The Interactive Fly
Genes involved in tissue and organ development
The somatic muscles, the heart, the fat body, the somatic part of the gonad and most of the visceral muscles are derived from a series of segmentally repeated primordia in the Drosophila mesoderm. This work describes the early development of the fat body and its relationship to the gonadal mesoderm, as well as the genetic control of the development of these tissues. The first sign of fat body development is the expression of serpent in segmentally repeated clusters within the trunk mesoderm in parasegments 4-9. Segmentation and dorsoventral patterning genes define three regions in each parasegment in which fat body precursors can develop. The primary and secondary dorsolateral fat body primordia are formed ventral to the visceral muscle primoridium in each parasegment. The ventral secondary cluster forms more ventrally in the posterior portion of each parasegment. Fat body progenitors in these regions are specified by different genetic pathways. Two dorsolateral regions require engrailed and hedgehog (within the even-skipped domain) for their development while the ventral secondary cluster is controlled by wingless. Ubiquitous mesodermal en expression leads to an expansion of the primary clusters into the sloppy-paired domain, resulting in a continuous band of serpent-expressing cells in parasegments 4-9. The observed effect of en on fat body development is seen not only on mesodermal overexpression but also when en is overexpressed in the ectoderm. Loss of wingless leads to an expansion of the dorsolateral fat body primordium. decapentaplegic and one or more unknown genes determine the dorsoventral extent of these regions. High levels of Dpp repress serpent, resulting in the formation of visceral musculature, an alternative cell fate (Reichmann, 1998).
In each of parasegments 10-12 one of these primary dorsolateral regions generates somatic gonadal precursors instead of fat body. The balance between fat body and somatic gonadal fate in these serially homologous cell clusters is controlled by at least five genes. A model is suggested in which tinman, engrailed and wingless are necessary to permit somatic gonadal develoment, while serpent counteracts the effects of these genes and promotes fat body development. In wg mutant embryos, all dorsolateral mesodermal cells, including those in parasegments 10-12, acquire fat body fate. This phenotype can be interpreted as the combined effects of two separate functions of wg: (1) wg is necessary to repress fat body development in the dorsolateral mesoderm underlying the wg domain in all parasegments; (2) wg is required in the primary cluster to permit somatic gonadal precursor instead of fat body development in parasegments 10-12. Loss of engrailed results in the absence of demonstrable somatic gonadal precursors, similar to the situation in tinman mutants. Ubiquitous mesodermal en expression leads to the formation of additional somatic gonadal precursor cells in parasegments 10-12. The homeotic gene abdominalA limits the region of serpent activity by interfering in a mutually repressive feed back loop between gonadal and fat body development. It is unlikely that abdA represses srp directly, since srp can be expressed in cells in which abdA is active. abdA might prevent srp from inhibition of a somatic gonadal precursor competence factor (Riechmann, 1998).
In newly hatched Drosophila larvae, quiescent cells reenter the cell cycle in response to dietary amino acids. To understand this process, larval nutrition was varied and effects on cell cycle initiation and maintenance were monitored in the mitotic neuroblasts and imaginal disc cells, as well as the endoreplicating cells in other larval tissues. After cell cycle activation, mitotic and endoreplicating cells respond differently to the withdrawal of nutrition: mitotic cells continue to proliferate in a nutrition-independent manner, while most endoreplicating cells reenter a quiescent state. Ectopic expression of Drosophila Cyclin E or the E2F transcription factor can drive quiescent endoreplicating cells, but not quiescent imaginal neuroblasts, into S-phase. Conversely, quiescent imaginal neuroblasts, but not quiescent endoreplicating cells, can be induced to enter the cell cycle when co-cultured with larval fat body in vitro. These results demonstrate a fundamental difference in the control of cell cycle activation and maintenance in these two cell types, and imply the existence of a novel mitogen generated by the larval fat body in response to nutrition (Britton, 1998).
These results suggest that multiple pathways are involved in regulating the onset of cell proliferation in different tissue types in response to the global nutritional cue. Mitotic and endoreplicating cell cycles are regulated differently in response to the nutritional state: the endoreplicating tissues (ERTs) require continuous nutrition to cycle, whereas the mitotic cells cycle in a nutrition-independent manner once activated. In addition, the mechanism of cell cycle arrest in the two types of quiescent cells is different: quiescent ERTs can be driven into S-phase by ectopic expression of either of the G1/S regulators E2F or Cyclin E, while neither of these regulators can induce quiescent neuroblasts to enter S-phase.Conversely, quiescent neuroblasts but not quiescent ERTs are induced to reenter the cell cycle in response to a mitogen produced by the larval fat body (Britton, 1998).
The differential responses of the mitotic and endoreplicative cell cycles to nutrient withdrawal may provide an important mechanism for survival of the organism and reproduction in the face of food shortages in the wild. When nutrients become limiting, available resources can be dedicated to maintaining growth and proliferation in the mitotic tissues which are required to form the reproductive adult. Indeed, larvae are capable of pupating at a much smaller size than they normally do. A 'critical size' has been defined at which larvae are able to pupariate without further feeding. The small pupae which are formed by these larvae produce normal, fertile, but small adult flies (Britton, 1998).
Embryonic neuroblasts have an intrinsic program of cell proliferation. Each type of neuroblast has a specific identity, expresses unique and dynamic combinations of sublineage genes, and will give rise to a precise number and type of progeny before exiting the cell cycle. Interestingly, temporal control of sublineage gene expression in embryonic neuroblasts can be independent of cell cycle progression. Thus arresting a proliferating neuroblast in mid-lineage could lead to the desynchronization of sublineage gene expression and the loss of certain types of progeny, a result which could have disastrous consequences for the developing CNS (Britton, 1998).
In a food withdrawal experiment it was observed that many activated neuroblasts continued to proliferate for up to 7 days after food withdrawal, however a subset of them did not. This observation was most striking in the abdominal region of the VNC. The abdominal neuroblast lineages are much shorter than those of the majority of brain and thoracic neuroblasts, with a single abdominal neuroblast producing as few as four neurons during its postembryonic period of proliferation. Since the abdominal neuroblasts generally complete their entire larval program of proliferation in less than 2 days, it is not surprising that after 7 days of culture on sucrose the majority of these neuroblasts have exited the cell cycle. It is suspected that the reduction in labeled neuroblasts observed in all regions of the CNS over the course of this experiment is due to a subset of neuroblasts completing their intrinsic program of proliferation and exiting the cell cycle (Britton, 1998).
