Schlank, a member of the ceramide synthase family controls growth and body fat in Drosophila

Article15 October 2009free access Schlank, a member of the ceramide synthase family controls growth and body fat in Drosophila Reinhard Bauer Corresponding Author Reinhard Bauer LIMES-Institute, Program Unit Development, Genetics & Molecular Physiology, Laboratory for Molecular Developmental Biology, University of Bonn, Bonn, Germany Search for more papers by this author André Voelzmann André Voelzmann LIMES-Institute, Program Unit Development, Genetics & Molecular Physiology, Laboratory for Molecular Developmental Biology, University of Bonn, Bonn, Germany Search for more papers by this author Bernadette Breiden Bernadette Breiden LIMES-Institute, Program Unit Membrane Biology & Lipid Biochemistry, c/o Kekulé Institute of Organic Chemistry and Biochemistry, University of Bonn, Bonn, Germany Search for more papers by this author Ute Schepers Ute Schepers LIMES-Institute, Program Unit Membrane Biology & Lipid Biochemistry, c/o Kekulé Institute of Organic Chemistry and Biochemistry, University of Bonn, Bonn, Germany Search for more papers by this author Hany Farwanah Hany Farwanah LIMES-Institute, Program Unit Membrane Biology & Lipid Biochemistry, c/o Kekulé Institute of Organic Chemistry and Biochemistry, University of Bonn, Bonn, Germany Search for more papers by this author Ines Hahn Ines Hahn LIMES-Institute, Program Unit Development, Genetics & Molecular Physiology, Laboratory for Molecular Developmental Biology, University of Bonn, Bonn, Germany Search for more papers by this author Franka Eckardt Franka Eckardt LIMES-Institute, Program Unit Development, Genetics & Molecular Physiology, Laboratory for Molecular Developmental Biology, University of Bonn, Bonn, Germany Search for more papers by this author Konrad Sandhoff Konrad Sandhoff LIMES-Institute, Program Unit Membrane Biology & Lipid Biochemistry, c/o Kekulé Institute of Organic Chemistry and Biochemistry, University of Bonn, Bonn, Germany Search for more papers by this author Michael Hoch Corresponding Author Michael Hoch LIMES-Institute, Program Unit Development, Genetics & Molecular Physiology, Laboratory for Molecular Developmental Biology, University of Bonn, Bonn, Germany Search for more papers by this author Reinhard Bauer Corresponding Author Reinhard Bauer LIMES-Institute, Program Unit Development, Genetics & Molecular Physiology, Laboratory for Molecular Developmental Biology, University of Bonn, Bonn, Germany Search for more papers by this author André Voelzmann André Voelzmann LIMES-Institute, Program Unit Development, Genetics & Molecular Physiology, Laboratory for Molecular Developmental Biology, University of Bonn, Bonn, Germany Search for more papers by this author Bernadette Breiden Bernadette Breiden LIMES-Institute, Program Unit Membrane Biology & Lipid Biochemistry, c/o Kekulé Institute of Organic Chemistry and Biochemistry, University of Bonn, Bonn, Germany Search for more papers by this author Ute Schepers Ute Schepers LIMES-Institute, Program Unit Membrane Biology & Lipid Biochemistry, c/o Kekulé Institute of Organic Chemistry and Biochemistry, University of Bonn, Bonn, Germany Search for more papers by this author Hany Farwanah Hany Farwanah LIMES-Institute, Program Unit Membrane Biology & Lipid Biochemistry, c/o Kekulé Institute of Organic Chemistry and Biochemistry, University of Bonn, Bonn, Germany Search for more papers by this author Ines Hahn Ines Hahn LIMES-Institute, Program Unit Development, Genetics & Molecular Physiology, Laboratory for Molecular Developmental Biology, University of Bonn, Bonn, Germany Search for more papers by this author Franka Eckardt Franka Eckardt LIMES-Institute, Program Unit Development, Genetics & Molecular Physiology, Laboratory for Molecular Developmental Biology, University of Bonn, Bonn, Germany Search for more papers by this author Konrad Sandhoff Konrad Sandhoff LIMES-Institute, Program Unit Membrane Biology & Lipid Biochemistry, c/o Kekulé Institute of Organic Chemistry and Biochemistry, University of Bonn, Bonn, Germany Search for more papers by this author Michael Hoch Corresponding Author Michael Hoch LIMES-Institute, Program Unit Development, Genetics & Molecular Physiology, Laboratory for Molecular Developmental Biology, University of Bonn, Bonn, Germany Search for more papers by this author Author Information Reinhard Bauer 1, André Voelzmann1, Bernadette Breiden2, Ute Schepers2, Hany Farwanah2, Ines Hahn1, Franka Eckardt1, Konrad Sandhoff2 and Michael Hoch 1 1LIMES-Institute, Program Unit Development, Genetics & Molecular Physiology, Laboratory for Molecular Developmental Biology, University of Bonn, Bonn, Germany 2LIMES-Institute, Program Unit Membrane Biology & Lipid Biochemistry, c/o Kekulé Institute of Organic Chemistry and Biochemistry, University of Bonn, Bonn, Germany *Corresponding authors. Program Unit Development, Genetics & Molecular Physiology, Laboratory for Molecular Developmental Biology, LIMES Institute, University of Bonn, Meckenheimer Allee 169, Bonn NRW 53115, Germany. Tel.: +49 228 73 6859; Fax: +49 228 73 4480; E-mail: [email protected] or Tel.: +49 228 73 4621; Fax: +49 228 73 4480; E-mail: [email protected] The EMBO Journal (2009)28:3706-3716https://doi.org/10.1038/emboj.2009.305 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Ceramide synthases are highly conserved transmembrane proteins involved in the biosynthesis of sphingolipids, which are essential structural components of eukaryotic membranes and can act as second messengers regulating tissue homeostasis. However, the role of these enzymes in development is poorly understood due to the lack of animal models. We identified schlank as a new Drosophila member of the ceramide synthase family. We demonstrate that schlank is involved in the de novo synthesis of a broad range of ceramides, the key metabolites of sphingolipid biosynthesis. Unexpectedly, schlank mutants also show reduction of storage fat, which is deposited as triacylglyerols in the fat body. We found that schlank can positively regulate fatty acid synthesis by promoting the expression of sterol-responsive element-binding protein (SREBP) and SREBP-target genes. It further prevents lipolysis by downregulating the expression of triacylglycerol lipase. Our results identify schlank as a new regulator of the balance between lipogenesis and lipolysis in Drosophila. Furthermore, our studies of schlank and the mammalian Lass2 family member suggest a novel role for ceramide synthases in regulating body fat metabolism. Introduction In all animals energy homeostasis is under tight control of evolutionarily conserved nutrient-sensing systems. These include the target of rapamycin (TOR) pathway (Martin and Hall, 2005) and several families of secreted peptide hormones, which regulate and fine-balance carbohydrate and lipid metabolism to match energy requirements. Particularly during growth, a coordinated regulation of the lipid metabolism is important on both the cellular and organismal level. On the cellular level, sterols in mammalian cells or phosphatidylethanolamine, the major phospholipid in Drosophila, control the release of sterol-regulatory element-binding protein (SREBP) from cell membranes, exerting feedback control on the synthesis of fatty acids (FAs) and phospholipids (Dobrosotskaya et al, 2002; Seegmiller et al, 2002; Kunte et al, 2006). SREBPs are membrane-bound transcription factors that monitor cell membrane composition and adjust lipid synthesis accordingly. The de novo synthesis of sphingolipids is also pivotal since sphingolipids are essential structural components of eukaryotic membranes and play important roles as second messengers regulating apoptosis, survival and differentiation (Spiegel and Milstien, 2000; Hannun et al, 2001; Acharya and Acharya, 2005). Misregulation of the sphingolipid metabolism is involved in the aetiology and pathology of a number of human diseases, including neurodegeneration, cancer, immunity, cystic fibrosis, emphysema, diabetes and sepsis (Kolter and Sandhoff, 2006; Lahiri and Futerman, 2007). The enzymes of the sphingolipid pathway are conserved in all genetically studied eukaryotes (Jiang et al, 1998; Hannun et al, 2001; Acharya and Acharya, 2005). However, in vivo information on how these enzymes are regulated in response to growth requirements or during starvation, is either limited by early lethality of the knockout animals reflecting the