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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Cell Metab. Author manuscript; available in PMC Nov 2, 2012.
Published in final edited form as:
PMCID: PMC3238792
NIHMSID: NIHMS332799
Modulation of longevity and tissue homeostasis by the Drosophila PGC-1 homolog
Michael Rera,1* Sepehr Bahadorani,1* Jaehyoung Cho,1* Christopher L. Koehler,2,3 Matthew Ulgherait,1,4 Jae H. Hur,1 William S. Ansari,3 Thomas Lo, Jr.,1 D. Leanne Jones,2,3# and David W. Walker1,5#
1Department of Integrative Biology and Physiology, University of California, Los Angeles, Los Angeles, California 90095
2Salk Institute for Biological Studies, Laboratory of Genetics, La Jolla, CA 92037
3Department of Biology, University of California, San Diego, La Jolla, CA 92037
4Department of Biological Chemistry, University of California, Los Angeles, Los Angeles, California 90095
5Molecular Biology Institute, University of California, Los Angeles, Los Angeles, California 90095
# Correspondence: D. Leanne Jones, Ph.D., Phone: 853-453-4100 x1869, ljones/at/salk.edu. David W. Walker, Ph.D., Phone: 310-825-7179, davidwalker/at/ucla.edu
*equal contribution
In mammals, the PGC-1 transcriptional co-activators are key regulators of energy metabolism, including mitochondrial biogenesis and respiration, which have been implicated in numerous pathogenic conditions including neurodegeneration and cardiomyopathy. Here, we show that overexpression of the Drosophila PGC-1 homolog (dPGC-1/spargel) is sufficient to increase mitochondrial activity. Moreover, tissue-specific overexpression of dPGC-1 in stem and progenitor cells within the digestive tract extends lifespan. Long-lived flies overexpressing dPGC-1 display a delay in the onset of aging-related changes in the intestine, leading to improved tissue homeostasis in old flies. Together, these results demonstrate that dPGC-1 can slow aging both at the level of cellular changes in an individual tissue and also at the organismal level by extending lifespan. Our findings point to the possibility that alterations in PGC-1 activity in high-turnover tissues, such as the intestine, may be an important determinant of longevity in mammals.
A progressive loss of mitochondrial energetic capacity is a common feature of multiple aspects of aging (Wallace, 2005). This may result from the age-related decline in the expression of genes important for mitochondrial electron transport chain (ETC) function observed in diverse organisms including humans (McCarroll et al., 2004; Zahn et al., 2006). A causal relationship is suggested by the fact that alterations in ETC activity are emerging as integrating phenomena in a number of lifespan-extending manipulations including dietary restriction (DR) (Guarente, 2008) and reduced insulin/TOR signaling (Bonawitz et al., 2007; Katic et al., 2007). More specifically, DR has been observed to result in an increase in mitochondrial biogenesis and/or respiratory activity in yeast, worms, flies, mice and humans (Bishop and Guarente, 2007; Civitarese et al., 2007; Lin et al., 2002; Lopez-Lluch et al., 2006; Nisoli et al., 2005; Zid et al., 2009). Furthermore, perturbation of mitochondrial ETC components has been shown to impair the ability of DR to promote longevity in yeast, worms and flies (Bahadorani et al., 2010; Bishop and Guarente, 2007; Lin et al., 2002; Zid et al., 2009). These findings suggest that strategies to enhance mitochondrial biogenesis and/or energy metabolism may promote healthy aging.
In mammals, the PGC-1 family of transcriptional co-activators plays a central role in the regulation of mitochondrial biogenesis, respiration and glucose homeostasis (Lin et al., 2005; Scarpulla, 2008b). Three members of this family have been identified based on sequence similarity to the founding member PGC-1α. PGC-1 family members promote mitochondrial biogenesis through co-activation of nuclear transcription factors, including nuclear respiratory factor-1 and -2 (NRF-1 and NRF-2) and estrogen-related receptor-α (ERRα) to induce the expression of genes encoding mitochondrial proteins (Puigserver and Spiegelman, 2003; Scarpulla, 2008a). Increased PGC-1 gene activity has been associated with health benefits in a number of pathogenic conditions, including various muscular (Handschin et al., 2007; Wenz et al., 2008) and neurodegenerative disorders (Cui et al., 2006; Zheng et al., 2010). Furthermore, increased expression of PGC-1α has been shown to protect against age-related sarcopenia (Wenz et al., 2009) and to improve respiration and gluconeogenesis under conditions of telomere dysfunction (Sahin et al., 2011). However, the role of PGC-1 co-activators in determining longevity remains poorly understood.
