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• W2017360512 abstract "Article12 November 2009free access The Drosophila PGC-1 homologue Spargel coordinates mitochondrial activity to insulin signalling Stefanie K Tiefenböck Stefanie K Tiefenböck ETH Zurich, Department of Biology, Zurich, Switzerland PhD Program in Molecular Life Sciences, Zurich, Switzerland Search for more papers by this author Claudia Baltzer Claudia Baltzer ETH Zurich, Department of Biology, Zurich, Switzerland PhD Program in Molecular Life Sciences, Zurich, Switzerland Search for more papers by this author Nicole A Egli Nicole A Egli ETH Zurich, Department of Biology, Zurich, SwitzerlandPresent address: [email protected]Search for more papers by this author Christian Frei Corresponding Author Christian Frei ETH Zurich, Department of Biology, Zurich, Switzerland Search for more papers by this author Stefanie K Tiefenböck Stefanie K Tiefenböck ETH Zurich, Department of Biology, Zurich, Switzerland PhD Program in Molecular Life Sciences, Zurich, Switzerland Search for more papers by this author Claudia Baltzer Claudia Baltzer ETH Zurich, Department of Biology, Zurich, Switzerland PhD Program in Molecular Life Sciences, Zurich, Switzerland Search for more papers by this author Nicole A Egli Nicole A Egli ETH Zurich, Department of Biology, Zurich, SwitzerlandPresent address: [email protected]Search for more papers by this author Christian Frei Corresponding Author Christian Frei ETH Zurich, Department of Biology, Zurich, Switzerland Search for more papers by this author Author Information Stefanie K Tiefenböck1,2, Claudia Baltzer1,2, Nicole A Egli1 and Christian Frei 1 1ETH Zurich, Department of Biology, Zurich, Switzerland 2PhD Program in Molecular Life Sciences, Zurich, Switzerland *Corresponding author. ETH Zurich, Department of Biology, HPM F38.2, 8093 Zurich, Switzerland. Tel.: +41 44 633 3394; Fax: +41 44 633 1069; E-mail: [email protected] The EMBO Journal (2010)29:171-183https://doi.org/10.1038/emboj.2009.330 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info 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 and PRC regulate multiple metabolic functions, including mitochondrial biogenesis. However, we know relatively little about their respective roles in vivo. Here we show 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 mediated mitochondrial activity, cell growth and transcription of target genes in response to insulin signalling. In this process, Spargel functioned in parallel to the insulin-responsive transcription factor, dFoxo, and provided a negative feedback loop to fine-tune insulin signalling. Taken together, our data place Spargel at a nodal point for the integration of mitochondrial activity to tissue and organismal metabolism and growth. Introduction 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 et al, 1998), as well as its homologues PGC-1β (Kamei et al, 2003) and PRC (PGC-1-related coactivator; Andersson and Scarpulla, 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 et al, 2004; Leone et al, 2005; Lelliott et al, 2006; Sonoda et al, 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 et al, 2006). Similarly, mice lacking both PGC-1α and PGC-1β showed defective mitochondrial biogenesis in the heart and the BAT (Lai et al, 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. 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 and Spiegelman, 2003; Scarpulla, 2008). Accordingly, NRF-1 and ERRα are known to be functionally important for PGC-1s to stimulate mitochondrial mass (Wu et al, 1999; Mootha et al, 2004; Schreiber et al, 2004). Similarly, NRF-2 promoter-binding sites were required for coactivation by PGC-1α and PRC in certain genes (Gleyzer et al, 2005), and PRC can coactivate NRF-2β (Vercauteren et al, 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. In flies and mammals, insulin signalling and TOR (target of rapamycin) function are known to link cellular growth and metabolism to nutrition (Grewal, 2008; Polak and Hall, 2009). Recent data showed that mammalian TOR (mTOR) and mitochondrial oxidative capacity are tightly linked (Schieke et al, 2006), 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 et al, 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 (Zinke et al, 2002; Gershman et al, 2007; Teleman et al, 2008). 