InteractiveFly: GeneBrief

slimfast: Biological Overview | References

Gene name - slimfast

Synonyms -

Cytological map position - 80B2-80B2

Function - transporter

Keywords - fat body, amino acid transport, TSC/TOR signaling, PI3-kinase signaling

Symbol - slif

FlyBase ID: FBgn0037203

Genetic map position - 3L:22,874,146..22,886,929 [+]

Classification - Transmembrane amino acid transporter protein

Cellular location - surface transmembrane

NCBI link: EntrezGene

Slif orthologs: Biolitmine
Recent literature
Jayakumar, S., Richhariya, S., Deb, B. K. and Hasan, G. (2018). A multi-component neuronal response encodes the larval decision to pupariate upon amino acid starvation. J Neurosci. PubMed ID: 30301757
Organisms need to co-ordinate growth with development, particularly in the context of nutrient availability. Thus, multiple ways have evolved to survive extrinsic nutrient deprivation during development. In Drosophila, growth occurs during larval development. Larvae are thus critically dependant on nutritional inputs but post critical weight they pupariate even when starved. How nutrient availability is coupled to the internal metabolic state for the decision to pupariate needs better understanding. Glutamatergic interneurons have been identified in the ventral ganglion that regulate pupariation on a protein-deficient diet. This study report that Drosophila third instar larvae (either sex) sense arginine to evaluate their nutrient environment using an amino-acid transporter Slimfast. The glutamatergic interneurons integrate external protein availability with internal metabolic state through neuropeptide signals. IP3 mediated calcium release and store-operated calcium entry are essential in these glutamatergic neurons for such integration and alter neuronal function by reducing the expression of multiple ion channels.

Organisms modulate their growth according to nutrient availability. Although individual cells in a multicellular animal may respond directly to nutrient levels, growth of the entire organism needs to be coordinated. This study provides evidence that in Drosophila, coordination of organismal growth originates from the fat body, an insect organ that retains endocrine and storage functions of the vertebrate liver. A genetic screen for growth modifiers discovered slimfast, a gene that encodes an amino acid transporter. Remarkably, downregulation of slimfast specifically within the fat body causes a global growth defect similar to that seen in Drosophila raised under poor nutritional conditions. This involves TSC/TOR signaling in the fat body, and a remote inhibition of organismal growth via local repression of PI3-kinase signaling in peripheral tissues. These results demonstrate that the fat body functions as a nutrient sensor that restricts global growth through a humoral mechanism (Colombani, 2003).

In multicellular organisms, the control of growth depends on the integration of various genetic and environmental cues. Nutrient availability is one of the major environmental signals influencing growth and, as such, has dictated adaptative responses during evolution toward multicellularity. In particular, complex humoral responses ensure that growth and development are properly coordinated with nutritional conditions (Colombani, 2003).

In isolated cells, amino acid withdrawal leads to an immediate suppression of protein synthesis, suggesting that cells are protected by active sensing mechanims that block translation prior to depletion of internal amino acid stores. In many mammalian cell types, changes in amino acid diet affect the binding of the translation repressor 4EBP1 to initiation factor eIF4E and the activity of ribosomal protein S6 kinase (S6K). These two signaling events require the activity of TOR (target of rapamycin), a conserved kinase recently shown to participate in a nutrient-sensitive complex both in mammalian cells and in yeast. Mutations in the Drosophila TOR homolog (dTOR) results in cellular and physiological responses characteristic of amino acid deprivation and establish that TOR is cell autonomously required for growth in a multicellular organism. Furthermore, the TSC (tuberous sclerosis complex) tumor suppressor, consisting of a TSC1 and TSC2 heterodimer (TSC1/2), as well as the small GTPase Rheb participate to the regulation of TOR function. Overall, these data suggest that TSC, Rheb, TOR, and S6K participate in a conserved pathway that coordinates growth with nutrition in a cell-intrinsic manner (Colombani, 2003).

In multicellular organisms, humoral controls are believed to buffer variations in nutrient levels. However, little is known about how growth of individual cells is coordinated. In vertebrates, growth-promoting action of the growth hormone (GH) is mostly relayed to peripheral tissues through the production of IGF-I. Binding of IGF-I to its cognate receptor tyrosine kinase (IGF-IR) induces phosphorylation of insulin receptor substrates (IRS), which in turn activate a cascade of downstream effectors. These include phospho-inositide 3-kinase (PI3K), which generates the second messenger phosphatidylinositol-3,4,5-P3 (PIP3), and thereby activates the AKT/PKB kinase. Genetic manipulation of IGF-I, IGF-IR, PI3K, and AKT in mice modulates tissue growth in vivo thus demonstrating a requirement of the IGF pathway for growth. In Drosophila, both loss- and gain-of function studies have also exemplified the role of a conserved insulin/IGF signaling pathway in the control of growth. Ligands for the unique insulin receptor (Inr) constitute a family of seven peptides related to insulin, the Drosophila insulin-like peptides (Dilps). Remarkably, three dilp genes (dilp2, dilp3, and dilp5) are expressed in a cluster of seven median neurosecretory cells (m-NSCs) in the larval brain, suggesting that they have an endocrine function. Indeed, ablation of the seven dilp-expressing mNSCs in larvae induces a systemic growth defect (Colombani, 2003).

Both in flies and mice, mutations in IRS provoke growth retardation as well as female sterility similar to what is observed in starved animals. Moreover, PI3K activity in Drosophila larvae depends on the availability of proteins in the food. Overall, this supports the notion that the insulin/IGF pathway might coordinate tissue growth with nutritional conditions. However, upon amino acid withdrawal, neither PI3K nor AKT/PKB activities are downregulated in mammalian or insect cells in culture, suggesting that this pathway does not directly respond to nutrient shortage. Hence, an intermediate sensor mechanism must link nutrient availability to insulin/IGF signaling (Colombani, 2003).

An intriguing possibility is that specific organs could function as nutrient sensors and induce a nonautonomous modulation of insulin/IGF growth signaling in response to changes in nutrient levels. This study used a genetic approach in Drosophila to assess both the cellular and humoral responses to amino acid deprivation in the context of a developing organism. The insect fat body (FB) has important storage and humoral functions associated with nutrition, comparable to vertebrate liver and adipose tissue. During larval stages, the FB accumulates large stores of proteins, lipids, and carbohydrates, which are normally degraded by autophagy during metamorphosis in order to supply the developing tissues but can also be remobilized during larval life to compensate transitory nutrient shortage. In addition to its storage function, the FB also has endocrine activity and supports growth of imaginal disc explants and DNA replication of larval brains in coculture. This study demonstrates that the FB operates as a sensor for variations in nutrient levels and coordinates growth of peripheral tissues accordingly via a humoral mechanism (Colombani, 2003).

In the course of a P[UAS]-based overexpression screen for growth modifiers, a P[UAS]-insertion line (UY681) was found to cause growth retardation upon ectopic activation. Sequence analysis revealed that P(UY)681 is inserted in a predicted gene (CG11128) that encodes a putative protein showing strong homology with amino acid permeases of the cationic amino acid transporter (CAT) family. The P[UAS] element is inserted in the first intron of the CG11128 gene, potentially driving transcription of an antisense RNA in a GAL4-dependent manner. To assess the function of this transporter, 3H-arginine uptake was measured in S2 cells. Results indicate that amino acid uptake is either enhanced by transfection of a CG11128 cDNA or suppressed by RNAi, indicating that the encoded protein presents CAT activity. In situ hybridization revealed basal levels of CG11128 expression in most larval tissues but much higher levels in the FB and the gut, two tissues involved in amino acid processing (Colombani, 2003).

By P element remobilization, an imprecise excision was obtained that deletes the sequences encoding the N-terminal half of the protein. 87% of homozygous mutant animals die during larval stages. The few viable adults emerged after a 2 day delay and were smaller and markedly slimmer than control animals. The associated gene was named slimfast (slif) and the excision allele slif1. Weight measurement indicated that homozygous slif1 adult males displayed a 16% mass reduction compared to control. Accordingly, adult wing size was reduced by 8% due to a reduction of both cell size and cell number. When the slif1 allele was in trans to Df(3L)Δ1AK, a deficiency covering the locus, larval lethality was slightly enhanced, suggesting that slif1 corresponds to a strong hypomorphic allele. The amino acid transporter function of slif, as well as the phenotypes observed upon reduction of slif function suggest that slif mutant animals might suffer amino acid deprivation. A major consequence of amino acid deprivation in larvae is the remobilization of nutrient stores in the FB, which typically results in aggregation of storage vesicles. Consistently, fusion of storage vesicles was observed in the FB of slif1 larvae and was indistinguishable from that observed in animals fed on protein-free media (Colombani, 2003).

