InteractiveFly: GeneBrief

I'm not dead yet: Biological Overview | References

BIOLOGICAL OVERVIEW

The Drosophila Indy (I'm not dead yet) gene encodes a plasma membrane transporter of Krebs cycle intermediates, with robust expression in tissues associated with metabolism. Reduced INDY alters metabolism and extends longevity in a manner similar to caloric restriction (CR); however, little is known about the tissue specific physiological effects of INDY reduction. This study focused on the effects of INDY reduction in the Drosophila midgut due to the importance of intestinal tissue homeostasis in healthy aging and longevity. The expression of Indy mRNA in the midgut changes in response to aging and nutrition. Genetic reduction of Indy expression increases midgut expression of the mitochondrial regulator spargel/dPGC-1, which is accompanied by increased mitochondrial biogenesis and reduced reactive oxygen species (ROS). These physiological changes in the Indy mutant midgut preserve intestinal stem cell (ISC) homeostasis and are associated with healthy aging. Genetic studies confirm that dPGC-1 mediates the regulatory effects of INDY, as illustrated by lack of longevity extension and ISC homeostasis in flies with mutations in both Indy and dPGC1. These data suggest INDY may be a physiological regulator that modulates intermediary metabolism in response to changes in nutrient availability and organismal needs by modulating dPGC-1 (Rogers, 2014).

Caloric restriction (CR) extends lifespan in nearly all species and promotes organismal energy balance by affecting intermediary metabolism and mitochondrial biogenesis. Interventions that alter intermediary metabolism are though to extend longevity by preserving the balance between energy production and free radical production Indy (I'm Not Dead Yet) encodes a plasma membrane protein that transports Krebs' cycle intermediates across tissues associated with intermediary metabolism (Birkenfeld, 2011; Fei, 2003; Knauf, 2006; Knauf, 2002). Reduced Indy-mediated transport extends longevity in worms and flies by decreasing the uptake and utilization of nutrients and altering intermediate nutrient metabolism in a manner similar to CR (Fei, 2004; Rogina, 2014; Rogina, 2000; Wang, 2009). Furthermore, it was shown that caloric content of food directly affects Indy expression in fly heads and thoraces, suggesting a direct relationship between INDY and metabolism (Rogers, 2014 and references therein).

dPGC-1/spargel is the Drosophila homolog of mammalian PGC-1, a transcriptional co-activator that promotes mitochondrial biogenesis by increasing the expression of genes encoding mitochondrial proteins. Upregulation of dPGC-1 is a hallmark of CR-mediated longevity and is thought to represent a response mechanism to compensate for energetic deficits caused by limited nutrient availability. Increases in dPGC-1 preserve mitochondrial functional efficiency without consequential changes in ROS. Previous analyses of Indy mutant flies revealed upregulation of mitochondrial biogenesis mediated by increased levels of dPGC-1 in heads and thoraces (Rogers, 2014 and references therein).

Recently, dPGC-1 upregulation in stem and progenitor cells of the digestive tract was shown to preserve intestinal stem cell (ISC) proliferative homeostasis and extend lifespan. The Drosophila midgut is regenerated by multipotent ISCs, which replace damaged epithelial tissue in response to injury, infection or changes in redox environment. Low levels of reactive oxygen species (ROS) maintain stemness, self-renewal and multipotency in ISCs; whereas, age-associated ROS accumulation induces continuous activation marked by ISC hyper-proliferation and loss of intestinal integrity (Rogers, 2014 and references therein).

This study describes a role for Indy as a physiological regulator that modulates expression in response to changes in nutrient availability. This is illustrated by altered Indy expression in flies following changes in caloric content and at later ages suggesting that INDY-mediated transport is adjusted in an effort to meet energetic demands. Further, role was characterized for dPGC-1 in mediating the downstream regulatory effects of INDY reduction, such as the observed changes in Indy mutant mitochondrial physiology, oxidative stress resistance and reduction of ROS levels. Longevity studies support a role for dPGC-1 as a downstream effector of Indy mutations as shown by overlapping longevity pathways and absence of lifespan extension without wild-type levels of dPGC-1. These findings show that Indy mutations affect intermediary metabolism to preserve energy balance in response to altered nutrient availability, which by affecting the redox environment of the midgut promotes healthy aging (Rogers, 2014).

Reduction of Indy gene activity in fruit flies, and homologs in worms, extends lifespan by altering energy metabolism in a manner similar to caloric restriction (CR). Indy mutant flies on regular food share many characteristics with CR flies and do not have further longevity extension when aged on a CR diet. Furthermore, mINDY-/- mice on regular chow share 80% of the transcriptional changes observed in CR mice, supporting a conserved role for INDY in metabolic regulation that mimics CR and promotes healthy aging. This study shifted from systemic to the tissue specific effects of INDY reduction, focusing on the midgut due to the high levels of INDY protein expression in wild type flies and the importance of regulated intestinal homeostasis during aging. The evidence supports a role for INDY as a physiological regulator that senses changes in nutrient availability and alters mitochondrial physiology to sustain tissue-specific energetic requirements (Rogers, 2014).

