InteractiveFly: GeneBrief

Spargel: Biological Overview | References


Gene name - spargel

Synonyms -

Cytological map position - 82B1-82B1

Function - transcription factor

Keywords - fat body; mediation of mitochondrial activity, cell growth and transcription of target genes in response to insulin signalling

Symbol - srl

FlyBase ID: FBgn0037248

Genetic map position - 3R: 248,205..255,051 [-]

Classification - RRM RNA-binding motif

Cellular location - nuclear



NCBI links: Precomputed BLAST | EntrezGene

Recent literature
Merzetti, E.M. and Staveley, B.E. (2015). spargel, the PGC-1α homologue, in models of Parkinson disease in Drosophila melanogaster. BMC Neurosci 16: 70. PubMed ID: 26502946
Summary:
Parkinson disease (PD) is a progressive neurodegenerative disorder presenting with symptoms of resting tremor, bradykinesia, rigidity, postural instability and additional severe cognitive impairment over time. These symptoms arise from a decrease of available dopamine in the striatum of the brain resulting from the breakdown and death of dopaminergic (DA) neurons. A process implicated in the destruction of these neurons is mitochondrial breakdown and impairment. Upkeep and repair of mitochondria involves a number of complex and key components including Pink1, Parkin, and the PGC family of genes. PGC-1α has been characterized as a regulator of mitochondria biogenesis, insulin receptor signalling and energy metabolism, mutation of this gene has been linked to early onset forms of PD. The mammalian PGC family consists of three partially redundant genes making the study of full or partial loss of function difficult. The sole Drosophila melanogaster homologue of this gene family, spargel (srl), has been shown to function in similar pathways of mitochondrial upkeep and biogenesis. This study shows that the directed expression of srl-RNAi in the D. melanogaster eye causes abnormal ommatidia and bristle formation while eye specific expression of srl-EY does not produce the minor rough eye phenotype associated with high temperature GMR-Gal4 expression. Ddc-Gal4 mediated tissue specific expression of srl transgene constructs in D. melanogaster DA neurons causes altered lifespan and climbing ability. Expression of a srl-RNAi causes an increase in mean lifespan but a decrease in overall loco-motor ability while induced expression of srl-EY causes a severe decrease in mean lifespan and a decrease in loco-motor ability. The reduced lifespan and climbing ability associated with a tissue specific expression of srl in DA neurons provides a new model of PD in D. melanogaster which may be used to identify novel therapeutic approaches to human disease treatment and prevention.

Merzetti, E. M. and Staveley, B. E. (2015). spargel, the PGC-1alpha homologue, in models of Parkinson disease in Drosophila melanogaster. BMC Neurosci 16: 70. PubMed ID: 26502946
Summary:
Parkinson disease (PD) is a progressive neurodegenerative disorder that arises from a decrease of available dopamine in the striatum of the brain resulting from the breakdown and death of dopaminergic (DA) neurons. PGC-1alpha has been characterized as a regulator of mitochondria biogenesis, insulin receptor signalling and energy metabolism, mutation of this gene has been linked to early onset forms of PD. The sole Drosophila homologue of this gene family, spargel (srl), has been shown to function in similar pathways of mitochondrial upkeep and biogenesis. Directed expression of srl-RNAi in the Drosophila eye causes abnormal ommatidia and bristle formation while eye specific expression of srl-EY does not produce the minor rough eye phenotype associated with high temperature GMR-Gal4 expression. Ddc-Gal4 mediated tissue specific expression of srl transgene constructs in Drosophila DA neurons causes altered lifespan and climbing ability. Expression of a srl-RNAi causes an increase in mean lifespan but a decrease in overall loco-motor ability while induced expression of srl-EY causes a severe decrease in mean lifespan and a decrease in loco-motor ability. The reduced lifespan and climbing ability associated with a tissue specific expression of srl in DA neurons provides a new model of PD in D. melanogaster which may be used to identify novel therapeutic approaches to human disease treatment and prevention.
Ng, C. H., Basil, A. H., Hang, L., Tan, R., Goh, K. L., O'Neill, S., Zhang, X., Yu, F. and Lim, K. L. (2017). Genetic or pharmacological activation of the Drosophila PGC-1alpha ortholog spargel rescues the disease phenotypes of genetic models of Parkinson's disease. Neurobiol Aging 55: 33-37. PubMed ID: 28407521
Summary:
Despite intensive research, the etiology of Parkinson's disease (PD) remains poorly understood and the disease remains incurable. However, compelling evidence gathered over decades of research strongly support a role for mitochondrial dysfunction in PD pathogenesis. Related to this, PGC-1&alpha, a key regulator of mitochondrial biogenesis, has recently been proposed to be an attractive target for intervention in PD. This study shows that silencing of expression of the Drosophila PGC-1α ortholog spargel results in PD-related phenotypes in flies and also seems to negate the effects of AMPK activation, which has been previously demonstrated to be neuroprotective; that is, AMPK-mediated neuroprotection appears to require PGC-1alpha. Importantly, this study further showed that genetic or pharmacological activation of the Drosophila PGC-1alpha ortholog spargel is sufficient to rescue the disease phenotypes of Parkin and LRRK2 genetic fly models of PD, thus supporting the proposed use of PGC-1alpha-related strategies for neuroprotection in PD.