fundamental necessity of these enzymes (Li et al, 2002; Hojjati et al, 2005; Mizugishi et al, 2005) or by the lack of mutants. The latter includes mutants for key enzymes of the pathway such as ceramide synthases, which produce the precursor metabolites for all sphingolipids. Mammals contain six family members of the ceramide synthase family, also called Lass (longevity assurance homologue-1 of yeast Lag1) proteins. Their function has mainly been studied in tissue culture cells since knockout models are not available yet, and it was shown that they control the synthesis of different ceramides (for reviews see Pewzner-Jung et al, 2006; Teufel et al, 2009). On the organismal level, energy homeostasis depends on the ability to control the balance between lipid synthesis, storage and lipid mobilization during conditions of energy abundance or deprivation, respectively (Hay and Sonenberg, 2004; Zechner et al, 2005). Storage fat is deposited as triacylglycerols (TAGs), in intracellular lipid droplets (Martin and Parton, 2006), which accumulate in specialized organs such as mammalian adipose tissue or the fat body of flies (Canavoso et al, 2001; Rosen and Spiegelman, 2006). Lipolysis of TAGs is induced by lipases and leads to release of free FAs or sn-1,2-diacylglycerol in mammals and insects, respectively, into the circulatory system (Arrese and Wells, 1997; Gibbons et al, 2000; Patel et al, 2005). Chronic dysregulation of the balance between lipolysis and lipogenesis may lead to metabolic abnormalities such as obesity, lipodystrophy syndromes or insulin resistance in humans (Kahn et al, 2006; Simha and Garg, 2006). We have identified a new regulator of growth and lipid homeostasis in Drosophila, the schlank gene. We show that schlank encodes a Drosophila member of the Lass/ceramide synthase family required for de novo synthesis of ceramides. Unexpectedly, schlank is also involved in the regulation of body fat metabolism and we show that it regulates the balance between lipogenesis and lipolysis during larval growth. Results Identification and molecular characterization of schlank Wild-type larvae hatch after embryogenesis and pass through three larval instar stages until puparium formation, which occurs at about 96–120 h after hatching. During larval development, the animals increase their body size by about 200-fold. In a search for genes controlling larval growth in Drosophila, we screened the Göttingen P-element collection (Peter et al, 2002) and identified four P-element insertion lines affecting a single gene locus on the X-chromosome, which we had previously named Drosophila longevitiy assurance gene-1 homologue (DLag1) due to its sequence homology to the yeast LAG1 ceramide synthase (for review see D'mello et al, 1994; Teufel et al, 2009). Since we now show that the P-element insertions into the DLag1 locus cause defects in larval growth and fat metabolism (see below), we propose to rename the gene schlank ('slim' in German) following the Drosophila nomenclature in which genes are named according to the phenotype of the respective mutants. The schlank gene locus maps to the X-chromosome (Supplementary Figure S1A). Hemizygous schlank mutants show a delay of larval development and pronounced growth defects, which depend on the strength of the alleles (Figure 1A–C). Mutants carrying the stronger schlankG0349 allele fail to grow in the larval stages, although they feed as determined by feeding assays. After about 3days, these animals die as small larvae, which morphologically correspond in size to first instar larvae (Figure 1A–C). In contrast, hemizygous animals carrying the weaker schlankG0061 allele are developmentally delayed; however, they reach the third instar larval stage and a fraction thereof even pupariates. Quantitative real-time PCR (qRT–PCR) experiments reveal that the level of schlank transcripts is reduced to less than 40% in hemizygous schlankG0061 larvae and to less than 10% in hemizygous schlankG0349 larvae as compared with control animals (Figure 1C). However, using an antibody against the C-terminus of the schlank protein (Supplementary Figure S4), we found that the level of schlank protein is only reduced by about 20% in the schlankG0061 and by 40% in the