The age-associated increase in midgut Indy mRNA levels that can be replicated by manipulations that accelerate aging such as increasing the caloric content of food or exposing flies to paraquat. Conversely, it was also shown that CR decreases Indy mRNA in control midgut tissues, which is consistent with previous findings in fly muscle and mouse liver. Diet-induced variation in midgut Indy expression suggests that INDY regulates intermediary metabolism by modifying citrate transport to meet tissue or cell-specific bioenergetic needs. Specifically, as a plasma membrane transporter INDY can regulate cytoplasmic citrate, thereby affecting fat metabolism, respiration, and via conversion to malate, the TCA cycle. Reduced INDY-mediated transport activity in the midgut could prevent age-related ISC-hyperproliferation by decreasing the available energy needed to initiate proliferation, thereby preserving tissue function during aging. This is supported by findings that nutrient availability affects ISC proliferation in adult flies and that CR can affect stem cell quiescence and activation (Rogers, 2014).

One of the hallmarks of CR-mediated longevity extension is increased mitochondrial biogenesis mediated by dPGC-1 (Spargel). Increased dPGC-1 levels and mitochondrial biogenesis have been described in the muscle of Indy mutant flies, the liver of mIndy-/- mice, and this study describes it in the midgut of Indy mutant flies. One possible mechanism for these effects can be attributed to the physiological effects of reduced INDY transport activity. Reduced INDY-mediated transport activity could lead to reduced mitochondrial substrates, an increase in the ADP/ATP ratio, activation of AMPK, and dPGC-1 synthesis. This is consistent with findings in CR flies and the livers of mINDY-/- mice. This study's analysis of mitochondrial physiology in the Indy mutant midgut shows upregulation of respiratory proteins, maintenance of mitochondrial potential and increased mitochondrial biogenesis, all of which are signs of enhanced mitochondrial health. The observed increase in dPGC-1 levels in Indy mutant midgut therefore appears to promote mitochondrial biogenesis and functional efficiency, representing a protective mechanism activated in response to reduced energy availability (Rogers, 2014).

Genetic interventions that conserve mitochondrial energetic capacity have been shown to maintain a favorable redox state and regenerative tissue homeostasis. This is particularly beneficial in the fly midgut, which facilitates nutrient uptake, waste removal and response to bacterial infection. Indy mutant flies have striking increases in the steady-state expression of the GstE1 and GstD5 ROS detoxification genes. As a result, any increase in ROS levels, whether from mitochondrial demise or exposure to external ROS sources can be readily metabolized to prevent accumulation of oxidative damage. Such conditions not only promote oxidative stress resistance, but also preserve ISC homeostasis as demonstrated by consistent proliferation rates throughout Indy mutant lifespan and preserved intestinal architecture in aged Indy mutant midguts. Thus, enhanced ROS detoxification mechanisms induced by Indy reduction and subsequent elevation of dPGC-1 contributes to preservation of ISC functional efficiency, and may be a contributing factor to the long-lived phenotype of Indy mutant flies (Rogers, 2014).

Several lines of evidence indicate that INDY and dPGC-1 are part of the same regulatory network in the midgut, in which dPGC-1 functions as a downstream effector of INDY. The similarity between dPGC-1 mRNA levels and survivorship of flies overexpressing dPGC-1 in esg-positive cells and Indy mutant flies suggests that Indy and dPGC-1 interact to extend lifespan. This is further supported by the lack of additional longevity extension when dPGC-1 is overexpressed in esg-positive cells of Indy mutant flies. Moreover, hypomorphic dPGC-1 flies in an Indy mutant background are similar to controls with respect to life span, declines in mitochondrial activity and ROS-detoxification. Together, these data suggest that dPGC-1 must be present to mediate the downstream physiological benefits and lifespan extension of Indy mutant flies (Rogers, 2014).

There are some physiological differences between the effects of Indy mutation and dPGC-1 overexpression in esg-positive cells. While Indy mutant flies are less resistant to starvation and more resistant to paraquat, a recent report showed that overexpressing dPGC-1 in esg-positive cells has no effect on resistance to starvation or oxidative stress. Additionally, mice lacking skeletal muscle PGC-1α were found to lack mitochondrial changes associated with CR but still showed other CR-mediated metabolic changes. In the fly INDY is predominantly expressed in the midgut, fat body and oenocytes, though there is also low level expression in the malpighian tubules, salivary glands, antenae, heart and female follicle cell membranes. Thus, the effects of INDY on intermediary metabolism and longevity could be partially independent from dPGC-1 or related to changes in tissues other than the midgut (Rogers, 2014).

This study suggests that INDY may function as a physiological regulator of mitochondrial function and related metabolic pathways, by modulating nutrient flux in response to nutrient availability and energetic demands. Given the localization of INDY in metabolic tissues, and importance of regulated tissue homeostasis during aging, these studies highlight INDY as a potential target to improved health and longevity. Reduced Indy expression causes similar physiological changes in flies, worms and mice indicating its regulatory role would be conserved. Further work should examine the interplay between Indy mutation and metabolic pathways, such as insulin signaling, which have been shown to promote stem cell maintenance and healthy aging in flies and mice. In doing so, the molecular mechanisms, which underlie Indy mutant longevity may provide insight for anti-aging therapies (Rogers, 2014).