The insect fat body is the source of the majority of hemolymph proteins, including lipid binding proteins, juvenile hormone binding proteins and esterases, peptides which mediate the insect immune response, and vitellogenins involved in oocyte maturation in the adult female. The fat body is also responsible for synthesizing the stores of protein, lipid and glycogen which sustain the animal throughout metamorphosis. Ultrastructurally, the fat body shows a dramatic response to starvation. In Calpodes larvae, starvation leads to a rapid reorganization of the fat body including loss of mitochondria and rough endoplasmic reticulum (RER) by autophagy and depletion of stored metabolites. Refeeding induces mitochondrial divisions andincreases in RER content as well as the eventual replenishment of depleted stores. This study observed dramatic changes in the larval fat body in the course of starvation experiments, including a loss of tissue cohesion and changes in opacity. These changes probably reflect the alteration in composition the fat body cells undergo as stores of metabolites are mobilized to support proliferating mitotic tissues during starvation (Britton, 1998).
Previous studies have demonstrated that the adult female fat body is able to regulate yolk gene transcription in response to the nutritional environment. Interestingly, there is evidence that a component of the adult female abdomen is also capable of supporting the proliferation of larval tissues in a nutrition dependent manner. It has been demonstrated that the proliferation of imaginal disc fragments transplanted into the abdominal cavity of adult female hosts is dependent onnutrition. This study has found that when quiescent central nervous systems from starved larvae are transplanted into the abdomens of fed adult female hosts, larval neuroblasts reenter the cell cycle in what appears to be a normal spatiotemporal pattern. An appealing hypothesis is that production of the neuroblast mitogen in the fat body is regulated at the transcriptional level under the control of nutritional enhancers similar to those identified in the regions upstream of yolk protein genes. The ability of something in the adult female abdomen to activate proliferation in quiescent neuroblasts suggests that similar fat body-derived mitogens are produced in the larval and adult female fat bodies. This adult mitogen could have a role in controlling proliferation in the adult, perhaps functioning to regulate some oogenic process in response to the nutritional state. Indeed, oogenesis is inhibited in adult females fed on sucrose (Britton, 1998).
The dramatic response of the fat body to starvation, the demonstration that there is a mechanism for nutritional controlof transcription in adult female fat body, and the similar abilities of the adult female abdomen and the larval fat body to support nutrition-dependent cell cycle activation lend support to the proposal that the fat body is responsible for mediating the nutritional response in larval neuroblasts. The results of co-culture experiment demonstrate that the fat body supplies a diffusible factor which stimulates larval neuroblasts to enter the cell cycle (Britton, 1998).
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 (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. 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. 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 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, 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. 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).
In response to starvation, eukaryotic cells recover nutrients through autophagy, a lysosomal-mediated process of cytoplasmic degradation. Autophagy is known to be inhibited by TOR signaling, but the mechanisms of autophagy regulation and its role in TOR-mediated cell growth are unclear. Signaling through TOR and its upstream regulators PI3K and Rheb is necessary and sufficient to suppress starvation-induced autophagy in the Drosophila fat body. In contrast, TOR's downstream effector S6K promotes rather than suppresses autophagy, suggesting S6K downregulation may limit autophagy during extended starvation. Despite the catabolic potential of autophagy, disruption of conserved components of the autophagic machinery, including ATG1 and ATG5, does not restore growth to TOR mutant cells. Instead, inhibition of autophagy enhances TOR mutant phenotypes, including reduced cell size, growth rate, and survival. Thus, in cells lacking TOR, autophagy plays a protective role that is dominant over its potential role as a growth suppressor (Scott, 2004).
Autophagy likely evolved in single-cell eukaryotes to provide an energy and nutrient source allowing temporary survival of starvation. In yeast, Tor1 and Tor2 act as direct links between nutrient conditions and cell metabolism. These proteins sense nutritional status by an unknown mechanism, and effect a variety of starvation responses including changes in transcriptional and translational programs, nutrient import, protein and mRNA stability, cell cycle arrest, and induction of autophagy. Autophagy thus occurs in the context of a comprehensive reorganization of cellular activities aimed at surviving low nutrient levels (Scott, 2004).
In multicellular organisms, TOR is thought to have retained its role as a nutrient sensor but has also adopted new functions in regulating and responding to growth factor signaling pathways and developmental programs. Thus in a variety of signaling, developmental, and disease contexts, TOR activity can be regulated independently of nutritional conditions. In these cases, autophagy may be induced in response to downregulation of TOR despite the presence of abundant nutrients and may potentially play an important role in suppressing cell growth rather than promoting survival. Identification of the tumor suppressors PTEN, and TSC1 and TSC2 as positive regulators of autophagy provides correlative evidence supporting such a role for autophagy in growth control. Alternatively, since TOR activity is required for proper expression and localization of a number of nutrient transporters, inactivation of TOR may lead to reduced intracellular nutrient levels, and autophagy may therefore be required under these conditions to provide the nutrients and energy necessary for normal cell metabolism and survival (Scott, 2004).
The results presented here provide genetic evidence that under conditions of low TOR signaling, autophagy functions primarily to promote normal cell function and survival, rather than to suppress cell growth. This conclusion is based on the finding that genetic disruption of autophagy does not restore growth to cells lacking TOR, but instead exacerbates multiple TOR mutant phenotypes. It is important to note that mutations in TOR do not disrupt larval feeding, and thus disruption of autophagy is detrimental in TOR mutants despite the presence of ample extracellular nutrients. The finding that autophagy is critical in cells lacking TOR further supports earlier studies suggesting that inactivation of TOR causes defects in nutrient import, resulting in an intracellular state of pseudo-starvation (Scott, 2004).
Can the further reduction in growth of TOR mutant cells upon disruption of autophagy be reconciled with the potential catabolic effects of autophagy? TOR regulates the bidirectional flow of nutrients between protein synthesis and degradation through effects on nutrient import, autophagy, and ribosome biogenesis. When TOR is inactivated, rates of nutrient import and protein synthesis decrease, resulting in a commensurate reduction in mass accumulation and cell growth. In addition, autophagy is induced to maintain intracellular nutrient and energy levels sufficient for normal cell metabolism. When autophagy is experimentally inhibited in cells lacking TOR, this reserve source of nutrients is blocked, leading to a further decrease in energy levels, protein synthesis, and growth. It is noted that autophagy may have additional functions in cells with depressed TOR signaling, including recycling of organelles damaged by the absence of TOR activity, or selective degradation of cell growth regulators, analogous to the regulatory roles of ubiquitin-mediated degradation (Scott, 2004).
Autophagy is required for normal developmental responses to inactivation of insulin/PI3K signaling in the nematode C. elegans. In response to starvation or disruption of insulin/PI3K signaling, C. elegans larvae enter a dormant state called the dauer. Autophagy has been observed in C. elegans larvae undergoing dauer formation: disruption of a number of ATG homologs interfers with normal dauer morphogenesis. Importantly, simultaneous disruption of insulin/PI3K signaling and autophagy genes results in lethality, similar to the results presented in this study. Thus despite significant differences in developmental strategies for surviving nutrient deprivation, autophagy plays an essential role in the starvation responses of yeast, flies, and worms (Scott, 2004).