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 (Guertin et al, 2006). Therefore, mechanisms in addition to TOR must exist to adapt mitochondrial mass and function to nutrient availability. We investigated the role of the Drosophila melanogaster PGC-1 homologue Spargel/CG9809 in the control of mitochondrial biogenesis and activity. The Drosophila genome encodes a single PGC-1 homologue (Gershman et al, 2007), thus providing a system in which PGC-1 function can be analysed without interfering redundancy. We focused on the larval fat body, the functional equivalent of the mammalian adipose tissue and liver (Baker and Thummel, 2007; Leopold and Perrimon, 2007). In this tissue, many genes encoding mitochondrial proteins were expressed in a nutrient-sensitive manner (Teleman et al, 2008; Baltzer et al, 2009). The larval fat body is, therefore, ideal to study how mitochondrial mass and activity are coordinated with cellular metabolism and nutrient supply. Here, we show 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 et al, 2009), Spargel and Delg were shown to function in two different pathways, each regulating mitochondrial in response to nutrients. Finally, we addressed the question how insulin signalling affected mitochondria, and found that Spargel is 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. Our data demonstrate a critical role for Spargel in the coordination of mitochondria with nutrients, and thus in cellular metabolism. Results The Drosophila PGC-1 homologue Spargel is required for growth The D. melanogaster genome encodes a single PGC-1 homologue, CG9809 (Gershman et al, 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 et al, 2007), yet its cellular function has not been addressed. To test whether CG9809 is a functional PGC-1 homologue in flies, we analysed mutants that have a P-element insertion (KG08646) in the 5′ UTR (Figure 1A). In comparison with controls (precise excision of the P-element), homozygous mutant larvae had a strong reduction in CG9809 mRNA levels (Figure 1B), adults were viable, and females were sterile. Both males and females had a 25% reduction in wet weight (Figure 1C and Supplementary Figure S1F), which correlated with reduced protein, lipid, glycogen and trehalose levels per animal (Supplementary Figure S1G). When normalized to body weight, reduced lipid and glycogen levels were observed in adult males, demonstrating metabolic defects (Supplementary Figure S1H). When external structures that are derived from imaginal discs were analysed, such as wings or legs, we did not observe large size defects in CG9809 mutants (Supplementary Figure S1A–E). These animals have, therefore, a lean phenotype, prompting us to term CG9809 ‘Spargel’, German for ‘asparagus’, and the KG08646 allele as srl1. To test the specificity of this allele, we used a second P-element insertion (d04518, termed srl2), which showed the same phenotypes as srl1 (Figure 1B and C), and created transgenic flies carrying a genomic rescue construct (SrlGR; Figure 1A), which suppressed all mutant phenotypes (Figure 1D and data not shown). As another gene, CG31525, is located within the first intron of Spargel, we also created a fly line expressing a Spargel cDNA under the control of the UAS promoter. When driven using heat-shock Gal4, UAS–Srl also suppressed the mutant phenotypes (Figure 1E and data not shown), 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 (Figure 1B), yet it did not lead to a further decrease in adult weight compared with srl1 homozygous mutant animals (Figure 1C). Taken together, srl1 is a strong hypomorphic allele, showing a ∼75% reduction in mRNA levels. Figure 1.Mutants of Spargel (CG9809), the Drosophila homologue to mammalian PGC-1 transcriptional coactivators, show growth defects. (A) Representation of the Spargel genomic locus. Black boxes represent coding regions of the respective genes (CG9809, CG31525 or eIF3-S10). Grey boxes indicate 5′ and 3′ untranslated regions of Spargel. The P-element insertion sites of srl1 and srl2 loss-of-function alleles are indicated as white triangles. The genomic rescue construct (SrlGR; 8700 bp) is represented as a hatched bar. (B) Spargel transcript levels determined by qRT–PCR. mRNA was isolated from whole, mid-third instar larvae: +/+: 4dAED; srl1/1, srl2/2 and