The fruit fly Drosophila melanogaster is an excellent model system to study the role of mitochondrial activity in the aging process (Cho et al., 2011). In addition, Drosophila has emerged as a premier model system to dissect the relationship between altered stem cell behavior, tissue homeostasis and aging (Wang and Jones, 2011). Several discrete populations of adult stem cells have been reported in Drosophila, such as germline stem cells and intestinal stem cells (ISCs) in the midgut (reviewed in (Wang and Jones, 2011)). These stem cells reside in defined niches and play active roles in maintaining local tissue homeostasis, resembling the behavior of mammalian stem cells.
Here, we report that up-regulation of the Drosophila PGC-1 homolog (dPGC-1/spargel) leads to an increase multiple markers of mitochondrial abundance and activity both during development and also in the adult stage. Furthermore, we find that targeted overexpression of dPGC-1 in the digestive tract, including restricted expression in somatic stem cells (including ISCs) and immediate daughter cells, can extend adult lifespan. Up-regulation of dPGC-1 abrogates the precocious activation of ISC proliferation and delays the accumulation of mis-differentiated cells in the intestinal epithelium- two hallmark of aging in this tissue. Furthermore, dPGC-1 up-regulation leads to improved intestinal integrity in old flies. Our findings demonstrate that dPGC-1 gene activity is an important determinant of aging both at the tissue and organismal level.
Overexpression of Drosophila PGC-1 leads to an increase in mitochondrial activity
The Drosophila genome contains a single PGC-1 homolog, CG9809/spargel/dPGC-1 (Gershman et al., 2007). A loss-of-function study reported that dPGC-1 is required for the normal expression of multiple genes encoding mitochondrial proteins in the larval fat body (Tiefenbock et al., 2010); therefore, we predicted that increased dPGC-1 might lead to an increase in mitochondrial abundance and/or activity. To investigate the physiologic and phenotypic consequences of overexpression of dPGC-1, we expressed dPGC-1 using the GAL4/UAS system (Brand and Perrimon, 1993). We transformed flies with UAS-constructs containing the dPGC-1 cDNA and performed twelve rounds of backcrossing into a w1118 background, which was used as a control strain in subsequent experiments. We confirmed that the dPGC-1 transcript was up-regulated in flies carrying both the dPGC-1 transgene and a ubiquitous GAL4 driver line, daughterless (da)-GAL4 (Figures S1A and S1B). Increased expression of dPGC-1 did not produce any gross changes in body size (Figure S1C) or obvious differences in size, morphology or cell number of external structures, such as wings (Figure S1D).
To determine whether up-regulation of dPGC-1 can increase mitochondrial activity, we measured three independent mitochondrial markers: the amount mitochondrial DNA (mtDNA), the enzymatic activity of citrate synthase, a key enzyme in the Krebs cycle and a widely used marker for mitochondrial density, and the abundance of HSP60, a mitochondrial matrix protein. Firstly, we measured the amount of mtDNA, relative to the amounts of a nuclear DNA (nDNA) amplicon, in 3rd instar larvae and observed a 2.5 fold increase in response to dPGC-1 up-regulation (Figure 1A). This data is consistent with a dPGC-1-mediated increase in mitochondrial density per cell. Thoraxes, which consist primarily of flight muscle (a rich source of mitochondria), were used for the measurement of changes in mitochondrial abundance in adult flies: a 60% increase in mtDNA in response to dPGC-1 up-regulation was observed in adult thoraxes (Figure 1B). In accordance, there was a significant increase in citrate synthase activity in both larvae (Figure 1C) and adult thoraxes in dPGC-1 overexpressing animals (Figure 1D). Similarly, Western blots using antibodies against a mitochondrial matrix protein, HSP60, showed significant increases of HSP60 levels in both whole larvae and adult thoraxes of animals overexpressing dPGC-1, when normalized to loading control (Figures 1E and 1F).
