The Drosophila Insulin receptor (InR) regulates cell growth and proliferation through the PI3K/Akt pathway, which is conserved in metazoan organisms. The Drosophila forkhead-related transcription factor Foxo is a key component of the insulin signaling cascade. Foxo is phosphorylated by Akt upon insulin treatment, leading to cytoplasmic retention and inhibition of its transcriptional activity. Mutant Foxo lacking Akt phosphorylation sites no longer responds to insulin inhibition, remains in the nucleus, and is constitutively active. Foxo activation in S2 cells induces growth arrest and activates two key players of the InR/PI3K/Akt pathway: the translational regulator d4EBP/Thor (eukaryotic initiation factor 4E binding protein) and the InR itself. Induction of d4EBP likely leads to growth inhibition by Foxo, whereas activation of InR provides a novel transcriptionally induced feedback control mechanism. Targeted expression of Foxo in fly tissues regulates organ size by specifying cell number with no effect on cell size. These results establish Foxo as a key transcriptional regulator of the insulin pathway that modulates growth and proliferation (Puig, 2003).
Foxo regulates cell cycle arrest possibly by transcriptionally activating genes implicated in cell division or in cell growth. As an initial attempt to identify target genes of Foxo, DNA microarrays were used to assess gene expression profiles in S2 cells stably transfected with mutant Foxo and grown in the presence of insulin. Cells expressing wild-type Foxo or untransfected S2 cells subjected to the same treatment were assayed as controls (Puig, 2003).
Two-hundered and seventy-seven genes were found to be up-regulated in Foxoa3-expressing cells when compared with Foxo-expressing cells or untransfected S2 cells. Interestingly, two genes that were consistently and specifically up-regulated in these conditions were the Drosophila InR gene (13.5-fold) and the Drosophila 4EBP gene (25-fold). Both genes have been implicated in the regulation of cell growth by insulin. To confirm that InR and 4EBP are bona fide transcriptional targets of Foxo, the same experiment described above was performed but in the presence of cycloheximide to inhibit translation. As expected, both InR and 4EBP continue to be transcriptionally activated (2.5- and 3.1-fold, respectively) by FOXOA3 but not Foxo in the insulin-repressed state. This result suggests that Foxo, when released from control by the insulin/dAkt cascade, is involved in transcription from the InR and 4EBP promoters (Puig, 2003).
To confirm these microarray results and to independently quantitate the increase in mRNA transcription, RNase protection assays were performed with mRNAs extracted from cells stably transfected with either Foxo or FoxoA3. Indeed, FoxoA3 stimulates transcription of Drosophila 4EBP and InR by 16.3- and 11-fold, respectively. A time-course experiment confirmed that Drosophila InR mRNA increases rapidly upon FoxoA3 expression: 3 h after CuSO4 addition, there is already an 8-fold increase, reaching 20-fold after 9 h of CuSO4 induction. Similar results were obtained for Drosophila 4EBP. These experiments suggest that Foxo expression specifically activates both Drosophila InR and 4EBP transcription, thus unmasking an important feedback control mechanism in this pathway involving Foxo and InR (Puig, 2003).
Having obtained evidence that exogenously transfected Foxo responds to insulin and regulates both the downstream target gene 4EBP and the feedback control target InR, it was of interest to know if endogenous Foxo would also activate transcription of these genes. The PI3K inhibitor LY294002 was used to activate endogenous Foxo or insulin to deactivate it. S2 cells grown in the absence of serum for 48 h were treated either with LY294002 or insulin. Total RNA was extracted and RNase protection was performed to detect Drosophila InR and 4EBP mRNAs. Both mRNA levels are significantly increased after LY294002 treatment (5.3-fold for dInR and 4-fold for d4EBP) when compared with insulin treatment. This result provides further evidence indicating that the PI3K–Akt pathway regulates InR and 4EBP transcription via Foxo (Puig, 2003).
It was of interest to determine whether Foxo directly binds to the promoters of Drosophila 4EBP and InR. To identify the DNA region recognized by Foxo in these two promoters, a 1708-bp fragment of the 4EBP promoter and a 1562-bp fragment of the InR promoter were inserted into a luciferase reporter vector. When transfected into S2 cells, these fragments responded to Foxo activation (3-fold for 4EBP, >200-fold for InR. A series of deletions lacking upstream sequences still responded to Foxo activation, albeit more weakly, suggesting that Foxo can bind the DNA in a region close to the start of transcription (485 bp for the d4EBP promoter and 194 bp for the dInR promoter). In contrast, Foxo completely fails to activate a reporter construct in which upstream activating sequences (UAS) for the transcription factor GAL4 are fused to the luciferase gene, confirming that transcription activation is specific for both 4EBP and InR promoters (Puig, 2003).
Interestingly, 125 bp upstream of the transcription start site of the d4EBP promoter there are three tandem copies of a putative FOXO4 recognition element (FRE). These elements are reminiscent of the ones present in the human glucose-6-phosphatase promoter, previously shown to bind FOXO4 (Yang, 2002). This was reassuring because Foxo and FOXO4 share 85% identity in the core of the forkhead DNA-binding domain. Similarly, several putative FRE sequences appear in the InR promoter in the region comprising nucleotides -1434 to -70 (Puig, 2003).
To determine whether Foxo binds these putative FREs, band shift experiments were performed with a 113-bp DNA probe encompassing the 4EBP FRE motifs and with 12 separate DNA probes (ranging from 100 to 150 bp) spanning a region of 1.4 kb from the InR promoter. Purified recombinant Foxo expressed in Escherichia coli efficiently binds the 113-bp FRE-containing fragment from the 4EBP promoter compared with control DNA fragments. Furthermore, Foxo binding to the 4EBP promoter fragment can be efficiently competed with an unlabeled 113-bp 4EBP promoter fragment but not with nonspecific DNA. Similarly, purified recombinant Foxo binds efficiently to 5 out of 12 of the DNA fragments located within the InR promoter. As expected, each of the five DNA fragments bound by Foxo contains putative FREs. Thus, Foxo can specifically bind to both promoters in vitro. To determine whether Foxo also binds these same DNA regions in vivo, chromatin immunoprecipitation (ChIP) experiments were performed with S2 cells expressing either Foxo or dFoxoA3. Cells were incubated with serum, and Foxo expression was induced with the addition of CuSO4. After 6 h, cells were cross-linked with formaldehyde, and extracts were prepared and immunoprecipitated. After reversal of cross-links, DNA was recovered, and PCR was performed with primers encompassing regions containing putative FREs in both promoters. The results indicate that Foxo can directly bind to both the 4EBP and InR promoters in vivo. These results establish that Foxo can specifically bind the 4EBP and InR promoters both in vitro and in vivo (Puig, 2003).
To demonstrate that Foxo can directly activate transcription of these promoters in vitro, the constructs were used that contain 485 bp of the 4EBP promoter region and 514 bp of the InR promoter region, respectively. Addition of purified recombinant Foxo to in vitro reactions activates transcription of these promoters by at least 3-fold (4EBP) and 5.5-fold (InR), which is comparable to the activation observed in vivo. Under in vitro transcription conditions, activation of the 4EBP promoter by Foxo becomes rapidly saturated with increasing amounts of Foxo. As expected, Foxo also activates (up to sixfold) a synthetic promoter bearing four FOXO4-binding sites placed upstream of the alcohol dehydrogenase distal promoter. Together these results show that transcription of 4EBP and InR can be directly activated by Foxo in vitro (Puig, 2003).
Studies in Drosophila have characterized Insulin receptor/Phosphoinositide 3-kinase (Inr/PI3K) signaling as a potent regulator of cell growth, but an understanding of its function during development has remained uncertain. Inhibiting Inr/PI3K signaling phenocopies the cellular and organismal effects of starvation, whereas activating this pathway bypasses the nutritional requirement for cell growth, causing starvation sensitivity at the organismal level. Consistent with these findings, studies using a pleckstrin homology domain-green fluorescent protein (PH-GFP) fusion as an indicator for PI3K activity show that PI3K is regulated by the availability of dietary protein in vivo. It is surmised that an essential function of insulin/PI3K signaling in Drosophila is to coordinate cellular metabolism with nutritional conditions (Britton, 2002).
To test whether PI3K is required for larval growth, its activity was inhibited by expressing p60, Deltap60, or Pten in large domains of the larva using the Gal4/UAS system. p60 is an adaptor that couples Inr to Pi3K92E (Dp110), and Deltap60 is a deletion variant lacking part of the Dp110 binding domain. These molecules and their mammalian homologs have dominant-negative effects on PI3K activity when overexpressed, presumably because they compete with endogenous Dp110/p60 complexes for binding sites on upstream activators such as insulin receptors and IRSs. When p60 is expressed under the control of Act-Gal4 (expressed ubiquitously) or Adh-Gal4 (expressed predominantly in fat body), larvae remain growth arrested in the first instar for as long as 2 weeks. Deltap60 and Pten had similar effects. Dissection of these animals revealed that all of their tissues and organs were proportionally reduced. This developmental arrest is indistinguishable from the effects of starvation or inhibition of protein synthesis (Britton, 2002).
To test whether PI3K is autonomously required for growth in the different larval cell types, the Flp/Gal4 technique was used to express p60, Deltap60, or Pten in scattered cells throughout the larva. This method employs the Act>CD2>Gal4 and hs-Flp transgenes to activate UAS-linked target genes, including the cell marker UAS-GFPnls, in cell clones. Heat shock-independent activation of Gal4 occurs prior to the onset of larval growth and DNA endoreplication in 1%-10% of cells (depending on organ) in the fat body, gut, salivary glands, renal (Malpigian) tubules, and epidermis. Cells overexpressing p60, Deltap60, or Pten in the salivary glands and fat body are greatly reduced in size and have much smaller nuclei with far less DNA than adjacent control cells. Similar effects are observed in other larval tissues. Despite their reduced growth, p60-, Deltap60-, and Pten-expressing cells are found at approximately the same frequencies as control GFP-marked cells. Apoptotic cells are not observed. Thus, reductions in PI3K activity are not incompatible with cell viability. Overt effects on cell morphology that might reflect changes in cell adhesion, motility, or identity are also not observed. It is concluded that reducing Inr/PI3K activity in the differentiated tissues of the larva has cell-autonomous effects that are limited to reducing cell growth and DNA replication (Britton, 2002).
These observations suggest that PI3K activity might be responsive to nutritional conditions. To directly test this possibility, a fusion protein was made for use as an in vivo reporter for PI3K activity. The pleckstrin homology (PH) domain of the Drosophila homolog of general receptor for phosphoinositides-1 (GRP1) was fused to green fluorescent protein (GFP), generating a protein called GPH (GFP-PH domain). PH domains from mammalian GRP1 genes bind specifically to phosphatidylinositol-3,4,5-P3 (PIP3), the second messenger generated by class I PI3-kinases. Since PIP3 generally resides in lipid membranes, particularly the plasma membrane, GRP1 is recruited to membranes when PI3-kinase activity raises cellular levels of PIP3. Fusion proteins containing the GRP1 PH domain are likewise recruited to plasma membranes by binding PIP3, and thus serve as in situ reporters for PI3K activity (Britton, 2002).
For in vivo studies, the GPH gene was placed under control of the Drosophila ß-tubulin promotor, generating a gene called 'tGPH' (tubulin-GPH), and introduced into Drosophila by P element-mediated transformation. Membrane localization of tGPH was observed in the larval epidermis, fat body, salivary glands, malpighian tubules, and wing imaginal discs. Cytoplasmic and nuclear tGPH was also visible in these cell types. The degree of membrane localization depends upon the developmental stage. Epidermal cells show little membrane localization of tGPH in embryos or newly hatched first instar (L1) larvae, but have strong membrane localization in second (L2) and early third (L3) instar larvae. Later, in wandering stage L3 larvae, membrane-associated tGPH is again diminished. Similar trends are observed in the fat body. These variations might reflect changes in the levels of Inr, PI3K, Pten, or insulin-like peptides (dILPs) in the larva as it feeds and grows (Britton, 2002).
To test whether tGPH localization was responsive to PI3K activity in vivo, Inr or Dp110 was overexpressed using the Gal4 system. Either gene causes a striking redistribution of tGPH to plasma membranes in cells of the fat body, epidermis, Malpighian tubules, gut, and imaginal discs. To determine whether endogenous PI3K is responsible for the membrane localization of tGPH, PI3K activity was suppressed by expressing p60 with the heat-inducible driver hs-Gal4. In the larval epidermis, partial loss of membrane-bound tGPH is apparent 1.5 hr post-heat shock (phs), and by 4 hr, phs tGPH is nearly completely lost from cell membranes. Similar results were obtained in the fat body when either p60 or dPten were expressed mosaically using the Flp/Gal4 method (Britton, 2002).
To determine whether PI3K activity is nutritionally modulated, tGPH localization was monitored after starvation. Early L2 larvae (48-60 hr AED) were deprived of dietary protein by culture on either 20% sucrose or Sang's defined media lacking casein. These treatments arrest cell growth in all of the differentiated larval tissues. L2 larvae fed on either protein-free diet survive for up to 14 days, but a marked shrinkage of cells in the epidermis and fat body is observed. In the epidermis of early L2 larvae, culture on either protein-free diet causes membrane-bound tGPH to be diminished after 24 hr, and to be nearly undetectable after 48 hr. Levels of total tGPH also decrease after protein deprivation, but nuclear and cytoplasmic tGPH remain detectable for more than 6 days. Loss of membrane-associated tGPH also occurs in fat body cells of protein-deprived L2 larvae, with similar kinetics. Starvation causes shrinkage of the nuclei and nonlipid cytoplasm in fat body cells, leaving large tGPH-negative lipid droplets that occupy most of the cell volume. To test whether membrane association of tGPH is reversible, L2 larvae were cultured on 20% sucrose for 8 days and then returned to whole food. In this experiment, tGPH levels rose and the protein reassociated with plasma membranes in epidermal cells between 24 and 48 hr after feeding. These results indicate that cellular levels of PIP3 drop as a consequence of starvation for dietary protein (Britton, 2002).
Terminally differentiated endoreplicating tissues (ERTs) constitute most of a Drosophila larva, and the growth of these tissues accounts for virtually all of the ~200-fold mass increase sustained by the animal during the larval stages. During larval life, the ERTs provide a physiologically nurturing environment for undifferentiated imaginal cells and neuroblasts, which generate much of the reproductive adult stage. Most of the biomass accumulated in the ERTs is eventually recycled into these progenitor cells as they form the adult. This study provides evidence that Inr/PI3K signaling coordinates nutritional status with ERT cell metabolism and growth. To determine whether Inr/PI3K signaling can maintain cell growth in the face of starvation, Flp/Gal4 was used to express Dp110 or Inr in scattered ERT cells, and then changes in cell size and DNA replication were assessed at time points during a protein starvation regime. At larval hatching, prior to starvation, cells expressing Dp110 or Inr in the gut, fat body, Malpighian tubules, and epidermis are only slightly larger than nonexpressing cells. After several days of starvation on 20% sucrose, Inr- or Dp110-expressing cells in these organs are much larger than adjacent control cells, and have visibly increased DNA content. BrdU incorporation indicated that gut and fat body cells expressing Dp110 or Inr continue to replicate their DNA for at least 2 to 3 days under starvation conditions. Normally, DNA endoreplication in these cells ceases within 1 to 2 days of starvation (Britton, 1998). A catalytically inactive PI3K, Dp110D945A, does not promote cell growth or DNA endoreplication, indicating that lipid kinase activity was required. These experiments indicate that active Inr/PI3K signaling is sufficient to bypass the nutritional requirement for cellular growth and DNA replication in many larval cell types, and that this effect is cell autonomous (Britton, 2002).