srl1/Df(3R)5046: 5dAED. Expression was normalized to Actin5C (CG4027) and +/+ was set to 1. (C) Wet weight from adult males, genotypes as indicated. (D) Complete rescue of the reduced weight phenotype of adult srl1/1 mutants by the genomic rescue construct, SrlGR, or with (E) hs-Gal4 driven UAS-Spargel (UAS-Srl) with one 1.5-h heat shock (37°C) per day. For (E), no rescue is seen in the absence of heat shocks (data not shown). (C–E) For all weight measurements, adult males were taken 2 days after eclosion. Number of flies per genotype ⩾10. Download figure Download PowerPoint Drosophila Spargel is required for proper expression of multiple genes encoding mitochondrial proteins To address a likely Spargel function as a transcriptional regulator, we performed genome-wide microarray analysis using dissected fat bodies. Genes encoding mitochondrial proteins are highly expressed during the larval feeding period but are repressed at the end of the last (third) instar, as the animals stop feeding and prepare for pupation (White et al, 1999; Arbeitman et al, 2002). Whereas control larvae enter the third instar 72 h after egg deposition (AED) and pupate 120 h AED, spargel mutants enter with a 12–14-h delay, and pupate with a 24-h delay (144 h AED; see Supplementary Figure S2). To avoid artefacts due to different developmental stages, mid-third instar larvae were taken for all experiments (96 h AED for control, and 120 h AED for spargel). As summarized in Table I, GO annotation showed that genes involved in several mitochondrial functions, in particular oxidative phosphorylation (OXPHOS; mostly electron transport complexes I, II and V), were expressed at reduced levels in the spargel mutant. In total, 44% of all nuclear genes encoding mitochondrial proteins were >1.5-fold downregulated in the spargel mutant fat body. In addition, few non-mitochondrial functions were downregulated, including translation, gene expression and RNA biology, suggesting that Spargel functions as a transcriptional regulator. As mammalian PGC-1 proteins are best characterized in respect to mitochondrial biogenesis, we focused on Spargel's role in respect to mitochondria. Table 1. GO term enrichment for genes that were significantly up- or downregulated (>1.5-fold) in the srl1/1 mutant fat body compared with wild-type GO ID P-value Term GO Biological Process, >1.5 × down in srl1/1 mutant fat body (P<0.05) Mitochondrial biogenesis and function GO:0022900 3.45E−25 Electron (e−) transport GO:0006119 8.14E−24 Oxidative phosphorylation GO:0042773 4.67E−23 ATP synthesis coupled e− transport GO:0006091 6.81E−22 Generation of precursor metabolites and energy GO:0006120 2.18E−14 Mitochondrial e− transport, NADH to ubiquinone GO:0006839 1.80E−03 Mitochondrial transport GO:0007005 1.57E−02 Mitochondrial organization GO:0006123 2.03E−02 Mitochondrial e− transport, cytochrome c to oxygen Transcription and translation GO:0006412 5.83E−49 Translation GO:0010467 2.91E−33 Gene expression GO:0019538 5.82E−15 Protein metabolic process GO:0042254 1.52E−30 Ribosomal biogenesis and assembly GO:0016072 1.27E−18 rRNA metabolic process GO:0006457 6.45E−03 Protein folding GO:0055086 1.04E−04 Nucleobase, nucleoside and nucleotide metabolic process Cell cycle GO:0051231 1.15E−18 Spindle elongation GO:0007052 4.69E−12 Mitotic spindle organization and biogenesis GO:0000226 3.93E−06 Microtubule cytoskeleton organization/biogenesis GO:0000278 3.98E−05 Mitotic cell cycle Cellular metabolic processes GO:0008152 4.31E−36 Metabolic process GO:0044249 3.04E−18 Cellular biosynthetic process GO:0009059 2.08E−17 Macromolecule biosynthetic process GO:0044267 5.42E−15 Cellular protein metabolic process GO:0006996 2.67E−06 Organelle organization and biogenesis GO Biological Process, >1.5 × up in srl1/1 mutant fat body (P<0.05) Developmental process GO:0007594 5.55E−03 Puparial adhesion GO:0007591 4.60E−02 Molting cycle, chitin-based cuticle Lipid metabolism GO:0016042 5.74E−05 Lipid catabolic process GO:0006635 9.40E−04 Fatty acid β-oxidation GO:0044242 1.44E−04 Cellular lipid catabolic process Others GO:0006810 1.03E−03 Transport GO:0051179 3.82E−03 Localization GO terms that are significantly enriched (P-value<0.05) are shown. First, we verified our microarray data by quantifying mRNA levels of selected genes using qRT–PCR on dissected