Figure 1
Figure 1
Overexpression of dPGC-1 increases mitochondrial markers
Next, we sought to address whether dPGC-1 can stimulate mitochondrial oxidative metabolism. We employed blue native polyacrylamide gel electrophoresis (BN-PAGE) to examine the impact of up-regulation of dPGC-1 on the abundance of the respiratory chain enzyme complexes. A significant increase in the abundance of respiratory complexes I, III, IV and V was observed in flies overexpressing dPGC-1 (Figure 2A). To analyze the effect of elevated dPGC-1 expression on respiratory chain activity, the rate of oxygen consumption was measured by using a Clark-type oxygen electrode. The steps in respiration were compared in mitochondria isolated from dPGC-1-overexpressing flies and controls by using substrates and inhibitors specific to individual respiratory complexes. In doing so, we observed that dPGC-1 up-regulation confers an increase in complex I-, II- and IV-dependent respiration (Figures 2B and S1E). Moreover, the respiratory control ratio (RCR), or state 3:state 4 respiration ratio, was significantly higher in mitochondria isolated from flies with increased dPGC-1 expression (Figure 2C). Taken together, our data suggest that dPGC-1 overexpression is sufficient to increase mitochondrial biogenesis and bioenergetic efficiency.
Figure 2
Figure 2
Regulation of oxidative metabolism, metabolic stores and glucose levels by dPGC-1
Drosophila PGC-1 modulates metabolic stores and free glucose levels
In mammals, PGC-1α regulates glucose homeostasis (Herzig et al., 2001; Yoon et al., 2001) and triglyceride (TAG) metabolism (Zhang et al., 2004). To determine whether dPGC-1 also regulates fuel homeostasis, we examined metabolic stores and free glucose levels in dPGC-1 overexpressing flies and controls. In Drosophila, metabolized nutrients are primarily stored as TAG and glycogen in the fat body, the insect equivalent of the mammalian liver and white adipose tissue. Using thin-layer chromatography, the most accurate method to measure stored TAG in Drosophila (Al-Anzi and Zinn, 2010), we observed ~20% reduction in TAG levels in dPGC-1 overexpressing flies (Figure 2D). In contrast, there was a significant increase in both the amount of stored glycogen (Figure 2E) and free glucose levels (Figure 2F) in dPGC-1 overexpressing flies. Our data suggests that dPGC-1 may play an important role in maintaining energy homeostasis in the fly-consistent with findings in mammals.
Effects of tissue-specific overexpression of Drosophila PGC-1 on longevity
As central regulators of energy homeostasis, PGC-1 co-activators provide an attractive target to modulate animal aging. Therefore, we examined the impact of targeted overexpression of dPGC-1 in major tissues on Drosophila lifespan. Firstly, we examined the effects of ubiquitous overexpression of dPGC-1 mediated by da-GAL4 and observed a moderate decrease in adult survival (Figures S2A and S2B). Similar effects were observed when dPGC-1 was ubiquitously expressed using the mifepristone (RU486) inducible driver Tubulin-Gene-Switch (Figures 3A and S3A). To begin to examine tissue-specific effects of dPGC-1, we used a panel of Gene-Switch driver lines with recently characterized age-related expression patterns (Poirier et al., 2008). Lifespan was significantly increased when dPGC-1 was induced with S1106 (Figures 3B and S3B), which is expressed in abdominal fat and the digestive tract (Poirier et al., 2008). To further narrow the tissue-specific requirements for dPGC-1-mediated longevity, we used the TIGS-2 driver, which is expressed in the digestive tract but not fat body (Poirier et al., 2008). Induced expression of dPGC-1 with this driver line resulted in a 33% increase in mean lifespan and a 37% increase in maximum lifespan in female flies (Figure 3C). Multiple independent dPGC-1 insertions were tested with TIGS-2 and each resulted in enhanced longevity (Figures S4A and S4B). However, no major longevity effects were observed in male flies with TIGS-2 (Figure S3C), nor in control flies in the presence of the inducer (Figures S5 and S6). Notably, adult survival was not improved when dPGC-1 was induced by the pan-neuronal driver ELAV-Gene-Switch (Figures 3D and S3D). Similarly, muscle-specific expression of dPGC-1 using a constitutive MHC-GAL4 driver did not promote life extension in male or female flies (Figures S7A and S7B).
Figure 3
Figure 3
Effects of tissue-specific overexpression of dPGC-1 on fly longevity
Together, the S1106 and TIGS-2 lifespan data support the idea that the digestive tract is an important target tissue in dPGC-1-mediated longevity.