BIOLOGICAL OVERVIEW

Mitochondrial mass and activity must be adapted to tissue function, cellular growth and nutrient availability. In mammals, the related transcriptional coactivators PGC-1α, PGC-1β, (Peroxisome proliferator-activated receptor gamma coactivator 1-α and -β) and PRC (PGC-1-related coactivator) regulate multiple metabolic functions, including mitochondrial biogenesis. However, relatively little is known about their respective roles in vivo. This study shows that the Drosophila PGC-1 family homologue, Spargel, is required for the expression of multiple genes encoding mitochondrial proteins. Accordingly, spargel mutants showed mitochondrial respiration defects when complex II of the electron transport chain was stimulated. Spargel, however, was not limiting for mitochondrial mass, but functioned in this respect redundantly with Delg, the fly NRF-2α/GABPα homologue. More importantly, in the larval fat body, Spargel mediates mitochondrial activity, cell growth and transcription of target genes in response to insulin signalling. In this process, Spargel functions in parallel to the insulin-responsive transcription factor, dFoxo, and provides a negative feedback loop to fine-tune insulin signalling. Taken together, these data place Spargel at a nodal point for the integration of mitochondrial activity to tissue and organismal metabolism and growth (Tiefenböck, 2010).

As animals grow, nutrients are taken up, leading to an increase in cellular mass. In this process, mitochondria have critical anabolic and catabolic functions in metabolizing nutrients and in adapting cellular physiology. Therefore, one would expect coordination of mitochondrial mass and activity with growth-promoting pathways and nutrient availability, yet this remains poorly understood. To study mitochondrial biogenesis, most studies focused on individual tissues that show an enormous increase in mitochondrial mass in response to external stimuli, for example, during the formation of the brown adipose tissue (BAT) or perinatal heart maturation. Such studies led to identification and characterization of the PGC-1 family of transcriptional coactivators, which are potent inducers of mitochondrial biogenesis: the founding member PGC-1α (PPARγ coactivator 1; Puigserver, 1998), as well as its homologues PGC-1β (Kamei, 2003) and PRC (PGC-1-related coactivator; Andersson, 2001). Mice lacking PGC-1α or PGC-1β showed reduced expression of multiple genes encoding mitochondrial proteins, yet mitochondrial mass and respiration activity were either not or only modestly reduced, depending on the tissue (Lin, 2004; Leone, 2005; Lelliott, 2006; Sonoda, 2007). These mild phenotypes could be because of redundancy. Indeed, RNAi-mediated downregulation of PGC-1β in a PGC-1α-/- background led to strong additive respiration defects in adipocytes (Uldry, 2006). Similarly, mice lacking both PGC-1α and PGC-1β showed defective mitochondrial biogenesis in the heart and the BAT (Lai, 2008). Although these studies clearly demonstrated critical functions for these proteins in mitochondria-rich tissues, a triple knockout of PGC-1α, PGC-1β and PRC has not been published; therefore it remains unclear whether PGC-1s are generally required for basal mitochondrial mass (Tiefenböck, 2010).