larval fat bodies: mtACP1, (mitochondrial acyl carrier protein of electron transport complex I), Scs-fp (succinyl CoA synthetase flavoprotein subunit of complex II), RFeSP (Rieske iron-sulfur protein of complex III), CoVa (subunit Va of complex IV), Bellwether (Blw; ATP synthase alpha subunit of complex V), Isocitrate dehydrogenase (Idh; tricarboxylic acid (TCA) cycle), and Cyt-c-p (encoding cytochrome c) were all expressed at significantly lower levels in spargel mutants, but not Glutamate dehydrogenase (GDH; amino-acid metabolism; Figure 2A). As explained above, we allowed spargel mutant larvae to grow for additional 24 h before dissection compared with controls. To test whether this shift in developmental timing would influence our results, we isolated fat body-specific mRNA from spargel mutants at the same timing as controls (96 h AED). Again RFeSP and Idh levels were strongly reduced compared with control (data not shown), demonstrating that the effects seen were not due to differential timing. Figure 2.Spargel and the NRF-2α homologue Delg share many putative target genes. (A) Larval fat bodies were dissected and mRNA levels of the genes indicated were determined using qRT–PCR (at least three biological replicates). Mid-third instar larvae were taken: 4d AED for +/+, 5d AED for spargel, 6d AED for delg and 8d AED for spargel–delg double mutant. Transcript levels were normalized to gammaTubulin23C (gTub, CG3157). In all cases, delg−/− refers to delg613/Df(3R)ro80b. Significance is indicated as compared with the control sample. (B) Comparison of microarray data from srl1/1 and delg−/− single mutants using mRNA from fat bodies dissected from mid-L3 larvae. Overlap of all nuclear-encoded mitochondrial genes that are downregulated >1.5 ×. Processes that are regulated in a Spargel- or Delg-specific manner, respectively, as well as overlapping gene sets are indicated below. Download figure Download PowerPoint Spargel and the NRF-2α homologue Delg share many putative target genes Mammalian PGC-1 proteins drive gene expression by coactivating multiple transcription factors, including NRF-2 (Puigserver and Spiegelman, 2003; Scarpulla, 2008). Drosophila Delg is the structural and functional homologue to mammalian NRF-2α, and multiple genes encoding mitochondrial proteins are expressed at lower levels in delg mutants (Baltzer et al, 2009). We therefore used Drosophila Spargel and Delg as a system to study the functional interaction of PGC-1 proteins with NRF-2. First, we compared our fat body-specific microarray data of spargel with delg single mutants (Baltzer et al, 2009) in more detail (Figure 2B and Supplementary Table S1). When focusing on genes encoding mitochondrial proteins that are downregulated in either the spargel or the delg mutant, about half (88 genes) overlapped, including many OXPHOS and TCA cycle genes. In contrast, 46 genes were downregulated in the spargel, but not in the delg mutant. These genes function in electron transport (complex I), DNA and RNA metabolism and mitochondrial protein synthesis and targeting. In addition, 27 genes were affected in a Delg-specific manner, including genes required for amino-acid and fatty-acid metabolism (Figure 2B and Supplementary Table S1). This demonstrates that Spargel and Delg share many putative target genes, but also affect transcription independently of each other. To study the Spargel–Delg interaction in more detail, we dissected mid-third instar larval fat bodies from delg single and spargel–delg double mutants, and measured transcript levels of the putative Spargel target genes using qRT–PCR. All genes tested above were also expressed in a Delg-dependent manner. Importantly, these genes did not show a further decrease in the spargel–delg double mutant when compared with single mutants (Figure 2A). We conclude that Spargel and Delg have a common role in the expression of many genes encoding mitochondrial proteins, possibly through Spargel-mediated coactivation of Delg. At the same time, either factor is required for expression levels of a subset of these genes independently of the other. Spargel and Delg function in parallel pathways in respect to mitochondrial mass To test whether the reduced expression of genes encoding mitochondrial proteins would result in reduced mitochondrial abundance, we