schlankG0349 mutants (Figure 1D), indicating a maternal supply of the gene products and/or an enhanced stability of the protein. Consistently, we found that schlank mRNA and protein are highly abundant in oocytes and during early embryogenesis (Supplementary Figure S5A and B). In order to generate a 'null' situation, we largely eliminated both schlank mRNA and protein expression by generating germline clones using the schlankG0349 allele. When both the zygotic and maternal contributions of schlank are largely eliminated, we did not obtain any eggs. This demonstrates that schlank has both a maternal and a zygotic supply and explains the residual mRNA and protein activity in schlankG0349-mutant animals. Furthermore, this is consistent with a fundamental function of schlank in the lipid metabolism (see below) and unfortunately impedes to work with complete schlank-null animals in Drosophila. Figure 1.Schlank is essential for larval growth. (A) Larval growth of schlankG0061 (61) mutants compared with wild-type w1118 larvae (control) at late L3 stage. schlankG0349 (349) hatch as first instar larvae and die after about 3 days as morphological first instar larvae. Food intake in mutants was controlled by feeding red-coloured yeast. (B) Average length of control [w1118] (n=123, n=81, n=48), schlankG0061 (n=54, n=187, n=63) and schlankG0349 (n=14, n=106) larvae after 24–25, 48–49 and 72–73 h after egg laying. (C) Reduced schlank mRNA expression in schlank mutants as compared with that in w1118 controls. (D) Determination of residual schlank protein (predicted size of about 46 kDa) in schlank mutants; reduction to 80% in schlankG0061 and 60% in schlankG349 mutants as compared with that in wild-type controls (100%; w1118) using schlank antibody (Supplementary Figure S4). An actin antibody was used to determine the loading control. (E,F) schlank knockdown using schlank RNAi (UASschlankRNAi) in combination with the daughterless-GAL4 (daGAL4) driver line phenocopies the schlank larval growth phenotype (E). Quantification of mRNA levels by qRT–PCR in panel F. Control: daGAL4∷w1118. Asterisks in panel B indicate significant differences to the wild type (P<0.001). (B–G) Error bars indicate s.e.m. Download figure Download PowerPoint To further provide evidence that the lethality and the growth phenotypes of the schlank P-alleles are caused by downregulation of the schlank gene, we used RNAi-mediated knockdown. Expression of a UASschlankRNAi transgene in combination with the ubiquitous daughterless-GAL4 (daGAL4) driver line phenocopied the growth defects observed in the schlank-mutant alleles (Figure 1E and F). Furthermore, transgenic flies carrying the full-length schlank cDNA were able to rescue schlankG0349-mutant animals to the third larval instar stage and some of the animals even to the pupal stage (Supplementary data and Supplementary Figure S1C). Together, molecular analysis of the schlank alleles, reversion of the phenotype by perfect excision of the P-elements (Supplementary data from Fly strains, and data not shown), RNAi-mediated knockdown and genetic rescue experiments demonstrate that the lethality and the growth phenotype of the schlank alleles are linked to the schlank gene function. Schlank encodes the Drosophila homologue of the Lass/ceramide synthase family The schlank gene locus codes for two transcripts, which differ in their 5′ UTR; however, they encode the same open reading frame (ORF; Supplementary Figure S1A). The schlank ORF codes for a transmembrane protein with high homology to ceramide synthases and a phylogenetic analysis indicates that schlank is closely related to the mammalian Lass family members both in sequence and protein domain structure (Figure 2A; Supplementary Table and Supplementary Figure 1B). Figure 2.Schlank is involved in de novo ceramide synthesis. (A) Phylogenetic tree of Lass family members and TRAM proteins. Protein sequences were derived from the Ensembl database. If more than one protein variant existed, the version with all domain features was used (for accession numbers see Supplementary Table SI). Sequences were aligned using EMBL-EBI ClustalW2 online service using standard settings. The alignment file was used to generate 100 bootstrapped data