The Drosophila FoxA Ortholog Fork Head Regulates Growth and Gene Expression Downstream of Target of Rapamycin

Forkhead transcription factors of the FoxO subfamily regulate gene expression programs downstream of the insulin signaling network. It is less clear which proteins mediate transcriptional control exerted by Target of rapamycin (TOR) signaling, but recent studies in nematodes suggest a role for FoxA transcription factors downstream of TOR. In this study we present evidence that outlines a similar connection in Drosophila, in which the FoxA protein Fork head (FKH) regulates cellular and organismal size downstream of TOR. We find that ectopic expression and targeted knockdown of FKH in larval tissues elicits different size phenotypes depending on nutrient state and TOR signaling levels. FKH overexpression has a negative effect on growth under fed conditions, and this phenotype is not further exacerbated by inhibition of TOR via rapamycin feeding. Under conditions of starvation or low TOR signaling levels, knockdown of FKH attenuates the size reduction associated with these conditions. Subcellular localization of endogenous FKH protein is shifted from predominantly cytoplasmic on a high-protein diet to a pronounced nuclear accumulation in animals with reduced levels of TOR or fed with rapamycin. Two putative FKH target genes, CG6770 and cabut, are transcriptionally induced by rapamycin or FKH expression, and silenced by FKH knockdown. Induction of both target genes in heterozygous TOR mutant animals is suppressed by mutations in fkh. Furthermore, TOR signaling levels and FKH impact on transcription of the dFOXO target gene d4E-BP, implying a point of crosstalk with the insulin pathway. In summary, our observations show that an alteration of FKH levels has an effect on cellular and organismal size, and that FKH function is required for the growth inhibition and target gene induction caused by low TOR signaling levels

Src tyrosine kinase signaling antagonizes nuclear localization of FOXO and inhibits its transcription factor activity

Biochemical experiments in mammalian cells have linked Src family kinase activity to the insulin signaling pathway. To explore the physiological link between Src and a central insulin pathway effector, we investigated the effect of different Src signaling levels on the Drosophila transcription factor dFOXO in vivo. Ectopic activation of Src42A in the starved larval fatbody was sufficient to drive dFOXO out of the nucleus. When Src signaling levels were lowered by means of loss-of-function mutations or pharmacological inhibition, dFOXO localization was shifted to the nucleus in growing animals, and transcription of the dFOXO target genes d4E-BP and dInR was induced. dFOXO loss-of-function mutations rescued the induction of dFOXO target gene expression and the body size reduction of Src42A mutant larvae, establishing dFOXO as a critical downstream effector of Src signaling. Furthermore, we provide evidence that the regulation of FOXO transcription factors by Src is evolutionarily conserved in mammalian cells.ISSN:2045-232

Peroxisomes in Immune Response and Inflammation.

International audienceThe immune response is essential to protect organisms from infection and an altered self. An organism’s overall metabolic status is now recognized as an important and long-overlooked mediator of immunity and has spurred new explorations of immune-related metabolic abnormalities. Peroxisomes are essential metabolic organelles with a central role in the synthesis and turnover of complex lipids and reactive species. Peroxisomes have recently been identified as pivotal regulators of immune functions and inflammation in the development and during infection, defining a new branch of immunometabolism. This review summarizes the current evidence that has helped to identify peroxisomes as central regulators of immunity and highlights the peroxisomal proteins and metabolites that have acquired relevance in human pathologies for their link to the development of inflammation, neuropathies, aging and cancer. This review then describes how peroxisomes govern immune signaling strategies such as phagocytosis and cytokine production and their relevance in fighting bacterial and viral infections. The mechanisms by which peroxisomes either control the activation of the immune response or trigger cellular metabolic changes that activate and resolve immune responses are also described