Overexpression of dPGC-1 in intestinal stem and progenitor cells extends lifespan
As noted above, our tissue-specific longevity studies indicate that increased expression of dPGC-1 in the digestive tract is sufficient to promote longevity. To validate and extend this finding, we set out to identify subsets of intestinal cells that are important in mediating this phenotype. Tissue homeostasis in the mid-gut is maintained by pluripotent intestinal stem cells (ISCs), which are distributed along the basement membrane (Micchelli and Perrimon, 2006; Ohlstein and Spradling, 2006). Division of an ISC gives rise to one daughter cell that retains stem cell fate and another daughter cell that becomes an enteroblast (EB), both expressing a transcription factor called Escargot (esg). Thus, expression of esg is often used as a surrogate marker for ISCs and EBs. After ISC division, the daughter EB does not divide again and differentiates into either a large, polyploid enterocyte (EC) or a small, diploid enteroendocrine (ee) cell that expresses Prospero (Pros) (Figure 6A). Cell cycle arrest and differentiation of EBs are controlled by Delta-Notch signaling. While the ligand Delta (Dl) specifically accumulates in ISCs, it is quickly lost in newly formed EBs, which is accompanied by an activation of Notch signaling.
Figure 6
Figure 6
dPGC-1 modulates tissue homeostasis in the aged intestine
To examine the impact of targeted expression of dPGC-1 in the ISCs and EBs, we used the constitutive esgGAL4 driver line to overexpress dPGC-1 and observed a significant lifespan extension in both male (Figure S8A) and female flies (Figure 4A) compared to isogenic control flies. Although esgGal4 expression is restricted to ISCs and EBs in the intestine, it is also expressed in stem cells within malpighian tubules, germline and somatic stem cells in the testis, and in salivary glands (Biteau et al., 2010). Therefore, we made use of the recently described RU486-inducible 5961GS, which recapitulates the esgGal4 expression pattern in the digestive tract (ISCs/EBs and malpighian tubule stem cells) (Biteau et al., 2010; Mathur et al., 2010) but is not expressed in salivary glands (Biteau et al., 2010) or testis (C.K., L.J., unpublished observations). Consistent with a previous report (Biteau et al., 2010), we failed to detect 5961GS expression in the brain, thorax or abdomen. 5961GS-mediated expression of dPGC-1, during both development and adulthood, results in a significant increase in lifespan (Figures 4B and S8B). No major longevity effects were observed in control flies exposed to RU486 (Figures S9A and S9B). Next, we took advantage of the inducible nature the 5961GS driver to determine whether adult-only induction of dPGC-1 is sufficient to promote longevity. Indeed, 5961GS > dPGC-1 flies were significantly longer lived when exposed to RU486 exclusively in the adult stage (Figure 4C); again no major effects on longevity were observed in control flies (Figure S9C). Taken together, our data with the constitutive esgGAL4 and the inducible 5961GS driver support a model whereby increased expression of dPGC-1 in stem cell lineages within the digestive tract of adults is sufficient to extend lifespan.
Figure 4
Figure 4
Overexpression of dPGC-1 in intestinal stem and progenitor cells extends lifespan
dPGC-1 extends lifespan independently of effects on reproduction or global stress resistance
To gain further insight into dPGC-1-mediated longevity, we examined a number of physiological and behavioral parameters in long-lived flies overexpressing dPGC-1. For these studies, we used esgGAL4 because expression in the ISCs/EBs is stronger than with 5961GS (Biteau et al., 2010), and the longevity effects are more pronounced with this driver. Importantly, there was no obvious difference in food consumption (Figure S10A) or body mass (Figure S10B) in long-lived flies compared to age-matched controls. As described earlier, we observed that ubiquitous up-regulation of dPGC-1 leads to alterations in metabolic stores and free glucose levels (Figures 2D–2F). Therefore, we examined these parameters in long-lived esgGAL4 > dPGC-1 flies. Interestingly, esgGAL4-mediated expression of dPGC-1 is sufficient to confer a decrease in triglyceride (TAG) levels in whole flies (Figure S10C). Although esgGAL4 > dPGC-1 flies display normal glycogen stores (Figure S10D), restricted expression of dPGC-1 is sufficient to produce a moderate increase in free glucose levels (Figure S10E).