sets with seqboot (PHYLIP 3.68 package; see also Felsenstein, 1989). The output was analysed using maximum likelihood (proml, PHYLIP package). An unrooted consensus tree was generated with consense (PHYLIP package). Bootstrap values are marked above each branch. The ceramide synthase family is highlighted in red and the TRAM family in yellow. Note that schlank falls into the Lass family and CG11642 into the TRAM family. (B) Biosynthesis of ceramides is significantly reduced in first instar schlankG0061 and schlankG0349 larvae as compared with that in w1118 wild-type controls. Sphingolipids were labelled by feeding larvae with radiolabelled L-[3-14C]-serine for 12 h. After lipid extraction equal amounts of radioactivity were applied to TLC plates, developed with chloroform/ methanol/ glacial acetic acid (190:9:1) and quantified. The total ceramide content in schlankG0061- and in schlankG0349-mutant larvae was reduced to 89 and 60%, respectively, as compared with that in the wild-type (100%). (C) The dose-dependent increase in the expression of immunoreactive schlankHA product correlates with an increase in ceramide synthase activity. Different amounts of eluted fractions after binding to a HA affinity matrix were used for immunoblotting with an HA antibody or determination of ceramide synthase activity (see also Supplementary data). Download figure Download PowerPoint Ceramide synthases use long-chain bases, sphinganine or sphingosine, and FA-CoAs with varying chain length to produce (dihydro)ceramide, which is a precursor metabolite for all sphingolipids. Sphingolipids are structural components of most cellular membranes and can act as signalling molecules in cell growth, differentiation and apoptosis (Spiegel and Milstien, 2000; Hannun et al, 2001; Acharya and Acharya, 2005). Lass family members contain four to seven predicted transmembrane domains (Venkataraman and Futerman, 2002), a catalytic Lag1 motif and most an N-terminal domain showing sequence homology to DNA-binding homeodomains (Hox domain) (Gehring et al, 1994; Venkataraman and Futerman, 2002). Structure predictions indicate that the putative schlank protein contains six transmembrane domains, a Lag1 motif and a Hox domain, which are highly conserved (Supplementary Figures 1B and 2C). The Lag1 motif of Lass proteins, which are found in organisms ranging from yeast to mammals, is functionally required for ceramide synthesis and is contained within a stretch of 52 amino acids (Pewzner-Jung et al, 2006; Spassieva et al, 2006). The Lag1 motif of schlank is highly conserved and shows sequence identity to the Lag1 consensus motif of 82.3% (Supplementary Figure S2A). Furthermore, it contains all of the conserved amino acids that were shown to be crucial for the catalytic function of Lag1 domains in ceramide synthesis (Spassieva et al, 2006 and Supplementary Figure S2A). In addition, schlank contains a putative homeodomain, which is also found in most vertebrate Lass proteins (Supplementary Figures S1B and S2C), but not in yeast, worms and plants (Venkataraman and Futerman, 2002). The function of Lass homeodomains is unknown. In addition to schlank, a second gene (CG11642) with some homology to Lass/ceramide synthase family members was identified in the Drosophila genome by sequence similarity searches (Acharya and Acharya, 2005). However, the sequence similarity of the putative Lag1 motif of the CG11642 gene product is much lower as compared with schlank, and it is of note that most of the amino acids, which were shown to be crucial for the function of the Lag1 motif in ceramide synthesis (Spassieva et al, 2006), are not conserved and changed in the CG11642 gene product (Supplementary Figure S2B). Rather, the gene product of CG11642 seems more homologous to members of the translocating chain-associated membrane (TRAM) protein family (Figure 2A), which are not involved in ceramide synthesis (Jiang et al, 1998; Winter and Ponting, 2002; Spassieva et al, 2006). This is further supported by RNAi-mediated knockdown of CG11642 showing no phenotype in ceramide synthesis (Supplementary Figure S3B). Together, these data suggest that schlank may encode the only ceramide synthase family member in Drosophila. Schlank is involved in de