GAL4 induction of P(UY)681 resulted in a growth-deficient phenotype similar to that of slif1 loss of function. The antisense orientation of P(UY)681 suggested that the growth defect following GAL4 induction was due to an RNAi effect. Indeed, Northern blot analysis revealed that ubiquitous GAL4-dependent activation of P(UY)681 using the daughterless-GAL4 (da-GAL4) driver strongly reduced slif mRNA levels. Only two of the three alternative first exons are potentially affected by the antisense RNA, possibly explaining the residual accumulation of slif mRNAs in da-GAL4; P(UY)681 animals. Most of these animals died at larval stage, similar to what was observed for slif1 mutants. Specific induction of P(UY)681in the wing disc using the MS1096-GAL4 driver provoked a reduction of the adult wing size, which could be either rescued by coactivation of a UAS-slif transgene or enhanced by reducing slif gene dosage with the heterozygous Df(3L)Δ1AK deficiency. Thus, GAL4-dependent activation of P(UY)681 reduces slif function and defines a conditional loss-of-function allele hereafter termed slifAnti (Colombani, 2003).

As expected, loss of slif function using the slifAnti allele also mimicked amino acid deprivation. Accordingly, ubiquitous slifAnti induction in growing larvae resulted in storage vesicle aggregation and strong reduction of global S6 kinase activity, similar to what was reported in animals raised on protein-free diet. Additionally, an increase in PEPCK1 gene transcription was observed, similar to the effect of amino acid withdrawal. In summary, this study has identified two loss-of-function alleles of the slif gene whose defects mimic physiological aspects of amino acid deprivation. Importantly, the conditional slifAnti allele provides a unique tool to mimic an amino acid deprivation in a tissue-specific manner (Colombani, 2003).

This study established that the FB is a sensor tissue for amino acid levels, as downregulation of the Slif amino acid transporter within the FB is sufficient to induce a general reduction in the rate of larval growth. In contrast, specific disruption of slif in imaginal discs, larval gut, or salivary glands did not induce a nonautonomous growth response, suggesting that these tissues do not participate in the systemic control of growth. The dilp-expressing median neurosecretory cells (m-NSCs) also affect growth control, since selective ablation of these cells in the larval brain induces an overall reduction of animal size. In response to complete sugar and protein starvation, the m-NSCs stop expressing dilp3 and dilp5 genes, suggesting that these neurons also sense nutrient levels. This study shows that the selective reduction of slif function in these cells has no obvious effect on tissue growth and animal development. This indicates that the seven dilp-expressing m-NSCs do not constitute a general amino acid sensor. In contrast, the role of m-NSCs in carbohydrate homeostasis and the observation that they stop expressing certain dilp genes when larvae are deprived of sugar rather suggests that these cells have a role in sensing carbohydrate levels (Colombani, 2003 and references therein).

This analysis also provides a framework in which to understand the phenotype of minidisc, a mutation in an amino acid transporter gene that exhibits nonautonomous growth defects in imaginal discs (Martin, 2000; Colombani, 2003).

In a number of model systems, both PI3K and TOR have been implicated in linking growth to nutritional status and, until recently, were considered as intermediates of a common regulatory pathway. In yeast, the TOR kinase is part of a cell-autonomous nutrient sensor, which controls protein synthesis, ribosome biogenesis, nutrient import, and autophagy. Genetic analysis in Drosophila indicates that dTOR is required for cell-intrinsic growth control. The results obtained using the slifAnti allele in the wing disc indicate that individual tissues have indeed the potential to respond to amino acid deprivation in a cell-autonomous manner. Nonetheless, this study also demonstrates that the TOR nutritional checkpoint participates in a systemic control of larval growth emanating from the FB. Within a developing organism, each cell may integrate these two distinct inputs regarding nutritional status, one originating from a systemically-acting FB sensor, and the other from TOR-dependent signaling in individual cells. One can further speculate that depending on the strength and duration of starvation, different in vivo nutritional checkpoints will be hierarchically recruited to protect the animal and that the systemic control might, in most physiological situations, override the cell-autonomous control. Indeed, as the data demonstrate, the FB sensor is sufficient to induce a general and coordinated response to starvation without calling individual cell-autonomous mechanisms into play (Colombani, 2003).

Several lines of evidence indicate that the PI3K pathway is not part of the sensor mechanism in FB cells. First, a sensor for PI3K activity in the FB is only marginally affected by amino acid deprivation in that tissue, indicating that the cell-autonomous response to amino acid starvation does not directly influence PI3K signaling. This is reminiscent of previous observations in mammalian cultured cells, showing that PI3K activity does not respond to variations in amino acid levels. Moreover, inhibition of PI3K signaling by dPTEN expression in the FB is not sufficient to trigger the sensing mechanism. Although, dPTEN overexpression causes a complete disappearance of the PI3K sensor accompanied by growth suppression of FB cells, the FB maintains a critical mass that allows for normal larval growth. In contrast, the regulatory subunit p60 whose overexpression potently inhibits PI3-kinase in flies has been shown to induce a systemic effect on larval growth when overexpressed in the FB using an Adh-Gal4 driver (Britton, 2002). This study found that a pumpless ppl-GAL4-directed expression of p60 also provokes a strong suppression of larval growth and a dramatic inhibition of FB development in young larvae. Thus, the systemic effect on growth observed upon p60 overexpression most likely results from a drastic reduction of FB mass, which then fails to support normal larval growth (Colombani, 2003).

These results further indicate that PI3K signaling is a remote target of the humoral message that originates from the FB in response to amino acid deprivation. This is in agreement with previous data showing that PI3K activity is downregulated by dietary amino acid deprivation and explains why global PI3-kinase inhibition mimics cellular and organismal effects of starvation (Britton, 2002). The existence of a humoral relay reconciles these in vivo studies with the absence of direct PI3K responsiveness to amino acid levels (Colombani, 2003).

The relative resistance of imaginal disc growth to the systemic control exerted by the FB correlates with maintenance of PI3K activity in these tissues. This is in agreement with previous observations (Britton, 1998) that cells in the larval brain and in imaginal discs maintain a slow rate of proliferation under protein starvation, while larval endoreduplicating tissues (ERTs) arrest. This difference might be attributed to the basal levels of dilp2 expression observed in imaginal discs, allowing a moderate growth rate of these tissues through an autocrine/paracrine mechanism. It was recently shown that clonal induction of PI3K potently induces cell-autonomous growth response even in fasting larvae (Britton, 2002), indicating that some nutrients are still accessible to support cell growth within a fasted larva. The main function of a general sensor could be to preserve these limited nutrients for use by high priority tissues. In this context, local PI3K activation through an autocrine loop in imaginal tissues could favor the growth of prospective adult structures in adverse food conditions. Thus, the FB would have an active role in controlling the allocation of resources depending on nutritional status. In this respect, it is noteworthy that FB cells are relatively resistant to the FB-derived humoral signal, since the PI3K sensor is not drastically affected in the FB of ppl>slifAnti animals. Thereby, essential regulatory functions of the FB could be preserved even in severely restricted nutritional conditions (Colombani, 2003).

How does the FB signal to other tissues? This study suggests that a humoral signal relays information from the FB amino acid sensor and systemically inhibits PI3K signaling. In addition, this downregulation is not due to a direct inhibition of dilp expression by neurosecretory cells in the brain. Nevertheless, it cannot be ruled out that the secretion of these molecules is subjected to regulation in the mNSCs. Both in vivo and in insect cell culture, several imaginal discs growth factors (IDGF) secreted by the FB have been proposed to function synergistically with Dilp signaling to promote growth (Kawamura, 1999). However, this study did not find any modification of IDGF expression in the FB of larvae raised on water- or sugar-only diet, or upon FB induction of slifanti. In vertebrates, the different functions of the circulating IGF-I are modulated through its association with IGF-BPs and acid labile subunit (ALS). In particular, the formation of a ternary complex with ALS leads to a considerable extension of IGF-I half-life. The finding that a Drosophila ALS ortholog is expressed within the FB in an amino acid-dependent manner provides a new avenue to study the molecular mechanisms of nonautonomous growth control mediated by the FB (Colombani, 2003).