Peroxisomes in Immune Response and Inflammation

The immune response is essential to protect organisms from infection and an altered self. An organism’s overall metabolic status is now recognized as an important and long-overlooked mediator of immunity and has spurred new explorations of immune-related metabolic abnormalities. Peroxisomes are essential metabolic organelles with a central role in the synthesis and turnover of complex lipids and reactive species. Peroxisomes have recently been identified as pivotal regulators of immune functions and inflammation in the development and during infection, defining a new branch of immunometabolism. This review summarizes the current evidence that has helped to identify peroxisomes as central regulators of immunity and highlights the peroxisomal proteins and metabolites that have acquired relevance in human pathologies for their link to the development of inflammation, neuropathies, aging and cancer. This review then describes how peroxisomes govern immune signaling strategies such as phagocytosis and cytokine production and their relevance in fighting bacterial and viral infections. The mechanisms by which peroxisomes either control the activation of the immune response or trigger cellular metabolic changes that activate and resolve immune responses are also described

Ceramide Synthase Schlank Is a Transcriptional Regulator Adapting Gene Expression to Energy Requirements

Maintenance of metabolic homeostasis requires adaption of gene regulation to the cellular energy state via transcriptional regulators. Here, we identify a role of ceramide synthase (CerS) Schlank, a multiple transmembrane protein containing a catalytic lag1p motif and a homeodomain, which is poorly studied in CerSs, as a transcriptional regulator. ChIP experiments show that it binds promoter regions of lipases lipase3 and magro via its homeodomain. Mutation of nuclear localization site 2 (NLS2) within the homeodomain leads to loss of DNA binding and deregulated gene expression, and NLS2 mutants can no longer adjust the transcriptional response to changing lipid levels. This mechanism is conserved in mammalian CerS2 and emphasizes the importance of the CerS protein rather than ceramide synthesis. This study demonstrates a double role of CerS Schlank as an enzyme and a transcriptional regulator, sensing lipid levels and transducing the information to the level of gene expression

Dietary rescue of lipotoxicity-induced mitochondrial damage in Peroxin19 mutants

<div><p>Mutations in peroxin (PEX) genes lead to loss of peroxisomes, resulting in the formation of peroxisomal biogenesis disorders (PBDs) and early lethality. Studying PBDs and their animal models has greatly contributed to our current knowledge about peroxisomal functions. Very-long-chain fatty acid (VLCFA) accumulation has long been suggested as a major disease-mediating factor, although the exact pathological consequences are unclear. Here, we show that a <i>Drosophila Pex19</i> mutant is lethal due to a deficit in medium-chain fatty acids (MCFAs). Increased lipolysis mediated by Lipase 3 (Lip3) leads to accumulation of free fatty acids and lipotoxicity. Administration of MCFAs prevents lipolysis and decreases the free fatty acid load. This drastically increases the survival rate of <i>Pex19</i> mutants without reducing VLCFA accumulation. We identified a mediator of MCFA-induced lipolysis repression, the ceramide synthase Schlank, which reacts to MCFA supplementation by increasing its repressive action on <i>lip3</i>. This shifts our understanding of the key defects in peroxisome-deficient cells away from elevated VLCFA levels toward elevated lipolysis and shows that loss of this important organelle can be compensated by a dietary adjustment.</p></div

Effects of dietary administration of M/LCFA on mitochondria and metabolism.

<p>(A-D) Staining of mitochondria with MitoTracker Red CM-H2XRos to show production of ROS. Scale bars represent 10 μm. <i>n</i> = 5. (E) Real-time qPCR analysis of genes encoding for metabolic enzymes. ΔCq values are normalized to w- (ΔΔC_q or fold regulation). (F, G) Oxygen consumption levels of wild-type and <i>Pex19</i> mutant larvae (G: glutamate, M: malate, Pro: proline, Pyr: pyruvate, D: ADP, G3P: glycerol 3 phosphate). <i>n</i> = 4. (H) Mitochondrial β-oxidation rate under control and rescue condition. (I) Addition of 25 μM etomoxir reduces the survival rate of coconut oil–fed <i>Pex19−/−</i>. <i>n</i> = 10 in groups of 25 individuals. Genotypes are w-: <i>w</i>^<i>1118</i>, <i>Pex19−/−</i>: <i>w; Pex19</i>^<i>ΔF7</i><i>/Pex19</i>^<i>ΔF7</i>. Error bars represent SD. *<i>p</i> < 0.05; **<i>p</i> < 0.01; ***<i>p</i> < 0.001. Significance tested with Student <i>t</i> test. Corresponding raw data can be found in supplemental file <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004893#pbio.2004893.s005" target="_blank">S1 Data</a>. Acsl, acyl-CoA synthetase long-chain; HexC, hexokinase C; IsoDH, isocitrate dehydrogenase; lip3, lipase 3; M/LCFA, medium- and long-chain fatty acid; mt comp I, mitochondrial complex I; qPCR, quantitative PCR; ROS, reactive oxygen species; ROX, residual oxygen consumption; yip2, yippee-interacting protein.</p