The prevailing view that S6K acts to suppress autophagy was founded on correlations between induction of autophagy and dephosphorylation of rpS6 in response to amino acid deprivation or rapamycin treatment. However, the genetic data presented in this study argue strongly against a role for S6K in suppressing autophagy: unlike other positive components of the TOR pathway, null mutations in S6K do not induce autophagy in fed animals. It is suggested that the observed correlation between S6K activity and suppression of autophagy is due to common but independent regulation of S6K and autophagy by TOR. Thus, autophagy suppression and S6K-dependent functions such as ribosome biogenesis represent distinct outputs of TOR signaling (Scott, 2004).
How might TOR signal to the autophagic machinery, if not through S6K? In yeast, this is accomplished in part through regulation of Atg1 kinase activity and ATG8 gene expression (Kamada, 2000 and Kirisako, 1999). The demonstration of a role for Drosophila ATG1 and ATG8 homologs [see TG8a (CG32672) and ATG8b (CG12334)] in starvation-induced autophagy, and the genetic interaction observed between ATG1 and TOR, are consistent with a related mode of regulation in higher eukaryotes. However, it is noted that other components of the yeast Atg1 complex such as Atg17 and Atg13, whose phosphorylation state is rapamycin sensitive, do not have clear homologs in metazoans, indicating that differences in regulation of autophagy by TOR are likely (Scott, 2004).
In addition to excluding a role for S6K in suppression of autophagy, these results reveal a positive role for S6K in induction of autophagy. S6K may promote autophagy directly, through activation of the autophagy machinery, or indirectly through its effects on protein synthesis. The latter possibility is consistent with previous reports that protein synthesis is required for expansion and maturation of autophagosomes. Interestingly, despite being required for autophagy, S6K is downregulated under conditions that induce it, including chronic starvation and TOR inactivation. Consistent with this, it was found that lysotracker staining is significantly weaker in chronically starved animals or in TOR mutants than in wild-type animals starved 3-4 hr. Furthermore, expression of constitutively activated S6K has no effect in wild-type, but restores lysotracker staining in TOR mutants to levels similar to those of acutely starved wild-type animals. It is suggested that downregulation of S6K may limit rates of autophagy under conditions of extended starvation or TOR inactivation and that this may protect cells from the potentially damaging effects of unrestrained autophagy (Scott, 2004).
Co-culture and conditioned media experiments have shown that the Drosophila fat body is a source of diffusible mitogens. The fat body has also been shown to act as a nutrient sensor through a TOR-dependent mechanism and to regulate organismal growth through effects on insulin/PI3K signaling. The results in this study extend these findings by showing that this endocrine response is accompanied by the regulated release of nutrients through autophagic degradation of fat body cytoplasm. Preventing this reallocation of resources, either through constitutive activation of PI3K or through inactivation of ATG genes, results in profound nutrient sensitivity. Thus, in response to nutrient limitation, the fat body is capable of simultaneously restricting growth of peripheral tissues through downregulation of insulin/PI3K signaling and providing these tissues with a buffering source of nutrients necessary for survival through autophagy (Scott, 2004).
Energy homeostasis, a fundamental property of all organisms, depends on the ability to control the storage and mobilization of fat, mainly triacylglycerols (TAG), in special organs such as mammalian adipose tissue or the fat body of flies. Malregulation of energy homeostasis underlies the pathogenesis of obesity in mammals including human. A screen was performed to identify nutritionally regulated genes that control energy storage in the Drosophila. The brummer (bmm) gene encodes the lipid storage droplet-associated TAG lipase Brummer, a homolog of adipocyte triglyceride lipase (ATGL). Food deprivation or chronic bmm overexpression depletes organismal fat stores in vivo, whereas loss of bmm activity causes obesity in flies. These study identifies a key factor of insect energy homeostasis control. Their evolutionary conservation suggests Brummer/ATGL family members to be implicated in human obesity and establishes a basis for modeling mechanistic and therapeutic aspects of this disease in the fly (Grönke, 2005).
Providing constant energy supply despite variability in food access and metabolic energy demand is a fundamental property of animals. Key to an individual's survival during food deprivation is the ability to mobilize stored energy resources accumulated during periods of excessive energy supply. In organisms as different as humans and the fruit fly Drosophila, energy-rich diet components are converted into glycogen and, to a larger extent, triacylglycerols (TAG), the storage forms of carbohydrate and fat, respectively. Storage fat is deposited in intracellular lipid droplets of specialized organs called the adipose tissue in mammals or the fat body in Drosophila. In mammals, adipose tissue cooperates with the digestive tract and the central nervous system to hardwire an integrated molecular communication network ensuring the lifelong integrity of an organism's energy homeostasis under varying environmental conditions. In the peripheral fat storage tissue, a regulated balance between lipogenesis and lipolysis is believed to continuously match acute energy needs by TAG mobilization and readjust organismal storage fat content to a genetically determined setpoint during periods of excessive energy supply. Chronic imbalance of energy storage control by the lack or malfunction of regulatory genes results in excessive fat accumulation and is causative to the obesity pandemics in human populations as well as to related phenotypes in rodent models (Grönke, 2005).
A key regulator of storage fat lipolysis in mammalian adipocytes is the hormone-sensitive lipase (HSL). Activated HSL interacts with perilipin at the lipid droplet membrane to eventually mobilize TAG. Acute HSL activation relies on posttranslational modification by protein kinase A (PKA) in response to hormonal β-adrenoceptor stimulation and subsequent activation of the cAMP second messenger signaling pathway. In addition, extensive fasting causes upregulation of mouse HSL mRNA and protein, supporting the enzyme's importance in acute and chronic TAG mobilization control. However, HSL knockout mice are viable and not obese, having substantial residual lipolytic activity. Accordingly, additional TAG lipases of the nutrin family, such as the most recently identified human adipose triglyceride lipase (ATGL), have been implicated in mammalian lipolysis. However, the in vivo relevance of this lipase family in fat storage control on the organismal level waits to be analyzed (Grönke, 2005 and references therein).
In insects, storage fat lipolysis is stimulated by adipokinetic hormone (AKH) in various species including the grasshopper Locusta migratoria, the tobacco hornworm Manduca sexta, and Drosophila, suggesting a general role in insect energy balance control. Like in mammalian TAG mobilization, AKH-stimulated lipolysis in the insect fat body relies on signaling via a G protein-coupled receptor (see Drosophila Gonadotropin-releasing hormone receptor/Adipokinetic hormone receptor), increase in intracellular cAMP, and activation of PKA. An insect TAG lipase, however, which makes the storage fat metabolically accessible for the energy-demanding target tissues, is currently unknown (Grönke, 2005).
Given the intriguing similarities in the regulatory mechanisms of TAG mobilization between mammals and insects, a genome-wide transcriptome profiling was performed in Drosophila to screen for nutritionally regulated and evolutionary conserved lipolysis effectors. This study presents the functional in vivo analysis of brummer, which encodes a TAG lipase of the nutrin family, whose lack causes obesity in the fly (Grönke, 2005).