This study highlights the contribution that genetics can provide to unravel the mechanisms of physiological control. Using a genetic tool to mimic amino acid deprivation, it was demonstrated that nutrition systemically controls body size through an amino acid sensor operating in the FB. It is proposed that (1) in metazoans, a systemic nutritional sensor modulates the conserved TOR-signaling pathway, and (2) the response to sensor activation is relayed by a hormonal mechanism, which triggers an Inr/PI3K-dependent response in peripheral tissues (Colombani, 2003).

TOR coordinates bulk and targeted endocytosis in the Drosophila melanogaster fat body to regulate cell growth

Target of rapamycin (TOR) is a central regulator of cellular and organismal growth in response to nutrient conditions. In a genetic screen for novel TOR interactors in Drosophila, the clathrin-uncoating ATPase Hsc70-4, which is a key regulator of endocytosis, was identified. Genetic evidence is presented that TOR signaling stimulates bulk endocytic uptake and inhibits the targeted endocytic degradation of the amino acid importer Slimfast. Thus, TOR simultaneously down-regulates aspects of endocytosis that inhibit growth and up-regulates potential growth-promoting functions of endocytosis. In addition, disruption of endocytosis leads to changes in TOR and phosphatidylinositol-3 kinase activity, affecting cell growth, autophagy, and rapamycin sensitivity. These data indicate that endocytosis acts both as an effector function downstream of TOR and as a physiologically relevant regulator of TOR signaling (Hennig, 2006; full text of article).

Inactivation of TOR causes an inhibition of cellular growth, a reduction in cell size, and a suppression of cell cycle progression. In addition to well described changes in protein synthesis and ribosome biogenesis, recent studies have suggested that other cell processes are likely to contribute to these growth effects of TOR. The present study identifies endocytosis as one such process. These results demonstrate that the clathrin-uncoating ATPase Hsc70-4 interacts genetically with TOR and Tsc1, and that bulk endocytosis is stimulated in cells with activated TOR signaling. Conversely, it was found that TOR activity inhibits the endocytic degradation of nutrient transporters such as Slimfast. Together, these endocytic effects of TOR promote both the bulk and targeted uptake of nutrients and other biomolecules required for cell mass increase. In addition to this direct role in cellular biosynthesis and growth, nutrients also act as potent regulators of TOR signaling. Indeed, Slimfast has been identified as an upstream activator of TOR (Colombani, 2003). These findings that disruption of endocytosis effects cell size, rapamycin sensitivity, and TOR kinase activity are consistent with an additional role for endocytosis upstream of TOR (Hennig, 2006).

Mutations that disrupt endocytosis are likely to have both positive and negative effects on nutrient uptake and cell growth because they inhibit bulk endocytic uptake, as well as degradation of nutrient transporters and other signaling molecules. Thus, the overall effects of endocytic disruption on nutrient uptake, cell growth, and TOR signaling are difficult to predict a priori. The results suggest that both the cellular context and the specific step at which endocytosis is blocked influence the growth response. Thus, in fat body cells, expression of ShiK44A resulted in an increase in cell size, whereas loss of Hsc70-4 function caused reduced cell size. It is noted that these changes mirror the effects of these mutations on Slimfast levels; whereas both ShiK44A expression and Hsc70-4 mutation decreased bulk endocytic uptake, only ShiK44A resulted in increased levels of Slimfast. In contrast, both ShiK44A and Hsc70-4 mutants led to the increased size of wing imaginal disc cells, suggesting that in these cells the growth-inhibitory effects of endocytic degradation of membrane proteins such as Slimfast predominate over the potential positive effects of increased bulk uptake. Similarly, the results indicate a complex effect of endocytosis on TOR signaling. Partial reduction in Hsc70-4 levels lead to an increase in TOR signaling, as was evident in an eyTOR interaction and rapamycin resistance. In contrast, larvae that are homozygous mutant for Hsc70-4 show a decrease in TOR kinase activity. These results suggest that modest inhibition of endocytosis may increase TOR signaling, whereas a complete block of endocytosis may reduce it (Hennig, 2006).

A striking parallel to the inverse regulation of bulk and targeted endocytic processes by TOR can be observed in its effects on autophagy in yeast. Through autophagy, random portions of cytoplasm are nonselectively engulfed within double membrane-bound vesicles for delivery to the lysosome. Activation of TOR causes this nonselective form of autophagy to be suppressed, and, instead, the autophagic machinery engages in a selective type of autophagy known as the cytoplasm-vacuole targeting (CVT) pathway, which is responsible for lysosomal delivery of specific hydrolases. Thus, TOR acts as a switch between selective and nonselective autophagy. TOR may also be involved in switching between clathrin-and caveolae/raft-mediated endocytosis in higher eukaryotes. A genome-wide survey of protein kinases found that RNAi-mediated inactivation of TOR in HeLa cells inhibited clathrin-dependent processes such as transferrin uptake and vesicular stomatitis virus infection, and stimulated cavelolae/raft-dependent events. Together, these findings suggest that TOR may control the specificity of membrane trafficking components. In addition, the results show that S6K, which is an important TOR substrate, acts downstream of TOR in promoting bulk endocytosis, but is not involved in the suppression of starvation-induced autophagy (Hennig, 2006).

The identification of endocytosis as a TOR-controlled function adds to the growing list of cell processes regulated by TOR, including protein synthesis, ribosome biogenesis, autophagy, metabolic gene expression, and cytoskeletal organization. How these distinct functions interact to achieve a coordinated growth response is only beginning to be understood. One likely mechanism involves the common use of molecular components and cellular substrates by different cell functions, as in the case of selective and nonselective autophagy, bulk endocytosis, and endocytic degradation. Two or more distinct branches of TOR signaling may also act cooperatively to control the same target, as in the case of Slimfast regulation by both translation and endocytosis, or may act in opposition, as previously observed for the role of S6K in limiting autophagy. Finally, distinct TOR complexes may converge on the same targets with opposing effects, as in the regulation of Akt by TOR-Raptor versus TOR-Rictor complexes. The finding that TOR signaling regulates the levels of Slimfast, which was previously shown to function upstream of TOR, adds another layer of complexity to the TOR signaling network (Hennig, 2006).

Specialized hepatocyte-like cells regulate Drosophila lipid metabolism

Lipid metabolism is essential for growth and generates much of the energy needed during periods of starvation. In Drosophila, fasting larvae release large quantities of lipid from the fat body but it is unclear how and where this is processed. This study identified the oenocyte as the principal cell type accumulating lipid droplets during starvation. Tissue-specific manipulations of the Slimfast amino-acid channel, the Lsd2 fat-storage regulator and the Brummer lipase indicate that oenocytes act downstream of the fat body. In turn, oenocytes are required for depleting stored lipid from the fat body during fasting. Hence, lipid-metabolic coupling between the fat body and oenocytes is bidirectional. When food is plentiful, oenocytes have critical roles in regulating growth, development and feeding behaviour. In addition, they specifically express many different lipid-metabolizing proteins, including Cyp4g1, an omega-hydroxylase regulating triacylglycerol composition. These findings provide evidence that some lipid-processing functions of the mammalian liver are performed in insects by oenocytes (Gutierrez, 2007).