Other <i>Pex</i> mutants show a phenotype similar to <i>Pex19</i> mutants and can also be rescued with coconut oil.

<p>(A) Survival of different <i>Pex</i> mutants on control and rescue diet. <i>n</i> = 5 in groups of 25 individuals. (B-I) TMRE staining of third-instar larval Malpighian tubules. Scale bars represent 10 μm. <i>n</i> = 5. (J) FAMEs comparing w- and Pex mutants fed with control and M/LCFA-enriched rescue food. <i>n</i> = 3. Genotypes are <i>Pex2</i>^{<i>HP35039</i>}<i>/Pex2</i>^{<i>f01899</i>}, <i>Pex3</i>^<i>2</i><i>/ Df(32)6262</i>, <i>Pex5</i>^{<i>MI06050</i>}<i>/ Pex5</i>^{<i>MI06050</i>}, <i>Pex10</i>^{<i>MI04076</i>}<i>/ Pex10</i>^{<i>MI04076</i>}, <i>Pex19</i>^<i>ΔF7</i><i>/ Df(2L)esc</i> ^<i>p3-0</i>, <i>Pex19</i>^<i>ΔF7</i><i>/ Pex19</i>^<i>ΔF7</i>. Error bars represent SD. Asterisks represent *<i>p</i> < 0.05, **<i>p</i> < 0.01, ***<i>p</i> < 0.001. Significance tested using ANOVA with Tukey posttest. Corresponding raw data can be found in supplemental file <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004893#pbio.2004893.s005" target="_blank">S1 Data</a>. FAME, fatty acid methyl ester; M/LCFA, medium- and long-chain fatty acid; NEFA, nonesterified fatty acid; Pex, peroxin; TMRE, tetramethylrhodamine ethyl ester.</p

<i>Pex19</i> mutants display shortage in M/LCFAs and survive better when fed with MCFA-rich oils.

<p>(A) GC/MS analysis of FAMEs of wild-typic and <i>Pex19</i> mutant pupae, concentration in nmol/mg. <i>n</i> = 4. Significance tested with ANOVA. (B) Survival upon addition of different natural oils with different fatty acid compositions. <i>n</i> = 5 in groups of 25 individuals. (C) Survival upon addition of M/LC-TAGs. <i>n</i> = 5 in groups of 25 individuals. -Nip: without nipagin. (D) Survival profile with percentage of pupae, adults, and surviving adults upon rescue diet feeding. <i>n</i> = 10 in groups of 25 individuals. Significance tested using Student <i>t</i> test. Genotypes are w-: <i>w</i>^<i>1118</i>, <i>Pex19−/−</i>: <i>w</i>^<i>1118</i>; <i>Pex19</i>^<i>ΔF7</i><i>/Pex19</i>^<i>ΔF7</i>. Error bars represent SD. *<i>p</i> < 0.05; **<i>p</i> < 0.01; ***<i>p</i> < 0.001. Corresponding raw data can be found in supplemental file <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004893#pbio.2004893.s005" target="_blank">S1 Data</a>. FAME, fatty acid methyl ester; GC/MS, gas chromatography/mass spectometry; LCFA, long-chain fatty acid; MCFA, medium-chain fatty acid; TAG, triacylglycerol.</p