To screen for nutritionally regulated genes, a genome-wide transcriptome analysis was performed, comparing gene expression of fed and food-deprived adult Drosophila flies. Sorting the total of 223 starvation-responsive genes according to their predicted function reveals that most of the starvation-induced genes are coding for metabolic enzymes (n = 44). In addition, genes coding for cytochromes (n = 10), metabolite transporters (n = 6), kinases (n = 5), and proteins involved in lipid metabolism (n = 7) are upregulated under starvation. Few metabolic enzymes (n = 7) are downregulated in response to starvation, whereas proteases and protease inhibitors form the largest group (n = 38). Nearly half of the starvation-induced metabolic enzymes are involved in carbohydrate catabolism, including key regulators like hexokinase (encoded by Hex-C), transketolase (CG8036), and phosphoglucomutase (Pgm) or enzymes involved in the breakdown of sugars like an α-Amylase (AmyD), a α-Glucosidase (CG11909), and six maltases (CG11669, CG8690, CG30359, CG30360, CG14934, CG14935). Protein degradation is reflected by the upregulation of genes involved in amino acid catabolism, including two aminotransferases encoded by got2 and spat, a phenylalanine-4-monooxygenase (henna), a 4-hydroxyphenylpyruvate-dioxygenase (CG11796), and a homogentisate-1,2-dioxygenase (hgo). The starvation-induced metabolic activation is further reflected by the transcriptional upregulation of five regulatory kinases or kinase subunits, which have all been implicated in energy homeostasis control. While the pyruvate dehydrogenase kinase encoded by pdk is critical for the regulation of oxidative glucose metabolism, the β subunit of the SNF1/AMP-activated protein kinase (AMPK) acts as a cellular energy sensor, and the cAMP-activated protein kinase A (PKA) promotes glycogen and TAG catabolism. The SNF4 γ subunit loechrig has been implicated in cholesterol homeostasis control. In addition, Lk6 kinase mutants have recently been described to have increased organismal TAG content, suggesting a function of the kinase in the control of organismal lipid storage (Grönke, 2005 and references therein).
Among the seven upregulated genes involved in lipid metabolism are genes encoding a putative TAG-lipase (CG5966), phospholipase A2 (CG1583), low-density lipoprotein receptor (LpR2), long chain fatty acid CoA ligase (CG9009), and carnitine-O-palmitoyltransferase (CPTI). Anabolic reactions of the lipid metabolism are repressed under starvation, as indicated by the transcriptional downregulation of a lipogenic 1-acylglycerol-3-P-O-acyltransferase (CG4753) and a long chain fatty acid elongase (CG6261). Moreover, the PAT domain containing lipid storage droplet-associated protein Lsd-1 and three TAG lipases are among the nine genes involved in lipid metabolism that are downregulated in response to starvation (Grönke, 2005).
Taken together, genome-wide transcriptome profiling of fed versus food-deprived flies displays various regulatory aspects of the metabolic starvation response in Drosophila, including carbohydrate, amino acid, and lipid catabolism. However, no function has been assigned to 25% of the 223 starvation-responsive genes. Among those, the gene CG5295 was found in region 70F5 on chromosome 3L, termed brummer (bmm), is upregulated upon starvation. The single bmm transcript, which encodes a 507 amino-acid-long protein (BMM) closely related to TTS-2/ATGL of mouse and human, is expressed during all ontogenetic stages of the fly. It is highly enriched in the energy storage tissue as well as the food-absorbing parts of the digestive tract, i.e., the larval midgut and gastric caeca. Quantitative Northern blot analysis confirms sustained transcriptional upregulation in response to food-deprivation and downregulation upon refeeding. The nutritional regulation and the patterns of bmm expression suggest that bmm participates in the control of energy homeostasis (Grönke, 2005).
BMM contains a patatin-like domain (PLD) including a serine hydrolase motif, originally described in plant acyl-hydrolases, and a so-called Brummer box (BB) of unknown function. The BB motif is found in a number of PLD-containing proteins, which are refered to as the Brummer/Nutrin subfamily. It includes the Anopheles BMM ortholog, a Drosophila paralogue called doppelgänger von brummer (dob; CG5560), the human proteins Adiponutrin, GS2-like, TTS-2/ATGL and GS2, Caenorhabditis elegans C05D11.7 and D1054.1 as well as Arabidopsis NP_174597 (Grönke, 2005).
PLD proteins are phospholipases in plants, human and Pseudomonas or TAG lipases, as recently shown for the human Brummer/Nutrin family members TTS-2.2/ATGL, GS2, and Adiponutrin. Recombinant BMM exhibits esterase activity on an esterified fatty acid (6,8-difluoro-4-methylumbelliferyl octanoate) as substrate but fails to catalyze the release of fatty acid from either the A2 position of a phospholipid (PAP), the glycosylphosphatidylinositol (GPI) membrane glycolipid membrane anchor of GPI-modified proteins (5′-nucleotidase, Gce1), or monoacylglycerol (MAG). However, it cleaves TAG in vitro, whereas the BMMS38A mutant, in which serine residue 38 of the catalytic center had been replaced by alanine, is enzymatically inactive. Thus, bmm as its mammalian homologs are candidates for nutritionally regulated in vivo effectors of TAG mobilization (Grönke, 2005).
To test whether bmm promotes fat mobilization in vivo, bmm loss-of-function mutant alleles (bmm1 and bmm2) were generated by mobilization of a transposable P element located in the first exon of bmm. Precise excision of the P element, as obtained with bmmrev, served as genetically matched control for phenotypic analysis. Embryos lacking both maternal and zygotic bmm activity are lethal, indicating that bmm carries an essential function. They develop pleiotropic degeneration phenotypes and have increased TAG levels in late embryogenesis. Embryonic lethality can be partially rescued by a paternally provided functional bmm gene and almost completely reverted by ubiquitous bmm expression from a cDNA-bearing transgene. Similar phenotypes and a reduced embryonic hatching rate have been reported for mutants of the perilipin-like fly gene Lsd-2. These results suggest that bmm fulfils a vital function in TAG mobilization during embryogenesis (Grönke, 2005).
Flies lacking only zygotic BMM lipase activity develop normally but show progressive obesity accumulating 17% (immature adults, <1 day old) to 101% (mature adults, 6 days old) more storage fat compared to control flies. Conversely, transgene-dependent bmm overexpression in fat body cells of fed flies, which mimics the effect of starvation-induced upregulation of bmm transcription, depletes the TAG content of immature and mature adults by 96% and 46%, respectively. These effects were not observed upon transgenic expression of the enzymatically inactive bmmS38A mutant, indicating that the TAG mobilization is caused by the lipase activity of BMM. bmm-dependent differences of organismal TAG content are also reflected by the lipid storage phenotype of fat body cells showing variously sized storage droplets in bmm1 mutant fat body cells and their reduction in size and number upon overexpression of the gene. The effect of BMM is specific for the fat-based aspect of energy storage, since the glycogen content is not affected in bmm mutant or bmm-overexpressing flies (Grönke, 2005).