PGC-1 family members drive mitochondrial biogenesis through coactivation of nuclear transcription factors, including nuclear respiratory factor-1 and -2 (NRF-1 and NRF-2), estrogen-related receptor-α (ERRα) and YY1, to enhance the expression of genes encoding mitochondrial proteins (Puigserver, 2003; Scarpulla, 2008). Accordingly, NRF-1 and ERRα are known to be functionally important for PGC-1s to stimulate mitochondrial mass (Wu, 1999; Mootha, 2004; Schreiber, 2004). Similarly, NRF-2 promoter-binding sites were required for coactivation by PGC-1α and PRC in certain genes (Gleyzer, 2005), and PRC can coactivate NRF-2β (Vercauteren, 2008). However, it is not known whether NRF-2 is required for PGC-1's effect on mitochondrial function, or whether NRF-2 is controlled through other factors (Tiefenböck, 2010).

In flies and mammals, insulin signalling and TOR (target of rapamycin) function are known to link cellular growth and metabolism to nutrition. Recent data showed that mammalian TOR (mTOR) and mitochondrial oxidative capacity are tightly linked, and mTOR inhibition reduced the association of PGC-1α with the transcription factor YY1, leading to lower expression of several genes encoding mitochondrial proteins (Cunningham, 2007). These data demonstrate that mTOR is a crucial regulator of mitochondrial function in mammals. In flies, nutrient starvation and subsequent inhibition of insulin signalling led to reduced expression of multiple genes encoding mitochondrial proteins. However, a direct role of TOR has not been addressed in these studies. Moreover, the majority of genes encoding mitochondrial proteins were not responsive to TOR inhibition in Drosophila cells. Therefore, mechanisms in addition to TOR must exist to adapt mitochondrial mass and function to nutrient availability (Tiefenböck, 2010).

This study investigated the role of the Drosophila PGC-1 homologue Spargel/CG9809 in the control of mitochondrial biogenesis and activity. The Drosophila genome encodes a single PGC-1 homologue (Gershman, 2007), thus providing a system in which PGC-1 function can be analysed without interfering redundancy. Focused was placed on the larval fat body, the functional equivalent of the mammalian adipose tissue and liver. In this tissue, many genes encoding mitochondrial proteins were expressed in a nutrient-sensitive manner (Teleman, 2008; Baltzer, 2009). The larval fat body is, therefore, ideal to study how mitochondrial mass and activity are coordinated with cellular metabolism and nutrient supply. This study shows that Spargel is required for proper expression of most genes encoding mitochondrial proteins. When complex II of the electron transport chain is stimulated, spargel mutants show respiration defects. Remarkably, Spargel is not required for basal mitochondrial mass, but becomes limiting in the absence of Delg, the fly NRF-2α homologue. Moreover, from these and previous experiments (Baltzer, 2009), Spargel and Delg were shown to function in two different pathways, each regulating mitochondrial in response to nutrients. Finally, the question how insulin signalling affected mitochondria was addressed; Spargel was found to be required for the stimulation of mitochondrial respiration, and to a large extent for the transcriptional control mediated by insulin signalling, including that of genes encoding mitochondrial proteins. Moreover, insulin signalling induces Spargel gene expression and protein levels, and Spargel mediates a negative feedback loop on insulin signalling. These data demonstrate a critical role for Spargel in the coordination of mitochondria with nutrients, and thus in cellular metabolism (Tiefenböck, 2010).