used MitoTracker, a mitochondrial-specific dye. In the larval fat body of control animals, mitochondrial staining was abundant throughout the cytoplasm. In spargel mutants, we detected no reduction in staining (Figure 3A and B). On the contrary, delg mutants showed a strong reduction in mitochondrial staining, in which residual mitochondria are concentrated around the nucleus (Baltzer et al, 2009). When quantified, delg mutants had a 24% decrease in MitoTracker staining (Figure 3B). Importantly, spargel–delg double mutants had a more severe phenotype compared with the delg single mutant, in which only few mitochondria are stained per cell. On average, these cells had a 56% reduction in MitoTracker staining compared with control or spargel single mutant cells (Figure 3B). To complement the MitoTracker stainings, we used NAO that specifically labels the mitochondrial phospholipid cardiolipin. Again we observed reduced mitochondrial abundance in the delg, but not the spargel mutant, and a more severe phenotype in the double mutant (Supplementary Figure S3A). To assess mitochondrial morphology, we used electron microscopy. Single mutants of Spargel or Delg did not show obvious morphological defects. In contrast, spargel–delg double mutants were rounded in shape, and inner-mitochondrial cristae were either missing or strongly reduced in size (Figure 3C and Supplementary Figure S3B). These data demonstrate that Spargel is not required for mitochondrial morphology and abundance under normal conditions, but becomes limiting in the absence of Delg. In addition, we observed enlarged lipid droplets in spargel mutant fat body cells. This phenotype has been described previously in studies on mobilization of lipid stores in response to nutrient starvation (Zhang et al, 2000; Colombani et al, 2003). Accordingly, GO annotations of microarray data demonstrated that genes involved in fatty-acid oxidation were increased in spargel mutants (Table I). Yet, larval lipid content was not altered (data not shown), thus further experiments are required to address a direct role for Spargel in this process. Figure 3.Spargel is required for mitochondrial respiration and functions in parallel to Delg. (A) Mitochondria-specific MitoTracker stainings of larval fat bodies, 5d AED. DAPI staining is shown in insets. Bar equals 20 μm. (B) Quantification of (A). Images were quantified in blind using ImageJ, n⩾20. (C) Electron microscopy images of larval fat bodies from mid-third instar larvae. Bar equals 400 nm. (D) Respiration assays using dissected, digitonin-permeabilized fat bodies. For complex I stimulation, serial addition of pyruvate and proline (state 2), ADP (state 3), atractyloside (state 4) and KCN was used. For complex II stimulation, rotenone was added to inhibit complex I, and respiration was stimulated by succinate (state 2). (E) Respiration assay as in (D), but KCN values were subtracted (see Supplementary Figure S3C for the same chart without subtraction). For (D) and (E), oxygen consumption was normalized to fat body protein content. Averages and s.d. values of three biological replicates are shown. (F) Mitochondrial DNA (mtDNA) content was quantified by qRT–PCR and normalized to nuclear DNA (nDNA) levels from dissected fat bodies (six biological replicates). (G) Mitochondrial-encoded COX level (subunit I) was determined by qRT–PCR from RNA isolated from dissected fat bodies (three biological replicates). For experiments in (C–G), mid-third instar larvae were taken as in Figure 2. (H) Shown are larvae 4d AED. Bar equals 1 mm. In all cases, delg−/− refers to delg613/Df(3R)ro80b. Significance is indicated as compared with the control sample. Download figure Download PowerPoint Respiration defects in spargel mutant fat bodies Mitochondria are best known to generate energy from nutrients through oxidative phosphorylation. During this process, NADH and FADH2, which are derived from the mitochondrial TCA cycle and fatty-acid oxidation, stimulate the electron transport chain at complex I (NADH dehydrogenase) and II (succinate dehydrogenase), respectively. Electrons are transferred through complex III (cytochrome bc1) to complex IV (cytochrome c oxidase), which consumes oxygen as the final electron acceptor. This generates a proton gradient across the inner-mitochondrial