Indy mutations and Drosophila longevity

Decreased expression of the fly and worm Indy genes extends longevity. The fly Indy gene and its mammalian homolog are transporters of Krebs cycle intermediates, with the highest rate of uptake for citrate. Cytosolic citrate has a role in energy regulation by affecting fatty acid synthesis and glycolysis. Fly, worm, and mice Indy gene homologs are predominantly expressed in places important for intermediary metabolism. Consequently, decreased expression of Indy in fly and worm, and the removal of mIndy in mice exhibit changes associated with calorie restriction, such as decreased levels of lipids, changes in carbohydrate metabolism and increased mitochondrial biogenesis. This study reports that several Indy alleles in a diverse array of genetic backgrounds confer increased longevity (Rogina, 2013).

Previous work has identified and characterized five independent mutations in the Indy gene in Drosophila that cause an increase in average and maximal life span for both male and female fruit flies (Rogina, 2000). The original five alleles were derived from three different mutageneses. Life spans of flies carrying one copy of P-element in the Indy gene were compared with their close genetically matched controls, flies from the same mutagenesis without a P-element insertion in the Indy gene. This study now shows that Indy²⁰⁶ heterozygous mutant flies also live longer when crossed into three different genetic backgrounds, Hk, short, and long-lived Luckinbill lines as compared to control flies from the same genetic background as Indy also crossed to these three different genetic backgrounds. Luckinbill short and long-lived lines have been generated by selective breeding for early and late female fecundity (Luckinbill, 1985). Presence of the yw;Indy²⁰⁶ mutant chromosome significantly extends longevity in the background of the Luckinbill short 1S9 line compared to the control line 1085. Moreover, the Indy²⁰⁶ mutation further extends longevity of two long-lived Luckinbill lines and does not cause shortening of life span of 2L18 long-lived line. At the same time, median longevity of control lines when crossed to Luckinbill long-lived lines are significantly shorter compared to homozygous Luckinbill lines. These data show that extension of life span by this Indy allele is not limited to the background of the short-lived lines, but further extends lines already selected for long life span (Rogina, 2013).

Extension of longevity also occurs with additional Indy mutant alleles. All Indy mutant alleles were treated by tetracycline to prevent any effects of Wolbachia and backcrossed to yw background. Wolbachia infection was proposed as a contributing factor to Indy longevity by Toivonen (2007). Indy^EY01442, Indy^EY01458, Indy^EY013297, Indy^KG07717 were generated by the Berkeley Drosophila Genome Project (BDGP) gene disruption project. The Indy gene region appears to be a 'hot spot' for P-element insertions illustrated by isolation of 5 KG, 28 EY, and 10 EP element insertions in the Indy region. P-element insertion in Indy²⁰⁶, Indy¹⁵⁹, Indy^EY01442, and Indy^EP3366 are within the Hoppel element in the first intron of the Indy gene, upstream of the putative translational start site. The conserved Hoppel element is present in the same position in wild type flies (Rogina, 2000). The insertion in Indy³⁰², Indy^EY013297, Indy^EY01458, Indy^KG07717, and Indy^EP3044 lines is upstream from putative transcriptional start sites. Indy encodes four putative transcripts, which have different 5′-exons. The positions of P-elements in Indy³⁰², Indy^EP3044, Indy^EY01458, Indy^EY013297, and Indy^KG07717 are located close to the three putative transcriptional start sites for three putative Indy transcripts (Indy-RA, Indy-RD, and Indy-RC) and about 5,000 bp upstream from the putative transcriptional start site in Indy-RB. Genomic organization of the Indy locus and positions of P-element insertion in different Indy alleles used in this manuscript can be seen in FlyBase. It was previously shown that the presence of the P-element in Indy²⁰⁶ and Indy³⁰² mutant alleles decreases the levels of Indy mRNA most likely by affecting transcription (Knauf, 2006; Wang, 2009). The levels of Indy mRNA are decreased about 95% in homozygous Indy²⁰⁶ and about 40% in homozygous Indy³⁰² alleles (Wang, 2009). The levels of INDY protein are also dramatically decreased in Indy²⁰⁶ homozygous mutant flies (Knauf, 2002). Similarly, the levels of Indy mRNA are decreased about 39% in the heterozygous Indy^EY01442/+ allele and about 49% in the heterozygous Indy²⁰⁶/+ allele compared to the levels of Indy mRNA found in yw flies. No significant decrease in the levels of Indy mRNA were observed in heterozygous Indy³³⁶⁶/+ flies, which correlates with the absence of longevity extension. It is likely that variation in longevity effects of different Indy alleles correlates to actual Indy mRNA levels and differential effects of P-elements on transcription. Male flies heterozygous for six Indy alleles have longevity extension ranging from 14.0 to 34.4%. Females heterozygous for seven Indy alleles show similar result having longevity extension ranging from 9.2 to 29.3%. The data further confirm the hypothesis that the level of Indy expression is central for longevity extension. When the levels of Indy mRNA are decreased approximately 49%, as in Indy²⁰⁶/+ heterozygous mutant flies, there is dramatic longevity extension of 34%. It has been previously reported that when the levels of Indy mRNA are radically reduced, as in Indy²⁰⁶ homozygous flies, longevity extension is less than extension of the Indy²⁰⁶/+ heterozygous flies (Wang, 2009). A smaller longevity effect of 17% was observed when Indy mRNA levels are moderately reduced, as in Indy^EY01442/+. Insignificant reduction of Indy mRNA levels, as in Indy^EP3366/+ mutant flies, resulted in no longevity effect. Besides Indy^EP3366/+, no longevity extension was found in another one of the new alleles, Indy^KG07717. In summary, maximal longevity in Indy mutant flies is associated with optimal reduction of Indy mRNA levels. When Indy levels are too low or close to normal, longevity effects are diminished. Although a recent report attributed life span extension in Indy to hybrid vigor, due to life span evaluation in an incorrect genetic background, and bacterial infection, the current data corroborate a link between the Indy mutations and longevity in flies (Toivonen, 2007; Wang, 2009). The effect of the Indy mutation on longevity was supported by findings that decreased activity of NaDC2, a C. elegans homolog of the Indy gene, extends the life span of worms (Fei, 2003; Fei, 2004). Similar effects of increased longevity associated with mutations in the fly and the worm Indy gene suggests a possibility of evolutionary conservation and a universal role of INDY in longevity (Rogina, 2013).