The D. melanogaster genome encodes a single PGC-1 homologue, CG9809 (Gershman, 2007). Sequence alignments have shown that the N-terminal acidic domain that mediates transcriptional coactivation for mammalian PGC-1s, and the C-terminal arginine-serine-rich and RNA recognition domains are highly conserved in the fly protein (Gershman, 2007), yet its cellular function has not been addressed. To test whether CG9809 is a functional PGC-1 homologue in flies, mutants that have a P-element insertion (KG08646) in the 5' UTR were examined. In comparison with controls (precise excision of the P-element), homozygous mutant larvae had a strong reduction in CG9809 mRNA levels, adults were viable, and females were sterile. Both males and females had a 25% reduction in wet weight, which correlated with reduced protein, lipid, glycogen and trehalose levels per animal. When normalized to body weight, reduced lipid and glycogen levels were observed in adult males, demonstrating metabolic defects. When external structures that are derived from imaginal discs were analysed, such as wings or legs, no large size defects were detected in CG9809 mutants. These animals have, therefore, a lean phenotype, prompting use of the name 'Spargel' German for 'asparagus', and the KG08646 allele as srl1. To test the specificity of this allele, a second P-element insertion (d04518, termed srl2), was tested; this showed the same phenotypes as srl1 and transgenic flies were created carrying a genomic rescue construct, which suppressed all mutant phenotypes. As another gene, CG31525, is located within the first intron of Spargel, a fly line was created expressing a Spargel cDNA under the control of the UAS promoter. When driven using heat-shock Gal4, UAS-Srl also suppressed the mutant phenotypes, demonstrating that loss of Spargel function was responsible for the observed phenotypes. Finally, a trans-heterozygous combination of srl1 with Df(3R)ED5046, a deficiency that deletes the Spargel locus, led to a further reduction in Spargel transcript levels, yet it did not lead to a further decrease in adult weight compared with srl1 homozygous mutant animals. Taken together, srl1 is a strong hypomorphic allele, showing a ~75% reduction in mRNA levels (Tiefenböck, 2010).

Mitochondrial mass and activity must adapt to cellular growth rates and nutrient availability, yet factors involved were poorly described. This study showed that Drosophila Spargel is critical for proper expression of genes encoding mitochondrial proteins, and that it mediates a link to the nutrient-sensitive insulin signalling pathway. In the larval fat body, Spargel was required for proper mitochondrial respiration when complex II was stimulated, both under normal and insulin signalling stimulated conditions. These data support the interpretation that the control of mitochondria represents an ancestral function of the PGC-1 proteins family. Importantly, it was shown that Spargel is not a master regulator of mitochondrial biogenesis, but becomes limiting in this respect in the absence of Delg. Furthermore, ectopic expression of Spargel was not sufficient to drive mitochondrial abundance, which contrasts mammalian data, where PGC-1's are potent stimulators of mitochondrial mass. Mammalian PGC-1 proteins are induced by external stimuli, including cold exposure in BAT or exercise in muscle tissues, thus adapting the cellular physiology in response to such stimuli (Puigserver, 2003). To test a similar function for Drosophila Spargel, larvae were exposed to cold shocks, and respiration rates from dissected fat bodies were measured by complex I stimulation. Although cold exposure led to reduced respiration, this response was not altered in the spargel mutant. Thus this function is not conserved in flies, which correlates with previous findings that the Drosophila fat body resembles the mammalian white adipose tissue, which is not involved in thermoregulation (Baltzer, 2009). Furthermore, genes involved in gluconeogenesis, β-oxidation and lipogenesis, all functions linked to mammalian PGC-1 proteins (Lin, 2005), were not reduced in the spargel mutant, at least not in the larval fat body. No altered lipid levels were detected in spargel mutant larvae, thus these functions might be vertebrate-specific. Potentially, these differences can be explained by the finding that Spargel lacks the canonical LXXLL motifs, which mediate binding to multiple transcription factors for mammalian PGC-1s (Gershman, 2007). Spargel however contains a conserved C-terminal FXXLL motif (Gershman, 2007), which could mediate transcription factor binding, yet such interactors remain elusive (Tiefenböck, 2010).