To explore the means by which Inr/PI3K signaling induces cell growth, an examination was carried out of the morphology of fat body cells in which PI3K activity had been manipulated. Fat body cells accumulate large stores of protein, carbohydrate, and lipids during larval life, and also produce growth factors. During the third larval instar, these accumulations of nutrients cause fat body cells to become opaque. These nutrients are normally utilized during metamorphosis, but if a larva is starved, they are precociously mobilized into the haemolymph to support the animal during the ensuing dietary crisis. This causes the fat body cells to shrink and become clear as they lose organelles by autophagy and deplete stored metabolites (Britton, 2002).
Expression of Inr or PI3K in fat body cells increases the opacity of the cytoplasm, and thus promotes nutrient storage. A similar cytoplasmic effect is observed in intestinal cells from L1 animals. Close inspection has revealed that in both cell types, ectopic Inr or PI3K decreases the size of prominent vesicles in the cytoplasm. Induction of p60 in early L3 larvae has opposite effects, causing fat body cells to become more translucent. A loss of opacity was observed in fat bodies from Dp110 mutants after their growth arrest at L3. In further tests, fat body cells were stained for lipids with Nile red or for protein with Texas red X succinimidyl ester. This revealed that the cytoplasm of Inr-expressing cells contains many more, but much smaller, lipid droplets than neighboring control cells. In summary, activation of the Inr/PI3K pathway has profound effects on cytoplasmic composition. These effects mimic changes in the fat body that normally take place late in the L3 stage when nutrient storage by these cells is maximal. Suppression of Inr/PI3K activity has opposite effects on cytoplasmic composition, and these appear to mimic the mobilization of nutrients that normally accompanies starvation (Britton, 2002).
Considering the above results, it should be advantageous to larvae to downregulate insulin/PI3K signaling when nutrients are limited, since this would suppress nutrient storage and cell growth and allow nutrient mobilization by tissues such as the fat body. This idea was tested by hyperactivating Inr/PI3K signaling and then tracking development under different nutritional conditions. Several Gal4 drivers were used to induce expression in large numbers of cells, including Adh-Gal4 (expressed in the fat body, trachea, and a few cells in the gut), en-Gal4 (expressed in posterior epidermal cells, the hindgut, and some neural cells), Act-Gal4 (expressed ubiquitously), and hs-Flp/Act>Cd2>Gal4 (induced by heat shock in all cells). In several cases, overexpressed Dp110 and Inr were tolerated in feeding animals. For instance, animals expressing Dp110 under Adh-Gal4 or en-Gal4 control developed without delay and eclosed at the same frequency as controls, giving viable fertile adults. Inr was more deleterious, but some animals expressing Inr under Adh-Gal4 control developed to the L3 stage and a few viable adults eclosed. Ubiquitous expression of Dp110 or Inr using the Act-Gal4 driver, however, was 100% lethal at prelarval stages (Britton, 2002).
In contrast, hyperactivating Inr/PI3K signaling under starvation conditions was catastrophic. When Adh-Gal4 was used to drive Dp110 or Inr expression, for instance, L1 larvae raised on the sucrose/PBS diet all perished within 3 to 4 days of hatching, whereas control animals survived 8 to 9 days. Animals expressing Dp110 under en-Gal4 control also perished within 2 to 3 days, 4 to 5 days before controls, when deprived of dietary protein. Suppressing PI3K activity by expressing p60, Deltap60, Pten, or Dp110D945A using Adh-Gal4, en-Gal4, or even Act-Gal4 had no effect on viability under starvation conditions. These results suggest that the starvation sensitivity caused by high Inr/PI3K activity is specifically related to nutrient uptake and storage, functions that appear to be unique to Inr and PI3K (Britton, 2002).
These results demonstrate that downregulation of Inr/PI3K activity is critical to maintaining metabolic homeostasis under starvation conditions. The remarkable ability of Inr/PI3K-expressing cells to continue stockpiling nutrients and grow, even in starved animals, may account for the starvation sensitivity observed at the organismal level. This idea was supported by observations made in starved tGPH larvae that overexpressed Dp110 in posterior compartment epidermal cells (genotype, en-Gal4 UAS-Dp110 tGPH). In these animals, high levels of membrane-bound tGPH persisted in Dp110-expressing epidermal cells until 2 days after nutrient withdrawal, at which point the animals died. In anterior (A) cells, which did not overexpress Dp110, tGPH was completely lost from plasma membranes within 18 hr after nutrient deprivation, and tGPH protein became undetectable by 36 hr. This is a much more rapid starvation response than observed in animals that did not contain Dp110-expressing cells. Anterior epidermal cells also shrank rapidly during starvation, whereas posterior, Dp110-expressing cells maintained their large size. These effects might result from the rapid depletion of nutrients by the PI3K-expressing cells, and a consequent drop in levels of hemolymph insulins (Britton, 2002).
In performing these experiments, it was noticed that larvae that overexpress Inr or Dp110 wander away from their food. To more carefully analyze this phenotype, animals expressing various PI3K signaling components under Adh-Gal4 control were cultured on agar plates with red-colored food (yeast paste) in the center for ~24 hr after hatching. These animals were then scored for the presence of red food in the gut as well as their proximity to the food source. Animals overexpressing Inr, Dp110, or Dp110CAAX feed poorly (i.e., often have no food in the gut) and frequently wander away from their food. Similar aberrant behaviors were observed when Dp110 was expressed ubiquitously using Flp/Gal4, in which case nearly all animals wandered out of the food and pupated precociously. Thus, elevated levels of Inr/PI3K signaling alter larval feeding behavior, perhaps by affecting the animal's perceived level of hunger (Britton, 2002).
Is nutrition sensed in Drosophila at the cell level or by the animal as a whole? Although animal cells can sense amino acids directly, these studies suggest that cells in Drosophila larvae sense and respond to changes in dietary protein indirectly, using secondary humoral signals -- most likely insulins -- long before they become acutely starved for amino acids. In support of this idea, some cells in the larva can continue to grow and replicate their DNA long after the animal is deprived of dietary protein. Imaginal cells and neuroblasts do this, as do ERT cells in which Inr or PI3K have been artificially switched on. This attests to the fact that starvation for dietary protein does not completely deplete the larval hemolymph of amino acids or cause a global shutdown of protein synthesis. This is probably possible because nutrients stored in ERTs such as the fat body are mobilized during starvation to maintain levels of hemolymph nutrients. Nevertheless, starvation does cause a rapid, global shutdown of ERT cell growth, and thus some essential signal is lost (Britton, 2002).
One factor that all animal cells use for nutritional sensing is TOR (target of rapamycin), a protein kinase that mediates diverse effects on cell metabolism including protein synthesis, amino acid import, ribosome biogenesis, and autophagy (Raught, 2001; Schmelzle, 2000). What role might Drosophila TOR (dTOR) play in the nutrition response system addressed here? The mechanism by which TOR 'senses' nutrition remains uncertain (Kleijn and Proud, 2000), but cellular levels of amino acids, aminoacylated tRNAs, and ATP have been suggested as direct inputs (Dennis, 2001; Iiboshi, 1999). Drosophila dTOR mutations or the TOR-specific inhibitor rapamycin inactivate the TOR target S6-kinase and phenocopy starvation in fed larvae (Zhang, 2000; Oldham, 2000). Starvation, however, does not completely inactivate S6K, suggesting that dTOR retains some activity under starvation conditions (Oldham, 2000). Consistent with this interpretation, overexpressed PI3K is a potent promotor of cell growth in starved larvae, but PI3K cannot drive cell growth in dTOR mutant larvae (J. Lande and T. Neufeld, personal communication to Britton, 2002). This suggests that although dTOR may act as a cell-autonomous nutrient-dependent checkpoint for metabolism, the larva's physiology is so effective in buffering cells against absolute starvation that this checkpoint is rarely if ever fully engaged. TOR is found in fungi and plants and so seems to be a metabolic regulator that was used prior to the advent of multicellularity. Insulin signaling, which is absent from fungi and plants, probably evolved later when multicellular animals required a system to coordinate and fine-tune metabolism in communities of cells. The insulin system is clearly advantageous, since animals in which it is 'short-circuited' by hyperactivation of Inr or PI3K are unable to tolerate even brief periods of starvation (Britton, 2002).
The most direct evidence that insulin signaling is nutritionally controlled was obtained using a cellular indicator of PI3K activity, tGPH, which is recruited to plasma membranes by the second messenger product of PI3K, PIP3. Subcellular tGPH distributions indicate that PIP3 levels are high in many cell types in fed larvae, but low in larvae that have been starved for protein. Although these changes in PIP3 levels might be due to altered expression of Inr, PI3K, or Pten, expression profiling experiments using cDNA microarrays indicate that levels of p60 and Dp110 mRNA are not depressed in L2 larvae that had been deprived of protein for 4 days. Perhaps the most attractive explanation for the apparent loss of PIP3 upon starvation is that some of the seven Drosophila insulin-like peptides (dILPs) are produced in a nutrition-dependent fashion. Several of the dilp genes are expressed in the larval gut (Brogiolo, 2001) which, as the conduit of nutritional influx, might be expected to mediate metabolic responses to feeding throughout the animal. Other dilps are expressed in the salivary glands, imaginal discs, and small numbers of cells in the central nervous system (Brogiolo, 2001; Britton, 2002).
In mammals, insulin promotes the cellular uptake and storage of carbohydrates, proteins, and lipids, and is the strongest anabolic inducer known. Insulin-mediated responses are indirectly antagonized by the hormone glucagon, which stimulates catabolic reactions and the mobilization of stored nutrients. Insulin and glucagon are produced in the pancreas, and their relative levels are constantly adjusted to maintain proper blood sugar levels. The mammalian liver is also a key player in the regulation of metabolic homeostasis. In humans, most of the accessible glycogen, the principle form of stored carbohydrate, is found in the liver. This glycogen can be mobilized in response to exercise or starvation. In Drosophila larvae, hyperactivation of the Inr/PI3K pathway leads to increased accumulation of nutrients in the fat body, an organ that resembles the mammalian liver as the principal site of stored glycogen. Conversely, inhibition of PI3K activity depletes stored nutrients from the fat body, as does starvation. This suggests that like mammals, insects regulate storage of metabolites in response to changes in levels of Inr/PI3K signaling. Direct assays of the levels of carbohydrates, storage proteins, and lipids in the fat body after starvation or manipulation of Inr/PI3K activity should prove informative. While there are as yet no known Drosophila homologs of glucagons, there must be some mechanism by which stored resources can be mobilized during starvation or at the transition from feeding to metamorphosis (Britton, 2002).
In the fruit fly Drosophila, four insulin genes are coexpressed in small clusters of cells [insulin-producing cells (IPCs)] in the brain. Ablation of these IPCs causes developmental delay, growth retardation, and elevated carbohydrate levels in larval hemolymph. All of the defects were reversed by ectopic expression of a Drosophila insulin transgene. On the basis of these functional data and the observation that IPCs release insulin into the circulatory system, it is concluded that brain IPCs are the main systemic supply of insulin during larval growth. It is proposed that IPCs and pancreatic islet beta cells are functionally analogous and may have evolved from a common ancestral insulin-producing neuron. Interestingly, the phenotype of flies lacking IPCs includes certain features of diabetes mellitus (Rulifson, 2002).
The Drosophila genome contains five Drosophila insulinlike peptide genes (dilp1 through -5) with significant homology to mouse and human insulins and two others with far less similarity (dilp6 and -7). These genes are expressed in tissues ranging from early embryonic mesoderm to small clusters of larval brain neurons, ventral nerve cord neurons, salivary gland, and midgut. Using messenger RNA (mRNA) in situ hybridization, it has been established that the most prominent insulin gene expression during larval stages, a period of intensive feeding and rapid growth, is within two bilaterally symmetric clusters of neurosecretory cells in the pars intercerebralis region of the protocerebrum (Rulifson, 2002).
An 859-base pair promoter fragment, comprised of sequences immediately 5' of dilp2, is sufficient to drive gene expression in the small clusters of larval brain neurons that express dilp1, -2, -3, and -5. To assess the role of the brain IPCs as an insulin-producing endocrine system, the brain IPCs were ablated using the dilp2 promoter to express the cell death-promoting factor, Reaper. The IPC ablation results in deficiency of brain neuron-derived insulin only. IPC ablation caused undergrowth phenotypes, developmental delays, and lethality similar to Drosophila insulin receptor (DInR) mutants. To rule out an underlying cause of these phenotypes other than insulin deficiency, such as non-IPC death from Reaper or loss of other essential brain IPC functions, a heat shock-inducible dilp2 transgene was used with ubiquitous expression to reverse the effect of the IPC ablation (Rulifson, 2002).
The defect in growth was quantified by comparing larval length after 120 hours of development, a time at which synchronized cultures of normal larvae will reach wandering third instar and puparium formation. After IPC ablation, larvae attained a mean length only 58% of normal size. Larvae with IPCs ablated but expressing the inducible dilp2 transgene had their mean length rescued to 88% of normal. The developmental time to reach wandering third instar and puparium formation was approximately 5 days in normal larvae but lengthened to 12 days in larvae after IPC ablation, a developmental rate similar to that observed in animals homozygous for loss-of-function mutations in DInR. Larvae with ablated IPCs that expressed the inducible dilp2 transgene require approximately 6 days to reach puparium formation. Thus, systemic DILP2 expression is sufficient to compensate for IPC ablation. The fact that brain IPC ablation is rescued by dilp2 alone suggests that insulin made by brain IPCs may be partially redundant (Rulifson, 2002).
IPC ablation produces small-sized adults of normal proportion. Examination of adult wings revealed reductions in both cell size and number after IPC ablation. Under the strongest condition of IPC ablation, mean wing size was reduced to 61% of normal, whereas wing cell number and size were reduced to 72% and 85% of normal, respectively. Under a less severe regimen of IPC ablation, mean wing size was reduced to 74% of normal, with reductions in cell number and size to 81% and 91% of normal, respectively. As in larval growth, the dilp2 transgene effectively reverses the effects of IPC ablation on wing growth and, in fact, caused a slight overgrowth effect, possibly due to the 20% lengthening of developmental time that allowed more growth. The overall reduction in cell size and number after IPC ablation is similar to that in DInR and IRS1-4 mutants. This, together with the observation that brain IPC-derived insulins can activate the DInR in vitro, suggests that brain IPCs are a key source of insulin for this growth control pathway (Rulifson, 2002).