Several studies have investigated the molecular mechanisms underlying the effects of the Indy mutation on longevity and health span of worms, flies, and mice (Fei, 2003; Marden, 2003; Neretti, 2009; Wang, 2009; Birkenfeld, 2011). INDY is a plasma membrane transporter that may mediate the movement of dicarboxylic acids through the epithelium of the gut and into organs important in intermediary metabolism and storage (Knauf, 2002; Knauf, 2006). Location of the INDY transporter in the fat body and oenocytes suggest a role in intermediary metabolism and expression in the gut suggests a role in uptake of nutrients. Reductions in INDY activity may alter uptake, utilization, or storage of important nutrients and affect normal metabolism. It has been hypothesized that reductions in Indy activity seen in Indy mutations might be altering the normal energy supply in flies resulting in life span extension through a mechanism similar to CR. CR has been shown to increase life span and delay the onset of age-related symptoms in a broad range of organisms. Consistent with the hypothesis that Indy is important in metabolism is the finding that Indy mutant worms, flies, and mice have disrupted lipid metabolism (Fei, 2003; Wang, 2009; Birkenfeld, 2011). Similarly to CR animals, Indy mutant flies have increased spontaneous physical activity, decreased starvation resistance, weight, egg production, and insulin signaling. Furthermore, wild type flies on CR have significantly decreased levels of Indy mRNA (Wang, 2009). Indy homozygous mutant flies live shorter on low calorie foods compared to controls, which is consistent with the hypothesis that Indy mutant flies are already in a state of reduced nutrition on normal food and when food is further reduced, life span is shortened due to starvation (Wang, 2009). In addition, Indy mutant flies have increased mitochondrial biogenesis in heads and thoraces similar to CR animals (Neretti, 2009). Similarly, mIndy knockout mice have increased mitochondrial biogenesis in the liver. The mechanism of the effect of a decrease in INDY on metabolism is likely from its physiological function as a citrate transporter. Cytosolic citrate is the main precursor for the synthesis of fatty acid, cholesterol, triacylglycerols, and low-density lipoproteins. In addition, cytosolic citrate inhibits glycolysis and fatty acid β-oxidation. Therefore, INDY by affecting the levels of cytosolic citrate may alter glucose and lipid metabolism in a manner that favors longevity. Additional support that Indy mutation mimics CR comes from the findings that mIndy knockout mice are protected against adiposity and insulin resistance when kept on high fat diet (Birkenfeld, 2011). The data from worm, fly, and mice studies highlight the importance of INDY in health span and longevity. New Indy alleles described here should provide additional tools to further explore the role of INDY in metabolism and its connection to extended longevity and health (Rogina, 2013).

Long-lived Indy and calorie restriction interact to extend life span

Calorie restriction (CR) improves health and extends life span in a variety of species. Despite many downstream molecules and physiological systems having been identified as being regulated by CR, the mechanism by which CR extends life span remains unclear. The Drosophila gene Indy (for I'm not dead yet), involved in the transport and storage of Krebs cycle intermediates in tissues important in fly metabolism, was proposed to regulate life span via an effect on metabolism that could overlap with CR. This study reports that CR down-regulates Indy mRNA expression, and that CR and the level of Indy expression interact to affect longevity. Optimal life span extension is seen when Indy expression is decreased between 25 and 75% of normal. Indy long-lived flies show several phenotypes that are shared by long-lived CR flies, including decreased insulin-like signaling, lipid storage, weight gain, and resistance to starvation as well as an increase in spontaneous physical activity. It is concluded that Indy and CR interact to affect longevity and that a decrease in Indy may induce a CR-like status that confers life span extension (Wang, 2009).

It is well recognized that in evaluating the effects of individual genes on complex biological phenomena such as aging the environmental context and genetic background of the organism needs to be taken into consideration. Genes that manifest their phenotype through alterations in metabolism are likely to lead to different effects based upon the environmental conditions (e.g., nutrition) in which they are examined. Given the central nature of metabolism and the large number of genes involved in setting the metabolic state of the organism, the effect of alterations in specific metabolically related genes on organismal function will be modulated by the specific genetic background of the organism (Wang, 2009).