In mammalian cells, overexpression of PGC-1α led to increased expression of NRF-2α/GABPα (Mootha, 2004), and NRF-2 binding sites in the promoters of mitochondrial transcription factors TFB1M and TFB2M were required for coactivation by PGC-1α and PRC (Gleyzer, 2005). Similarly, PRC was shown to coactivate NRF-2β-dependent transcription (Vercauteren, 2008), thus it was assumed that PGC-1 proteins and NRF-2 would function in the same pathway. Very importantly, these studies focused on individual genes, but not on mitochondrial function and mass, thus it is still unclear whether NRF-2 is functionally required for PGC-1 proteins in respect to mitochondrial mass. Although Drosophila Spargel and Delg may share many putative target genes, these factors function in parallel pathways in respect to mitochondrial mass, morphology and OXPHOS activity (Tiefenböck, 2010).

The respiration defects in spargel single mutants can be explained by reduced expression of genes involved in oxidative phosphorylation. Spargel mutants however did not show a reduction in mitochondrial abundance, which appears surprising. In wild-type larvae, the fat body does not attract tracheoles for gas exchange, and shows physiological low oxygen levels, suggesting low rates of mitochondrial respiration. Indeed, compared to other larval tissues, a low inner-mitochondrial membrane potential and reduced oxygen consumption was detected (Baltzer, 2009). This suggests that oxidative phosphorylation might not be a critical function of the fat body mitochondria. Rather, this tissue releases lipids and amino acids, in particular proline and glutamine, providing energy sources for other tissues. Since proline and glutamine are synthesized from the mitochondrial TCA cycle intermediate 2-oxoglutarate, it is proposed that such a function is rate limiting for mitochondrial mass in the fat body. Delg but not Spargel was required for proper expression of genes involved in proline and glutamine metabolism (Baltzer, 2009), thus explaining the mitochondrial abundance defect in the delg mutant, and the absence of such defects in the spargel mutant (Tiefenböck, 2010).

Spargel and Delg are distinct in respect to upstream signalling: Spargel is functionally required for insulin signalling, which was not seen for Delg. In contrast, Delg, but not Spargel, is required for Cyclin D/Cdk4 to stimulate mitochondrial abundance (Baltzer, 2009). Thus the data provided genetic evidence that Spargel and Delg represent two different pathways, each regulating mitochondria in response to nutrients. Although several recent reports have demonstrated a functional link between insulin signalling and PGC-1 proteins in mammalian cells, the interaction appears to be complex, and is most likely tissue and/or context dependent: Some studies showed that PGC-1 proteins were required for insulin signalling (Vianna, 2006; Pagel-Langenickel, 2008), whereas other studies found an inhibitory function for PGC-1α (Koo, 2004; Choi, 2008). In flies, the insulin signalling pathway is well conserved, and since Spargel is the only PGC-1 protein, Drosophila is an ideal organism to study a functional interaction between insulin signalling and PGC-1 proteins in vivo (Tiefenböck, 2010).

Spargel is required for insulin signalling stimulated growth, functioning downstream of Dp110, but presumably independently of Akt, and balances signalling activity through a negative-feedback loop. Very remarkably, Spargel mediates ~40% of the transcriptional control in response to insulin signalling, emphasizing a major function in the control of cellular growth and metabolism. Moreover, these data suggest that Spargel functions independently of dFoxo, therefore representing a novel transcriptional output of insulin signalling in the larval fat body. This appears to contradict recent microarray data, which showed repressed Spargel mRNA levels upon expression of an activated form of dFoxo in culture Drosophila cells (Gershman, 2007). The discrepancy could be due to tissue-inherent differences. In this study, INR expression led to an increase in Spargel mRNA and protein abundance, further suggesting that Spargel has a direct role within the insulin signalling pathway. Future experiments are required to establish a molecular mechanism how Spargel function and subcellular localization is mediated by insulin signalling components. Analogous to the current results, reduced PGC-1α protein levels were observed upon knockdown of the insulin receptor InsRβ in mammalian cells (Pagel-Langenickel, 2008), suggesting a conserved mechanism between flies and mammals. Thus the data are further genetic evidence for a functional interaction between PGC-1 proteins and insulin signalling. Although flies and mammals are several hundred millions apart in evolution, Spargel and its mammalian counterparts PGC-1α, PGC1-1β and PRC have conserved functions in respect to mitochondria and insulin signalling. In the future, this will allow use of Drosophila as a powerful system to understand regulatory circuitries that control homeostasis of cellular metabolism (Tiefenböck, 2010).