Excessive fat storage in flies lacking bmm function reduces the median lifespan by only 10%. Acute TAG mobilization is impaired but not abolished in bmm mutants. While controls deplete their storage TAG during starvation, bmm mutants are able to consume 73% of their prestarvation fat content. Accordingly, food-deprived bmm mutants outlive controls by 56% on the expense of their increased prestarvation fat storage. The lipolytic activity present in bmm mutants allows fuelling their extended survival under food deprivation by metabolizing in total 65% more TAG than controls. Thus, as in mammals, mobilization of TAG storage in flies is controlled by more than one TAG lipase. Candidate effectors of bmm-independent TAG mobilization are the bmm paralogue dob and the genes CG5966 and CG11055, which code for a starvation-induced putative TAG lipase and a Drosophila HSL homolog, respectively (Grönke, 2005).
To possibly extend the functional similarity between mammalian and Drosophila TAG lipases, it was asked whether BMM localizes at the surface of lipid droplets. Transgenic flies expressing BMM:EGFP fusion protein variants in their fat body cells allow examination of BMM intracellular localization and lipolytic activity in vivo. Ubiquitous expression of BMM:EGFP or BMM reverts the obese phenotype of bmm mutant flies. Targeting of BMM:EGFP but not BMMS38A:EGFP expression to the fat body of otherwise wild-type flies depletes the organismal TAG storage and reduces both the number and size of lipid droplets in fat body cells. BMM:EGFP localizes at islands on the droplet surface, often at interdroplet contact sites). In contrast, nonfunctional BMMS38A:EGFP distributes homogenously over the droplet surfaces. The evolutionary conserved part of BMM including the Brummer box is sufficient to properly localize the protein on lipid droplets, likely to represent active sites of BMM-dependent TAG mobilization. Other BMM-related lipases, such as hamster desnutrin and human TTS-2.1/ATGL, also localize on lipid droplets, but their localization sequences are presently unknown (Grönke, 2005).
The results indicate that the surface of lipid droplets is an evolutionary conserved intracellular compartment boundary for organismal TAG storage control, as has been suggested for mammalian adipocytes where perilipin modulates activity of HSL and possibly non-HSL lipases such as ATGL. Lack of perilipin results in lean mice with increased lipolysis and reverses the obese phenotype of leptin receptor-deficient mutants. The perilipin-like LSD-2 of fly localizes to lipid droplets of fat cells and adjusts organismal TAG content in a dosage-dependent manner, suggesting that it functions as an evolutionary conserved modulator of lipolysis. In fact, bmm− Lsd-2− double mutants have wild-type TAG levels, indicating that loss of Lsd-2 activity compensates for the lack of bmm. Conversely, combined overexpression of bmm and Lsd-2 in the fat body can partially revert the complementary phenotypes caused by the overexpression of each of the two genes. These data demonstrate that the lipid droplet-associated factors Brummer and LSD-2, which have opposite roles in organismal fat storage, act in an antagonistic manner (Grönke, 2005).
This first in vivo analysis of any insect lipase demonstrates a remarkable conservation of effectors controlling organismal fat storage in mammals and flies, emphasizing the value of Drosophila for research in energy homeostasis. On the basis of these results in the fly, it is speculated that mammalian members of the brummer/nutrin gene family like ATGL play an essential role in organismal fat mobilization and that malfunction of Brummer-homologous TAG lipases might contribute to mammalian obesity. Accordingly, stimulating Brummer-like lipase activity is a potential therapeutic approach to control TAG release from adipose tissue in obese patients, and lipase activators could be tested in the fly model (Grönke, 2005).
Energy homeostasis is a fundamental property of animal life, providing a genetically fixed balance between fat storage and mobilization. The importance of body fat regulation is emphasized by dysfunctions resulting in obesity and lipodystrophy in humans. Packaging of storage fat in intracellular lipid droplets, and the various molecules and mechanisms guiding storage-fat mobilization, are conserved between mammals and insects. A Drosophila mutant was generated lacking the receptor (AKHR; FlyBase name -- Gonadotropin-releasing hormone receptor or GRHR) of the adipokinetic hormone signaling pathway, an insect lipolytic pathway related to ss-adrenergic signaling in mammals. Combined genetic, physiological, and biochemical analyses provide in vivo evidence that AKHR is as important for chronic accumulation and acute mobilization of storage fat as is the Brummer lipase, the homolog of mammalian adipose triglyceride lipase (ATGL). Simultaneous loss of Brummer and AKHR causes extreme obesity and blocks acute storage-fat mobilization in flies. These data demonstrate that storage-fat mobilization in the fly is coordinated by two lipocatabolic systems, which are essential to adjust normal body fat content and ensure lifelong fat-storage homeostasis (Grönke, 2007).
Expression studies in a heterologous tissue culture system and in Xenopus oocytes identified AKH-responsive G protein-coupled receptors in Drosophila, such as the one encoded by the AKHR (or CG11325) gene. AKHR is expressed during all ontogenetic stages of the fly. It consists of seven exons, which encode a predicted protein of 443 amino acids. In late embryonic and larval stages, AKHR is expressed in the fat body. This finding is consistent with its predicted role as transmitter of the lipolytic AKH signal in this organ (Grönke, 2007).
In order to examine the effect of AKHR signaling on fat storage and mobilization in vivo, two different P element-insertion mutants were used, CG11188A1332 and AKHRG6244, which are located close to and within the AKHR gene, respectively. CG11188A1332 flies carrying the transposable element integration designated A1332 allow for the transcriptional activation of the adjacent AKHR gene. This ability was used for AKHR gain-of-function studies by overexpression of AKHR in the fat body of flies. Overexpression of AKHR in response to a fat body-specific Gal4 inducer causes dramatic reduction of organismal fat storage. This finding could be recapitulated by fat body-targeted AKHR expression from a cDNA-based upstream activation sequence (UAS)-driven AKHR transgene. These gain-of-function results suggest a critical in vivo role for AKHR in storage-lipid homeostasis of the adult fly (Grönke, 2007).
Flies of strain AKHRG6244, which carry a P element integration in the AKHR untranslated leader region, were used to generate the AKHR deletion mutants AKHR1 and AKHR2, as well as the genetically matched control AKHRrev, which possesses a functionally restored AKHR allele. As exemplified for embryonic and larval stages, AKHR1 mutants lack AKHR transcript. Ad libitum-fed flies without AKHR function are viable, fertile, and have a normal lifespan. However, such flies accumulate lipid storage droplets in the fat body and have 65%-127% more body fat than the controls. These results indicate that AKHR1 mutants develop an obese phenotype. The same result was obtained with AKHR2 and AKHR1/AKHR2 transheterozygous mutant flies, as well as with flies lacking the AKH-producing cells of the neuroendocrine system due to targeted ablation by the cell-directed activity of the proapoptotic gene reaper. Conversely, chronic overexpression of AKH provided by a fat body-targeted AKH transgene of otherwise wild-type flies largely depletes lipid storage droplets and organismal fat stores. However, the obese phenotype of AKHR mutants is unresponsive to AKH, indicating that AKHR is the only receptor transmitting the lipolytic signal induced by AKH in vivo. Collectively, these data demonstrate that AKH-dependent AKHR signaling is critical for the chronic lipid-storage homeostasis in ad libitum-fed flies (Grönke, 2007).