membrane, which is dissipated either at complex V, producing ATP, or through uncoupling proteins. To measure oxidative phosphorylation capacity, we had previously adapted an assay using dissected and digitonin-permeabilized fat bodies (Baltzer et al, 2009). Strikingly, we observed severe respiration defects in spargel mutants when complex II was stimulated: oxygen consumption was strongly reduced after the addition of the substrate succinate (state 2), ADP stimulation (state 3) and the addition of atractyloside (an ADP/ATP transporter inhibitor; state 4). In contrast, when oxygen consumption was measured in the presence of cyanide, a potent complex IV inhibitor, we did not observe a difference between spargel mutants and controls, demonstrating that non-mitochondrial oxygen consumption was not affected (Figure 3D). This correlates with mammalian data that showed reduced complex II-mediated respiration in tissues from PGC-1α- or PGC-1β-knockout mice (Leone et al, 2005; Lelliott et al, 2006). Next, we repeated these assays using pyruvate and proline, as well as glutamate and malate as substrates, which all stimulate complex I of the electron transport chain. Surprisingly, we did not detect any differences in respiration between spargel mutants and control fat bodies (Figure 3D and data not shown). We conclude that complex II must be particularly affected in the spargel mutant. Accordingly, genes encoding three of the four complex II subunits were expressed at lower levels in spargel mutants (Supplementary Table S1). Nonetheless, these results are surprising given the reduced expression of other electron transport complexes. However, these data are similar to those from delg mutants, which showed comparable low expression levels of many OXPHOS genes, yet did not have respiration defects when complex I was stimulated (Baltzer et al, 2009). As postulated before, post-transcriptional mechanisms might exist to compensate for reduced expression rates (Baltzer et al, 2009). Alternatively, factors that are rate-limiting for complex I activity might not be affected in spargel or delg single mutants. Given the strong synergistic functions of Spargel and Delg in respect to mitochondrial morphology and abundance, we anticipated respiration defects in the spargel–delg double mutant, which are not seen in either of the single mutant. Indeed, compared with heterozygous controls, respiration was strongly defective when complex I was stimulated using pyruvate and proline (Figure 3E). A similar strong defect was also observed when complex II was stimulated (data not shown). As an additional control, we re-expressed Spargel using the genomic rescue construct in the double mutant background. This led to suppression of the phenotype (Figure 3E). Importantly, not all genes involved in electron transport required Spargel for proper expression. Of the Spargel-independent genes, some showed lower expression in the delg mutant (Supplementary Table S1). The synergistic respiration defects of the double mutant, compared with single mutants, can therefore be explained by a more complete downregulation of electron transport genes. To further characterize OXPHOS activity, we quantified the mitochondrial DNA (mtDNA), which encodes several factors required for electron transport, and levels of which correlate with OXPHOS activity (Rocher et al, 2008). When normalized to nuclear DNA, we did not detect any change in the spargel single mutant, but increased levels were observed in delg single and spargel-delg double mutants (Figure 3F; Baltzer et al, 2009). Importantly, this did not lead to enhanced mtDNA transcription, as we detected reduced transcript levels of mitochondria-encoded COX subunit I (Figure 3G). Given the general correlation between mtDNA replication and transcription in mammalian cells, this seems surprising. However, a similar discrepancy has been observed recently on downregulation of mitochondrial transcription factor" @default.
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• W2017360512 title "The Drosophila PGC-1 homologue Spargel coordinates mitochondrial activity to insulin signalling" @default.
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• W2017360512 doi "https://doi.org/10.1038/emboj.2009.330" @default.
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