Increased mitochondrial biogenesis preserves intestinal stem cell homeostasis and contributes to longevity in Indy mutant flies

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. 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. 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).

High-resolution dynamics of the transcriptional response to nutrition in Drosophila: a key role for dFOXO

A high-resolution time series of transcript abundance was generated to describe global expression dynamics in response to nutrition in Drosophila. Nonparametric change-point statistics revealed that within 7 h of feeding upon yeast, transcript levels changed significantly for approximately 3,500 genes or 20% of the Drosophila genome. Differences as small as 15% were highly significant, and 80% of the changes were <1.5-fold. Notably, transcript changes reflected rapid downregulation of the nutrient-sensing insulin and target of rapamycin pathways, shifting of fuel metabolism from lipid to glucose oxidation, and increased purine synthesis, TCA-biosynthetic functions and mitochondria biogenesis. To investigate how nutrition coordinates these transcriptional changes, feeding-induced expression changes were compared with those induced by the insulin-regulated transcription factor dFOXO in Drosophila S2 cells. Remarkably, 28% (995) of the nutrient-responsive genes were regulated by activated dFOXO, including genes of mitochondrial biogenesis and a novel homolog of mammalian peroxisome proliferator-gamma coactivator-1 (PGC-1), a transcriptional coactivator implicated in controlling mitochondrial gene expression in mammals. These data implicate dFOXO as a major coordinator of the transcriptional response to nutrients downstream of insulin and suggest that mitochondria biogenesis is linked to insulin signaling via dFOXO-mediated repression of a PGC-1 homolog (Gershman, 2007).

Given the change in mRNA encoding proteins involved in intermediary metabolism upon refeeding, transcriptional changes were expected in genes underlying mitochondrial function. Indeed, many genes whose products directly participate in mitochondrial substrate transport and ATP synthesis were coordinately upregulated. These included at least 13 genes encoding proteins with functions in electron transport and thirteen substrate carriers. Indeed, of 232 genes assigned Gene Ontology (GO) functions associated with mitochondria, 54% changed with refeeding, and 91% of these were increased (Gershman, 2007).

Most strikingly, there was a marked and rapid increase in transcripts encoding genes with roles in mitochondrial biogenesis, such as membrane preprotein translocases, chaperones, mitochondrial DNA binding proteins, and nuclear encoded mitochondrial ribosome proteins. Of 60 nuclear encoded mitochondrial ribosomal genes on the array, expression of 54 was increased an average of 44% at 5-7 h after refeeding. The induction of mitochondrial ribosomal proteins contrasts with the relative stasis of transcripts encoding proteins of the cytoplasmic ribosome. Of the 39 cytoplasmic ribosome genes represented on the array, mRNA for seven were moderately increased and two were reduced. An expansion of mitochondria within cells may reflect de novo adipogenesis in adult fat body when females first feed on yeast, similar to what is observed in mammalian 3T3-L1 cells that increase mitochondrial density as they differentiate into adipocytes. The observed increase in mitochondrial gene expression may also reflect activity in skeletal muscle cells, given that nutrients have been shown to regulate expression of mitochondrial oxidative phosphorylation genes in mammalian muscle (Gershman, 2007).

How nutrients regulate mitochondrial capacity is poorly understood. Many nutrient-induced transcripts encoding proteins of mitochondria biogenesis were broadly repressed in cells expressing dFOXO-A3. Of the 54 mRNAs for mitochondrial ribosome proteins that increased upon refeeding, 42 were downregulated by dFOXO-A3. Likewise, of the 25 upregulated genes with functions in mitochondrial biogenesis, 14 were repressed in dFOXO-A3 cells. In contrast, only six of the 53 nutrient-regulated, nuclear-encoded genes for mitochondrial function were inversely regulated by dFOXO-A3. Therefore, insulin, presumably via dFOXO, has a strong and selective impact on genes associated with mitochondrial biogenesis (Gershman, 2007).