In mammals, specialized cells of the adipose tissue and liver are critical for coordinating fat metabolism. This physiological axis regulates a complex set of lipid uptake, storage, synthesis, modification and degradation reactions essential for normal growth and development. Lipid metabolism also has a particularly critical role in providing energy during periods of starvation. Food deprivation (fasting) stimulates the lipolysis of triglycerides (also called triacylglycerol, TAG) stored in adipocyte fat droplets via increases in hormone-sensitive lipase activity and adipose triglyceride lipase (ATGL) expression (Zechner, 2005). A large proportion of the fatty acids and other lipids thereby released into the circulation are then captured, modified and broken down by the major cell type of the liver, the hepatocyte. An intriguing feature of the starvation response is that, in contrast to many other cell types, hepatocytes accumulate large numbers of fat droplets, resulting in hepatic steatosis. Fatty acids are released from hepatic lipid droplets during starvation and oxidized into shorter chain fatty acids and ultimately into soluble ketone bodies that can be discharged into the circulation for use as an energy source by many tissues. This fatty acid oxidation process involves chain shortening by α- and β-oxidation pathways active in peroxisomes and mitochondria. Although lipid catabolism predominates during starvation, in the postprandial state, hepatocytes are highly active in synthesizing fatty acids for incorporation into triglycerides. These can then be assembled into lipoprotein particles, delivered to adipocytes and stored in lipid droplets. One critical step for incorporating newly synthesized fatty acids into TAG is catalysed by stearoyl CoA-desaturase-1 (SCD-1), a hepatic enzyme converting palmitic (C16:0) and stearic (C18:0) acids into monounsaturated palmitoleic (C16:1) and oleic (C18:1) acids, respectively. The importance of maintaining an appropriate balance between hepatic fatty acid synthesis and oxidation is highlighted by human diseases arising from mutations in fatty acid oxidation enzymes, and also by widespread diet-influenced pathologies such as non-alcoholic fatty liver disease and metabolic syndrome (Gutierrez, 2007).

Invertebrate model organisms offer a powerful means to identify and functionally analyse lipid-metabolising genes. In Caenorhabditis elegans, fat is stored by intestinal epithelial cells and many regulators of this process have been identified using reverse genetic screens. In contrast, Drosophila and other insects store lipid in a specialized tissue that resembles the adipose tissue of mammals, the fat body. Diet-derived lipids, exported from the midgut as lipoproteins, are taken up from the haemolymph by the fat body via a mechanism involving Low-Density Lipoprotein (LDL) receptor-like molecules called Lipophorin receptors. These lipids accumulate in fat body cells in the form of intracellular droplets but, when larvae are food-deprived, there is a net efflux of lipid into the haemolymph. The mobilization process is regulated by TSC/TOR signalling and a nutrient sensor in the fat body that monitors amino-acid levels via the Slimfast (Slif) amino-acid channel. Fasting-induced fat release is accompanied by increased lipolysis, at least in part associated with upregulation of Brummer, an ATGL-related lipase localized to lipid droplets. Fat mobilization is also influenced by Lsd2, a lipid droplet protein related to a mammalian negative regulator of TAG hydrolysis called perilipin. In addition to its involvement in lipid storage and release, the fat body produces a humoral signal regulating larval tissue growth in response to food availability. Thus far, efforts to harness the power of Drosophila genetics to model human fat metabolism have been limited by the lack of information on how and where insect lipids are processed once they have been released from the fat body. For example, it is not known whether there is a specialized Drosophila tissue that synthesizes, modifies and oxidizes fatty acids in a similar way to the mammalian liver, nor is it clear to what degree the mammalian biochemical pathways metabolizing fatty acids are conserved in Drosophila. This study addresses both of these issues using a combination of bioinformatics, genetics and integrative physiology (Gutierrez, 2007).

How fat is redistributed throughout the larval body after food deprivation was studied. Using Oil Red O staining, three cell types in the third instar (L3) larva were found to contain numerous large (0.5-2.5 microm) lipid droplets under fed and/or fasting conditions: fat body cells, midgut epithelial cells and larval oenocytes. The fat body of L3 larvae has such a large capacity for lipid storage that, despite lipid loss over a 14-h period of fasting, intense Oil Red O staining persists. Lipid release from the L3 fat body during starvation correlates with lipid droplet aggregation. However, droplet aggregation is not a reliable indicator of fasting at some other larval ages and durations of fasting. In contrast to the fat body, regions of the gut (including the proventriculus and anterior midgut) staining strongly with Oil Red O under fed conditions have only limited storage capacity, losing most lipid droplets after 14 h of fasting. The third cell type, larval oenocytes (distinct from adult oenocytes but abbreviated hereafter as oenocytes), are large cells of unknown function that are attached to the basal surface of the lateral epidermis in clusters of ~6 cells per abdominal hemi-segment. L3 oenocytes do not stain strongly with Oil Red O under fed conditions but they do contain numerous large lipid droplets after a 14-h fast. This change in droplet abundance is consistent from oenocyte-to-oenocyte within one cluster and also from one cluster to another. Thus, oenocytes are highly atypical cells, in that they accumulate large numbers of lipid droplets specifically during fasting. As this is a hallmark of hepatocytes, the possibility is raised that insect oenocytes might process lipids in a similar way to the mammalian liver (Gutierrez, 2007).

Next, whether the accumulation of lipid droplets in oenocytes is regulated by the fat body nutrient sensor was tested. An antisense transgene directed against the amino-acid transporter Slimfast was expressed in the fat body (ppl-GAL4 driving UAS-slifAnti; hereafter called ppl>slifAnti). As reported previously (Colombani, 2003), it was observed that ppl>slifAnti larvae raised to L3 on a standard diet resemble starved wild-type larvae in that lipid droplets aggregate in the fat body. Notably, it was also found that oenocytes contain numerous lipid droplets, regardless of whether ppl>slifAnti larvae are fed on a standard diet or food-deprived for 14 h. This indicates that amino-acid monitoring via Slif in the fat body is required to ensure that lipid accumulation in oenocytes is kept low under standard nutritional conditions. TSC/TOR signalling, another component of the fat body nutrient sensor, is also involved; overexpressing TSC1 and TSC2 (ppl>Tsc1+2) leads to a marked accumulation of large lipid droplets in the oenocytes of 100% (n = 11) of fed larvae. Similarly, inhibiting the phosphatidylinositol-3 kinase pathway, which intersects with TOR signalling, by overexpressing the lipid phosphatase PTEN, also produces a build up of lipid droplets in the oenocytes of 89% of fed ppl>PTEN larvae. Hence, the fat body nutrient sensor regulates lipid accumulation in oenocytes but this could be directly via lipid release or indirectly, in response to a TSC/TOR-dependent signal (Gutierrez, 2007).

To assess directly the effect of lipid mobilization from the fat body, the balance between TAG storage and hydrolysis was altered in two ways. First, Brummer (Bmm) lipase, which is normally limiting for lipid release from the fat body, was overexpressed. This is sufficient to produce specific accumulation of lipid droplets in the oenocytes of 92% of fed ppl>bmm larvae. Second, TAG release from lipid droplets was decreased by overexpressing Lsd2. This reduces the accumulation of oenocyte lipid droplets in 78% of starved ppl>Lsd2 larvae, with ~4-fold fewer large droplets per oenocyte. A second driver, Lsp2-GAL4, was used that unlike ppl-GAL4 is activated in the fat body only at the mid-L3 stage. This temporally restricted driver nevertheless suffices to induce oenocyte lipid droplet accumulation in 100% of fed Lsp2>bmm larvae and also in fed Lsp2>slifAnti animals. Together, the Slif, TSC, PTEN, Lsd2 and Bmm results demonstrate metabolic regulation from the fat body to the oenocytes, although they do not exclude the involvement of intermediate tissues such as the gut. Either way, these results strongly suggest that, when nutrition is poor, falling amino-acid levels stimulate lipid release from the fat body and subsequent lipid uptake from the haemolymph by oenocytes (Gutierrez, 2007).

To determine the in vivo roles of oenocytes during fasting and normal development, a targeted binary cell ablation system was developed. Larvae carrying sal[BO,7.6kb]GAL4, a purpose-built oenocyte driver, and also UAS-reaper, an inducible pro-apoptotic transgene, lack 100% of oenocytes from L1 onwards and die before reaching pupariation (hereafter called BO>rpr larvae). As a specificity control, BO>rpr animals were rescued to viable adults by expressing Gal80, an inhibitor of Gal4, under the regulation of an independent oenocyte enhancer from seven up (svp). As svp[3kb]GAL80 suppresses sal[BO,7.6kb]GAL4 activity in oenocytes but not in secondary larval sites, BO>rpr lethality results from the ablation of oenocytes and not some other cell type (Gutierrez, 2007).