The role of brain IPCs and insulin in the regulation of carbohydrate metabolism was also investigated. Trehalose is a disaccharide composed of two glucose molecules and is the principal blood sugar in many insects. Using the same heat shock regimen, the combined concentration was examined of glucose and trehalose in the hemolymph of wandering third instar larvae, a brief and discrete developmental stage before puparium formation when feeding has ceased. The IPC-ablated larvae had an average combined glucose and trehalose level of 38% above normal, and these levels returned to normal when DILP2 was provided systemically by the transgene. Elevated hemolymph carbohydrate levels in larvae lacking IPCs indicate that insulin is an essential regulator of energy metabolism in Drosophila. This accumulation of carbohydrate in the blood is reminiscent of that seen in human diabetes mellitus, although it should be noted that carbohydrate levels were measured during development rather than in adults (Rulifson, 2002).
To investigate how central nervous system (CNS)-derived insulin regulates systemic functions, the Drosophila IPC contacts outside the CNS were examined. The morphology of the brain IPCs was examined with the use of the dilp2 promoter to drive expression of mGFP, a membrane bound green fluorescent protein (GFP). The IPC clusters within the pars intercerebralis extend processes that terminate at the lateral protocerebrum and subesophageal ganglion. IPC processes also terminate on the heart and in the corpora cardiaca (CC) compartment of the ring gland, after crossing the midline and exiting the CNS. Labeling of the IPC processes with mGFP and DILP2 antibody revealed localization of DILP2 peptide within the processes that contact the heart and ring gland. DILP2 peptide is concentrated in a graded distribution outside the cells of synthesis on the heart, and colabeling with myosin heavy chain antibody, which labels the columnar heart epithelium, showed that the IPC processes and DILP2 are localized outside the lumen of the heart. These results suggest the heart surface may be the site of insulin release to the openly circulating hemolymph. It is proposed that brain IPCs are essential for organism-wide growth control and carbohydrate homeostasis through release of insulin peptides into circulating hemolymph. These cellular functions are notably similar to those of mammalian pancreatic ß cells (Rulifson, 2002).
In Drosophila and other insects, a fraction of CC cells synthesize adipokinetic hormone (AKH). AKH resembles glucagon in its activation of glycogen phosphorylase through heteromeric GTP-binding protein (G protein) and adenosine 3',5'-monophosphate (cAMP) signaling to elevate blood sugar, and the two proteins have some limited sequence similarity. Double labeling of AKH mRNA and DILP2 peptide shows that IPCs extend processes to the CC and that AKH-expressing cells contain DILP2. CC cells accumulate DILP2 within membrane-bound particles of the perinuclear space, suggesting the possibility that DILP2 is taken up by AKH cells. The possibility that dilp2 is transiently or minutely transcribed by AKH cells cannot be ruled out, although expression of either the dilp2 promoter or dilp1, -2, -3, and -5 mRNAs in the AKH cells has not been detected. Thus, in addition to contacts between IPCs themselves, the primary sites of IPC contact outside the CNS are the heart and the CC. Though they lack strict morphological homology, these intercellular contacts are analogous to the association of pancreatic ß cells with other ß cells, with glucagon-expressing a cells, and with blood vessels in the islets of Langerhans and may reflect underlying evolutionary conservation (Rulifson, 2002).
Thus, there is remarkable similarity of the organ systems underlying conserved insulin function in diptera and mammals. Moreover, the presence of IPCs in the nervous systems of other invertebrate and protochordate species and in primary cell cultures from mammalian fetal brain provides further evidence for a common ancestral insulin-producing organ of neural origin. These results also raise the possibility that common mechanisms of cell specification regulate development of pancreatic a cells and Drosophila brain IPCs (Rulifson, 2002).
The insulin/IGF-1 signaling pathway controls cellular and organismal growth in many multicellular organisms. In Drosophila, genetic defects in components of the insulin signaling pathway produce small flies that are delayed in development and possess fewer and smaller cells as well as female sterility, reminiscent of the phenotypes of starved flies. This study establishes a causal link between nutrient availability and insulin-dependent growth. In addition to the Drosophila insulin-like peptide 2 (dilp2) gene, overexpression of dilp1 and dilp3-7 is sufficient to promote growth. Three of the dilp genes are expressed in seven median neurosecretory cells (m-NSCs) in the brain. These m-NSCs possess axon terminals in the larval endocrine gland and on the aorta, from which DILPs may be released into the circulatory system. Although expressed in the same cells, the expression of the three genes is controlled by unrelated cis-regulatory elements. The expression of two of the three genes is regulated by nutrient availability. Genetic ablation of these neurosecretory cells mimics the phenotype of starved or insulin signaling mutant flies. These results point to a conserved role of the neuroendocrine axis in growth control in multicellular organisms (Ikeya, 2002).
The insulin/IGF signaling pathway plays a key role in the control of growth in vertebrates and invertebrates. In mammals, the primary role of insulin and the insulin receptor is energy homeostasis by the regulation of blood glucose levels. But mutations in the human insulin receptor gene also cause embryonic growth retardation. The primary growth regulatory function in mammals, however, is mediated by the IGF-1 and IGF-2 growth factors and the IGF-1 receptor. In Drosophila, there is a single insulin-like receptor, and it regulates postembryonic growth, reproduction, and aging. In vertebrates and invertebrates, intracellular signal transduction from the insulin/IGF receptors depends on insulin receptor substrate (IRS) proteins. In mammals, loss of IRS1 function causes severe reduction in embryonic and postembryonic growth (Araki, 1994; Tamemoto, 1994). Loss of IRS2 leads only to a moderate reduction in growth, but mice become hyperglycemic and contain increased body fat, and females are sterile (Withers, 1998). In Drosophila, flies mutant for chico, which encodes the single Drosophila homolog of IRS1-4, are developmentally delayed, have a severely reduced body size and increased fat, and females are sterile. This demonstrates a striking conservation of the role of the insulin/IGF signaling pathway during evolution (Ikeya, 2002 and references therein).
Several lines of evidence suggest a link between the activity of the insulin/IGF signaling pathway and nutrient availability. The female sterility and the growth retardation phenotypes of IRS2-/- and IRS1-/- mice, respectively, are similar to those observed in starved mice (Butler, 2001, Tiessen, 1999). In Drosophila, growth retardation, reduced body weight due to fewer and smaller cells, and female sterility are phenotypes not only characteristic of chico mutant flies but also of flies that have been starved during development. In fact, oogenesis is blocked during stem cell divisions and before the onset of vitellogenesis in starved flies and in chico mutant flies. Furthermore, starvation reduces the activity of the insulin signaling pathway in vivo. In nematodes, starvation or mutations in the insulin signaling pathway arrest development at the so-called Dauer stage. Moreover, caloric restriction and mutations in the insulin/IGF1 signaling system extend life span in vertebrates and invertebrates. It is not known, however, whether the link between nutrient availability and the activity of the insulin signaling pathway is direct, and how the nutritional status is translated into insulin/IGF receptor activity in the target tissues (Ikeya, 2002 and references therein).
One obvious hypothesis for a connection between nutrient availability and insulin/IGF activity is that nutrients control the expression of the insulin/IGF growth factors. In mammals, blood glucose levels not only regulate the release of insulin from the pancreatic ß cells, but also regulate the insulin gene expression via an autocrine loop (Xu, 2001). The regulation of IGF-1 levels that control postnatal growth is more complex. Growth hormone synthesized by the pituitary controls IGF-1 expression in the liver, accounting for approximately one-third of the postnatal growth promoting activity. Growth also depends on GH-independent expression of IGF-1 and GH activity that is independent of IGF-1 (Ikeya, 2002 and references therein).
The role of insulin-like growth factors in invertebrates in the regulation of growth and their regulation in response to starvation is less well defined. In Drosophila, the search for insulin-like genes (dilps) in the genome has revealed seven genes that show highly regulated temporal and spatial expression. Ubiquitous overexpression of one of the dilp genes, dilp2, is sufficient to increase body size. This growth-promoting activity of DILP2 is dependent on the insulin receptor signaling pathway. Three dilp genes are expressed in small cell clusters in the central region of the brain. These three genes are expressed in the same median neurosecretory cells (m-NSCs) possessing axon terminals in the ring gland and the corpora cardiaca. Although all three peptides can promote growth when overexpressed, they are regulated differentially in response to starvation. Furthermore, genetic ablation of the m-NSCs produces a phenotype reminiscent of chico mutant flies (Ikeya, 2002).
Expression of dilp2, 3, and 5 genes is detected in the same two clusters of cells. The position of these cell clusters in the pars intercerebralis of the larval brain suggests that they correspond to the m-NSC clusters that stain with an anti-Bombyxin antibody. Indeed, the expression of GFP under the control of the dilp2 cis-regulatory elements permits the visualization of the axonal projections of the dilp expressing cells. Axon projections are seen in the corpora cardiaca of the ring gland and on the aorta, the site from which neuropeptides are released into the hemolymph. Therefore, dilp2, 3, and 5 are coexpressed in the cluster of m-NSCs identified in larger insects and in Drosophila. Although expressed in the same cells, the temporal expression pattern of dilp 2, 3, and 5 in the m-NSCs differs. While dilp2 is expressed already in the first instar stage, dilp2 and dilp5 expression is detectable in the second instar stage, while dilp3 expression starts at the mid to late third instar stage. The successive activation of dilp genes in the m-NSCs correlates with the increasing growth that occurs during the last larval instar (Ikeya, 2002).
The dilp2, 3, and 5 genes are located together with dilp1 and dilp4 in a gene cluster spanning 26 kb on the third chromosome. To search for enhancer elements controlling the expression of the three genes in the m-NSCs, a series of lacZ reporter genes containing various amounts of upstream genomic sequences was constructed. Upstream fragments of 1.0 kb, 1.7 kb, and 450 bp derived from the dilp2, 3, and 5 genes, respectively, are sufficient to drive reporter gene expression specifically in the m-NSC. Thus, each of the three genes possesses its own m-NSC-specific enhancer. A further dissection of these upstream sequences has revealed that a 394 bp fragment located at position -540 to -146 of dilp2 is sufficient to recapitulate the expression of dilp2 in the m-NSCs. This construct, however, is no longer expressed in imaginal discs, suggesting that different enhancer elements control expression in this tissue. The cis-regulatory elements that control dilp3 expression are more complex. The m-NSC-specific expression depends on two separate elements located between -763 to -1167 and between +1 to -165, including the putative transcription start site. The fragment located between the m-NSC enhancers drives expression of dilp3 in gut muscles (Ikeya, 2002).
Surprisingly, sequence comparison of the genomic sequences required for m-NSC expression of dilp2, 3, and 5 did not reveal obvious stretches of similarity that may identify a common m-NSC-specific enhancer element in the different genes. It therefore appears that even within this small group of cells, the three dilp genes are regulated by different combinations of transcription factors (Ikeya, 2002).
A causal link between starvation-induced reduction in growth and reduced insulin receptor activity may involve the regulation of circulating DILP levels by nutrient availability. To test this hypothesis, the expression of dilp2, 3, and 5 was examined in third instar larvae that had been starved for 24 hr. In nonstarved larvae, mRNA transcripts of dilp2, 3 and dilp5 were detected in the m-NSCs. Upon starvation, dilp3 and dilp5 transcript levels are reduced, while dilp2 transcript levels remain unchanged. Similar results were obtained when the dilp5-lacZ reporter construct was used. In these larvae, lacZ mRNA is severely reduced upon starvation, while LacZ activity is still detectable owing to the stability of the LacZ protein. Therefore, the enhancer elements that respond to starvation are located in the 450 bp fragment used to analyze dilp5 expression. These results demonstrate that at least part of the nutrient-dependent regulation of growth is mediated by the regulated expression of the dilp3 and dilp5 genes (Ikeya, 2002).
During larval development, the seven dilp genes are expressed in a variety of different tissues in addition to the m-NSCs. To begin to address the role of DILP production in the m-NSCs, these cells were specifically ablated. The dilp2-Gal4 line, which is exclusively expressed in the m-NSCs starting at the late third instar stage, was used to drive expression of the proapoptotic gene reaper (rpr) in these cells. dilp2:rpr flies are viable but eclose one day later than control flies. Freshly eclosed flies show a slight but significant reduction in body weight. The size difference is also observed in the reduced wing area. Interestingly, the largest difference between control and dilp2:rpr flies is observed in the size of the abdomen of females. This difference becomes further enhanced during the first three days of adult life. During this phase, egg production is stimulated by feeding and mating in wild-type females. Comparison of the ovaries of three-day-old wild-type and dilp2:rpr females revealed a striking difference in ovary size. Each ovary is composed of approximately 15 ovarioles. Ovarioles are oocyte tubes containing stem cells at the tip and oocytes of increasing maturity toward the oviduct. While wild-type females possess multiple vitellogenic oocytes in each ovariole, dilp2:rpr females possess at most a single vitellogenic oocyte. This reduced size of the ovary of dilp2:rpr flies is reflected in the reduced fecundity of these females. While control flies lay on average 60 eggs per day, dilp2:rpr females produce only 10 eggs per day. The dilp2:rpr flies exhibit a developmental delay, reduced body size, and reduced fecundity of females owing to a partial block in production of vitellogenic oocytes. These phenotypes resemble those of weak mutations in the genes coding for components of the insulin signaling pathway. While chico females are almost completely sterile and severely reduced in size, certain combinations of Inr alleles produce females very similar to the dilp2:rpr flies. It is possible that the partially penetrant phenotype of m-NSC ablation is due to the late onset of expression of the dilp2-Gal4 driver line, resulting in only a partial ablation of the m-NSCs. Since dilp5 expression is also observed in the follicle cells of the female ovary, this m-NSC independent source of DILP may provide an alternative explanation for the partially penetrant phenotype of m-NSC ablation (Ikeya, 2002).
Nutrient-dependent regulation of growth and reproduction is observed in all multicellular organisms. The results presented here provide further support for an evolutionary conserved signaling pathway involved in this regulation. In mammals, insulin secretion from the pancreatic ß cells is regulated by the concentration of glucose in the blood, and thereby regulates energy homeostasis in response to food intake. Embryonic and postembryonic growth is regulated by IGF-1 production that in part depends on GH synthesized from the pituitary. In insects, the m-NSCs appear to play a role in both functions. The release of insulin-like peptides from the corpora cardiaca in Bombyx is regulated by carbohydrate levels in the hemolymph (Masumura, 1997) in a way analogous to the release of glucose from pancreatic ß cells in mammals. Expression of dilp3 and dilp5 is repressed by food withdrawal. Furthermore, ablation of the m-NSCs results in growth retardation. These results are consistent with a recent study showing that early Reaper-induced ablation of the m-NSC cells using a multimerized dilp2-Gal4 construct severely reduced growth and led to a concomitant increase in glucose and trehalose levels in the hemolymph of these animals (Rulifson, 2002). Given the data from Bombyx and Drosophila together, it is suggested that the m-NSCs possess functions similar to those of the pancreas and the pituitary in mammals (Ikeya, 2002).