Studies on various insect species helped elucidate several components and mediators of the lipolytic AKH/AKHR signal transduction pathway. However, the identity of the TAG lipase(s) executing the AKH-induced fat mobilization program remained elusive. Besides the Drosophila homolog of the TG lipase from the tobacco hornworm Manduca sexta, the recently identified Brummer lipase, a homolog of the mammalian ATGL, is a candidate member of the AKH/AKHR pathway. This is based on the striking similarity between the phenotypes of AKHR and bmm mutants. Ad libitum-fed flies lacking either AKHR or bmm activity, store excessive fat. Both mutants show incomplete storage-fat mobilization (Grönke, 2005) and starvation resistance (Grönke, 2005) in response to food deprivation. Starvation resistance of these mutants might be caused by their increased metabolically accessible fat stores and/or changes in their energy expenditure due to locomotor activity reduction as described for flies with impaired AKH signaling. Despite the phenotypic similarities of their mutants, however, AKHR and bmm are members of two different fat-mobilization systems in vivo. Several lines of evidence support this conclusion. On one hand, AKH overexpression reduces the excessive TAG storage of bmm mutants, while on the other, bmm-induced fat mobilization can be executed in AKHR mutants. Thus, AKH/AKHR signaling is not a prerequisite for Brummer activity. Moreover, genetic epistasis experiments support this idea that AKHR and bmm belong to different control systems of lipocatabolism in vivo. Double-mutant analysis reveals that the obesity of AKHR and bmm single mutants is additive. Accordingly, AKHR bmm double-mutant flies store about four times as much body fat as control flies and accumulate excessive lipid droplets in their fat body cells (Grönke, 2007).
Thin layer chromatography (TLC) analysis was used to compare the storage-fat composition of AKHR and bmm single mutants with AKHR bmm double-mutant and control flies. Excessive body fat accumulation in AKHR bmm double mutants is on the one hand due to TAG, which is increased compared to AKHR and bmm single-mutant flies. Additionally, an uncharacterized class of TAG (TAGX) appears exclusively in AKHR bmm double mutants. In contrast to TAG, changes in diacylglycerol (DAG) content do not substantially contribute to the differences in body fat content in any of the analyzed genotypes. Taken together a quantitative increase and a qualitative change in the TAG composition account for the extreme obesity in AKHR bmm double-mutant flies (Grönke, 2007).
To address the in vivo response of AKHR bmm double mutants to induced energy-storage mobilization, flies were starved and their survival curve monitored. AKHR bmm double mutants die rapidly after food deprivation. In contrast to the starvation-resistant obese AKHR and bmm single mutants, the double mutants are not capable of mobilizing even part of their excessive fat stores. AKHR bmm double mutants do not, however, suffer from a general block of energy-storage mobilization because they can access and deplete their carbohydrate stores. These data demonstrate that energy homeostasis in AKHR bmm double-mutant flies is imbalanced by a severe and specific lipometabolism defect, which cannot be compensated in vivo (Grönke, 2007).
The nature of Brummer as a TAG lipase and AKHR as a transmitter of lipolytic AKH signaling suggests that the extreme storage-fat accumulation and starvation sensitivity of ad libitum-fed AKHR bmm double mutants is due to severe lipolysis dysfunction. To address this possibility in vitro, lipolysis rate measurements on fly fat body cell lysates and lysate fractions of control flies were performed. Results show that the cytosolic fraction of fat body cells contains the majority of basal and starvation-induced lipolytic activity against TAG, similar to the activity distribution in mammalian adipose tissue. Little basal and induced total TAG cleavage activity localizes to the lipid droplet fraction, whereas the pellet fraction including cellular membranes shows low basal, non-inducible TAG lipolysis. Lipolysis activity against DAG is similarly distributed between fat body cell fractions. However, in accordance with the function of DAG as major transport lipid in Drosophila, DAG lipolysis in fat body cells is not induced in response to starvation (Grönke, 2007).
Based on the lysate fraction analysis of control flies, cytosolic fat body cell extracts were used to assess the basal and starvation-induced lipolytic activity of mutant and control flies on TAG, DAG, and cholesterol oleate substrates. Whereas DAG and cholesterol oleate cleavage activity of fat body cells is comparable between all genotypes and physiological conditions tested, TAG lipolysis varies widely. Compared to control flies, basal TAG lipolysis of AKHR bmm double mutants is reduced by 80% and induced TAG cleavage is completely blocked, consistent with the flies' extreme obesity and their inability to mobilize storage fat. The impairment of basal lipolysis in the double mutants is largely due to the absence of bmm function, because it is also detectable in bmm single-mutant cells, whereas basal lipolysis in AKHR mutants is not reduced. Interestingly, bmm mutants mount a starvation-induced TAG lipolysis response after short-term (6 h), but not after extended (12 h), food deprivation. Conversely, AKHR mutant cells lack an early lipolysis response, but exhibit strong TAG cleavage activity after extended food deprivation. These data suggest that induced storage-fat mobilization in fly adipocytes relies on at least two lipolytic phases: an early, AKH/AKHR-dependent phase and a later, Brummer-dependent phase. Accordingly, it is speculated that the obesity of bmm and AKHR mutant flies is caused by different mechanisms: chronically low basal lipolysis in bmm mutants and, in AKHR mutants, lack of induced lipolysis during short-term starvation periods that is characteristic of organisms with discontinuous feeding behavior. It is acknowledged, however, that in vitro lipolysis assays on artificially emulsified substrates allow only a limited representation of the lipocatabolism in vivo, because lipid droplet-associated proteins modulate the lipolytic response in the insect fat body and mammalian tissue. Moreover, excessive fat accumulation in AKHR mutants may be in part due to increased lipogenesis because AKH signaling has been demonstrated to repress this process in various insects (Grönke, 2007).
Fat body cells of control flies (AKHRrev bmmrev) exhibit basal TAG lipolysis, which is doubled by starvation-induced lipolysis after 6 h or 12 h of food deprivation. bmm mutant cells have reduced basal lipolysis and lack induced lipolysis after 12 h starvation. AKHR mutant cells lack early (6 h) induced lipolysis, but show strong starvation-induced lipolysis after 12 h food deprivation. AKHR bmm double mutants have reduced basal lipolysis and lack starvation-induced lipolysis altogether (Grönke, 2007).