The Indy gene product has been postulated to be involved in normal metabolism, as it is a plasma membrane transporter of Kreb's cycle intermediates found primarily in tissues responsible for uptake, utilization, and storage of nutrients in the fly. This study found that the life span extension seen with the long-lived Indy mutation is sensitive to both food conditions and genetic background. When living on the typical Drosophila laboratory-culturing food conditions, similar to normal- or high-calorie food, reduction of Indy leads to significant life span extension. Under low-calorie food conditions however, the life span extending effects of calorie restriction mask the life span extending effect of Indy. The conclusion by Toivenon (2007) that the Indy mutation plays no role in life span extension is likely due to the use of low-calorie food conditions in their studies. Unlike the conclusions of Toivenon, this study directly shows that reduction of Indy transcription, between a range of 25%-75% of normal, has a strong positive effect on life span extension in high- and normal-calorie food conditions (Wang, 2009).

Genetic background is known to affect the calorie restriction life span-extension response in Drosophila. For example, the w1118 strain has a severely blunted life span-extension response to CR compared with other wild-type strains such as Canton-S. Similarly, life span extension induced by a decrease in Indy expression is also dependent upon genetic background. Toivenon (2007) backcrossed Indy into the w1118 strain, found a loss of Indy related life span extension, and interpreted this as demonstrating Indy expression is not involved in life span extension. This study independently backcrossed Indy into w1118 and found that the w1118 does suppress the life span extending effect of Indy mutants. However, when the Indy mutation is in a Canton-S background (the original strain it was isolated in) or yw background, a significant life span extension is seen. The study by Toivenon (2007) rather than disproving that mutations in Indy are causally involved in life span extension provide evidence supporting a strong interaction between food conditions, genetic background, and Indy expression on longevity (Wang, 2009).

The current studies suggest an intimate relationship between food calorie content and Indy expression. Food calorie conditions directly affect the level of Indy transcription. A reduction of food calorie content, such as CR, causes a 20% or greater decrease in Indy mRNA expression in normal or heterozygous mutant Indy flies. Examination of life span, food conditions, and Indy mRNA expression, demonstrates the strong interaction between Indy and food calorie content in the determination of longevity in flies. The data support the hypothesis that the level of Indy expression plays an important role in life span determination regardless of whether the reduction in Indy mRNA expression is through the insertion of a P-element into the Indy region or a reduction of Indy mRNA via a change in food calorie content. These data suggest that the amount of Indy mRNA and food calorie content interact to achieve the significant life span extension seen (Wang, 2009).

The finding that food calorie content directly affects the level of Indy expression and that either CR or Indy expression can affect longevity suggested that there may be some overlap in the mechanisms by which CR and Indy mutations extend life span. In support of this, it was found that normal flies on low-calorie food (CR) and Indy heterozygous mutant flies on normal- or high-calorie food share several physiological and behavioral changes. Both normal animals on low-calorie conditions and Indy long-lived heterozygotes on normal-calorie conditions: (1) have a decrease in insulin signaling as measured by a decrease in transcription of Dilp2, 3, and 5 and an increase in dFOXO nuclear expression in fat body cells; (2) a decrease in total triglycerides and total fat storage in fat body cells; (3) do not gain weight; (4) are sensitive to starvation; and (5) have a higher rate of spontaneous physical activity. The induction of these changes is not the result of a decrease in food intake in Indy long-lived heterozygotes that could secondarily cause CR since Indy long-lived heterozygotes take in as much food as normal flies, if not more. This is in agreement with findings that functional knockdown of nac-2, a nematode transporter with sequence homology to Indy, has extended longevity, smaller body size, and decreased levels of fat (Wang, 2009).

The nature of the relationship between Indy life span extension and CR life span extension is not clear. The data show that Indy long-lived heterozygote flies manifest a number of physiological and behavioral changes that occur when normal animals are placed under CR life span-extending conditions. The lack of an additive effect on life span extension when Indy long-lived heterozygote flies are placed on CR conditions, coupled with the finding that CR directly leads to a decrease in Indy transcription, provide genetic and molecular epistasis evidence, suggesting that CR and Indy interact to extend life span. The finding that CR reduces Indy expression suggests that a decrease in Indy may be one of the 'downstream' components of the normal CR life span extending pathway in the fly (Wang, 2009).

The manipulation of food content, CR, and the level of Indy expression appear to interact to attain an optimal balance for achieving life span extension. The possibility that Indy is one of the downstream effectors of CR life span extension suggests that identification of the downstream physiological and molecular targets shared between these two, CR and Indy, life span-extending interventions may provide further insights into the mechanisms of CR life span extension. A better understanding of the interaction between these 2 related interventions as well as other shared downstream elements could be of great benefit in developing interventions that can extend healthy life span and lead to other positive health benefits without the need for some of the unacceptable effects of severe CR. The realization that Indy and CR interact to extend life span suggests that a simultaneous modest modification of both could obviate the need for a more severe CR regime (Wang, 2009).

Long-lived Indy induces reduced mitochondrial reactive oxygen species production and oxidative damage

Decreased Indy activity extends lifespan in D. melanogaster without significant reduction in fecundity, metabolic rate, or locomotion. To understand the underlying mechanisms leading to lifespan extension in this mutant strain, the genome-wide gene expression changes in the head and thorax of adult Indy mutant were compared with control flies over the course of their lifespan. A signature enrichment analysis of metabolic and signaling pathways revealed that expression levels of genes in the oxidative phosphorylation pathway are significantly lower in Indy starting at day 20. It was confirmed experimentally that complexes I and III of the electron transport chain have lower enzyme activity in Indy long-lived flies by Day 20 and predicted that reactive oxygen species (ROS) production in mitochondria could be reduced. Consistently, it was found that both ROS production and protein damage are reduced in Indy with respect to control. However, no significant differences were detected in total ATP, a phenotype that could be explained by the finding of a higher mitochondrial density in Indy mutants. Thus, one potential mechanism by which Indy mutants extend life span could be through an alteration in mitochondrial physiology leading to an increased efficiency in the ATP/ROS ratio (Neretti, 2009).