In insects, the growth regulatory function of DILPs is 2-fold. (1) Circulating levels of DILPs in the hemolymph activate growth in the target tissues by the activation of the insulin receptor PI3K pathway in each cell. This action is complemented by the local production of DILPs in the target tissues in a manner similar to the expression of IGF-1 in target tissues. (2) DILPs exert their effect on growth indirectly. The m-NSCs project their axon terminals into the ring gland where ecdysone and JH are synthesized. The stimulation of ecdysone synthesis by insulin-like peptides is well documented in many insects. Furthermore, JH levels are reduced in long-lived insulin receptor mutant flies, suggesting that DILPs also regulate JH synthesis. Through the regulation of the levels of one or both of these two hormones, DILPs may regulate growth indirectly. Under starvation conditions, reduced JH levels may result in the premature increase in ecdysone titer in third instar larvae, leading to the precocious initiation of metamorphosis and thus producing flies with fewer cells. Alternatively, a precocious rise in ecdysone titer may be caused by the increase in the local concentration of DILPs in the ring gland due to the increased retention of insulin-like peptides in the corpora cardiaca during starvation (Ikeya, 2002).
In nematodes, nutrient availability regulates the developmental program and fertility without having a direct effect on cell size or cell number. In the absence of food, the larvae enter the immature long-lived Dauer stage. This response is controlled by two pathways, the insulin signaling pathway and the daf-4/TGF-ß pathway. Each of these pathways acts nonautonomously in the nervous system, and they converge on the nuclear hormone receptor daf-12. This implies an intermediate steroid or lipid hormone signal. Indeed, daf-9 that acts genetically between daf-2 and daf-4 signaling encodes a cytochrome P450 enzyme involved in steroid and fatty acid metabolism. It is interesting to note that the two activities of the Prothoracicotropic hormone (PTTH) that regulate ecdysone synthesis in Bombyx involve a member of the TGF-ß superfamily and an insulin-related peptide synthesized in distinct sets of neurosecretory cells in the brain. Therefore, it is likely that the nutrient-dependent growth regulation in nematodes and Drosophila is conserved in spite of the absence of an autonomous requirement of insulin signaling in cell growth in C. elegans (Ikeya, 2002).
Egg maturation is blocked by starvation in many species. In insects, ecdysone produced by the ovary is required for yolk protein production in the fat body and oocyte maturation. Ecdysone production is stimulated by insulin-like peptides in vitro and in vivo. Ablation of m-NSCs significantly slows down oogenesis. In humans, brain-specific knockouts of the insulin receptor or IRS2 also block oocyte maturation by affecting the synthesis of gonadotropins. Furthermore, steroidogenesis in the female gonad is required for oocyte maturation and is regulated by the expression of IGF-1 in different gonadal cells. Interestingly, expression of dilp5 is also detected in the ovarian follicle cells in Drosophila. Local production of DILP5 may stimulate ecdysone production in the female ovary directly. The similar roles of insulin-related peptides in growth regulation, energy homeostasis, and oogenesis in nematodes, insects, and mammals are striking. How far the underlying mechanisms are also conserved remains to be investigated (Ikeya, 2002).
Multicellular organisms must integrate growth and differentiation precisely to pattern complex tissues. Despite great progress in understanding how different cell fates are induced, it is poorly understood how differentiation decisions are temporally regulated. In a screen for patterning mutants, alleles were isolated of tsc1, a component of the insulin receptor (InR) growth control pathway. Loss of tsc1 disrupts patterning due to a loss of temporal control of differentiation. tsc1 controls the timing of differentiation downstream or in parallel to the RAS/MAPK pathway. Examination of InR, PI3K, PTEN, Tor, Rheb, and S6 kinase mutants demonstrates that increased InR signaling leads to precocious differentiation while decreased signaling leads to delays in differentiation. Importantly, cell fates are unchanged, but tissue organization is lost upon loss of developmental timing controls. These data suggest that intricate developmental decisions are coordinated with nutritional status and tissue growth by the InR signaling pathway (Bateman, 2004).
Thus InR/Tor signaling has a novel role in controlling the timing of differentiation. In both loss-of-function and ectopic expression experiments, it was found that activation of the InR/Tor pathway leads to the precocious acquisition of neuronal cell fate, while loss of signaling through this pathway delays (but does not block) differentiation. Importantly, InR and Tor signaling does not alter cell fates, only the time at which these cell fate decisions are made. This characteristic is important to a temporal control mechanism and ensures that only timing is regulated and not the actual cell fate decision (Bateman, 2004).
Mutants in tsc1 were isolated in a screen for genes that affect adhesion and PCP. Loss of tsc1 causes defects in ommatidial rotation due to precocious differentiation which is accompanied by the precocious initiation of rotation and hence ommatidial overrotation. Although cell fate is not affected by perturbations in InR/Tor signaling, developmental timing and tissue patterning are aberrant. Therefore, the precise control of timing of differentiation is essential for correct formation of complex tissues such as the Drosophila compound eye. The data show that the action of InR/Tor pathway on differentiation allows fine-tuning of binary switching mechanisms such as EGF signaling. This novel mechanism allows the organism to use humoral signals such as insulin-like molecules to temporally regulate differentiation. Under conditions of nutrient deprivation when growth rate slows, it is essential that differentiation keep pace with growth to maintain accurate patterning. The use of the InR/Tor pathway to control both growth and the timing of differentiation is an elegant solution to this challenge during development (Bateman, 2004).
The pattern of MAPK activation is unaffected by loss of tsc1. The EGF ligand, Spitz, is secreted by the R8 photoreceptor and diffuses to nearby cells, causing their recruitment and differentiation by activating the RAS/MAPK pathway. These data indicate that Spitz production in the R8 photoreceptor is unaffected by loss of tsc1, as is the transduction of the EGFR signal as far as the activation of MAPK in the recruited photoreceptors. In addition, the expression of regulators of photoreceptor differentiation downstream of MAPK (such as Lozenge, Yan, and Ttk), have been examined and no alteration in their levels or distribution in tsc1 mutant clones was found. Therefore, the temporal control of differentiation by InR/Tor signaling, acts downstream (or in parallel) to known components of photoreceptor differentiation (Bateman, 2004).
Studies of birth order-dependent cell fate specification in the Drosophila CNS have revealed that neuroblasts express a series of transcription factors in a set sequence, and both overexpression and loss-of-function studies have demonstrated that transcription factors present at the birth of neuroblasts are necessary and sufficient to direct differential cell lineages that are linked to different birth dates. Progression through the cell cycle is required for the temporal transition of these transcription factors. Although loss of tsc1 has been shown to lead to an acceleration through G1, alteration of the cell cycle by overexpression of cyclin E or cyclin D/CDK4 does not induce precocious differentiation. Therefore, precocious differentiation cannot be simply due to the alterations in the cell cycle. Another hallmark of tsc1 mutant cells is increased cell size. However, increasing cell size by overexpressing cyclin D/CDK4 or by overexpression of myc did not induce precocious differentiation, indicating that although cell size is increased in cases of overactive InR/Tor signaling, it is not an increase in cell mass that triggers premature differentiation. Moreover, compensating for the decreases in overall cellular growth rate caused by loss of InR signaling in clones by making clones in a Minute heterozygous background does not affect the slowing of differentiation caused by loss of the InR, confirming that InR/Tor signaling regulates timing of differentiation by a mechanism that is independent of and genetically separable from its effects on growth (Bateman, 2004).
Importantly, the InR/Tor pathway is found to control the timing of neuronal cell fate decisions in the eye and leg but does not appear to affect the timing of epithelial prehair initiation. The temporal control of differentiation by the InR/Tor pathway may be especially important for neurons since their axons must contact targets that are often far away. During normal development of the embryonic CNS, pioneer neurons are the first to differentiate and provide spatial cues for later-born neurons. If pioneer neurons are absent, targeting defects can occur. Tight temporal control of differentiation ensures that neurons are born in an environment that has the correct spatial cues for pathfinding. Intriguingly, disrupting insulin signaling results in defects in axonal targeting from the eye to the brain in Drosophila. The data suggest that these results may in part be due to precocious differentiation of the neurons (Bateman, 2004).
What is the mechanism by which InR/Tor signaling controls the timing of differentiation? Regulation of growth by InR/Tor signaling is mediated through translational control. This control is achieved though phosphorylation of S6 kinase (which phosphorylates the ribosomal protein S6) and 4E binding protein, an inhibitor of the translational initiation factor 4E. Ribosomal proteins and many protein synthesis elongation factors contain 5' oligopyrimidine tracts at their transcriptional start site, known as 5'TOPs. Translation of 5'TOP-containing transcripts is increased in response to PI3K/Tor signaling, thereby allowing coordinate expression of all ribosomal components. It is proposed that there is a 5'TOP present in the mRNA of an unknown proneural factor(s) that undergoes increased translation in response to InR/Tor signaling. This increased translation would lead to higher levels of proneural factors, speeding neural differentiation, which would allow for the precise coordination of growth and differentiation needed during the development of complex neural structures. Supporting this model is the finding that none of the InR/Tor signaling mutants tested gives rise to ectopic differentiation of neurons. Alterations are observed only in the timing of differentiation, at the correct location, in both the eye and leg imaginal discs. This observation is consistent with a mechanism involving translational regulation (via a 5'TOP) of hypothetical proneural factor(s), i.e., modulation of the level of such a factor or factors can only occur once the proneural transcript is already present. A corollary of this model is that InR/Tor signaling would act to modulate the gap between transcription and translation of the hypothetical factor(s). The importance of the gap length between transcription and translation has recently been demonstrated for Notch signaling in the presomitic mesoderm during somite formation (Bateman, 2004).
Interestingly, a hallmark of the tumors that arise from loss of TSC1 is that they are highly differentiated and largely benign. This characteristic is in contrast to tumors that are malignant, arising from loss of PTEN. This malignancy may be due to the role of PTEN in many other pathways aside from growth signaling, while TSC1 has a more restricted function in the growth control pathway. The precocious differentiation induced by loss of TSC1 may contribute to their low malignancy, since high levels of differentiation are generally considered an indication of low metastatic potential. However, the exact causes of some of the most debilitating symptoms of tuberous sclerosis, such as neurological abnormalities and epilepsy, are still unclear. Future work will determine if precocious and hence inappropriate differentiation decisions contribute to the pathology of tuberous sclerosis in man (Bateman, 2004).
The insulin signaling pathway evolved to allow a fast response to changes in nutrient availability while keeping glucose concentration constant in serum. This study shows that, both in Drosophila and mammals, insulin receptor (InR) represses its own synthesis by a feedback mechanism directed by the transcription factor dFOXO/FOXO1. In Drosophila, dFOXO is responsible for activating transcription of dInR, and nutritional conditions can modulate this effect. Starvation up-regulates mRNA of dInR in wild-type but not dFOXO-deficient flies. Importantly, FOXO1 acts in mammalian cells like its Drosophila counterpart, up-regulating the InR mRNA level upon fasting. Mammalian cells up-regulate the InR mRNA in the absence of serum, conditions that induce the dephosphorylation and activation of FOXO1. Interestingly, insulin is able to reverse this effect. Therefore, dFOXO/FOXO1 acts as an insulin sensor to activate insulin signaling, allowing a fast response to the hormone after each meal. These results reveal a key feedback control mechanism for dFOXO/FOXO1 in regulating metabolism and insulin signaling (Puig, 2005).
It is well known that the expression and activity of the InR can be regulated by a wide variety of factors and that changes in the numbers of receptor molecules plays a pivotal role in several physiologic and pathologic states. The lowered sensitivity of cells to insulin and the hyperinsulinemia observed in obesity and type II diabetes mellitus is often accompanied by a reduced number of insulin receptors. Insulin is thought to down-regulate its own receptor by a variety of mechanisms that can influence its synthesis as well as degradation. Interestingly, it has been shown that the number of InR molecules correlates with nutritional conditions both in tissue culture cells and in animals. Thus, levels of InR in growing HepG2 cells are relatively low, and they increase substantially if cells are starved. In addition, states of chronic hyperinsulinemia produce a reduction in the number of InR present in the plasma membrane. InR mRNA levels also change in animals depending on fasting-feeding conditions. For example, rats fed a high-fat diet display a decreased number of InR molecules in liver plasma membranes, and InR mRNA levels in rat skeletal muscle or liver increase after fasting, returning to normal levels after insulin treatment or refeeding. Interestingly, tissues other than muscle or liver might have similar regulation. For example, mRNA and protein levels of rat intestinal InR increase up to 230% in fasting conditions, and these effects are fully reversed by refeeding. Similar observations have been made in other organisms. These effects indicate a nutritional influence on the abundance of the InR. Importantly, insulin levels in serum change in parallel to nutrient availability, both in flies and mammals. Thus, when nutrients are high—that is, after a meal—insulin levels increase, while they decrease upon fasting. In Drosophila it has been shown that the InR/PI3K pathway coordinates cellular metabolism with nutritional conditions. Inhibiting this pathway phenocopies the cellular and organismal effects of starvation, while activating it bypasses the nutritional requirements for cell growth. The InR/PI3K pathway regulates the activity of FOXO1 in mammals, C. elegans, and Drosophila, so nutrient activation of the PI3K pathway results in inactivation of FOXO1 by phosphorylation. However, despite this accumulated base of information, the molecular mechanism linking FOXO1 and InR expression had not been revealed (Puig, 2005).
This study shows that mammalian FOXO1 and its Drosophila counterpart dFOXO directly regulate insulin-signaling response to nutritional conditions through a feedback mechanism that involves activation of transcription from the InR promoter. Incubating C2C12 cells with a balanced salt solution or with serum-free medium up-regulates insulin receptor mRNA. Under these conditions, FOXO1 becomes dephosphorylated and actively binds to the InR promoter. When insulin is added to the medium, InR mRNA is down-regulated, even in the absence of serum, vitamins, amino acids, and glucose. Concomitantly, phosphorylation of FOXO1 increases and binding to InR promoter decreases. These results indicate that FOXO1 regulates InR transcription through a direct feedback mechanism that senses insulin levels in serum, which is, in turn, a reflection of nutrient load. It is important to note that, at this point, it cannot be ruled out that the increased InR protein levels caused by FOXO1 could be due to other mechanisms in addition to increased transcription from the InR promoter (i.e., affecting mRNA stability, or protein translation) (Puig, 2005).