novo ceramide synthesis in Drosophila Since schlank appears to be a member of the Lass/ceramide synthase family, we first tested whether schlank is involved in ceramide metabolism and found that the total ceramide content in schlankG0061 and in schlankG0349-mutant larvae was significantly reduced as compared with wild-type animals (Figure 2B). To specifically address whether de novo synthesis of ceramides is affected in the mutant animals, we fed schlankG0061 and schlankG0349-mutant larvae for 12 h with radiolabelled L-[3-14C]-serine, a precursor of sphingolipid biosynthesis, and we analysed the incorporation of this label into de novo generated ceramide. schlankG0061 and schlankG0349 mutants as well, as the wild-type controls, incorporated the same amount of total radioactivity per dry weight (schlankG0349 19 882 cpm/mg, schlankG0061 18 930 cpm/mg and wild-type controls 20 685 cpm/mg; s.e.m.±5%). After the extraction of lipids, ceramides were separated by thin-layer chromatography (TLC) and identified and quantified using commercially available reference standards (Supplementary Figure S3A and Supplementary data). We found in our metabolic labelling studies that the levels of de novo generated ceramides were significantly decreased in schlankG0061 (69.25%) and schlankG0349 (39%) mutants as compared with wild-type controls (100%; Figure 2B), in line with the reduced schlank protein levels in the mutants (Figure 1D). To further establish a role of schlank in the de novo synthesis of ceramides, we generated transgenic flies expressing a C-terminally HA-tagged, full-length schlank protein (UASschlankHA). We induced the tagged protein in larvae using a heat shock-GAL4 (hsGAL4) driver line, prepared extracts to purify it partially (Materials and methods) and performed standard in vitro ceramide synthase assays (Wang and Merrill, 1999). We found that increasing amounts of purified schlank led to a dose-dependent increase in ceramide synthase activity and increased ceramide levels (Figure 2C). Manipulation of schlank activity in loss- and gain-of-function experiments To further support a role of schlank as ceramide synthase, we reduced or elevated its activity in loss- and gain-of-function experiments and analysed the effects on ceramide levels. To this end, we fed radiolabelled L-[3-14C]-serine to larvae carrying transgenic UASschlankRNAi or UASschlankHA in combination with the hsGAL4 driver line. Upon a short heat shock (1 h) to induce schlank RNAi knockdown or schlank overexpression, we observed a decrease or increase of ceramide levels, respectively (Figure 3A and B). Most interestingly, overexpression of the murine Lass2 homologue for which ceramide synthase activity has been shown previously (Mizutani et al, 2005), resulted in a similar increase in ceramide levels (Figure 3A and B). In contrast, an increase in ceramide levels could not be observed when we overexpressed a schlank protein variant, schlankH215D (Supplementary Figure S3B), which contains a point mutation in the Lag1 motif shown to inhibit ceramide synthase function in Lass1 and 5 (Spassieva et al, 2006; change of a highly conserved histidine at position 215 into glutamate; Supplementary Figure S2A). Figure 3.Modulation of schlank activity correlates with the rate of ceramide de novo synthesis (A, B) Larvae carrying hsGAL4 and either UASschlankRNAi (schankRNAi), UASschlankHA (schlankHA) or UASlass2HA (lass2HA) were heat shocked for 1 h and subsequently fed L-[3-14C]-serine. Its incorporation of L-[3-14C]-serine into de novo ceramide was analysed in larvae 12 h after heat shock by TLC with chloroform/methanol/glacial acetic acid (190:9:1) (A) and quantified (B). Asterisks in panel B indicate significant differences to wild-type controls [hsGAL4∷w1118] (P<0.001). (C, D) Treatment of SL-2 cells in the presence of [14C]serine with schlank dsRNA or schlank overexpression after transfection of SL-2 cells shows significant downregulation or upregulation of (dihydro)ceramide, respectively. Ceramides and dihydroceramides were separated on TLC plates impregnated with borate with chloroform/methanol 9:1. The main (dihydro)ceramide bands depicted in panel C correspond to the main ceramide marked with an asterisk in panel A (see