BO>rpr larvae raised on a standard diet attain a similar mass to UAS-rpr controls during L1 but, after the L1-to-L2 transition, they grow at a much slower rate. Notably, reduced growth correlates with aberrant feeding behaviour, with most BO>rpr larvae dispersing away from the yeast food source during L2. This dispersal is distinct from premature wandering behaviour; BO>rpr larvae enter and exit the yeast source multiple times, retain food in the gut and do not pupariate precociously. Since BO>rpr larvae spend less time in the food source and grow more slowly than L2 controls, whether they show increased mouth-hook contractions, a behavioural response to hunger, was investigated. However, reduced mouth-hook contractions were observed that are not significantly increased by the motivation of a 2-h period of food deprivation. Thus, rather than stimulating hunger-driven feeding behaviour, oenocyte ablation seems to block it, although this effect could be very indirect. Either way, reduced feeding is likely to contribute to the slow growth rate of BO>rpr larvae during L2 (Gutierrez, 2007).

Since reduced growth resulting from inadequate nutrition before 70 h after egg laying (just before the L2/L3 moult) is associated with larval arrest rather than smaller-than-normal adult flies, morphological criteria were used to stage oenocyte-ablated animals. It was observed that BO>rpr larvae arrest at several different stages after the L1/L2 transition, thus displaying a polyphasic lethality profile. Although arrested development can result from reduced signalling by ecdysteroids, the BO>rpr polyphasic lethality profile is not significantly altered by adding 20-hydroxyecdysone or its precursor ecdysone. Therefore, a deficiency in these ecdysteroids is not the sole reason for BO>rpr arrest, but the possibility cannot be excluded that it, together with some other oenocyte deficit, contributes to the moulting phenotype (Gutierrez, 2007).

Unlike many larval tissues, oenocytes persist for much of pupal development. To address whether oenocytes are required for metamorphosis, a temperature-sensitive version of Gal80 (GAL80ts ) was used to attenuate Gal4 activity, thus bypassing BO>rpr larval lethality. Combining tub-GAL80ts with BO>rpr suppresses apoptosis in approximately 50% of oenocytes at 25 °C (from L1 onwards) and allows developmental progression until pupal stages. However, no animals complete pupal development, with many failing to separate from the puparial case during eclosion. Together, the 50% and 100% oenocyte ablation phenotypes demonstrate that oenocytes are required for growth and developmental progression during both larval and pupal stages (Gutierrez, 2007).

Whether lipid metabolism is altered in larvae lacking all oenocytes was examined. At early L2, when BO>rpr larvae are the same size as controls, no significant abnormalities in TAG content or in the relative amounts of the major long-chain fatty acids were detected. The fat storage capacity of larvae at early L2 is much less than at L3 such that a 12-h period of food withdrawal is sufficient to deplete ~60% of stored TAG in control animals. However, during this same fasting period, BO>rpr larvae only lose ~10% of total TAG. This deficit in TAG depletion correlates with a higher density of fat-body lipid droplets in 100% of starved BO>rpr larvae compared to controls after fasting. Since ~80% of larval fatty acids are stored as TAG, the proportions of individual fatty acids were examined in fasting BO>rpr larvae. In early L2 controls, lauric (C12:0) and myristic (C14:0) acids are depleted more efficiently than longer-chain (C16-C20) fatty acids such that their mass, relative to stearic acid (C18:0), is reduced twofold after 12 h fasting. However, in fasted BO>rpr larvae, the C12:0/C18:0 and C14:0/C18:0 ratios remain close to those before starvation, corresponding to approximately twice the value of starved controls. Together, these results indicate that oenocytes are required for efficiently depleting fatty acids, stored largely in the fat body as TAG, during nutrient deprivation. With the previous Slif, TSC, PTEN, Bmm and Lsd2 results, it is proposed that lipid-metabolic coupling between the fat body and oenocytes is bidirectional (Gutierrez, 2007).

To identify the metabolic pathways processing lipids within oenocytes, 51 genes expressed selectively or exclusively in oenocytes were identified. About 40% of these encode orthologues of known human lipid-metabolizing/processing proteins. The high degree of conservation of most Drosophila proteins, together with some previous functional studies, suggests that oenocytes express lipid metabolic pathways strikingly similar to those of hepatocytes. By analogy, oenocytes would capture lipid from lipophorin in the haemolymph via LpR1 and LpR2, two Lipophorin receptors. Fatty acids released from lipid droplets by lipases such as the CG17292 product, could then be modified by a variety of enzymes, including the Desat1 and CG9743 acyl-CoA desaturases, the CG18609 and CG6921 fatty acid elongases and the microsomal lipid omega-hydroxylase, Cytochrome P450-4g1 (Cyp4g1). Fatty acids could also be chain shortened, at least partially, by the actions of peroxisomal β-oxidation components including those encoded by CG11151 (similar to Sterol carrier protein 2), CG12428 (Carnitine O-octanoyl transferase), CG9527 (Pristanoyl-CoA oxidase) and Catalase, the peroxisomal enzyme inactivating oxygen free radicals produced by pristanoyl-CoA oxidases. In addition, oenocytes strongly express Hnf4 and Svp, orthologues of the mammalian nuclear receptors Hnf4-α and COUP-TF, known regulators of hepatocyte differentiation and lipid-metabolic genes. Thus, the oenocyte/hepatocyte analogy includes a shared set of lipid-metabolizing genes and at least two of their transcriptional regulators (Gutierrez, 2007).

To explore the functions of fatty acid metabolism specifically within Drosophila oenocytes, two lethal protein-null alleles were generated for the predicted lipid omega-hydroxylase encoded by Cyp4g1. Cyp4g1 is known to be expressed in oenocytes, and it was found that this is the only site of detectable expression in embryos and larvae. Animals homozygous for either the Cyp4g1Delta4 or Cyp4g1Delta4-9 allele develop normally through larval and early pupal stages but arrest during mid-to-late pupal stages, with many failing during adult eclosion. This pupal phenotype is strikingly similar to the 50% oenocyte ablation phenotype. Moreover, although late-L3 Cyp4g1 mutant larvae appear morphologically indistinguishable from controls, they manifest a twofold increase in the oleic acid:stearic acid ratio (C18:1/C18:0). Notably, this imbalance in fatty acid desaturation is found in the TAG fraction but not in the phospholipid fraction. This selectivity strongly suggests that the Cyp4g1 defect is specific to fatty acids in metabolic storage form, most of which reside in the fat body, rather than fatty acids present in the structural lipids of all cell membranes. Taken together, the metabolic profiles of oenocyte-ablated and Cyp4g1 mutant larvae provide two independent lines of evidence that oenocytes regulate the lipid composition of the fat body (Gutierrez, 2007).

Functions of larval oenocytes, described in insects over 140 yr ago, have remained unclear, with largely descriptive studies implicating them in processes such as cuticle synthesis and the regulation of haemolymph composition. Using cell ablation to test their functions directly for the first time, clear requirements for larval growth and pupal development were found. Although the subset of oenocyte genes mediating the larval developmental functions remains to be identified, for pupal development it was shown that the lipid omega-hydroxylase Cyp4g1 is required. At least one important role of Cyp4g1 is to downregulate the ratio of oleic-to-stearic acid, widely used as a marker of SCD-1 activity in mammals. This prompts speculation that Cyp4g1 may repress the activity of stearoyl CoA-desaturases like Desat1, thereby inhibiting inappropriate monounsaturated fatty acid synthesis during long non-feeding periods such as late L3 and pupal stages (Gutierrez, 2007).

Four lines of evidence argue that at least some of the lipid-metabolizing roles of insect oenocytes are analogous to those of mammalian hepatocytes: (1) oenocytes express 22 orthologues of human fat-metabolizing genes expressed in hepatocytes; (2) like hepatocytes, they are atypical cells in that they accumulate fat droplets during starvation; (3) like the liver, oenocytes lie downstream of a nutrient sensor in a major fat depot; (4) Brummer lipase and Lsd2 in the fat body regulate oenocyte lipid content in a broadly similar way as ATGL and perilipin in adipose tissue regulate hepatic fat influx. However, whereas hepatocytes store large quantities of glycogen, this role in Drosophila is primarily carried out by the fat body. Thus, mammalian liver functions in glycogen storage and lipid processing seem to be divided in Drosophila between the fat body and oenocytes (Gutierrez, 2007).