In Drosophila a similar mechanism occurs. Incubation of S2 cells with complete medium keeps dFOXO phosphorylated and inactive, while incubation in HBSS dephosphorylates dFOXO. dInR mRNA is up-regulated only when dFOXO is dephosphorylated and active. In addition, wild-type flies starved for 4 d up-regulate dInR, and this effect requires an intact dfoxo gene. These studies indicate that in Drosophila, the PI3K/Akt pathway also senses insulin levels and regulates binding of dFOXO to the dInR promoter accordingly. These results underscore the importance of the InR/PI3K/Akt pathway in sensing nutrients and insulin, a function that has been conserved during evolution. They also highlight the role of FOXO1 as a sensor for insulin levels, promoting accumulation of InR in the absence of insulin, thereby allowing a fast response to the hormone after each meal. Under conditions in which insulin levels are chronically elevated, for example, in obese animals or patients, down-regulation of InR transcription would occur and insulin sensitivity would be impaired. These results establish the FOXO1 transcription factor as a key player in a feedback control mechanism that regulates metabolism and insulin signaling (Puig, 2005).
The results show that in conditions in which insulin levels are low, mammalian FOXO1 activates InR. Interestingly, it was observed that FOXO1 also activates the insulin receptor substrate-2 (IRS-2) promoter under fasting conditions, and, since it occurs with InR, insulin is sufficient to reverse this effect. FOXO1 binds IRS-2 promoter in vitro and in vivo and activates IRS-2 transcription when muscle or liver cells are fasted. In addition, FOXO1 activates IRS-2 promoter in luciferase assays, and this activation depends on the presence of a consensus FRE present in the IRS-2 promoter, because mutating this FRE abolishes FOXO1-dependent activation. Thus, FOXO1 regulation of IRS-2 is parallel to InR regulation. It has also been reported that SREBPs compete with FOXO transcription factors for binding to the IRS-2 promoter in liver; while SREBPs inhibit IRS-2 production, FOXO1 was found to activate IRS-2 transcription. It was also found that fasting promotes binding of FOXO1 to the FRE of the IRS-2 promoter. Therefore, these findings strongly support the conclusions that FOXO1 regulates insulin signaling through a feedback mechanism that impinges on the insulin receptor and at least one of its substrates, IRS-2. After a meal, high levels of insulin peptide hormone activate its cognate receptor, which leads to repression of InR and IRS-2 transcription, resulting in subsequent dampening of the pathway by reducing the number of receptors on the cell surface and by limiting its ability to signal downstream through IRS-2. Conversely, fasting causes reduced levels of InR signaling, which in turn activates FOXO1, leading to increased transcription of InR and IRS-2. Once this transcription mechanism is activated, feedback regulation and phosphorylation of FOXO1 via the insulin signaling cascade automodulates InR expression. Insulin sensitivity could, therefore, be significantly affected by FOXO1 regulation. Regulation of insulin sensitivity by a feedback loop through FOXO1 would allow the cells to keep an exquisite metabolic balance between feeding and fasting states, permitting a faster response of the tissues to insulin changes. This feedback mechanism could well be disrupted in pathological states with abnormally increased insulin levels as is found in the case of insulin-resistant diabetes (Puig, 2005).
Hunger elicits diverse, yet coordinated, adaptive responses across species, but the underlying signaling mechanism remains poorly understood. This study reports on the function and mechanism of the Drosophila insulin-like system in the central regulation of different hunger-driven behaviors. Overexpression of Drosophila insulin-like peptides (DILPs) in the nervous system of fasted larvae suppresses the hunger-driven increase of ingestion rate and intake of nonpreferred foods (e.g., a less accessible solid food). Moreover, up-regulation of Drosophila p70/S6 kinase activity in DILP neurons leads to attenuated hunger response by fasted larvae, whereas its down-regulation triggered fed larvae to display motivated foraging and feeding. Finally, evidence is provided that neural regulation of food preference but not ingestion rate may involve direct signaling by DILPs to neurons expressing neuropeptide F receptor 1, a receptor for neuropeptide Y-like neuropeptide F. This study reveals a prominent role of neural Drosophila p70/S6 kinase in the modulation of hunger response by insulin-like and neuropeptide Y-like signaling pathways (Wu, 2006).
The relatively simple Drosophila larva offers a genetically tractable model to define and characterize different neuronal signaling pathways that constitute a complete central feeding apparatus. Younger third-instar larvae forage actively and use their mouth hooks for food intake. Larvae normally feed on liquid food, and their food ingestion can be quantified by measuring the contraction rate of the mouth hooks. This study examined how food deprivation affects larval feeding response to a liquid (e.g., 10% glucose-agar paste) and less accessible solid food (e.g., 10% glucose agar blocks). To extract embedded glucose from the solid food, larvae have to pulverize the food by scraping agar surface with mouth hooks. Unless stated otherwise, synchronized third-instar larvae (74 h after egg laying) were used for the assays (Wu, 2006).
When fed ad libitum, normal larvae (w1118) display significant feeding activity in the liquid food with an average mouth-hook contraction frequency of ~30 times in a 30-s test period; in contrast, these larvae declined the solid food. However, larvae withheld from food (on a wet tissue) for 40 or 120 min display increased intake of both liquid and solid foods. For example, larvae fasted for 120 min show a 100% and >500% increase in mouth-hook contraction rate in liquid and solid food, respectively. Thus, deprivation not only enhances feeding rate in a graded fashion, but also triggers motivated foraging on the less accessible food normally rejected by fed larvae. In addition, larvae display virtually identical feeding responses to liquid and solid foods containing 10% glucose, apple juice, or 10% glucose/yeast under deprived and nondeprived conditions. Therefore, these paradigms appear to provide a general assessment of larval feeding response (Wu, 2006).
dS6K is a cell-autonomous effector of nutrient-sensing pathways. This study investigated a possible role of neural dS6K in coupling peripheral physiological hunger signals and neuronal activities critical for hunger-driven behaviors. The transcripts of dilp1, dilp2, dilp3, and dilp5 are predominantly expressed in two small clusters of medial neurosecretory cells that project to the ring gland, the fly heart, and the brain lobes. A gal4 driver containing a 2-kb fragment from the dilp2 promoter (dilp2-gal4) was generated that directs the specific expression of a GFP reporter in those cells. Using dilp2-gal4, two transgenes, UAS-dS6KDN, encoding a dominant negative, and UAS-dS6KACT, a constitutively active form of dS6K, were expressed. When fed ad libitum, control larvae (w x UAS-dS6KDN or UAS-dS6KACT) behave like w larvae. However, dilp2-gal4 x UAS-dS6KDN larvae displayed a 50% increase in the rate of liquid-food intake and significant feeding of the solid food. Conversely, fasted larvae overexpressing dS6K activity (dilp2-gal4 x UAS-dS6KACT) showed attenuated feeding response to both liquid and solid foods. These findings reveal that dS6K in DILP neurons mediates hunger regulation of approaching/consumptive behaviors, controlling both quality and quantity of food for ingestion. The body size and the developmental rate of all four groups of larvae were measured, and no significant differences were detected (Wu, 2006).
DILPs act as neurohormones in Drosophila larvae. Down-regulation of dS6K activity in DILP neurons may reduce DILP release, thereby promoting increased food intake that is normally triggered only by hunger. A corollary of this interpretation is that overproduction of DILPs in the nervous system should interfere with hunger response by deprived animals. To test this idea, a neural-specific elav-gal4 driver was used to direct dilp expression in the larval nervous system. Three UAS-dilp lines (UAS-dilp2, UAS-dilp3, and UAS-dilp4) were chosen for the analysis. The elav-gal4 x UAS-dilp2 and UAS-dilp4 larvae displayed normal feeding response when fed ad libitum. However, the same larvae fasted for 120 min displayed significantly attenuated feeding rates, similar to those of dilp2-gal4 x UAS-dS6KACT larvae. For example, the comparative analysis of the elav-gal4 x UAS-GFP control and elav-gal4 x UAS-dilp2 and UAS-dilp4 experimental larvae showed that the latter were ~30% and 33–45% lower in the ingestion rate of the liquid and solid food, respectively; surprisingly, elav-gal4 x UAS-dilp3 and UAS-GFP larvae showed virtually identical feeding responses. Therefore, DILP2 and DILP4 negatively regulate hunger-driven feeding activities. Taken together, these results suggest that a high level of dS6K activity in DILP neurons may suppress hunger response by reducing DILP release (Wu, 2006).
Attempts were made to delineate the signaling mechanism that couples the dS6K activity in DILP neurons with its broad impact on hunger-driven feeding activities. A previous study showed that fasted larvae ablated of NPF or its receptor (NPFR1) neurons are deficient in motivated feeding of the less-preferred solid food but normal in feeding of richer liquid food. It was of interest to enquire whether the NPF/NPFR1 neuronal pathway might be one of the downstream effectors of the DILP pathway. To test this hypothesis, the function of three components of the dInR signaling pathway were analyzed in NPFR1 neurons: dInR, phosphatidylinositol 3,4,5-trisphosphate 3-phosphatase (dPTEN), and phosphatidylinositol 3-kinase (dPI3K). Five different transgenes were used: UAS-dInRACT and UAS-dInRDN encode a constitutively active and a dominant-negative form of dInR, respectively; UAS-Dp110 and UAS-dPI3KDN encode a catalytic subunit and a dominant-negative form of dPI3K, respectively; and UAS-dPTEN encodes a functional enzyme. When fed ad libitum, npfr1-gal4 x UAS-dInRDN, UAS-dPTEN, or UAS-dPI3KDN larvae display hyperactive feeding of the solid food, similar to w larvae deprived for 40 min. In contrast, fasted larvae overexpressing dInR or dPI3K (npfr1-gal4 x UAS-dInRACT or UAS-Dp110) display attenuated feeding response to the solid food. Importantly, larvae with up- or down-regulated dInR signaling in NPFR1 neurons do not exhibit significant changes in the intake rate of the richer liquid food relative to the paired controls. Taken together, these findings suggest that the dInR pathway negatively regulates the activity of NPFR1 neuron and mediates the DILP-regulated change in food preference but not ingestion rate. Furthermore, the results suggest that NPFR1 neurons are the direct targets of DILPs (Wu, 2006).
A possible role of dS6K in hunger regulation of the functioning of NPFR1 neurons was evaluated, by expressing UAS-dS6KDN and UAS-dS6KACT using npfr1-gal4. When fed ad libitum, npfr1-gal4 x UAS-dS6KDN larvae display hyperactive feeding of the solid food, similar to npfr1-gal4 x UAS-dInRDN larvae. However, these larvae, unlike dilp2-gal4 x UAS-dS6KDN animals, display no increases in the ingestion rate of the richer liquid food. Conversely, fasted larvae overexpressing dS6K (npfr1-gal4 x UAS-dS6KACT) display attenuated feeding response to the solid food. These findings suggest that dS6K also negatively regulates the activity of NPFR1 neurons in food preference, but does not mediate the regulation of feeding rate by DILP signaling (Wu, 2006).
The food response was evaluated of the solid and liquid food by larvae overexpressing an npfr1 cDNA under the control of an npfr1-gal4 driver. In the presence of the liquid food, both experimental (npfr1-gal4 x UAS-npfr1) and control larvae (e.g., npfr1-gal4 x UAS-ANF-GFP), fed or fasted, show similar intake rates and comparable increases in feeding response to hunger. However, when forced to feed on the solid food, fed experimental larvae exhibit significant intake of the solid food (30 times per 30 s), whereas fed controls rejected the same food. Thus, NPFR1 overexpression selectively promotes change in food preference without increasing ingestion rate. It was also observed that the feeding responses of NPFR1-overexpressing larvae and controls fasted for 120 min were indistinguishable. Thus, the effect of NPFR1 overexpression on food preference is detectable only in fed or mildly fasted larvae, suggesting hunger-activated NPFR1 signaling approaches a plateau in severely fasted animals (Wu, 2006).
npfr1 activity was selectively knocked down by expressing npfr1 dsRNA in the nervous system. The UAS-npfr1dsRNA lines were previously used to functionally disrupt npfr1 activity. It was found that 120-min fasted larvae expressing npfr1 dsRNA in NPFR1 or the nervous system (npfr1-gal4, elav-gal4, or appl-gal4 x UAS-npfr1dsRNA) were deficient in motivated feeding of the solid but not liquid food. In contrast, all control larvae, including those expressing npfr1dsRNA in muscle cells (MHC82-gal4 x UAS-npfr1dsRNA), showed normal feeding responses. These results indicate that neural NPFR1 mediates hunger regulation of food selection (Wu, 2006).
A potential problem of the previous transgenic studies is that NPF/NPFR1 signaling is likely to be disrupted in a relatively early stage of larval development. Conceivably, the NPF/NPFR1 neuronal pathway could be essential for ad libitum or hunger-driven feeding of richer liquid foods, but such an activity might be masked by some yet-unidentified compensatory mechanism triggered by its early loss. To test this idea, attempts were made to disrupt NPF/NPFR1 neuronal signaling in a temporally controlled manner by expressing a temperature-sensitive allele of shibire (shits1) driven by npf-gal4 or npfr1-gal4. The shits1 allele encodes a semidominant-negative form of dynamin that blocks neurotransmitter release at a restrictive temperature (>29°C). At the permissive temperature of 23°C, 120-min-fasted experimental larvae (npf-gal4 and npfr1-gal4 x UAS-shits1) and paired controls (y w x UAS-shits1 and npf-gal4 and npfr1-gal4 x w1118) displayed normal feeding responses to both liquid and solid foods. However, if larvae were incubated at 30°C for 15 min, controls still displayed normal feeding activities, whereas the experimental larvae showed attenuated feeding response to the solid but not liquid food. Therefore, there was no detectable developmental or physiological compensation for the loss of NPF signaling in Drosophila larvae. These results also suggest that the NPF/NPFR1 neuronal pathway is acutely required to initiate and maintain larval hunger response. The foraging activity of the experimental larvae was completely restored when the assay temperature was reduced to 23°C, suggesting that the NPF system can modulate the intensity and duration of feeding response (Wu, 2006).
This study has shown that dS6K regulates different, yet coordinated, behaviors controlling quantitative and qualitative aspects of hunger-adaptive food response. Evidence is provided that dS6K mediates hunger regulation of two opposing insulin- and NPY-like signaling activities, dynamically modifying larval food preference and feeding rate based on the nutritional state. For example, hunger stimuli may cause a reduction of dS6K activity in DILP neurons, resulting in the suppression of DILP signaling that negatively regulates a downstream NPF/NPFR1-dependent and another NPF-independent neuronal pathway. The DILP/NPFR1 neuronal pathway selectively mediates hunger-adaptive change in food preference, possibly by overriding the high threshold of food acceptance set by a separate default pathway, enabling hungry animals to be receptive to less preferred foods. The NPF/NPFR1-independent pathway promotes a general increase in the ingestion rate of preferred/less preferred foods, enabling animals to compete effectively for limited food sources. This study also implicates the presence of a separate default pathway for mediating the selective intake of preferred foods (baseline feeding) in larvae fed ad libitum. This default pathway may be largely insensitive to DILP or NPF signaling, because overexpression of dS6K, DILPs, or NPFR1 in nondeprived larvae does not affect ad libitum feeding in the liquid food. It is suggested that the conserved S6K pathway may be critical for regulating behavioral adaptation to hunger in diverse organisms, including humans, and its components are potential drug targets for appetite control (Wu, 2006).