Interventions that extend lifespan are often associated with a decline in reproductive output (Partridge et al., 2005). However, long-lived esgGAL4 > dPGC-1 flies display normal fertility compared to age-matched isogenic controls (Figure S11A). Interestingly, we also failed to detect fertility defects in long-lived dPGC-1 flies mediated by either S1106 or TIGS-2 (SB & DW, unpublished observations). Another hallmark of extended longevity is an increase in the ability to withstand extrinsic stress (Lithgow and Walker, 2002). Therefore, we tested the ability of esgGAL4 > dPGC-1 flies to survive under conditions of starvation and oxidative stress. However, we observed no difference in survival times when long-lived dPGC-1 flies and controls were maintained on an agar-only diet to induce starvation (Figure S11B). In addition, long-lived dPGC-1 flies displayed only a marginal increase in survival under hyperoxia (100% O2; Figure S11C). Together, these data indicate that life extension mediated by overexpression of dPGC-1 via esgGAL4 acts independently of effects on reproduction or global stress resistance.
dPGC-1 modulates mitochondrial activity and ROS levels in the aged intestine
To better understand the relationship between dPGC-1 gene activity and longevity, we examined dPGC-1 mRNA levels in the intestine of young and aged flies. We observed that dPGC-1 expression was dramatically decreased (~60%) in the aged intestine of control flies (Figure 5A), whereas esgGAL4-mediated expression of dPGC-1 conferred ~ a 2-fold increase in dPGC-1 mRNA levels in the intestine in both young and aged flies (Figure 5A). Next, we examined whether increased dPGC-1 expression in the intestine impacts the activities of mitochondrial respiratory chain enzymes in the target tissue. Indeed, esgGAL4 > dPGC-1 flies display an increase in both mitochondrial complex I (Figure 5B) and complex II (Figure 5C) activities in the aged intestine. Interestingly, complex IV activity was increased in the intestines of esgGAL4 > dPGC-1 flies at 10 days but not 30 days (Figure 5D).
Figure 5
Figure 5
dPGC-1 modulates mitochondrial activity and ROS levels in the aged intestine
The cationic dye JC-1 can be used to measure mitochondrial membrane potential at a cellular level. In healthy cells, the negative charge established by the intact mitochondrial membrane potential allows this lipophilic dye to enter the mitochondrial matrix where it accumulates and fluoresces red. When mitochondrial membrane potential is low, the dye remains monomeric in the cytoplasm and fluoresces green. Therefore, membrane potential can be determined by the presence of J-aggregates and measured by the ratio of green: red fluorescence. Using this approach, we examined mitochondrial activity in the mid-gut epithelium (Figure S12). In doing so, we discovered that aging results in a progressive loss of mitochondrial membrane potential in this region of the intestine; however, intestines of long-lived esgGAL4 >dPGC-1 flies showed significant maintenance of mitochondrial membrane potential, when compared to isogenic controls (Figures S12A-S12C).
PGC-1α is a potent regulator of reactive oxygen species (ROS) metabolism required for the induction of several ROS-detoxifying enzymes (St-Pierre et al., 2006). Therefore, we speculated that up-regulation of dPGC-1 may reduce ROS levels in the target tissue. To test this idea, we examined the endogenous levels of ROS in the intestines of control and esgGAL4 >dPGC-1 flies using dihydro-ethidium (DHE), a redox-sensitive dye that exhibits increased fluorescence intensity when oxidized (Owusu-Ansah and Banerjee, 2009; Owusu-Ansah et al., 2008). Targeted expression of dPGC-1 in ISCs/EBs led to a reduction of DHE fluorescence in these cells and throughout the aged intestine (Figures 5E, F and S13A, B). Therefore, up-regulation of dPGC-1 in stem cells and immediate daughter cells is sufficient to lower ROS levels throughout the intestinal epithelium in aged flies.
dPGC-1 modulates tissue homeostasis in the aged intestine
Given the ability of dPGC-1 expression in the intestine to maintain mitochondrial activity and lower ROS levels, we wanted to determine whether targeted expression of dPGC-1 in ISCs/EBs was sufficient to delay the onset of previously characterized aging-related phenotypes in the intestine. In the Drosophila intestine, aging or stress results in a dramatic increase in ISC proliferation, which is accompanied by an accumulation of mis-differentiated daughter cells that express markers of both ISCs and terminally differentiated daughter cells (Biteau et al., 2008; Choi et al., 2008; Park et al., 2009). These cells retain expression of the stem cell markers, Esg and Dl, yet they are polyploid, suggesting that this population comprises EBs that that are blocked in the ability to terminally differentiate into functional enterocytes (Biteau et al., 2008; Choi et al., 2008; Park et al., 2009). The age-related increase in mis-differentiated cells disrupts epithelial integrity and tissue architecture, as revealed by staining for the membrane marker Armadillo (Arm), the Drosophila homolog of β-catenin. This leads to a loss of normal tissue homeostasis and severe deterioration of the midgut epithelium in aged flies (Biteau et al., 2008), which may impact gut function or integrity.