This study suggests the existence of two-way metabolic coupling between the fat body and oenocytes. Analogous to the mammalian adipose-liver axis, lipid mobilization from the fat body during starvation produces lipid droplet accumulation in oenocytes, a metabolic change resembling hepatic steatosis. In Drosophila, a reciprocal regulation was also found, namely that oenocytes are required for efficiently depleting lipid from the fat body during fasting. This suggests a feedback mechanism for matching lipid supply to demand, whereby oenocytes keep haemolymph lipids low and also promote lipid mobilization from the fat body. Thus, in oenocyte-less larvae, excess circulating lipids might underlie the behavioural syndrome of larval dispersal and reduced feeding in a similar way as reported for elevated amino-acid levels (Zinke, 1999). Central to the proposed feedback model is the signal acting on the lipogenesis/lipolysis balance within the fat body. The data presented in this study are equally compatible with this signal corresponding to a haemolymph lipid/metabolite or to a separate signal generated by oenocytes. Regarding the latter possibility, it is interesting that recent work in mammals indicates that the liver secretes signalling factors (hepatokines) that promote lipolysis in adipose tissue (Oike, 2005). This suggests that Drosophila may prove useful, not only for modelling hepatic steatosis, but also some regulatory roles of the liver in metabolic homeostasis (Gutierrez, 2007).

Fat cells reactivate quiescent neuroblasts via TOR and glial insulin relays in Drosophila

Many stem, progenitor and cancer cells undergo periods of mitotic quiescence from which they can be reactivated. The signals triggering entry into and exit from this reversible dormant state are not well understood. In the developing Drosophila central nervous system, multipotent self-renewing progenitors called neuroblasts undergo quiescence in a stereotypical spatiotemporal pattern. Entry into quiescence is regulated by Hox proteins and an internal neuroblast timer. Exit from quiescence (reactivation) is subject to a nutritional checkpoint requiring dietary amino acids. Organ co-cultures also implicate an unidentified signal from an adipose/hepatic-like tissue called the fat body. This study provides in vivo evidence that Slimfast amino-acid sensing and Target of rapamycin (TOR) signalling activate a fat-body-derived signal (FDS) required for neuroblast reactivation. Downstream of this signal, Insulin-like receptor signalling and the Phosphatidylinositol 3-kinase (PI3K)/TOR network are required in neuroblasts for exit from quiescence. Nutritionally regulated glial cells provide the source of Insulin-like peptides (ILPs) relevant for timely neuroblast reactivation but not for overall larval growth. Conversely, ILPs secreted into the haemolymph by median neurosecretory cells systemically control organismal size but do not reactivate neuroblasts. Drosophila thus contains two segregated ILP pools, one regulating proliferation within the central nervous system and the other controlling tissue growth systemically. These findings support a model in which amino acids trigger the cell cycle re-entry of neural progenitors via a fat-body-glia-neuroblasts relay. This mechanism indicates that dietary nutrients and remote organs, as well as local niches, are key regulators of transitions in stem-cell behaviour (Sousa-Nunes, 2011).

In fed larvae, Drosophila neuroblasts exit quiescence from the late first instar (L1) stage onwards. This reactivation involves cell enlargement and entry into S phase, monitored in this study using the thymidine analogue 5-ethynyl-2'-deoxyuridine (EdU). Reactivated neuroblast lineages (neuroblasts and their progeny) reproducibly incorporated EdU in a characteristic spatiotemporal sequence: central brain --> thoracic --> abdominal neuromeres. Mushroom-body neuroblasts and one ventrolateral neuroblast, however, are known not to undergo quiescence and to continue dividing for several days in the absence of dietary amino acids. This indicates that dietary amino acids are more than mere 'fuel', providing a specific signal that reactivates neuroblasts. However, explanted central nervous systems (CNSs) incubated with amino acids do not undergo neuroblast reactivation unless co-cultured with fat bodies from larvae raised on a diet containing amino acids. Therefore the in vivo requirement for a fat-body-derived signal (FDS) in neuroblast reactivation was tested by blocking vesicular trafficking and thus signalling from this organ using a dominant-negative Shibire dynamin (SHIDN). This strongly reduced neuroblast EdU incorporation, indicating that exit from quiescence in vivo requires an FDS. One candidate tested was Ilp6, known to be expressed by the fat body, but neither fat-body-specific overexpression nor RNA interference of this gene significantly affected neuroblast reactivation. Fat-body cells are known to sense amino acids via the cationic amino-acid transporter Slimfast (SLIF), which activates the TOR signalling pathway, in turn leading to the production of a systemic growth signal. Fat-body-specific overexpression of the TOR activator Ras homologue was shown to be enriched in brain (RHEB), or of an activated form of the p110 PI3K catalytic subunit, or of the p60 adaptor subunit, had no significant effect on neuroblast reactivation in fed animals or in larvae raised on a nutrient-restricted diet lacking amino acids. In contrast, global inactivation of Tor, fat-body-specific Slif knockdown or fat-body-specific expression of the TOR inhibitors Tuberous sclerosis complex 1 and 2 (Tsc1/2) all strongly reduced neuroblasts from exiting quiescence. Together, these results show that a SLIF/TOR-dependent FDS is required for neuroblasts to exit quiescence and that this may be equivalent to the FDS known to regulate larval growth (Sousa-Nunes, 2011).

Next, the signalling pathways essential within neuroblasts for their reactivation were investigated. Nutrient-dependent growth is regulated in many species by the interconnected TOR and PI3K pathways. In fed larvae, it was found that neuroblast inactivation of TOR signalling (by overexpression of TSC1/2), or PI3K signalling (by overexpression of p60, the Phosphatase and tensin homologue PTEN, the Forkhead box subgroup O transcription factor FOXO or dominant-negative p110), all inhibited reactivation. Conversely, stimulation of neuroblast TOR signalling (by overexpression of RHEB) or PI3K signalling [by overexpression of activated p110 or Phosphoinositide-dependent kinase 1 (PDK1)] triggered precocious exit from quiescence. RHEB overexpression had a particularly early effect, preventing some neuroblasts from undergoing quiescence even in newly hatched larvae. Hence, TOR/PI3K signalling in neuroblasts is required to trigger their timely exit from quiescence. Importantly, neuroblast overexpression of RHEB or activated p110 in nutrient-restricted larvae, which lack FDS activity, was sufficient to bypass the block to neuroblast reactivation. Notably, both genetic manipulations were even sufficient to reactivate neuroblasts in explanted CNSs, cultured without fat body or any other tissue. Together with the previous results this indicates that neuroblast TOR/PI3K signalling lies downstream of the amino-acid-dependent FDS during exit from quiescence (Sousa-Nunes, 2011).

To identify the mechanism bridging the FDS with neuroblast TOR/PI3K signalling, the role of the Insulin-like receptor (InR) in neuroblasts was tested. Importantly, a dominant-negative InR inhibited neuroblast reactivation, whereas an activated form stimulated premature exit from quiescence. Furthermore, InR activation was sufficient to bypass the nutrient restriction block to neuroblast reactivation. This indicates that at least one of the potential InR ligands, the seven ILPs, may be the neuroblast reactivating signal(s). By testing various combinations of targeted Ilp null alleles and genomic Ilp deficiencies, it was found that neuroblast reactivation was moderately delayed in larvae deficient for both Ilp2 and Ilp3 (Df(3L)Ilp2-3) or lacking Ilp6 activity. Stronger delays, as severe as those observed in InR31 mutants, were observed in larvae simultaneously lacking the activities of Ilp2, 3 and 5 [Df(3L)Ilp2-3, Ilp5] or Ilp1-5 [Df(3L)Ilp1-5]. Despite the developmental delay in Df(3L)Ilp1-5 homozygotes, neuroblast reactivation eventually begins in the normal spatial pattern -- albeit heterochronically -- in larvae with L3 morphology. Together, the genetic analysis shows that Ilp2, 3, 5 and 6 regulate the timing but not the spatial pattern of neuroblast exit from quiescence. However, as removal of some ILPs can induce compensatory regulation of others, the relative importance of each cannot be assessed from loss-of-function studies alone (Sousa-Nunes, 2011).