also Supplementary Figure S2A). Error bars indicate s.d. Download figure Download PowerPoint To further test whether the effects on larval lipid composition were due to the influence of schlank on the metabolism and catabolism of endogenous lipids, rather than, for example, on an impaired uptake from the yeast food, we also analysed de novo synthesis of ceramide in SL-2 cells. Upon RNAi-mediated knockdown of schlank (dihydro)ceramide levels were significantly downregulated, whereas they were upregulated upon schlank overexpression (Figure 3C and D), consistent with the results obtained in larvae. Together, the analysis of schlank mutants, knockdown animals and overexpression studies in vivo and in vitro strongly support a role of schlank as a ceramide synthase in Drosophila. TAG levels are reduced in schlank mutants While hemizygous animals carrying the stronger schlankG0349 allele fail to grow in the larval stages and die with a morphology of first instar larvae, the schlankG0061 mutants are developmentally delayed; however, some of them pass through the third instar larval stage and die later after eclosion. When analysing third instar larvae of schlankG0061 mutants, we noticed that they appeared much slimmer and somewhat transparent, indicating loss of storage fat in the fat body (Figure 1A). A function in regulating organismal fat storage or mobilization has previously not been observed for Lass/ceramide synthase family members due to lack of animal models. We, therefore, investigated this phenotype in more detail. The larval period is critical for the control of animal growth. Regulation of larval growth by fat body has been demonstrated previously (Britton et al, 2002; Colombani et al, 2005). The larval stage is characterized by extensive feeding, which supports rapid growth of the animal and allows accumulation of energy stores, primarily in the larval fat body (Aguila et al, 2007). Immunohistochemical analysis indicates that schlank is strongly expressed in the fat-body cells (Supplementary Figure S5C–F). To study whether schlank may play a role in fat metabolism, we analysed TAG, diacylglycerol (DAG) and FA levels in schlankG0061- and schlankG0349-mutant larvae of the same developmental stage and compared them with those in wild-type animals. Both schlankG0061 and schlankG0349 mutants showed significantly reduced TAG levels to 63 and 13%, and FA and DAG levels were also altered (Figure 4A; Table I for quantification). The specificity of this phenotype was further confirmed by induction of schlank RNAi knockdown showing also strong TAG reduction (Supplementary Figure S3C). In contrast, we observed an increase of TAG, DAG and FA levels upon overexpression of schlank and the murine Lass2 homologue (Figure 4B; Table I for quantification), suggesting a conserved role for Lass proteins in lipid homeostasis. When we overexpressed the schlankH215D variant containing a mutation, which severely affects ceramide synthase activity (Supplement Figure S3B; Materials and methods), we observed a similar increase of TAG, DAG and FA levels (Figure 4B and Table I). This effect was comparable to the increase seen when overexpressing wild-type schlank or murine Lass2, suggesting that there are also effects of schlank on fat metabolism that may be independent of its ceramide synthase function. In summary, these data indicate an important role of schlank in regulating TAG levels and body fat metabolism, consistent with the strong expression of schlank protein in the larval fat body. To study how this effect occurs, we analysed the expression of key regulators of lipogenesis and lipolysis in schlank mutants and in animals overexpressing schlank. Figure 4.A role for schlank in TAG regulation. (A) Comparison of in vivo TAG levels in 38- to 42-h-old schlankG0061- and schlankG0349-mutant animals of the same age with w1118 controls. In both mutants TAG and FA levels were reduced to a different extent correlating well with the severity of the mutant schlank alleles. (B) TAG and FAs are elevated in 42- to 46-h-old larvae upon overexpression of either schlankHA (hsGAL4∷UASschlankHA), murine lass2HA (hsGAL4∷UASlass2HA) or schlankH215D (hsGAL4∷UASschlankH215D), which cannot upregulate ceramide synthesis