Longevity of Indy mutant Drosophila not attributable to Indy mutation

In a recent study in PNAS, Neretti (2009) identified mitochondrial defects in male Drosophila melanogaster that were heterozygous for the mutation Indy²⁰⁶. This allele lowers the expression of Indy, which encodes a Krebs cycle intermediate transporter and was previously proposed to increase adult fly lifespan. The authors propose an interesting model in which mitochondria work at lower rate but are present in greater density. This model results in unchanged ATP levels but less ROS production, potentially accounting for the longevity of this strain (Toivonen, 2009).

Although this model may be correct, the role of the Indy gene in these effects on lifespan is highly doubtful. Although males of the mutant strain Neretti used (Indy206 in the Canton-S genetic background) are verifiably long-lived, it was previously demonstrated that the longevity of this strain does not segregate with the Indy mutation (Toivonen, 2007). Instead, it largely depends on the presence of a tetracycline-dependent agent (probably Wolbachia), plus some other X-chromosomal locus (or loci) (3). Regrettably, it seems that Neretti have attempted to brush these inconvenient facts under the rug, and this note should draw attention to this. Clearly, if mutation of Indy does not slow aging, then the mitochondrial defects in the Indy strain are either not caused by Indy or, if they are, they do not cause the longevity (Toivonen, 2009).

No influence of Indy on lifespan in Drosophila after correction for genetic and cytoplasmic background effects

To investigate whether alterations in mitochondrial metabolism affect longevity in Drosophila melanogaster, this paper describes a study of lifespan in various single gene mutants, using inbred and outbred genetic backgrounds. As positive controls the two most intensively studied mutants of Indy, which encodes a Drosophila Krebs cycle intermediate transporter, were included. It has been reported that flies heterozygous for these Indy mutations, which lie outside the coding region, show almost a doubling of lifespan. Only one of the two mutants lowers mRNA levels, implying that the lifespan extension observed is not attributable to the Indy mutations themselves. Moreover, neither Indy mutation extended lifespan in female flies in any genetic background tested. In the original genetic background, only the Indy mutation associated with altered RNA expression extended lifespan in male flies. However, this effect was abolished by backcrossing into standard outbred genetic backgrounds, and was associated with an unidentified locus on the X chromosome. The original Indy line with long-lived males is infected by the cytoplasmic symbiont Wolbachia, and the longevity of Indy males disappeared after tetracycline clearance of this endosymbiont. These findings underscore the critical importance of standardisation of genetic background and of cytoplasm in genetic studies of lifespan, and show that the lifespan extension previously claimed for Indy mutants was entirely attributable to confounding variation from these two sources. In addition, no effects were seen on lifespan of expression knockdown of the Indy orthologues nac-2 and nac-3 in the nematode Caenorhabditis elegans (Toivonen, 2007).

The life-extending gene Indy encodes an exchanger for Krebs-cycle intermediates

A longevity gene called Indy, with similarity to mammalian genes encoding sodium-dicarboxylate cotransporters, was identified in Drosophila melanogaster. Functional studies in Xenopus oocytes showed that INDY mediates the flux of dicarboxylates and citrate across the plasma membrane, but the specific transport mechanism mediated by INDY was not identified. To test whether INDY functions as an anion exchanger, this study examined whether substrate efflux is stimulated by transportable substrates added to the external medium. Efflux of [¹⁴C]citrate from INDY-expressing oocytes was greatly accelerated by the addition of succinate to the external medium, indicating citrate-succinate exchange. The succinate-stimulated [¹⁴C]citrate efflux was sensitive to inhibition by DIDS (4,4'-di-isothiocyano-2,2'-disulphonic stilbene), as demonstrated previously for INDY-mediated succinate uptake. INDY-mediated efflux of [14C]citrate was also stimulated by external citrate and oxaloacetate, indicating citrate-citrate and citrate-oxaloacetate exchange. Similarly, efflux of [14C]succinate from INDY-expressing oocytes was stimulated by external citrate, alpha-oxoglutarate and fumarate, indicating succinate-citrate, succinate-alpha-oxoglutarate and succinate-fumarate exchange respectively. Conversely, when INDY-expressing Xenopus oocytes were loaded with succinate and citrate, [14C]succinate uptake was markedly stimulated, confirming succinate-succinate and succinate-citrate exchange. Exchange of internal anion for external citrate was markedly pH(o)-dependent, consistent with the concept that citrate is co-transported with a proton. Anion exchange was sodium-independent. It is concluded that INDY functions as an exchanger of dicarboxylate and tricarboxylate Krebs-cycle intermediates. The effect of decreasing INDY activity, as in the long-lived Indy mutants, may be to alter energy metabolism in a manner that favours lifespan extension (Knauf, 2006).