The finding of the dual lipolytic control in the fly raises the question of whether the two systems involved act independently of each other or whether one system responds to the impairment of the other. Modulation of transcription is an evolutionarily conserved regulatory mechanism of lipases from the ATGL/Brummer family. ATGL is transcriptionally up-regulated in fasting mice, as is bmm transcription in starving flies. Moreover bmm overexpression depletes lipid stores in the fat body of transgenic flies. Accordingly, bmm transcription was analyzed in response to modulation of AKH/AKHR signaling to assess a potential regulatory interaction between the two lipolytic systems. Compared to the moderate starvation-induced up-regulation of bmm in control flies, the gene is hyperstimulated in flies with impaired AKH signaling. As early as 6 h after food deprivation, bmm transcription is up-regulated by a factor of 2.5-3 in flies lacking the AKH-producing neuroendocrine cells (AKH-ZD) or in AKHR mutant flies. Conversely, chronic expression of AKH in the fat body suppresses bmm transcription. Bmm hyperstimulation in AKHR mutants is consistent with a subsequent strong increase of starvation-induced TAG lipolysis observed 12 h after food deprivation. Taken together, these data demonstrate an AKH/AKHR-independent activation mechanism of bmm and suggest the existence of compensatory regulation between bmm and the AKH/AKHR lipolytic systems, the mechanism of which is currently unknown (Grönke, 2007).
The results presented in this study provide in vivo evidence that the fly contains two induced lipolytic systems. One system confers AKH/AKHR-dependent lipolysis, a signaling pathway, which assures rapid fat mobilization by cAMP signaling and PKA activity. Drosophila's second lipolytic system involves the Brummer lipase, which is responsible for most of the basal and part of the induced lipolysis in fly fat body cells, likely via transcriptional regulation. Currently, it is unknown whether Brummer activity is post-translationally modulated by an α/β hydrolase domain-containing protein like the regulation of its mammalian homolog ATGL by CGI-58. Homology searches between mammalian and Drosophila genomes identify the CGI-58-related fly gene CG1882 and the putative Hsl homolog CG11055, providing additional support for the evolutionary conservation of fat-mobilization systems. However, differences in lipid transport physiology (i.e., DAG transport in Drosophila, and FFA in mammals) suggest a different substrate specificity or tissue distribution of fly Hsl compared to its mammalian relative (Grönke, 2007).
Future studies will not only unravel the crosstalk between the two Drosophila lipocatabolic systems, but also disclose the identity of additional genes involved in this process, such as the upstream regulators of bmm. This study substantiates the emerging picture of the evolutionary conservation between insect and mammalian fat-storage regulation and emphasizes the value of Drosophila as a powerful model system for the study of human lipometabolic disorders (Grönke, 2007).
Insulin-like peptides (ILPs) couple growth, metabolism, longevity, and fertility with changes in nutritional availability. In Drosophila, several ILPs called Dilps are produced by the brain insulin-producing cells (IPCs), from which they are released into the hemolymph and act systemically. In response to nutrient deprivation, brain Dilps are no longer secreted and accumulate in the IPCs. The larval fat body, a functional homolog of vertebrate liver and white fat, couples the level of circulating Dilps with dietary amino acid levels by remotely controlling Dilp release through a TOR/RAPTOR-dependent mechanism. Ex vivo tissue coculture was used to demonstrate that a humoral signal emitted by the fat body transits through the hemolymph and activates Dilp secretion in the IPCs. Thus, the availability of nutrients is remotely sensed in fat body cells and conveyed to the brain IPCs by a humoral signal controlling ILP release (Géminard, 2009).
Due to the lack of immunoassay, the study of the regulation of Dilp levels in Drosophila has been limited so far to the analysis of their expression level in response to nutritional conditions. This study presents evidence that the secretion of Dilp2 and Dilp5 as well as a GFP linked to a signal peptide (secGFP) is controlled by the nutritional status of the larva. The data also indicate that the IPCs have the specific ability to couple secretion with nutritional input. This suggests that all Dilps produced in the IPCs could be subjected to a common control on their secretion that could therefore override differences in their transcriptional regulation. It was further shown that the regulation of Dilp secretion plays a key role in controlling Dilp circulating levels and biological functions, since blocking neurosecretion in the IPCs led to growth and metabolic defects, and conversely, expression of Dilp2 in nonregulated neurosecretory cells is lethal upon starvation. Interestingly, previous reports suggest that Dilp release could also be controlled in the adult IPCs, raising the possibility that this type of regulation contributes to controlling metabolic homeostasis, reproduction, and aging during adult life (Géminard, 2009).
Dilp release is not activated by high-carbohydrate or high-fat diets, but rather depends on the level of amino acids and in particular on the presence of branched-chain amino acids like leucine and isoleucine. This finding is consistent with the described mechanism of TOR activation by leucine in mammalian cells (Avruch, 2009: Nicklin, 2009). In particular, it was recently shown that Rag GTPases can physically interact with mTORC1 and regulate its subcellular localization in response to L-leucine (Sancak, 2008). Interestingly, the present work indicates that amino acids do not directly signal to the IPCs, but rather they act on fat-body cells to control Dilp release. TOR signaling has been previously shown to relay the nutritional input in fat-body cells. Tor signaling is required for the remote control of Dilp secretion, since inhibition of Raptor-dependent TOR activity in fat cells provokes Dilp retention. Surprisingly, activation of TOR signaling in fat cells of underfed larvae is sufficient to induce Dilp release, indicating that TOR signaling is the major pathway relaying the nutrition signal from the fat body to the brain IPCs. In contrast, inhibition of PI3K activity in fat cells does not appear to influence Dilp secretion in the brain. This result is in line with previous in vivo data showing that reduction of PI3K levels in fat cells does not induce systemic growth defects. Altogether, this suggests that the nutritional signal is read by a TOR-dependent mechanism in fat cells, leading to the production of a secretion signal that is conveyed to the brain by the hemolymph (Géminard, 2009).
Ex vivo brain culture experiments demonstrate that hemolymph or dissected fat bodies from fed larvae constitute an efficient source for the Dilp secretion factor. This signal is absent in underfed animals, suggesting that it could be identified by comparative analysis of fed and underfed states. The nature of the secretion signal is unknown. It is produced and released in the hemolymph by fat cells, and its production relies on TORC1 function. Given the role of TORC1 in protein translation, one could envisage that the secretion factor is a protein or a peptide for which translation is limited by TORC1 activity and relies on amino acid input in fat-body cells. In mammals, fatty acids and other lipid molecules have the capacity to amplify glucose-stimulated insulin secretion in pancreatic β cells. The fly fat body carries important functions related to lipid metabolism, and a recent link has been established between TOR signaling and lipid metabolism in flies (Porstmann, 2008), leaving open the possibility that a TOR-dependent lipid-based signal could also operate in this regulation. Interestingly, carbohydrates do not appear to contribute to the regulation of insulin secretion by brain cells in flies. This finding is reminiscent of the absence of expression of the Sur1 ortholog in the IPCs and suggests that global carbohydrate levels are controlled by the glucagon-like AKH produced by the corpora cardiaca cells (Géminard, 2009).