In order to determine whether increased expression of dPGC-1 leads to a delay in the aging-related phenotypes and improved tissue homeostasis in the gut, we quantified esg-positive cells (GFP+ as a consequence of a UAS-gfp reporter) in guts of flies overexpressing dPGC-1 under control of the esg promoter. For quantification of ISCs/EBs, GFP+ cells were counted in control flies (genotype: esgGAL4, UAS-gfp/+) or dPGC-1 flies (genotype: esgGAL4, UAS-gfp/+; UAS-dPGC-1/+), and at least 25 guts were examined for each time point (See Experimental Procedures for details). Both the increase in mis-differentiated cells, as well as the characteristic changes in tissue architecture were delayed in older animals expressing dPGC-1 in ISCs/EBs (Figures 6B and S14A). The average number of GFP+ cells per FOV in 50-day old control females was 115.1±6.4 (SEM) (n=26), whereas the average for 50-day old dPGC-1 females was 69±5.5 (n=27) (Figure 6C). The same trend was observed in male flies (Figure S14B).
In addition to a decrease in the accumulation of mis-differentiated cells, we also observed a delay in the precocious activation of ISC proliferation, as measured by phosphorylation of histone H3 (pHH3), a marker of cell cycle progression through mitosis. Forty-eight day old female flies expressing dPGC-1 under the control of the inducible 5961GS driver, induced from the onset of adulthood, contained significantly fewer pHH3+ cells, when compared to uninduced age-matched sibling controls (Figure 6D). No difference in the number of pHH3+ cells was observed in 10 day old flies, indicating that the expression of dPGC-1 delays the age-related increase in ISC proliferation. Furthermore, our data demonstrates that dPGC-1 acts during the adult stage to abrogate the precocious activation of ISC proliferation, which occurs during aging.
Finally, we sought to determine whether dPGC-1 expression in the digestive tract affects intestinal integrity as a function of age. To develop an assay of intestinal integrity, we examined flies of different ages that had consumed a non-absorbable blue food dye (F D & C Blue Dye no. 1). As expected, we observed that in young flies (10 days) the dye is restricted to the proboscis and digestive tract post-feeding (Figure 7A). However, in aged flies (>30 days) we observed a fraction of animals that displayed a strikingly different phenotype. In these animals, the blue dye was clearly visible throughout the body post-feeding; subsequently, these flies were referred to as ‘Smurf’ flies (Figure 7B). To exclude the possibility that this phenotype was due to a unique property of this dye, we fed aged flies a different non-absorbable red food dye (F D & C Red Dye no. 40) and observed a fraction of individuals that displayed red food dye throughout the body post-feeding (MR & DW, unpublished observations). Therefore, we interpret the ‘Smurf’ phenotype, ie., the leakage of dye into the haemolymph and consequently all tissues, to reflect a defect(s) in intestinal integrity.
Figure 7
Figure 7
dPGC-1 modulates intestinal integrity in old flies
We next quantified the increase in ‘Smurf’ flies as a function of age in control and long-lived esgGAL4 > dPGC-1 flies (Figure 7C). In control flies, the fraction of ‘Smurf’ flies in the population increases dramatically with age; from 0% at 10 days to ~35% at 45 days of age. Strikingly, esgGAL4-mediated activation of dPGC-1 retards the age-related onset of the ‘Smurf’ phenotype. Therefore, an increase in dPGC-1 expression within the digestive tract results in improved intestinal integrity in aged flies, which is consistent with the delay in disruption of apical-basal polarity in intestines from aging flies, as revealed by Arm staining (Figure 6B). Together, our analysis of proliferative homeostasis and tissue integrity strongly support a model whereby dPGC-1 activity in somatic stem cell lineages within the digestive tract regulates tissue homeostasis in the aged intestine.