Conditional tradeoffs between aging and organismal performance of Indy long-lived mutant flies

Alterations that extend the life span of animals and yeast typically involve decreases in metabolic rate, growth, physical activity, and/or early-life fecundity. This negative correlation between life span and the ability to assimilate and process energy, to move, grow, and reproduce, raises questions about the potential utility of life span extension. Tradeoffs between early-life fitness and longevity are central to theories of the evolution of aging, which suggests there is necessarily a price to be paid for reducing the rate of aging. It is not yet clear whether life span can be extended without undesirable effects on metabolism and fecundity. This study reports that the long-lived Indy mutation in Drosophila causes a decrease in the slope of the mortality curve consistent with a slowing in the rate of aging without a concomitant reduction in resting metabolic rate, flight velocity, or age-specific fecundity under normal rearing conditions. However, Indy mutants on a decreased-calorie diet have reduced fecundity, suggesting that a tradeoff between longevity and this aspect of performance is conditional, i.e., the tradeoff can occur in a stressful environment while being absent in a more favorable environment. These results provide evidence that there do exist mechanisms, albeit conditional, that can extend life span without significant reduction in fecundity, metabolic rate, or locomotion (Marden, 2003).

Functional characterization and immunolocalization of the transporter encoded by the life-extending gene Indy

Caloric restriction extends life span in a variety of species, highlighting the importance of energy balance in aging. A new longevity gene, Indy, which doubles the average life span of flies without a loss of fertility or physical activity, was postulated to extend life by affecting intermediary metabolism. This study reports that functional studies in Xenopus oocytes show INDY is a metabolite transporter that mediates the high-affinity, disulfonic stilbene-sensitive flux of dicarboxylates and citrate across the plasma membrane by a mechanism that is not coupled to Na⁺, K⁺, or Cl^-. Immunocytochemical studies localize INDY to the plasma membrane with most prominent expression in adult fat body, oenocytes, and the basolateral region of midgut cells and show that life-extending mutations in Indy reduce INDY expression. It is concluded that INDY functions as a novel sodium-independent mechanism for transporting Krebs and citric acid cycle intermediates through the epithelium of the gut and across the plasma membranes of organs involved in intermediary metabolism and storage. The life-extending effect of mutations in Indy is likely caused by an alteration in energy balance caused by a decrease in INDY transport function (Knauf, 2002).

Deletion of the mammalian INDY homolog mimics aspects of dietary restriction and protects against adiposity and insulin resistance in mice

Reduced expression of the Indy (I'm Not Dead Yet) gene in D. melanogaster and its homolog in C. elegans prolongs life span and in D. melanogaster augments mitochondrial biogenesis in a manner akin to caloric restriction. However, the cellular mechanism by which Indy does this is unknown. This study reports on the knockout mouse model of the mammalian Indy (mIndy) homolog, SLC13A5. Deletion of mIndy in mice (mINDY^-/-) mice) reduces hepatocellular ATP/ADP ratio, activates hepatic AMPK, induces PGC-1alpha, inhibits ACC-2, and reduces SREBP-1c levels. This signaling network promotes hepatic mitochondrial biogenesis, lipid oxidation, and energy expenditure and attenuates hepatic de novo lipogenesis. Together, these traits protect mINDY^-/- mice from the adiposity and insulin resistance that evolve with high-fat feeding and aging. These studies demonstrate a profound effect of mIndy on mammalian energy metabolism and suggest that mINDY might be a therapeutic target for the treatment of obesity and type 2 diabetes (Birkenfeld, 2011).

Relevance of NAC-2, an Na+-coupled citrate transporter, to life span, body size and fat content in Caenorhabditis elegans

An Na+-coupled citrate transporter has been cloned and functionally characterized from Caenorhabditis elegans (ceNAC-2). This transporter shows significant sequence homology to Drosophila Indy and the mammalian Na+-coupled citrate transporter NaCT (now known as NaC2). When heterologously expressed in a mammalian cell line or in Xenopus oocytes, the cloned ceNAC-2 mediates the Na+-coupled transport of various intermediates of the citric acid cycle. However, it transports the tricarboxylate citrate more efficiently than dicarboxylates such as succinate, a feature different from that of ceNAC-1 (formerly known as ceNaDC1) and ceNAC-3 (formerly known as ceNaDC2). The transport process is electrogenic, as evidenced from the substrate-induced inward currents in oocytes expressing the transporter under voltage-clamp conditions. Expression studies using a reporter-gene fusion method in transgenic C. elegans show that the gene is expressed in the intestinal tract, the organ responsible for not only the digestion and absorption of nutrients but also for the storage of energy in this organism. Functional knockdown of the transporter by RNAi (RNA interference) not only leads to a significant increase in life span, but also causes a significant decrease in body size and fat content. The substrates of ceNAC-2 play a critical role in metabolic energy production and in the biosynthesis of cholesterol and fatty acids. The present studies suggest that the knockdown of these metabolic functions by RNAi is linked to an extension of life span and a decrease in fat content and body size (Fei, 2004).