These experiments demonstrate that Dilp secretion is linked to the polarization state of the IPC membrane, suggestive of a calcium-dependent granule exocytosis, like the one observed for insulin and many other neuropeptides. The nature of the upstream signal controlling membrane depolarization is not known. Recent data concerning the function of the nucleostemin gene ns3 in Drosophila suggest that a subset of serotonergic neurons in the larval brain act on the IPCs to control insulin secretion (Kaplan, 2008). Therefore, it remains to be known whether the IPCs or upstream serotonergic neurons constitute a direct target for the secretion signal. So far, no link has been established between the serotonergic stimulation of IPC function and the nutritional input (Géminard, 2009).
In 1998, J. Britton and B. Edgar presented experiments where starved brain and fed fat bodies were cocultured, allowing arrested brain neuroblasts to resume proliferation in the presence of nutrients (Britton and Edgar, 1998). From these experiments, the authors proposed that quiescent neuroblasts were induced to re-enter the cell cycle by a mitogenic factor emanating from the fed fat bodies. The present data extend these pioneer findings and suggest the possibility that the factor sent by the fed fat bodies is the secretion factor that triggers Dilp release from the IPCs, allowing neuroblasts to continue their growth and proliferation program through paracrine Dilp-dependent activation (Géminard, 2009).
In conclusion, this work combines genetic and physiology approaches on a model organism to decipher key physiological regulations and opens the route for a genetic study of the molecular mechanisms controlling insulin secretion in Drosophila (Géminard, 2009).
The cAMP-responsive transcription factor CREB functions in adipose tissue and liver to regulate glycogen and lipid metabolism in mammals. While Drosophila has a homolog of mammalian CREB, dCREB2, its role in energy metabolism is not fully understood. Using tissue-specific expression of a dominant-negative form of CREB (DN-CREB), this stud examined the effect of blocking CREB activity in neurons and in the fat body, the primary energy storage depot that functions as adipose tissue and the liver in flies, regulating energy balance, stress resistance and feeding behavior. It was found that disruption of CREB function in neurons reduces glycogen and lipid stores and increases sensitivity to starvation. Expression of DN-CREB in the fat body also reduces glycogen levels, while it does not affect starvation sensitivity, presumably due to increased lipid levels in these flies. Interestingly, blocking CREB activity in the fat body increased food intake. These flies do not show a significant change in overall body size, suggesting that disruption of CREB activity in the fat body causes an obese-like phenotype. Using a transgenic CRE-luciferase reporter, it was further demonstrated that disruption of the adipokinetic hormone receptor, which is functionally related to mammalian glucagon and beta-adrenergic signaling, in the fat body reduces CRE-mediated transcription in flies. This study demonstrates that CREB activity in either neuronal or peripheral tissues regulates energy balance in Drosophila, and that the key signaling pathway regulating CREB activity in peripheral tissue is evolutionarily conserved (Iijima, 2009).
This study provides in vivo evidence that both neuronal and peripheral CREB activities are involved in the regulation of energy balance in flies. Blocking CREB activity in neurons causes reductions in both glycogen and lipid stores and a higher sensitivity to starvation stress. In contrast, while disruption of CREB function in the fat body also reduces glycogen levels, it increases lipid stores, and does not affect starvation sensitivity. Since there was no significant change in overall body size in these flies, disruption of CREB activity in the fat body causes an obese-like phenotype. These results also indicate that CREB activity can both increase and reduce lipid stores in flies depending on its site of action. Recently, two distinct populations of Drosophila brain neurons that regulate fat deposition were identified in Drosophila (Al-Anzi, 2009). It will be interesting to determine in which neurons CREB functions to regulate energy metabolism in flies (Iijima, 2009).
In a recent study, TORC-mediated CREB activity in neurons was shown to positively regulate glycogen and lipid stores in flies. This is based on results showing that expression of TORC in neurons rescues the starvation sensitivity of TORC mutant flies. In addition, expression of TORC in neurons partially rescues the lower energy stores of these mutants. While supporting the conclusions of this study with respect to the role of neuronal CREB activity, the current results also provide evidence that CREB in the fat body plays roles in energy balance. Moreover, in contrast to the normal feeding behavior of a TORC mutant, it was found that blocking CREB activity in the fat body increases food intake. Thus, disruption of CREB functions has a broader impact on energy metabolism and feeding behavior than the loss of TORC. It is likely that not all CREB functions depend on TORC. In support of this, although a TORC null mutant is viable and fertile, CREB mutants are lethal (Iijima, 2009).
This study found that adipokinetic hormone (AKH/AKHR) signaling in the fat body, which is thought to be functionally related to glucagon/glucogon receptor signaling in the mammalian liver, positively regulates CRE-mediated transcription. In the mammalian liver, CREB activates the gluconeogenic program following a glucagon stimulus. Recent studies reported that promoting AKH signaling in the fat body significantly reduces, while loss of AKHR function modestly increases, glycogen levels in flies, presumably through AKH/AKHR-mediated carbohydrate catabolism in the fat body (Gronke, 2007; Bharucha, 2008). However, this study found that blocking CREB activity in the fat body significantly reduces glycogen levels, which would seem to contradict the proposed role of AKH/AKHR in mediating carbohydrate catabolism in the fat body. One possibility is that CREB activity in the fat body regulates multiple aspects of glucose/glycogen metabolism in addition to the AKH/AKHR-mediated pathway, and that blocking all CREB functions in the fat body reduces total glycogen levels as a net effect. In fact, significant CREB activity was remaining in AKHR mutant flies, suggesting that other signaling pathways might contribute to the activation of CREB activity in the fat body. Further studies will be required to delineate the role of CREB activity in the fat body in carbohydrate metabolism and its relationship with the AKH signaling pathway (Iijima, 2009).
This study found that blocking CREB activity in the fat body increased lipid stores. AKH/AKHR is also thought to be functionally related to β-adrenergic signaling in mammalian adipose tissue, which activates protein kinase A (PKA) and stimulates lipolysis by phosphorylating hormone-sensitive lipase and perilipin. In Drosophila, the promotion of AKH signaling in the fat body reduces lipid levels, whereas loss of AKHR function has the opposite effect; this is partly ascribed to altered activity in lipocatabolic systems. In addition, AKH signaling has been shown to repress the lipogenesis pathway in various insects. Interestingly, blocking CREB activity in mammalian liver causes excessive fat accumulation, resulting in 'fatty liver' through overactivation of liposynthesis. Future analysis will unravel whether CREB activity in the fat body represses liposynthesis and/or promotes lipid catabolism under the control of AKH/AKHR signaling (Iijima, 2009).
In summary, these results demonstrate that CREB is involved in both central and peripheral regulation of energy balance and feeding behavior in Drosophila. Future studies of CREB in flies hold great promise for revealing the mechanisms underlying energy balance and feeding behavior. Such studies will likely contribute to understanding of human metabolic disorders (Iijima, 2009).
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See also: Genes expressed in mesoderm
Genes involved in tissue development
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