Brain median neurosecretory cells (mNSCs) are an important source of ILPs, secreted into the haemolymph in an FDS-dependent manner to regulate larval growth. They express Ilp1, 2, 3 and 5, although not all during the same development stages. However, this study found that none of the seven ILPs could reactivate neuroblasts during nutrient restriction when overexpressed in mNSCs. Similarly, increasing mNSC secretion using the NaChBac sodium channel or altering mNSC size using PI3K inhibitors/activators, which in turn alters body growth, did not significantly affect neuroblast reactivation under fed conditions. Surprisingly, therefore, mNSCs are not the relevant ILP source for neuroblast reactivation. Nonetheless, Ilp3 and Ilp6 messenger RNAs were detected in the CNS cortex, at the early L2 stage, in a domain distinct from the Ilp2+ mNSCs. Two different Ilp3-lacZ transgenes indicate that Ilp3 is expressed in some glia (Repo+ cells) and neurons (Elav+ cells). An Ilp6-GAL4 insertion indicates that Ilp6 is also expressed in glia, including the cortex glia surrounding neuroblasts and the glia of the blood-brain barrier (BBB) (Sousa-Nunes, 2011).

Next the ability of each of the seven ILPs to reactivate neuroblasts when overexpressed in glia or in neurons was assessed. Pan-glial or pan-neuronal overexpression of ILP4, 5 or 6 led to precocious reactivation under fed conditions. Each of these manipulations also bypassed the nutrient restriction block to neuroblast reactivation, as did overexpression of ILP2 in glia or in neurons, or ILP3 in neurons. In all of these ILP overexpressions, and even when ILP6 was expressed in the posterior Ultrabithorax domain, the temporal rather than the spatial pattern of reactivation was affected. Importantly, experiments blocking cell signalling with SHIDN indicate that glia rather than neurons are critical for neuroblast reactivation. Interestingly, glial-specific overexpression of ILP3-6 did not significantly alter larval mass. Thus, in contrast to mNSC-derived ILPs, glial-derived ILPs promote CNS growth without affecting body growth (Sousa-Nunes, 2011).

Focusing on ILP6, CNS explant cultures were used to demonstrate directly that glial overexpression was sufficient to substitute for the FDS during neuroblast exit from quiescence. In vivo, ILP6 was sufficient to induce reactivation during nutrient restriction when overexpressed via its own promoter or specifically in cortex glia but not in the subperineurial BBB glia, nor in many other CNS cells that were tested. Hence, cortex glia possess the appropriate processing machinery and/or location to deliver reactivating ILP6 to neuroblasts. Ilp6 mRNA is known to be upregulated rather than downregulated in the larval fat body during starvation and, accordingly, Ilp6-GAL4 activity is increased in this tissue after nutrient restriction. Conversely, it was found that Ilp6-GAL4 is strongly downregulated in CNS glia during nutrient restriction. Thus, dietary nutrients stimulate glia to express Ilp6 at the transcriptional level. Consistent with this, an important transducer of nutrient signals, the TOR/PI3K network, is necessary and sufficient in glia (but not in neurons) for neuroblast reactivation. Together, the genetic and expression analyses indicate that nutritionally regulated glia relay the FDS to quiescent neuroblasts via ILPs (Sousa-Nunes, 2011).

This study used an integrative physiology approach to identify the relay mechanism regulating a nutritional checkpoint in neural progenitors. A central feature of the fat-body --> glia --> neuroblasts relay model is that glial insulin signalling bridges the amino-acid/TOR-dependent fat-body-derived signal (FDS) with InR/PI3K/TOR signalling in neuroblasts. The importance of glial ILP signalling during neuroblast reactivation is also underscored by an independent study, published while this work was under revision (Chell, 2010). As TOR signalling is also required in neuroblasts and glia, direct amino-acid sensing by these cell types may also impinge upon the linear tissue relay. This would then constitute a feed-forward persistence detector, ensuring that neuroblasts exit quiescence only if high amino-acid levels are sustained rather than transient. This study also showed that the CNS 'compartment' in which glial ILPs promote growth is functionally isolated, perhaps by the BBB, from the systemic compartment where mNSC ILPs regulate the growth of other tissues. The existence of two functionally separate ILP pools may explain why bovine insulin cannot reactivate neuroblasts in CNS organ culture, despite being able to activate Drosophila InR in vitro. Given that insulin/PI3K/TOR signalling components are highly conserved between insects and vertebrates, it will be important to address whether mammalian adipose or hepatic tissues signal to glia and whether or not this involves an insulin/IGF relay to CNS progenitors. In this regard, it is intriguing that brain-specific overexpression of IGF1 can stimulate cell-cycle re-entry of mammalian cortical neural progenitors, indicating utilization of at least part of the mechanism identified by this study in Drosophila (Sousa-Nunes, 2011).

Cheng, L. Y., Bailey, A. P., Leevers, S. J., Ragan, T. J., Driscoll, P. C. and Gould, A. P. (2011). Anaplastic lymphoma kinase spares organ growth during nutrient restriction in Drosophila. Cell 146: 435-447. PubMed ID: 21816278

Shi, P., Lai, R., Lin, Q., Iqbal, A. S., Young, L. C., Kwak, L. W., Ford, R. J. and Amin, H. M. (2009). IGF-IR tyrosine kinase interacts with NPM-ALK oncogene to induce survival of T-cell ALK+ anaplastic large-cell lymphoma cells. Blood 114: 360-370. PubMed ID: 19423729

Sousa-Nunes, R., Yee, L. L. and Gould, A. P. (2011). Fat cells reactivate quiescent neuroblasts via TOR and glial insulin relays in Drosophila. Nature 471: 508-512. PubMed ID: 21346761

Anaplastic lymphoma kinase spares organ growth during nutrient restriction in Drosophila

Developing animals survive periods of starvation by protecting the growth of critical organs at the expense of other tissues. This study used Drosophila to explore the as yet unknown mechanisms regulating this privileged tissue growth. As in mammals, it was observed in Drosophila that the CNS is more highly spared than other tissues during nutrient restriction (NR). Anaplastic lymphoma kinase (Alk) efficiently protects neural progenitor (neuroblast) growth against reductions in amino acids and insulin-like peptides during NR via two mechanisms. First, Alk suppresses the growth requirement for amino acid sensing via Slimfast/Rheb/TOR complex 1. And second, Alk, rather than insulin-like receptor, primarily activates PI3-kinase. Alk maintains PI3-kinase signaling during NR as its ligand, Jelly belly (Jeb), is constitutively expressed from a glial cell niche surrounding neuroblasts. Together, these findings identify a brain-sparing mechanism that shares some regulatory features with the starvation-resistant growth programs of mammalian tumors (Cheng, 2011).

This study found that CNS progenitors are able to continue growing at their normal rate under fasting conditions severe enough to shut down all net body growth. Jeb/Alk signaling was identified as a central regulator of this brain sparing, promoting tissue-specific modifications in TOR/PI3K signaling that protect growth against reduced amino acid and Ilp concentrations. These findings highlight that a 'one size fits all' wiring diagram of the TOR/PI3K network should not be extrapolated between different cell types without experimental evidence. The two molecular mechanisms by which Jeb/Alk signaling confers brain sparing is discussed, and how they may be integrated into an overall model for starvation-resistant CNS growth (Cheng, 2011).

One mechanism by which Alk spares the CNS is by suppressing the growth requirement for amino acid sensing via Slif, Rheb, and TORC1 components in neuroblast lineages. An important finding of this study is that in the presence of Alk signaling Tor has no detectable growth input (evidence from Tor clones), but in its absence (evidence from UAS-AlkDN; Tor clones) Tor reverts to its typical role as a positive regulator of both growth and proliferation. The growth requirement for Slif/TORC1 is clearly much less in the CNS than in other tissues such as the wing disc but a low-level input cannot be ruled out due to possible perdurance inherent in any clonal analysis. Although Slif, Rheb, Tor, and Raptor mutant neuroblast clones attain normal volume, this reflects increased cell numbers offset by reduced average cell size. Atypical suppression of proliferation by TORC1 has also been observed in wing discs, where partial inhibition with rapamycin advances G2/M progression (Cheng, 2011).