The functional differences of DILP1–7 have not been reported previously. In this study, dilp2, dilp3, and dilp4 were shown to be functionally distinct. DILP2 and DILP3 both are produced in the same medial neurosecretory cells. However, unlike DILP2, DILP3 is apparently not involved in suppressing deprivation-motivated feeding. It is still unclear whether the differential activities of DILP2 and DILP3 reflect their structural divergence or are caused by the presence of yet-unidentified dInR isoforms. DILP4 is not expressed within the two medial clusters of DILP neurons. Under acute deprivation, the level of dilp4 transcripts showed a 5-fold reduction in the larval CNS. Thus, it is possible that DILP4 may play a localized role in promoting feeding response inside the CNS (Wu, 2006).
Feeding is a reward-seeking behavior, and deprivation strengthens the reinforcing effect (reward value) of food. These studies suggest a previously uncharacterized role of the DILP/dInR signaling pathway in regulating an animal's perception of food quality. The DILP/NPF neural network may regulate an animal's incentive to acquire lower-quality foods by modifying the reward circuit. This hypothesis is interesting in light of the findings that foods and abused substances may act on the same reward circuit, and highly palatable foods can reduce drug-seeking behaviors. It is also possible that the DILP/NPF system might represent a specialized neural circuit that positively alters the reward value of lesser-quality foods. Conceivably, a better understanding of the action of this signaling system may provide fresh insights into neural mechanisms for controlling eating and drug-seeking behaviors (Wu, 2006).
Given its prominent role in behavioral adaptation to hunger, the insulin/NPY-like neural network is likely of primary importance to animal evolution. In addition, insulin and NPY family molecules have been found in a wide range of animals from humans to worms. Therefore, the insulin/NPY-like network may be a useful model for studying comparatively how diverse animals have evolved distinct ways of adapting an ancestral neural system to suit their respective lifestyles (Wu, 2006).
Insulin signaling pathways are implicated in several physiological processes in invertebrates, including the control of growth and life span; the latter of these has also been correlated with juvenile hormone (JH) deficiency. In turn, JH levels have been correlated with sex-specific differences in locomotor activity. This study examined the involvement of the insulin signaling pathway in sex-specific differences in locomotor activity in Drosophila. Ablation of insulin-producing neurons in the adult pars-intercerebralis was found to increase trehalosemia and to abolish sexual dimorphism relevant to locomotion. Conversely, hyper-insulinemia induced by insulin injection or by over-expression of an insulin-like peptide decreases trehalosemia but does not affect locomotive behavior. Moreover, this study also showed that in the head of adult flies, the insulin receptor (InR) is expressed only in the fat body surrounding the brain. While both male and female InR mutants are hyper-trehalosemic, they exhibit similar patterns of locomotor activity. These results indicate that first, insulin controls trehalosemia in adults, and second, like JH, it controls sex-specific differences in the locomotor activity of adult Drosophila in a manner independent of its effect on trehalose metabolism (Belgacem, 2006).
In Drosophila, sexual dimorphism has been reported for the number of activity/inactivity phases (start/stops) during locomotion. Feminizing cells (FCs) of the mid-anterior part of the pars intercerebralis (PI), and JH
have been implicated in the control of this dimorphism. This study showed that insulin-producing cells (IPCs) are also located in the mid-anterior part of the PI in adult flies, in the same cluster as, but distinct from, the PI neurons termed feminizing cells (FCs). Second, this study showed by conditional genetic ablation that these cells are involved in the control of sex-specific differences
in locomotion: flies without IPCs present the
same number of start/stops, such that males have a
feminine activity profile. Third, perturbation of insulin
pathways by mutations affecting the insulin receptor
(InR) also has a similar effect on sex-specific
locomotion. This result corroborates the idea that
control of the number of start/stops by IPCs is mediated
by insulin-like peptides and not by another product
from these cells. Indeed, the ablation of IPCs
removes the cells and everything in them, like putative
unrelated but co-expressed peptides or transmitters.
However, the finding that the InR mutation leads
to a similar phenotype than the IPCs ablation is in
accordance with the statement that the number of
start/stop might be mediated by the insulin-like peptides.
In contrast, hyper-insulinemia induced by insulin
injection or by over-expression of the dilp2 gene (hsdilp2)
does not affect the number of start/stops in
males and thus is not implicated in this sexual dimorphism (Belgacem, 2006).
Another physiological parameter that was investigated
is the carbohydrate level in hemolymph. IPCs control the trehalose level at the
organismal level via the secretion of insulin analogues.
Indeed, a decrease in the insulin level (caused
by ablation of IPCs or disruption of insulin signaling
with InR mutations) increases the trehalose level,
while augmentation of the insulin level (caused by
insulin injection or over-expression of dilp2) reduces
it. These results clearly show that insulin has an endocrine
function in adult Drosophila, as in mammals. In some other insects, insulin
and/or insulin analogues have been shown to influence
carbohydrate levels, particularly as hypoglycemic hormones (Belgacem, 2006).
This study also reports that the insulin receptor is
expressed at the brain periphery in the fat body (FB), as well as
in the corpus allatum, the well established site for the
JH synthesis. Surprisingly, no InR was found on
neuronal cells of the brain. Perhaps InR is not expressed
at all in the central nervous system of adult Drosophila,
or it was not detected because a heterologous
antibody (anti-human) was used to detect it (Belgacem, 2006).
The antibody used was directed against the α-subunit
of human insulin receptor, and precisely from the
sequence of the third exon, which encodes the insulin-binding
domain. A sequence homology performed between this human InR third exon and Drosophila, reveals 36% of homology over 102 amino-acid residues,
suggesting that this domain is well conserved.
Moreover, the strong detection of the over-expressed
InR in the muscle is in accordance with the
specificity of the antibody used. Alternatively and in
an independent way, physiological approaches have
also shown that injection of heterologous insulin (from
bovine) is able to activate its invertebrate homologue,
both in blowflies as well as in Drosophila, again suggesting a well-conserved
domain between different species. Finally, a third
argument in accordance with the specificity of the InR
is supported by the similar results obtained from the
two independent approaches: immunohistological
staining analysis and injection of labeled insulin leading
to a similar localization in the brain fat body (Belgacem, 2006).
Although InR is expressed both in the head fat body and in the corpus allatum, physiological and behavioral results suggest that the observed effects on sex-specific differences in locomotor activity probably result from disruption of the
insulin pathway in the corpus allatum, rather than in
the fat body. Indeed, the disruption of the cc-ca
gland, by the ablation of the cc, which leads, in
males, to a female-like activity profile supports this
assumption. Conversely, it is suspected that altered trehalose
metabolism phenotype might be due to the distortion
of the insulin pathway in the fat body. However,
sex-specific differences in locomotor activity
under control of signal arising from the fat body
could not yet be totally excluded, since two recent
studies have suggested that genetic disruption and/or
manipulation of the FB affects behavior. The findings
that the InR is specifically expressed in FB cells and
that the locomotor activity of males lacking InR function
is feminized could also correlate with a role for the FB in a sexually dimorphic behavior. Obviously, further experiments, as for instance, tissue-specific
targeting of InR disruption, which will require specific
GAL4 drivers either in the corpus allatum or fat
body, will be necessary for such fine differential dissection (Belgacem, 2006).
The molecular linkage of the insulin pathway to
locomotor activity patterns, which also depend on JH
levels, remains to be elucidated. Mammalian hydroxymethylglutaryl-CoA
reductase (HMGCR), a key enzyme in cholesterol
and sterol synthesis, is transcriptionally regulated by
the insulin pathway. Drosophila HMGCR is also a central enzyme in the JH biosynthetic pathway and likely plays an important role in JH regulation. Thus, transcriptional regulation of HMGCR by insulin-dependent regulatory elements may link the JH and insulin pathways (Belgacem, 2006).
In conclusion, this study has shown that the insulin signaling
pathway is implicated in both males and females in the regulation of trehalose levels, since hypo- and hyper-insulinemia affects both sexes.
Moreover, this pathway is also implicated in sex-specific differences in locomotor activity, since perturbations resulting in hypo-insulinemia
feminize the locomotor behavior of males, whereas hyper-insulinemia has no effect. Therefore, this new insulin-dependent effect seems to be distinct
from the hormonal role of insulin in trehalose level
regulation and is likely mediated by either a different
intracellular signaling pathway or under control of
different tissues. Finally, the identification of the JH
target, and more specifically its receptor, is the next
crucial step in understanding how brain structures and neurons differentially control sex-specific aspects of locomotor activity (Belgacem, 2006).
It is generally accepted that the growth rate of an organism is modulated by the availability of nutrients. One common mechanism to control cellular growth is through the global down-regulation of cap-dependent translation by eIF4E-binding proteins (4E-BPs). Evidence is reported for a novel mechanism that allows eukaryotes to coordinate and selectively couple transcription and translation of target genes in response to a nutrient and growth signaling cascade. The Drosophila insulin-like receptor (dINR) pathway incorporates 4E-BP resistant cellular internal ribosome entry site (IRES) containing mRNAs, to functionally couple transcriptional activation with differential translational control in a cell that is otherwise translationally repressed by 4E-BP. Although examples of cellular IRESs have been previously reported, their critical role mediating a key physiological response has not been well documented. These studies reveal an integrated transcriptional and translational response mechanism specifically dependent on a cellular IRES that coordinates an essential physiological signal responsible for monitoring nutrient and cell growth conditions (Marr, 2007).
Coupled transcription and protein synthesis is a hallmark of prokaryotic gene expression. The advantages of such a linked system are well recognized as it provides smooth coordination to ensure that cells respond appropriately to signals such as nutrient availability. A rapid response to such environmental signals also allows for multiple points of regulation and a fine-tuning mechanism for controlling gene expression. In eukaryotic organisms, the compartmentalization of the cell nucleus makes the direct coupling of transcription and translation problematic. Nevertheless, like prokaryotes, the metazoan cell must respond to many external as well as internal signals, and a coupled response would be highly advantageous. However, there is currently little evidence for such a direct linkage, either physical or functional, in metazoans. In attempts to dissect the transcriptional regulatory circuitry of the insulin-like signaling cascade in Drosophila, a potentially new mechanism that functionally links transcription and translation has been identified (Marr, 2007).
Metazoan organisms must strictly control both body and organ size during development. Thus, cell size and cell number are tightly controlled to determine the final size of an animal. One of the cues used in determining growth regulation is nutrient availability. The insulin receptor (INR) and insulin-like growth factor (IGF) receptor pathways have evolved as key sensors of nutrient availability and play an important role in both cell-autonomous and nonautonomous decisions controlling cellular proliferation, cell size determination, and the response to nutrient availability. In Drosophila, this pathway is critical for determining body and organ size as well as metabolic homeostasis and life span. Perhaps most notably, misregulation of this pathway in humans can lead to type 2 diabetes and all of its associated pathologies, which is becoming a rapidly escalating worldwide epidemic (Marr, 2007).
The INR/IGF pathway is highly conserved, with homologs of the key molecular players present in metazoan organisms from flies to humans. The downstream targets of this signaling cascade are thought to separately modulate both transcription and translation to potentiate signals for either growth or stasis. In the presence of insulin or insulin-like peptides, the signaling cascade activates the oncogenic protein kinase Akt. To control RNA synthesis, Akt phosphorylates the Forkhead-box-binding protein (dFOXO) family of transcription factors, sequestering them in the cytoplasm and thus effectively inactivating them. This in turn prevents activated transcription of the dFOXO target genes. In addition, Akt stimulates the modification of the target of rapamycin (TOR) protein, which in turn phosphorylates and inactivates the translation initiation inhibitor eIF4E-binding protein (d4E-BP). In its unphosphorylated and active state, d4E-BP binds to the 7-methyl-guanosine (m7G) cap-binding protein eIF4E. This prevents formation of the translation initiation complex eIF4F, thereby inhibiting cap-dependent translation. This combination of inactivated dFOXO and inactive d4E-BP efficiently drives the cell toward growth and proliferation. Conversely, active dFOXO and d4E-BP conspire to arrest cell growth until the cell receives favorable nutrient and physiological signals to continue proliferation (Marr, 2007).
Drosophila melanogaster has proven to be a valuable model organism for working out the molecular details of this conserved pathway. In the absence of insulin or insulin-like peptides, dFOXO activates the transcription of both the insulin-like receptor (dINR) gene and the gene for Drosophila 4E-BP, establishing a transcriptional signaling loop that sensitizes the cell to receive further nutrient-dependent signals while preventing the cell from proliferating. In order to investigate this intriguing transcriptional feedback control, the start site of transcription for the dINR gene was precisely mapped using a modification of the cap-trapping cDNA synthesis method. This method, which depends on an intact m7G cap for capture of the mRNA, when combined with rapid amplification of five prime (5') cDNA ends (5' RACE) maximizes the yield of full-length 5' untranslated regions (UTRs). The use of this methodology allowed detection of critical UTRs associated with the mRNA that had previously gone undocumented. The dINR gene is actually controlled by a complex set of three distinct promoters (P1, P2, and P3) spread over 38 kb of the Drosophila genome. These combined promoters and associated introns and exons encompass the entire region between the Drosophila E2F gene and the currently annotated dINR gene. This complex control region fills a gap in the genome annotation that contains no other annotated genes or gene predictions (Marr, 2007).
Each of the dINR promoters produces a transcript with a unique and unusually long 5'UTR spliced to a short common exon that is in turn spliced to the first coding exon. The UTR originating from P1 is 1118 bases, the UTR originating from P2 is 419 bases, and the UTR originating from P3 is 485 bases. In contrast, the average 5'UTR in Drosophila is only 256 bases. All three UTRs contain multiple AUG initiator codons upstream of the legitimate INR initiator codon. In the case of the transcript that originates from P1, there are 12 AUGs before the legitimate translational start signal (Marr, 2007).
The DNA sequences immediately upstream of the mapped transcript start sites contain easily recognizable sequences similar to the computationally and biochemically determined common core promoter elements. P1 contains a TATA box, an Initiator element, and a downstream promoter element (DPE). P2 contains a TATA-like box and a DPE but no recognizable Initiator. P3 contains a recognizable Initiator but no recognizable TATA box or DPE. Importantly, a constitutively active form of dFOXO (dFOXO-A3) activates all three promoters in Drosophila Schneider line 2 (S2) cells, and this increased RNA synthesis can produce dINR protein even in the presence of insulin. The transcript originating at P1 is by far the most abundant transcript under both unactivated and activated conditions. P2 is present at an intermediate level, and P3 is a low-abundance transcript. Interestingly, the level of transcription correlates with the number of recognized core promoter elements, illustrating the important role these different elements play in determining the total level of transcription from a gene in both activated and unactivated states (Marr, 2007).