Aging is associated with a decline of function at the organismal level that has origins in cellular deterioration and the loss of tissue homeostasis. Considerable attention has been focused separately on the roles of stem cells (Rando, 2006) and mitochondria (Guarente, 2008; Wallace, 2005) in the aging process, yet fundamental questions remain regarding the interplay between mitochondrial metabolism, stem cell behavior and lifespan determination. In this study, we demonstrate that the Drosophila PGC-1 homolog is a potent inducer of mitochondrial activity and that overexpression in somatic stem cells within the digestive tract can slow aging at both the tissue and organismal level.
It is interesting to speculate upon the tissue-specific requirements for dPGC-1-mediated longevity. Although we failed to observe life extension in response to ubiquitous, muscle or neuronal activation of dPGC-1, we cannot exclude the possibility that expression in subsets of muscle and/or neuronal cells or different levels of expression in these tissues could promote longevity. That said, we observed robust dPGC-1-mediated life extension with multiple driver lines that are expressed in the digestive tract. Consistent with our own findings, it was recently reported that intestinal homeostasis correlates with lifespan in a number of different genotypes including flies with altered Jun-N-terminal Kinase (JNK) or insulin/IGF signaling (IIS) activities (Biteau et al., 2010). One plausible explanation for such findings is that maintaining healthy intestinal function and/or integrity is an important determinant of fly lifespan. In our own study, using a non-absorbed food dye, we demonstrate that there is a loss of intestinal integrity as a function of age. It is possible that this phenomenon could impact the survival of the animal by exposing the internal tissues and organs to toxins or pathogens. However, although the intestine appears to play an important role in modulating longevity, we cannot rule out the role of cell non-autonomous effects in dPGC-1-mediated longevity. In this regard, we observed that esg-GAL4-mediated expression of dPGC-1 resulted in changes in lipid/carbohydrate metabolism in whole flies.
In this study, we report that dPGC-1 expression declines in the aged intestine of control flies, whereas directed expression of dPGC-1 in somatic stem cells within the digestive tract is sufficient to retard aging in this tissue. Importantly, our data indicate that manipulating dPGC-1 in ISCs/EBs leads to maintenance of mitochondrial activity throughout the midgut, rather than exclusively in stem cells. This is not unexpected, as there is no transit amplifying population of daughter cells; the EBs differentiate directly into one of two lineages. Therefore, any manipulation of stem cell physiology could easily be passed onto directly differentiating daughter cells. The enterocytes are the predominant cell type in the intestine; therefore, changes in these cells likely account for most of the phenotypic differences that we observe. In the case of ROS levels, we observe that dPGC-1 expression in ISCs/EBs leads to a reduction in ROS levels in the stem cells, as well as in the enterocytes (Figures 5E, F and S13A, B).
Given the diverse roles that PGC-1 plays in metabolism (Lin et al., 2005), the relative contribution of each of these processes in modulating tissue homeostasis and longevity remains to be determined. However, our finding that dPGC-1 modulates ROS levels in the aging intestine may provide mechanistic insight. ROS levels have been demonstrated to influence stem cell self-renewal and the onset of differentiation in multiple systems (Ito et al., 2004; Owusu-Ansah and Banerjee, 2009; Smith et al., 2000; Tothova and Gilliland, 2007). Indeed, it was recently reported that Nrf2, a master regulator of the cellular redox state, specifically controls the proliferative activity of ISCs, promoting intestinal homeostasis (Hochmuth et al., 2011). Our findings support a model whereby dPGC-1-mediated alterations in metabolism, including ROS metabolism, can retard aging of the intestine with significant consequences for animal lifespan. It will be interesting to determine whether PGC-1 family members in other species also regulate tissue homeostasis in high-turnover tissues such as intestine.
Analysis of survivorship, fertility, stress resistance, food intake, lipid/carbohydrate levels, mtDNA amount, enzyme activity assays, oxygen consumption, qRT-PCR, BN-PAGE and western blotting were conducted by standard methods and are available in the Supplemental Information.
Drosophila strains
Tubulin-GS was provided by S. Pletcher. Elav-GS was provided by H. Keshishian. S1106 and TIGS-2 were provided by L. Seroude. 5961GS was provided by H. Jasper. esgGAL4 was provided by A. Christiansen. All other stocks were provided by the Bloomington Drosophila Stock Center. We transformed flies with pUAST plasmids containing dPGC-1 and performed 12 rounds of backcrossing into a w1118 background.