One candidate to mediate mitochondrial biogenesis is PGC-1. PGC-1α of mammals stimulates mitochondrial biogenesis in muscle, brown adipose tissue, and adipogenic 3T3-L1 cells. Interestingly, a mRNA of the single ortholog of a PGC-like peptide in D. melanogaster (CG9809, Spargel) increases upon refeeding, and declines in dFOXO-A3 cells. CG9809 encodes a predicted protein of 1,088 amino acids that exhibits 68 or 52% homology with mammalian PGC-1α or PGC-1ß, respectively, in their COOH-terminal RNA-binding motif. Other domains in common with its mammalian homologs include an arginine-serine-rich domain located NH2-terminal to the RNA-binding motif, an acidic NH2-terminal domain and leucine-rich motifs, although the canonical LXXLL nuclear receptor binding motif is not present. However, the LXXLL motif is not absolutely required for coactivator binding, and CG9809 does contain a variant of this motif near its COOH terminus, FXXLL, which has been reported to function in nuclear receptor binding. Notably, this sequence is conserved in all mammalian PGC-1 family members. Beside a parallel based on sequence, orthologous targets of PGC-1 were elevated in refed flies and were reduced by dFOXO-A3 in S2 cells, including mitochondrial transcription factor A. Consistent with these data, insulin has been reported to upregulate PGC-1α and PGC-1ß mRNA levels in human muscle, although the mechanism was unclear. These data suggest that insulin signaling may be a conserved regulator of normal mitochondrial biogenesis via FOXO-mediated control of PGC-1-like cofactors. Importantly, this suggests a mechanistic basis for the prevalent mitochondrial dysfunction in humans with insulin resistance and diabetes (Gershman, 2007).


REFERENCES

Search PubMed for articles about Drosophila Spargel

Andersson, U. and Scarpulla, R. C. (2001). Pgc-1-related coactivator, a novel, serum-inducible coactivator of nuclear respiratory factor 1-dependent transcription in mammalian cells. Mol. Cell. Biol. 21: 3738-3749. PubMed ID: 11340167

Baltzer, C., Tiefenbock, S. K., Marti, M. and Frei, C. (2009). Nutrition controls mitochondrial biogenesis in the Drosophila adipose tissue through Delg and cyclin D/Cdk4. PLoS One 4: e6935. PubMed ID: 19742324

Choi, C. S., et al. (2008). Paradoxical effects of increased expression of PGC-1alpha on muscle mitochondrial function and insulin-stimulated muscle glucose metabolism. Proc. Natl. Acad. Sci. 105: 19926-19931. PubMed ID: 19066218

Cunningham, J. T., et al. (2007). mTOR controls mitochondrial oxidative function through a YY1-PGC-1alpha transcriptional complex. Nature 450: 736-740. PubMed ID: 18046414

Gershman, B., et al. (2007). High-resolution dynamics of the transcriptional response to nutrition in Drosophila: a key role for dFOXO. Physiol. Genomics 29: 24-34. PubMed ID: 17090700

Gleyzer, N., Vercauteren, K. and Scarpulla, R. C. (2005). Control of mitochondrial transcription specificity factors (TFB1M and TFB2M) by nuclear respiratory factors (NRF-1 and NRF-2) and PGC-1 family coactivators. Mol. Cell. Biol. 25: 1354-1366. PubMed ID: 15684387

Kamei, Y., et al. (2003). PPARgamma coactivator 1beta/ERR ligand 1 is an ERR protein ligand, whose expression induces a high-energy expenditure and antagonizes obesity. Proc. Natl. Acad. Sci. 100: 12378-12383. PubMed ID: 14530391

Koo, S. H., et al. (2004). PGC-1 promotes insulin resistance in liver through PPAR-alpha-dependent induction of TRB-3. Nat. Med. 10: 530-534. PubMed ID: 15107844

Lai, L., et al. (2008). Transcriptional coactivators PGC-1alpha and PGC-lbeta control overlapping programs required for perinatal maturation of the heart. Genes Dev. 22: 1948-1961. PubMed ID: 18628400