In the animal, all three transcripts are detectable in multiple developmental stages. They are present in whole animal extracts in the same relative order of abundance that is detected in S2 cells (P1 >> P2 > P3). When compared with the Rp49 transcript, a common control transcript that changes little over the stages tested, all three transcripts fluctuate in abundance. Notably, all three transcripts diminish significantly in the L3 larva, a time when the animal is voraciously eating. In contrast, these dINR transcripts peak in the pupae, a time when the animal is fasting and expending much of the energy gained during the larval stage. This observation is consistent with a previous finding that dINR expression is linked to nutrient availability (Marr, 2007).
Strikingly, dINR is not only transcriptionally up-regulated but also robustly translated. Growing S2 cells in the absence of serum and insulin causes a marked decrease in the rate of incorporation of radiolabeled cysteine and methionine consistent with a global decrease in the rate of translation. Despite this slowing of overall translation, dINR protein accumulates in S2 cells. This is detectable by immunoblot of whole cell extracts with antisera raised against the dINR protein. The increase in dINR protein levels is at least partially due to the absence of insulin itself and not another component of serum because the accumulation of dINR protein is inhibited by addition of insulin to media containing insulin-depleted serum. In addition, the increased dINR protein level is most likely due to increased synthesis since serum starved cells contain more radiolabeled receptor that binds to insulin-agarose. This raises the intriguing question of how translation of dINR can proceed in the presence of a quantitatively dephosphorylated, potently active, and up-regulated inhibitor of protein synthesis, d4E-BP. This paradoxical finding that the dINR pathway transcriptionally up-regulates both dINR and d4E-BP combined with the newly discovered unusually long 5'UTRs of these transcripts suggest that perhaps the INR gene engages the translation machinery in an unconventional manner that bypasses the need for eIF4E. A potential d4E-BP resistant internal ribosome entry site (IRES) exists in these Drosophila genes that contain long UTRs, as has been seen in other instances. For example, both the Antennapedia and Ultrabithorax long 5'UTRs contain IRESs, although their physiological role has remained undetermined (Marr, 2007).
As a first test of whether the dINR 5'UTRs also contain an IRES activity, a bicistronic construct, commonly used to assess IRES activity, was generated. The various 5'UTRs of dINR were inserted in both the forward and reverse orientations between the Renilla and firefly luciferase genes. The reverse orientation was used as a spacer length control equivalent. The ratio of Renilla luciferase expression to firefly luciferase expression should provide an indication of the cap-independent translational potential of the various 5'UTRs. Since resistance to d4E-BP is most relevant to this pathway, these experiments were carried out in the presence and absence of a constitutively active form of d4E-BP. Because the Renilla luciferase ORF is the first in the mRNA, it should be uniquely sensitive to inhibition of cap-dependent translation, while the firefly gene expression, if any, should be dependent on internal ribosome entry. The data are expressed as a ratio of the activity in the presence of d4E-BP to the activity in the absence of d4E-BP. Therefore, a number close to 1 indicates that there is no resistance to d4E-BP. In these cell-based assays, the 5'UTR from both P1 and P2 showed significant resistance to d4E-BP (about fourfold better than the reverse orientation in both cases), but only when inserted in the forward direction. Curiously, the 5'UTR from P3 showed unusual resistance to d4E-BP in either orientation. Indeed, the P3 UTR showed a perplexing increase in expression of the firefly ORF in the presence of d4E-BP compared with no UTR in both orientations. This finding reveals a potential limitation of using the bicistronic assays since interfering effects from cryptic promoters, cryptic splicing, or secondary effects of expression of d4E-BP cannot be ruled out with this assay (Marr, 2007).
To circumvent some of the inherent idiosyncrasies of the bicistronic constructs, monocistronic constructs were used that more closely mimic the situation of the endogenous dINR gene. Potential IRES activity esd measured in two complementary ways. First, in a DNA-based transient transfection, either the constitutively active form of d4E-BP or a control protein, green fluorescent protein (GFP), was expressed and resistance to d4E-BP was measure as the ratio of luciferase activity (provided by a second plasmid) in the presence of d4E-BP to the activity in the presence GFP. In this set of experiments, the minimal Antennapedia IRES, a Drosophila 5'UTR known to support cap-independent initiation of translation, was included as a positive control. Under these cell-based assay conditions, the P1 and P2 UTRs again displayed robust resistance to d4E-BP, while P3 and the common exons showed little resistance. Notably, the P2 5'UTR is as efficient as the minimal Antennapedia IRES, and the P1 5'UTR is actually significantly more efficient than the control IRES. Taken together, these two cell-based assays suggest that the 5'UTRs of at least the P1 and P2 transcripts can direct substantial IRES activity, while the P3 UTR appears to have much less if any such activity in S2 cells. Second, to complement these plasmid-based assays and directly investigate the contribution of the UTRs to translation, an RNA-based transfection assay was used. The RNAs contained either a m7G cap or an ApppG cap mimic. Only the 7mG cap allows cap-dependent translation. The ApppG cap stabilizes the transcript but does not allow cap-dependent translation, so it is a direct measure of the contribution of IRES activity. In this assay, the UTRs again showed significant IRES activity. The P1 UTR confers the same activity with or without a m7G cap, indicating a strong IRES activity. The P2 and P3 UTRs also confer cap-independent translation activity, although the level of activity is not equal to UTR plus cap. In contrast, the common exon or nonspecific UTR retains only 20% of their translation potential without the m7G cap. Taken together, these cell-based assays provide encouraging evidence for IRES activity of the dINR 5'UTRs (Marr, 2007).
However, given the well-recognized limitations inherent with using cell-based assays to establish IRES activity, a Drosophila embryo-derived cap-dependant in vitro translation system was used to test more directly the putative IRES activity and more specifically the potential d4E-BP resistance of the INR UTRs. The translation extracts were treated with micrococcal nuclease to destroy the bulk of competing endogenous transcripts so that translation would be largely dependent on exogenously added RNA. As expected, addition of normal capped transcripts results in robust translation from all of the UTR-containing RNAs as well as the common UTR and a short nonspecific UTR control RNA. To test the dependence of translation on eIF4E, exogenous m7G cap analog was added as a competitor. This excess free cap efficiently binds and sequesters the available eIF4E, preventing this essential initiation factor from binding capped RNA, thus effectively blocking the nucleation of the eIF4F complex and cap-dependent initiation. Remarkably, only the transcripts containing the P1, P2, and P3 UTRs are resistant to exogenously added competitor cap analog, whereas the common UTR fragment and the short nonspecific leader are effectively inhibited. This finding strongly suggests that the various dINR-specific UTRs, indeed, provide a cap-independent mechanism of translation initiation. To directly test the resistance of these transcripts to d4E-BP-mediated translation inhibition, recombinant d4E-BP was added to the reactions. Whereas the common exon and control RNAs are efficiently inhibited by this blocker of eIF4E-mediated translation initiation, the P1, P2, and P3 UTR-containing transcripts are highly resistant to d4E-BP. These findings taken together with cell-based assays suggest that, indeed, dINR protein synthesis can proceed via an IRES-mediated eIF4E-independent mechanism of initiation both in vitro and in vivo (Marr, 2007).
What purpose might a cap-independent translation activity serve beyond simple resistance to the active d4E-BP in the absence of insulin? Perhaps by functionally coupling transcription and translation, such a mechanism could serve to amplify the signal received from the insulin receptor pathway. To test this idea, in vitro translation experiments were used. In the absence of miccrococal nuclease treatment, the endogenous transcripts present in the translation extract should effectively compete with the experimental dINR transcripts for limiting amounts of the translation machinery. Advantage was taken of this inevitable competition for translation machinery to test the response of the various UTRs in a situation that may more closely reflect the cellular environment, where multiple variable abundant transcripts must compete for a limited supply of the translational apparatus. Under these competitive conditions, addition of either m7G or d4E-BP actually results in an even more robust increase in translation of the dINR UTR-containing RNAs relative to the unchallenged state. This finding suggests that these RNAs that contain dINR UTRs, and presumably IRES activity, are highly effective at out-competing other transcripts for access to the translational machinery when m7G cap-dependent initiation is inhibited. While the molecular mechanism of 4E-BP resistance of the dINR transcripts have not been unequivocally defined, it is clear that the UTRs allow significant translation in conditions when cap-dependent translation is inhibited (Marr, 2007).
These data allowed formulation of a new model to explain the effects of nutrients and insulin levels on dINR feedback regulation. In times of high nutrients and therefore high insulin-like peptides, both dFOXO and d4E-BP are phosphorylated and inactive. Under these 'rich' conditions, dFOXO is sequestered in the cytoplasm and phosphorylated d4E-BP is unable to interact with eIF4E. This situation allows efficient translation of most cellular transcripts regardless of the mechanism of initiation (cap-dependent vs. cap-independent). In contrast, in low nutrient conditions or in the absence of insulin or insulin-like peptides, both dFOXO and d4E-BP become dephosphorylated and active. Activated dFOXO directs a robust increase in the transcription of both dINR and d4E-BP (among other genes). Additionally, the active and up-regulated d4E-BP effectively inhibits cap-dependent translation, freeing up the protein synthesis machinery to selectively translate IRES-containing transcripts like dINR. These two coordinated mechanisms consequently orchestrate the integration of a specific transcriptional response and simultaneously a translational response that greatly amplifies the signal and sensitizes the cell for detection of small changes in nutrient availability as well as, possibly, developmental and environmental cues (Marr, 2007).
Interestingly, the dFOXO-responsive dINR promoters produce three distinct transcripts. Why such a complex regulatory network? A hint may be that the P3 UTR does not seem to have detectable IRES activity in the S2 cells but shows substantial activity in vitro with extracts derived from whole Drosophila embryos. It is likely that the three transcripts are produced in a tissue- or temporal-specific manner during development, and it is speculated that each may depend on cell-specific IRES trans-acting factors (ITAFs) that are required for activity. This would direct tissues to respond differentially to dINR signaling. In tissues lacking specific ITAFs, the IRES activity would be diminished and the tissue may produce only a moderate level of dINR protein (Marr, 2007).
An interesting parallel was found between mechanisms for reprogramming the gene expression machinery in a cell to respond to physiological cues and the more commonly observed viral takeover of the cellular macromolecular synthesis machinery. When some viruses, such as polio, infect a cell, they target the translation initiation machinery (either eIF4G or 4E-BP) so that there is a switch from cap-dependent synthesis to IRES-dependent synthesis. This leads to a robust and specific stimulation of viral protein synthesis at the expense of most cellular protein synthesis. By the evolution of cellular mechanisms that activate 4E-BP and simultaneously produce transcripts containing cellular IRESs, a critical physiological signaling cascade can evidently adopt a similar mechanism to effectively usurp the macromolecular synthesis machinery to drive cellular physiology in a very specific direction. Indeed, viruses may have merely co-opted the mechanism from cells in the eternal battle between host and virus (Marr, 2007).
Although the initial characterization of the INR transcriptional feedback loop was carried out in Drosophila, a similar regulatory circuit has been found in vertebrates. It is interesting to note that the transcripts for human insulin receptor and IGF-2 receptor remain associated with polysomes when cap-dependant translation is inhibited by poliovirus infection. Although the level of INR mRNA up-regulation by FOXO in mouse muscle cells is only twofold, the levels of INR protein increase much more dramatically (six- to eight-fold), consistent with a coupled transcription/translation mechanism of the signal in vertebrates. It seems likely, given the findings report in this study, that the same type of coupling between the transcriptional program of FOXO proteins and translational control by IRES activity is also occurring in vertebrate systems. Understanding this novel mechanism that couples transcription and translation may provide new insight into disease states such as insulin-resistant type 2 diabetes (Marr, 2007).
The glycosylation of the Drosophila insulin receptor (InR) has been compared to
that of the rat insulin receptor by means of an examination of the binding of receptors to the
lectins wheat germ agglutinin, Concanavalin-A, and lentil lectin. Although rat
insulin receptors bind and are specifically eluted from all three lectins,
only a small fraction of the InR (< 5%) is retained on wheat germ agglutinin.
In contrast, the InR binds strongly to Concanavalin-A and lentil lectin and is
recovered from lentil lectin columns after elution with alpha-methyl-mannoside.
The pattern of lectin binding indicates that glycosylation of the InR and rat
insulin receptors differs, with the InR containing primarily high mannose-type
oligosaccharides. After lectin chromatography, the InR exhibits an elevated
level of basal autophosphorylation and kinase activity, which can be restored
to a low level by incubation with 0.5 mM dithiothreitol (DTT). DTT does not,
however, affect ligand-stimulated kinase activity. The ability of low
concentrations of DTT to deactivate the InR kinase suggests that, like the
mammalian receptor, beta-subunit thiols may be involved in regulation of
conformational changes between activated and unactivated receptor states.
Interestingly, DTT-induced deactivation of the InR is blocked by preincubation
with an antipeptide antibody against the carboxy-terminal domain of the InR.
This suggests that the InR carboxyl terminus undergoes a conformational change
during the activation-inactivation cycle of the kinase, which can be sterically
hindered by the antibody. Conformational changes in this region of the mammalian
receptor have been observed, and these data suggest that features of the insulin
receptor activation mechanism have been substantially conserved during
evolution (Marin-Hincapie, 1995).
Chimeric receptors encoding either the whole or a portion of the cytoplasmic domain of the Drosophila insulin receptor (InR) with the extracellular domain of the human insulin receptor (IR) were expressed either transiently in COS cells or stably in Chinese hamster ovary cells and compared with the wild-type human IR. All three receptors bind insulin equally and exhibit an insulin-activated tyrosine kinase activity. The ability of the Drosophila cytoplasmic domain to mediate the tyrosine phosphorylation of insulin receptor substrate 1, stimulate cell proliferation, and activate MAP kinase is indistinguishable from that of the human IR. The chimeric Drosophila receptors do not bind more phosphatidylinositol 3-kinase (see Phosphotidylinositol 3 kinase 92E) than the human IR, despite containing a C-terminal extension with potential tyrosine phosphorylation sites in the motif recognized by the SH2 domain of this enzyme. Thus, the essential signal-transducing abilities of the IR appear to have been conserved from invertebrates to mammals, despite the considerable differences in the sequences of these receptors (Yamaguchi, 1995).
The InR proreceptor [M(r) 280 kDa] is processed proteolytically to generate an insulin-binding alpha subunit [M(r) 120 kDa] and a beta subunit [M(r) 170 kDa] with a protein tyrosine kinase domain. The InR beta 170 subunit contains a novel domain at the carboxyterminal side of the tyrosine kinase, in the form of a 60 kDa extension that contains multiple potential tyrosine autophosphorylation sites. This 60 kDa C-terminal domain undergoes cell-specific proteolytic cleavage that leads to the generation of a total of four polypeptides (alpha 120, beta 170, beta 90, and a free 60 kDa C-terminus) from the inr gene. These subunits assemble into mature InR receptors with the structures alpha 2(beta 170)2 or alpha 2(beta 90)2. Mammalian insulin stimulates tyrosine phosphorylation for both types of beta subunits; in turn, the phosphorylation allows the beta 170, but not the beta 90 subunit, to bind directly to p85 SH2 domains of PI-3 kinase. It is likely that the two different isoforms of InR have different signaling potentials. Loss of function mutations in the InR gene, induced by either a P-element insertion occurring within the predicted ORF, or by ethylmethane sulfonate treatment, renders pleiotropic recessive phenotypes that lead to embryonic lethality. The activity of InR appears to be required in the embryonic epidermis and nervous system among organ systems, since development of the cuticle, as well as the peripheral and central nervous systems are affected by InR mutations (Fernandez, 1995).