Dihydroethidium (DHE) Staining
ROS levels were detected in live tissue based on previously described methods (Hochmuth et al., 2011; Owusu-Ansah and Banerjee, 2009; Owusu-Ansah et al., 2008). In brief, guts were dissected directly in Schneider’s medium, then incubated, protected from light, in 60uM dihydroethidium (Invitrogen Molecular Probes) in Schneider’s medium for 7 minutes (Dye freshly reconstituted each time in anhydrous DMSO). Three washes were performed for 5 minutes each in Schneider’s medium at room temperature before mounting in ProlongGold antifade reagent containing DAPI (Invitrogen Molecular Probes). Midguts were imaged immediately following the staining procedure. For total ROS production from the midgut, Z-stacks of regions 200–500uM anterior to the pylorus were measured for mean signal intensity at 568nM in Image J. For ROS output of ISCs/EBs, mean intensity of DHE signal was measured only in cell clusters identified by their esgGFP expression, their size, and their basal location within the intestinal epithelium. Pixel intensities of Z-stacks, spanning from the basal to apical cell layers, for a minimum of 10 midguts were used for each of the quantifications. Statistical analysis was conducted on mean DHE intensities averaged from each midgut using a two-tailed, unpaired Student’s T-test.
Immunofluorescence, quantification of ISCs/EBs, and pHH3+ cell counts
Fixation of Drosophila intestines was carried out according to (Boyle et al., 2007), prepared according to standard procedures, and mounted in Vectashield mounting medium containing 4′,6-diamidino-2-phenylindole (DAPI) from Vector Laboratories. Primary antibodies used in this study included rabbit anti-GFP (1:5,000) from Molecular Probes, rabbit anti-phospho-histone H3 (1:200) from Millipore (06–570), and mouse anti-Armadillo (N2 7A1) (1:20) and mouse anti-Prospero (MR1A) (1:100), both obtained from the Developmental Studies Hybridoma Bank at the University of Iowa. For the quantification of GFP+ cells, images were acquired from sections imaged 250–500 microns anterior to the pyloric ring in the posterior midgut, and MetaMorph software (Molecular Devices, Downingtown, PA) was used to quantify GFP+ cells. The average number of GFP+ cells was obtained from at least 25 guts per treatment; means were compared using one-way ANOVA followed by Tukey’s HSD post-test. Due to the smaller size of male posterior midgut, the field of view (FOV) was divided into 9 equal sections, and the central region was used to quantify the average number of GFP+ cells. To quantify pHH3+ cells, images were acquired using a 20X objective 1–2 fields of view anterior to the pylorus. The numbers of pHH3+ cells were normalized to gut area for each gut (n>21 samples per treatment). Statistical significance was determined with a Kruskall-Wallis test followed by a Dunn’s post hoc test.
Analysis of Intestinal Integrity
Quantification of intestinal integrity was based upon the distribution of a blue food dye (FD&C blue dye #1) post-feeding. Briefly, two vials of female flies of each genotype were transferred onto fresh medium containing blue dye (2.5% w/v) at 9 a.m. for 150 min. Flies showing an extended blue coloration (not limited to the proboscis and crop) were considered ‘Smurf’ flies.
Statistical analysis
Unless indicated otherwise, significance was determined using a two-tailed, unpaired t-test from at least three independent experiments and expressed as P values. Unless indicated otherwise, error bars reflect standard error of the mean (SEM).
Highlights
  • Up-regulation of dPGC-1 increases mitochondrial gene expression and activity
  • dPGC-1 modulates fuel homeostasis in the adult fly
  • dPGC-1 protects against the age-related loss of intestinal homeostasis and integrity
  • Up-regulation of dPGC-1 in the digestive tract extends lifespan
Supplementary Material
Acknowledgments
The authors would like to thank Kevin Vu, Kent Vu, Holly Vu and Jeff Copeland for help with fly work and generation of UAS-constructs. We also thank H. Keshishian, S. Pletcher, L. Seroude, H. Jasper and the Drosophila Stock Center (Bloomington) for fly stocks. D.L.J. is funded by the Emerald Foundation, the G. Harold and Leila Y. Mathers Charitable Foundation, the ACS, the California Institute for Regenerative Medicine (CIRM), and the NIH (R01 AG028092). C.K. was funded by an NIH Developmental Biology training grant (2T32HD007495, C. Kintner). M.U. is supported by a Ruth L. Kirschstein National Research Service Award (GM07185). D.W.W is funded by the National Institute on Aging (RO1 AG037514) and the Ellison Medical Foundation. D.W.W is an Ellison Medical Foundation New Scholar in Aging.
Footnotes
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