Lelliott, C. J., et al. (2006). Ablation of PGC-1beta results in defective mitochondrial activity, thermogenesis, hepatic function, and cardiac performance. PLoS Biol. 4: e369. PubMed ID: 17090215

Leone, T. C., et al. (2005). PGC-1alpha deficiency causes multi-system energy metabolic derangements: muscle dysfunction, abnormal weight control and hepatic steatosis. PLoS Biol 3: e101. PubMed ID: 15760270

Lin, J., Handschin, C. and Spiegelman, B. M. (2005). Metabolic control through the PGC-1 family of transcription coactivators. Cell Metab 1: 361-370. PubMed ID: 16054085

Mootha, V. K., et al. (2004). Erralpha and Gabpa/b specify PGC-1alpha-dependent oxidative phosphorylation gene expression that is altered in diabetic muscle. Proc. Natl. Acad. Sci. 101: 6570-6575. PubMed ID: 15100410

Pagel-Langenickel, I., et al. (2008). PGC-1alpha integrates insulin signaling, mitochondrial regulation, and bioenergetic function in skeletal muscle. J. Biol. Chem. 283: 22464-22472. PubMed ID: 18579525

Puigserver, P., et al. (1998). A cold-inducible coactivator of nuclear receptors linked to adaptive thermogenesis. Cell 92: 829-839. PubMed ID: 9529258

Puigserver, P. and Spiegelman, B. M. (2003). Peroxisome proliferator-activated receptor-gamma coactivator 1 alpha (PGC-1 alpha): transcriptional coactivator and metabolic regulator. Endocr. Rev. 24: 78-90. PubMed ID: 12588810

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

Scarpulla, R. C. (2008). Transcriptional paradigms in mammalian mitochondrial biogenesis and function. Physiol. Rev. 88: 611-638. PubMed ID: 18391175

Schreiber, S. N., et al. (2004). The estrogen-related receptor alpha (ERRalpha) functions in PPARgamma coactivator 1alpha (PGC-1alpha)-induced mitochondrial biogenesis. Proc. Natl. Acad. Sci. 101: 6472-6477. PubMed ID: 15087503

Sonoda, J., et al. (2007). PGC-1beta controls mitochondrial metabolism to modulate circadian activity, adaptive thermogenesis, and hepatic steatosis. Proc. Natl. Acad. Sci. 104: 5223-5228. PubMed ID: 17360356

Teleman, A. A., Hietakangas, V., Sayadian, A. C. and Cohen, S. M. (2008). Nutritional control of protein biosynthetic capacity by insulin via Myc in Drosophila. Cell. Metab. 7: 21-32. PubMed ID: 18177722

Tiefenböck, S. K., Baltzer, C., Egli, N. A. and Frei, C. (2010). The Drosophila PGC-1 homologue Spargel coordinates mitochondrial activity to insulin signalling. EMBO J. 29(1): 171-83. PubMed ID: 19910925

Uldry, M., et al. (2006). Complementary action of the PGC-1 coactivators in mitochondrial biogenesis and brown fat differentiation. Cell. Metab. 3: 333-341. PubMed ID: 16679291

Vercauteren, K., Gleyzer, N. and Scarpulla, R. C. (2008). PGC-1-related coactivator complexes with HCF-1 and NRF-2beta in mediating NRF-2(GABP)-dependent respiratory gene expression. J. Biol. Chem. 283: 12102-12111. PubMed ID: 18343819

Vianna, C. R., et al. (2006). Hypomorphic mutation of PGC-1beta causes mitochondrial dysfunction and liver insulin resistance. Cell. Metab. 4: 453-464. PubMed ID: 17141629

Wu, Z., et al. (1999). Mechanisms controlling mitochondrial biogenesis and respiration through the thermogenic coactivator PGC-1. Cell 98: 115-124. PubMed ID: 10412986


Biological Overview

date revised: 2 March 2016

Home page: The Interactive Fly © 2009 Thomas Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.