Alk signaling in neuroblast lineages does not override the growth requirements for all TOR pathway components. The downstream effectors S6k and 4E-BP retain functions as positive and negative growth regulators, respectively. 4E-BP appears to be particularly critical in the CNS as mutant animals have normal mass, but mutant neuroblast clones are twice their normal volume. In many tissues, 4E-BP is phosphorylated by nutrient-dependent TORC1 activity. In CNS progenitors, however, 4E-BP phosphorylation is regulated in an NR-resistant manner by Alk, not by TORC1. Hence, although the pathway linking Alk to 4E-BP is not yet clear, it makes an important contribution toward protecting CNS growth during fasting (Cheng, 2011).

A second mechanism by which Alk spares CNS growth is by maintaining PI3K signaling during NR. Alk suppresses or overrides the genetic requirement for InR in PI3K signaling, which may or may not involve the direct binding of intracellular domains as reported for human ALK and IGF-IR (Shi, 2009). Either way, in the CNS, glial Jeb expression stimulates Alk-dependent PI3K signaling and thus neural growth at similar levels during feeding and NR. In contrast, in tissues such as the salivary gland, where PI3K signaling is primarily dependent upon InR, falling insulin-like peptides concentrations during NR strongly reduce growth (Cheng, 2011).

The finding that Alk signals via PI3K during CNS growth differs from the Ras/MAPK transduction pathway described in Drosophila visceral muscle. However, a link between ALK and PI3K/Akt/Foxo signaling during growth is well documented in humans, both in glioblastomas and in non-Hodgkin lymphoma. Similarities with mammals are less obvious with regard to Alk ligands, as there is no clear Jeb ortholog and human ALK can be activated, directly or indirectly, by the secreted factors Pleiotrophin and Midkine (Cheng, 2011).

A comparison of these results with those of previous studies indicates that CNS super sparing only becomes fully active at late larval stages. Earlier in larval life, dietary amino acids are essential for neuroblasts to re-enter the cell cycle after a period of quiescence. This nutrient-dependent reactivation involves a relay whereby Slif-dependent amino acid sensing in the fat body stimulates Ilp production from a glial cell niche (Sousa-Nunes, 2011). In turn, glial-derived Ilps activate InR and PI3K/TOR signaling in neuroblasts thus stimulating cell cycle re-entry. Hence, the relative importance of Ilps versus Jeb from the glial cell niche may change in line with the developmental transition of neuroblast growth from high to low nutrient sensitivity (Cheng, 2011).

The results of this study suggest a working model for super sparing in the late-larval CNS. Central to the model is that Jeb/Alk signaling suppresses Slif/ RagA/Rheb/TORC1, InR, and 4E-BP functions and maintains S6k and PI3K activation, thus freeing CNS growth from the high dependence upon amino acid sensing and Ilps that exists in other organs. The CNS also contrasts with other spared diploid tissues such as the wing disc, in which PI3K-dependent growth requires a positive Tor input but is kept in check by negative feedback from TORC1 and S6K. Alk is both necessary (in the CNS) and sufficient (in the salivary gland) to promote organ growth during fasting. However, both Alk manipulations produce organ-sparing percentages intermediate between the 2% salivary gland and the 96% neuroblast values, arguing that other processes may also contribute. For example, some Drosophila tissues synthesize local sources of Ilps that could be more NR resistant than the systemic supply from the IPCs. In mammals, this type of mechanism may contribute to brain sparing as it has been observed that IGF-I messenger RNA (mRNA) levels in the postnatal CNS are highly buffered against NR. It will also be worthwhile exploring whether mammalian neural growth and brain sparing involve Alk and/or atypical TOR signaling. In this regard, it is intriguing that several studies show that activating mutations within the kinase domain of human ALK are associated with childhood neuroblastomas. In addition, fetal growth of the mouse brain was recently reported to be resistant to loss of function of TORC1. Finally, a comparison between the current findings and those of a cancer study, highlights that insulin/IGF independence and constitutive PI3K activity are features of NR-resistant growth in contexts as diverse as insect CNS development and human tumorigenesis (Cheng, 2011).

Novel male-biased expression in paralogs of the aphid slimfast nutrient amino acid transporter expansion

A major goal of molecular evolutionary biology is to understand the fate and consequences of duplicated genes. In this context, aphids are intriguing because the newly sequenced pea aphid genome harbors an extraordinary number of lineage-specific gene duplications relative to other insect genomes (see Phylogeny of the slimfast gene family). Though many of their duplicated genes may be involved in their complex life cycle, duplications in nutrient amino acid transporters appear to be associated rather with their essential amino acid poor diet and the intracellular symbiosis aphids rely on to compensate for dietary deficits. Past work has shown that some duplicated amino acid transporters are highly expressed in the specialized cells housing the symbionts, including a paralog of an aphid-specific expansion homologous to the Drosophila gene slimfast. Previous data provide evidence that these bacteriocyte-expressed transporters mediate amino acid exchange between aphids and their symbionts. This study reports that some nutrient amino acid transporters show male-biased expression. Male-biased expression characterizes three paralogs in the aphid-specific slimfast expansion, and the male-biased expression is conserved across two aphid species for at least two paralogs. One of the male-biased paralogs has additionally experienced an accelerated rate of non-synonymous substitutions. This is the first study to document male-biased slimfast expression. The data suggest that the male-biased aphid slimfast paralogs diverged from their ancestral function to fill a functional role in males. Furthermore, the results provide evidence that members of the slimfast expansion are maintained in the aphid genome not only for the previously hypothesized role in mediating amino acid exchange between the symbiotic partners, but also for sex-specific roles (Duncan, 2011).


Search PubMed for articles about Drosophila Slimfast

Britton, J. S. and Edgar, B. A. (1998). Environmental control of the cell cycle in Drosophila: nutrition activates mitotic and endoreplicative cells by distinct mechanisms. Development 125: 2149-2158. PubMed ID: 9570778

Britton, J. S., Lockwood, W. K., Li, L., Cohen, S. M. and Edgar, B. A. (2002). Drosophila's insulin/PI3-kinase pathway coordinates cellular metabolism with nutritional conditions. Dev. Cell 2: 239-249. PubMed ID: 11832249

Chell, J. M. and Brand, A. H. (2010). Nutrition-responsive glia control exit of neural stem cells from quiescence. Cell 143: 1161-1173. PubMed ID: 21183078

Colombani, J., et al. (2003). A nutrient sensor mechanism controls Drosophila growth. Cell 114: 739-749. PubMed ID: 14505573

Duncan, R. P., Nathanson, L. and Wilson, A. C. C. (2011). Novel male-biased expression in paralogs of the aphid slimfast nutrient amino acid transporter expansion. BMC Evol. Biol. 11: 253. PubMed ID: 21917168

Gutierrez, E., Wiggins, D., Fielding, B. and Gould, A. P. (2007). Specialized hepatocyte-like cells regulate Drosophila lipid metabolism. Nature 445(7125): 275-80. PubMed ID: 17136098

Hennig, K. M., Colombani, J. and Neufeld, T. P. (2006). TOR coordinates bulk and targeted endocytosis in the Drosophila melanogaster fat body to regulate cell growth. J. Cell Biol. 173(6): 963-74. PubMed ID: 16785324

Kawamura, K., et al. (1999). A new family of growth factors produced by the fat body and active on Drosophila imaginal disc cells. Development 126: 211-219. PubMed ID: 9847235

Martin, J. F., Hersperger, E., Simcox, A. and Shearn, A. (2000). minidiscs encodes a putative amino acid transporter subunit required non-autonomously for imaginal cell proliferation Mech. Dev. 92: 155-167. PubMed ID: 10727855

Sousa-Nunes, R., Yee, L. L. and Gould, A. P. (2011). Fat cells reactivate quiescent neuroblasts via TOR and glial insulin relays in Drosophila. Nature 471(7339): 508-12. PubMed ID: 21346761

Zinke, I., et al. (1999). Suppression of food intake and growth by amino acids in Drosophila: the role of pumpless, a fat body expressed gene with homology to vertebrate glycine cleavage system. Development 126(23): 5275-84. PubMed ID: 10556053

Biological Overview

date revised: 20 April 2012

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