Structural and functional characteristics of two sodium-coupled dicarboxylate transporters (ceNaDC1 and ceNaDC2) from Caenorhabditis elegans and their relevance to life span

Two Na⁺-coupled dicarboxylate transporters, namely ceNaDC1 and ceNaDC2, have been cloned and functionally characterized from Caenorhabditis elegans. These two transporters show significant sequence homology with the product of the Indy gene identified in Drosophila melanogaster and with the Na⁺-coupled dicarboxylate transporters NaDC1 and NaDC3 identified in mammals. In a mammalian cell heterologous expression system, the cloned ceNaDC1 and ceNaDC2 mediate Na⁺-coupled transport of various dicarboxylates. With succinate as the substrate, ceNaDC1 exhibits much lower affinity compared with ceNaDC2. Thus, ceNaDC1 and ceNaDC2 correspond at the functional level to the mammalian NaDC1 and NaDC3, respectively. The nadc1 and nadc2 genes are not expressed at the embryonic stage, but the expression is detectable all through the early larva stage to the adult stage. Tissue-specific expression pattern studies using a reporter gene fusion approach in transgenic C. elegans show that both genes are coexpressed in the intestinal tract, an organ responsible for not only the digestion and absorption of nutrients but also for the storage of energy in this organism. Independent knockdown of the function of these two transporters in C. elegans using the strategy of RNA interference suggests that NaDC1 is not associated with the regulation of average life span in this organism, whereas the knockdown of NaDC2 function leads to a significant increase in the average life span. Disruption of the function of the high affinity Na⁺-coupled dicarboxylate transporter NaDC2 in C. elegans may lead to decreased availability of dicarboxylates for cellular production of metabolic energy, thus creating a biological state similar to that of caloric restriction, and consequently leading to life span extension (Fei, 2003).

Extended life-span conferred by cotransporter gene mutations in Drosophila

Aging is genetically determined and environmentally modulated. In a study of longevity in the adult fruit fly, Drosophila melanogaster, five independent P-element insertional mutations were found 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 (Rogina, 2000).

Search PubMed for articles about Drosophila Indy

Birkenfeld, A. L., (2011). Deletion of the mammalian INDY homolog mimics aspects of dietary restriction and protects against adiposity and insulin resistance in mice. Cell Metab 14: 184-195. PubMed ID: 21803289

Fei, Y. J., Inoue, K. and Ganapathy, V. (2003). Structural and functional characteristics of two sodium-coupled dicarboxylate transporters (ceNaDC1 and ceNaDC2) from Caenorhabditis elegans and their relevance to life span. J Biol Chem 278: 6136-6144. PubMed ID: 12480943

Fei, Y. J., Liu, J. C., Inoue, K., Zhuang, L., Miyake, K., Miyauchi, S. and Ganapathy, V. (2004). Relevance of NAC-2, an Na+-coupled citrate transporter, to life span, body size and fat content in Caenorhabditis elegans. Biochem J 379: 191-198. PubMed ID: 14678010

Knauf, F., Rogina, B., Jiang, Z., Aronson, P. S. and Helfand, S. L. (2002). Functional characterization and immunolocalization of the transporter encoded by the life-extending gene Indy. Proc Natl Acad Sci U S A 99: 14315-14319. PubMed ID: 12391301

Knauf, F., Mohebbi, N., Teichert, C., Herold, D., Rogina, B., Helfand, S., Gollasch, M., Luft, F. C. and Aronson, P. S. (2006). The life-extending gene Indy encodes an exchanger for Krebs-cycle intermediates. Biochem J 397: 25-29. PubMed ID: 16608441

Luckinbill, L. S. and Clare, M. J. (1985). Selection for life span in Drosophila melanogaster. Heredity (Edinb) 55 ( Pt 1): 9-18. PubMed ID: 3930429

Marden, J. H., Rogina, B., Montooth, K. L. and Helfand, S. L. (2003). Conditional tradeoffs between aging and organismal performance of Indy long-lived mutant flies. Proc Natl Acad Sci U S A 100: 3369-3373. PubMed ID: 12626742

Neretti, N., Wang, P. Y., Brodsky, A. S., Nyguyen, H. H., White, K. P., Rogina, B. and Helfand, S. L. (2009). Long-lived Indy induces reduced mitochondrial reactive oxygen species production and oxidative damage. Proc Natl Acad Sci U S A 106: 2277-2282. PubMed ID: 19164521

Rogers, R.P. and Rogina, B. (2014). Increased mitochondrial biogenesis preserves intestinal stem cell homeostasis and contributes to longevity in Indy mutant flies. Aging (Albany NY). 6: 335-350. 24827528

Rogina, B., Reenan, R. A., Nilsen, S. P. and Helfand, S. L. (2000). Extended life-span conferred by cotransporter gene mutations in Drosophila. Science 290: 2137-2140. PubMed ID: 11118146

Rogina, B. and Helfand, S. L. (2013). Indy mutations and Drosophila longevity. Front Genet 4: 47. PubMed ID: 23580130

Toivonen, J. M., Walker, G. A., Martinez-Diaz, P., Bjedov, I., Driege, Y., Jacobs, H. T., Gems, D. and Partridge, L. (2007). No influence of Indy on lifespan in Drosophila after correction for genetic and cytoplasmic background effects. PLoS Genet 3: e95. PubMed ID: 17571923

Toivonen, J. M., Gems, D. and Partridge, L. (2009). Longevity of Indy mutant Drosophila not attributable to Indy mutation. Proc Natl Acad Sci U S A 106: E53; author reply E54. PubMed ID: 19435842

Wang, P. Y., Neretti, N., Whitaker, R., Hosier, S., Chang, C., Lu, D., Rogina, B. and Helfand, S. L. (2009). Long-lived Indy and calorie restriction interact to extend life span. Proc Natl Acad Sci U S A 106: 9262-9267. PubMed ID: 19470468

date revised: 22 February 2022

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