenocytes (A andB) and the gut (C and D). (A) Low-power view of oenocytes in the (v) ventral and (d) dorsal abdominal segments. (B) High-power view of dorsal midline oenocytes. (D) A 5-μm section showing X-Gal staining within the cells of the gut. Whole-mount staining was performed as in (20). After whole-mount X-gal staining, the tissue in (D) was postfixed in 6.25% glutaraldehyde, embedded in paraffin, and then sectioned. Scale bar in (A) through (C), 100 μm; in (D), 10 μm.

To exclude the possibility that the extended life-span of theIndy mutants was due to the rescue of uncharacterized deleterious mutations accumulating in our wild-type Canton-S stock, we crossed the Indy mutation into several different genetic backgrounds that were distinct from our Canton-S stock. These included the Hyperkinetic, Shaker, and drop deadstocks, each of which was isolated from other wild-type stocks 25 to 30 years ago, as well as the long-lived laboratory-selected lines of Luckinbill (21, 22). In all cases, there was an extension in life-span. For all genetic backgrounds, mean life-span was extended by 40 to 80%, except in the case of the long-lived lines of Luckinbill, in which life-span was additionally extended by only 15%. These data indicate that the mechanism by which Indymutations extend life-span is a positive effect of the mutation on life-span and not simply of the rescue of deleterious mutations. The smaller increase in life-span seen with the laboratory-selected long-lived lines provides additional evidence that the mechanisms by which Indy acts to increase life-span may represent physiological systems already partially optimized by laboratory selection.

A decline in fertility (23, 24) or a reduction in physical activity (25) can lead to an extension of life-span in flies. Indy long-lived heterozygote males and females were compared to controls and found to be normal or superior in fertility and fecundity (Table 1). Qualitative observations of flight, courtship, feeding behavior, and negative geotaxis revealed no significant differences betweenIndy long-lived males and females and control flies during early life. Differences occurred later in life when physical measures of behavior and locomotor function were maintained at high levels in the Indy long-lived animals but not in normal-lived controls. For instance, one physiological milestone of aging in flies is the onset of female infertility. Indy heterozygous long-lived females continued to produce viable adult offspring 40% longer on average than did control flies (23.2 versus 16.5 days). This was a true extension of the period of fertility and was not associated with a compensatory delay in fertility during early life, as is seen in laboratory-selected long-lived lines (26).Indy long-lived females showed the same early peak of egg laying and fertility as control females but sustained the ability to produce larger numbers of offspring for a longer period of time (Table 1). There was no alteration in the rate or timing of developmental events in Indy long-lived mutant animals, as in the C. elegans clock mutants (3). The time from egg to adult at 25°C was the same as that for normal-lived controls (9 to 10 days).

Table 1

Fertility and fecundity of long-lived Indymutant animals. CS indicates the Canton-S control, 302/CS indicates heterozygotes of 302 and Canton-S, and 206/CS indicates heterozygotes of 206 and Canton-S. Single-pair mates were kept in individual vials. Each day throughout their entire life-span, they were passed to new vials, the number of eggs from the vial of the previous day was counted, and the vial was saved in order to determine the number of adult offspring.

View this table:

The expression of Indy in the fat body, gut, and oenocytes, and the amino acid sequence similarity of the Indy protein to dicarboxylate cotransporters, suggest that Indy may play a role in both the absorption of metabolites and in intermediary metabolism. Thus, insertion of a P element in the Indy gene may alter normal metabolism. In mammals, a moderate caloric restriction increases life-span, whereas a more severe restriction leads to starvation and decreased life-span (27). We postulate that the level of Indy expression is critical for life-span extension and that P-element insertions in Indyreduce Indy expression. When the level of Indy is mildly reduced, as in flies heterozygous for Indy insertions, there is a large extension in life-span. The extension in life-span is less dramatic with a further reduction in Indy activity, such as that seen inIndy homozygous flies, in which a 10 to 20% increase in mean life-span was seen (12). This model predicts that a further reduction in Indy activity would shorten life-span, and indeed this was observed when Indy gene activity was further reduced by placing an Indy mutation over a chromosome deleted for the Indy region. Flies with only a single copy of a mutant Indy gene and no normal copy of Indy(Indy mutant over deletion for the Indy region) had a 10 to 20% shorter mean and maximum life-span than did homozygousIndy mutants (12).

The mechanism by which caloric restriction mediates life-span extension is not understood, but it is likely to involve alterations in energy utilization. In contrast to many of the previously identified genes associated with life-span extension in metazoans, which have indirect effects on metabolism (5–9), Indy appears to be directly involved in intermediary metabolism and thus may represent a new class of longevity genes. A genetically induced reduction in the amount or efficiency of a dicarboxylic acid cotransporter in theIndy mutants may be creating a metabolic state similar to caloric restriction. Further characterization of the Indymutants may provide direct genetic insight into the role of energy balance and aging, and a point of access for genetic and pharmacological interventions for extending life-span.

  • * To whom correspondence should be addressed. E-mail: shelfand{at}neuron.uchc.edu

REFERENCES AND NOTES

View Abstract

Navigate This Article