Stimulation of the activity of protein kinase C by pretreatment of cells with phorbol esters was tested for its ability to inhibit signaling by four members of the insulin receptor family, including the human insulin and insulin-like growth factor-I receptors, the human insulin receptor-related receptor, and the Drosophila insulin
receptor. Activation of overexpressed protein kinase Calpha results in a subsequent inhibition of the ligand-stimulated increase in antiphosphotyrosine-precipitable
phosphatidylinositol 3-kinase mediated by the kinase domains of all four receptors. This inhibition varies from 97% for the insulin receptor-related receptor to 65%
for the Drosophila insulin receptor. In addition, the activation of protein kinase Calpha inhibits the in situ ligand-stimulated increase in tyrosine phosphorylation of the GTPase-activating protein-associated p60 protein as well as Shc mediated by these receptors. The mechanism for this inhibition was further studied in the case of the
insulin-like growth factor-I receptor. Although the in situ phosphorylation of insulin-receptor substrate-1 and p60 by this receptor is inhibited by prior stimulation of protein kinase Calpha, the in vitro tyrosine phosphorylation of these two substrates by this receptor is not decreased by prior stimulation of the protein kinase Calpha in the cells that served as a source of the substrates. Finally, the insulin-like growth factor-I-stimulated increase in cell proliferation was found to be inhibited by prior activation of protein kinase Calpha. These results indicate that the ability of activated protein kinase Calpha to antagonize signaling by the human insulin receptor is shared by the other members of the insulin receptor family despite their considerable differences in amino acid sequence. Moreover, the present study shows that this antagonism is exerted at a very early step, the initial tyrosine phosphorylation of three distinct endogenous substrates. Finally, the present study indicates that this inhibition is not caused by an increased Ser/Thr phosphorylation of these two substrates (Danielsen, 1996).
A monoclonal antibody has been produced that immunoprecipitates 58- and 53-kDa
proteins that are rapidly tyrosine phosphorylated in insulin-treated cells.
These proteins can also be tyrosine phosphorylated in vitro by the isolated
human insulin receptor. Increased tyrosine phosphorylation of these proteins is
also observed in cells expressing a transforming chicken c-Src (mutant Phe-527)
and in cells with the activated tyrosine kinase domains of the Drosophila
insulin receptor, human insulin-like growth factor I receptor, and human insulin
receptor-related receptor. P58/53 does not appear to associate with either the
GTPase activating protein of Ras (called GAP) or the phosphatidylinositol
3-kinase by either co-immunoprecipitation experiments or in Far Westerns with
the SH2 domains of these two proteins. Since p58/53 does not appear, by
immunoblotting, to be related to any previously described tyrosine kinase
substrate such as the SH2 containing proteins SHC and the tyrosine phosphatase
Syp, the protein was purified in sufficient amounts to obtain peptide sequence.
This sequence was utilized to isolate a cDNA clone that encodes a previously
uncharacterized 53-kDa protein that, when expressed in mammalian cells, is
tyrosine phosphorylated by the insulin receptor (Yeh, 1996).
Drosophila contain an insulin receptor homolog, encoded by the InR gene located at position 93E4-5 on the third chromosome. The receptor protein is strikingly homologous to the human receptor, exhibiting the same alpha2beta2 subunit structure and containing a ligand-activated tyrosine kinase in its cytoplasmic domain. Chemical mutagenesis was used to induce mutations in the inr gene. Six independent mutations that lead to a loss of expression or function of the receptor protein have been identified. These mutations are recessive, embryonic, or early larval lethals, but some alleles exhibit heteroallelic complementation to yield adults with a severe developmental delay (10 days), growth-deficiency, female-sterile phenotype. Interestingly, the severity of the mutant phenotype correlates with biochemical measures of loss of function of the receptor tyrosine kinase. The growth deficiency appears to be due to a reduction in cell number, suggesting a role for InR in regulation of cell proliferation during development. The phenotype is reminiscent of those seen in syndromes of insulin-resistance or IGF-I and IGF-I receptor deficiencies in higher organisms, suggesting a conserved function for this growth factor family in the regulation of growth and body size (Chen, 1996).
The Drosophila insulin receptor (InR) contains a 368-amino-acid COOH-terminal extension that contains several tyrosine phosphorylation sites in YXXM motifs. This extension is absent from the human insulin receptor but resembles a region in insulin receptor substrate (IRS) proteins that binds to the phosphatidylinositol (PI) 3-kinase and mediates mitogenesis. The function of a chimeric InR containing the human insulin receptor binding domain (hDIR) was investigated in 32D cells, which contain few insulin receptors and no IRS proteins. Insulin stimulates tyrosine autophosphorylation of both the human insulin receptor and hDIR, and both receptors mediate tyrosine phosphorylation of Shc and activate mitogen-activated protein kinase. IRS-1 is required by the human insulin receptor to activate PI 3-kinase and p70s6k (see Drosophila RPS6-p70-protein kinase), whereas hDIR associates with PI 3-kinase and activates p70s6k without IRS-1. However, both receptors required IRS-1 to mediate insulin-stimulated mitogenesis. These data demonstrate that the InR possesses additional signaling capabilities when compared with its mammalian counterpart but still requires IRS-1 for the complete insulin response in mammalian cells (Yenush, 1996).
Like the mammalian insulin receptor, the Drosophila insulin receptor (INR)1 is a
tetramer formed by two alpha subunits and two beta subunits. INR alpha and
beta subunits are synthesized together as a proreceptor precursor, proteolytically processed, and linked together by disulfide bonds. The alpha subunits, with a molecular mass of 110-120 kDa, are
extracellular and contain the ligand binding domains that are capable
of binding mammalian insulin with a Kd of 15 nM. The beta subunits traverse the plasma membrane and have an insulin-stimulated tyrosine kinase in the cytoplasmic portion. DNA sequence analysis and expression of the INR beta subunit
in mammalian and Drosophila cells indicate that the
INR beta subunit is larger than its mammalian homolog and exhibits an
apparent molecular mass of ~180 kDa. The increased mass is due to the
presence of a 400-amino acid carboxyl-terminal extension. However,
the majority of INR beta subunits are processed to 92/102-kDa forms in Drosophila embyros and some cell lines, the difference being
due to proteolytic cleavage of the carboxyl-terminal extension.
Both truncated and full-length beta subunits are autophosphorylated on
tyrosine residues in response to insulin binding (Marin-Hincapie, 1999 and references therein).
The 400-amino acid carboxyl-terminal extension of the beta INR contains
clusters of motifs known to be involved in the interaction with SH2 and
PTB domain-containing proteins, suggesting a role for this domain
in signaling through interaction with other signaling molecules.
Interestingly, four tyrosines are found in 'hybrid' amino acid
motifs in which residues amino-terminal to each tyrosine form the motif
NP X Y, resembling known PTB domain binding sites, and
residues carboxyl-terminal to the same tyrosines form the motifs
YXXM, YMXM, or YXLLD -- all known to be
involved in binding to SH2 domains. Thus, tyrosines 1993 and 2030 appear in the motif
SXNPXYXX M;
tyrosine 2009 is part of
S X NPXYMXM, and tyrosine
1969 appears in the sequence SDNPXYRLLD.
Whether these motifs serve to bind SH2 or PTB domain-containing
proteins upon tyrosine phosphorylation and whether one is preferred
over the other is not clear. The cytoplasmic domain of the INR
expressed in cells lacking IRS-1 has been shown to bind PI3-kinase.
However, a similar construct expressed in Chinese hamster ovary cells
that contain IRS-1 fails to do so. Since a significant percentage
of the INR beta subunit undergoes tissue- or stage-specific proteolytic
processing in Drosophila embryos to remove the
carboxyl-terminal extension and once it is removed it appears not
to be phosphorylated, its role in signal transduction by the INR is
not clear. Therefore, the signaling capacity conferred by the beta INR
carboxyl-terminal extension has been explored by
expressing either full-length or truncated INR beta subunit forms in
mammalian cells and determining the effect on protein-protein
interactions and cell growth (Marin-Hincapie, 1999 and references therein).
In order to explore the role of the 400 AA extention in INR function, mammalian
expression vectors encoding either the complete INR beta subunit (beta-Myc) or the INR beta subunit without the carboxyl-terminal extension (betaDelta) were
constructed, and the membrane-bound beta subunits were expressed in 293 and Madin-Darby canine kidney (MDCK) cells in the absence of the ligand-binding alpha
subunits. beta-Myc and betaDelta proteins are constitutively active tyrosine kinases of 180 and 102 kDa, respectively. INR beta-Myc co-immunoprecipitates a
phosphoprotein of 170 kDa identified as insulin receptor substrate-1 (IRS-1, Flipper or Chico), whereas INR betaDelta does not, suggesting that the site of interaction is within the
carboxyl-terminal extension. IRS-1 is phosphorylated on tyrosine to a much greater extent in cells expressing INR beta-Myc than in parental or INR betaDelta
cells. Despite this, a variety of PTB or SH2 domain-containing signaling proteins, including IRS-2, mSos-1, Shc, p85 subunit of phosphatidylinositol 3-kinase,
SHP-2, Raf-1, and JAK2, are not associated with the INR beta-Myc.IRS-1 complex. Overexpression of INR beta-Myc and betaDelta kinases confers an
equivalent increase in cell proliferation in both 293 and Madin-Darby canine kidney cells, indicating that this growth response is independent of the carboxyl-terminal
extension. However, INR beta-Myc-expressing cells exhibit enhanced survival, relative to parental and betaDelta cells, suggesting that the carboxyl-terminal
extension, through its interaction with IRS-1, plays a role in the regulation of cell death (Marin-Hincapie, 1999).
Thus, overexpression of constitutively active INR beta and betaDelta receptors in
293 and MDCK cells promotes cell proliferation, indicating that the INR
can engage the mammalian proliferation pathways. The equivalent
proliferative responses induced by INR beta-Myc and betaDelta kinases
suggests that the growth-promoting function of the INR in these cells
is independent of the carboxyl-terminal extension. In contrast, cells
expressing the full-length INR beta subunit exhibit significantly
enhanced survival as compared with cells expressing the betaDelta INR.
Relative to the parental 293 and MDCK cells, the INR beta-Myc and betaDelta
proteins confer somewhat different behavior; beta-Myc clearly
promotes survival in 293 cells, whereas betaDelta more dramatically accelerates cell death in MDCK cells. Nonetheless, a clear difference in the behavior of cells expressing the full-length or truncated INR
beta subunits is evident in both backgrounds. Despite the presence of a
juxtamembrane NPXY motif predicted to interact with IRS-1 in
both beta-Myc and betaDelta proteins, IRS-1 is not highly phosphorylated in
betaDelta cells. This suggests that the
carboxyl-terminal extension of the INR beta subunit is required for
sustained association and phosphorylation of IRS-1. This persistent
IRS-1 phosphorylation distinguishes beta-Myc from betaDelta cells and may
be of primary importance in promoting cell survival. Without this
sustained interaction, cell death may actually be accelerated, as
observed in MDCK cells transfected with the INR betaDelta kinase (Marin-Hincapie, 1999).
IRS-1 that is bound to the INR beta subunit is phosphorylated on
tyrosine; however, no evidence has been found for increased association of
PI3-kinase or other candidate signaling molecules with this complex.
Therefore, the mechanism whereby this association leads to increased cell
survival is unclear at present. Interestingly, a recent report
demonstrates that expression of a truncated IRS-1 containing only the
pleckstrin homology and phosphotyrosine binding domains, without any
tyrosine phosphorylation sites, mediates PI3-kinase and
phosphotyrosine-independent signals that contribute to the regulation
of cell survival and apoptosis. IRS-1 that is bound to the
carboxyl-terminal extension of INR in 293 and MDCK cells may have
similarly activated pathways that promote cell survival in the absence
of PI3-kinase activation (Marin-Hincapie, 1999 and references therein).
Thus, two isoforms of an activated INR beta subunit have been expressed
in mammalian cells, and a functional difference between them has been
demonstrated. The data presented here indicate that the stimulation of
cell proliferation by INR is mediated by the kinase domain independent
of the carboxyl-terminal extension. In contrast, the carboxyl-terminal
extension mediates an interaction with IRS-1 and influences cell
survival. Since an IRS homolog is present in Drosophila, this may reflect an inherent function of the INR which, in flies,
is modulated by tissue- or stage-specific processing of the receptor.
These data also suggest that in mammalian cells, persistent localization of IRS-1 to membranes via the interaction of IRS-1 with receptors and/or persistent tyrosine phosphorylation generates signals
independent of association with PI3-kinase (Marin-Hincapie, 1999 and references therein).
Insulin receptor substrate (IRS) proteins are phosphorylated by multiple
tyrosine kinases, including the insulin receptor. Phosphorylated IRS proteins
bind to SH2 domain-containing proteins, thereby triggering downstream signaling
pathways. The Drosophila insulin receptor (InR) C-terminal extension contains
potential binding sites for signaling molecules, suggesting that InR might not
require an IRS protein to accomplish its signaling functions. However, a cDNA encoding Drosophila IRS (Chico, but referred to in this study as dIRS) has been obtained and one for Chico in a Drosophila cell line has also been demonstrated. Like mammalian IRS proteins, the N-terminal
portion of Chico contains a pleckstrin homology domain and a phosphotyrosine
binding domain that binds to phosphotyrosine residues in both human and
Drosophila insulin receptors. When coexpressed with Chico in COS-7 cells, a
chimeric receptor (the extracellular domain of human IR fused to the cytoplasmic
domain of InR) mediates the insulin-stimulated tyrosine phosphorylation of Chico.
Mutating the juxtamembrane NPXY motif markedly reduces the ability of the
receptor to phosphorylate Chico. In contrast, the NPXY motifs in the C-terminal
extension of InR are required for stable association with Chico.
Coimmunoprecipitation experiments demonstrate insulin-dependent binding of Chico
to phosphatidylinositol 3-kinase and SHP2. However,
interactions with Grb2, SHC, or phospholipase C-gamma were not detected. Taken together with
published genetic studies, these biochemical data support the hypothesis that
Chico functions directly downstream from the insulin receptor in Drosophila (Poltilove, 2000).
Perturbation in the Dystroglycan (Dg)-Dystrophin (Dys) complex results in muscular dystrophies and brain abnormalities in human. Drosophila is an excellent genetically tractable model to study muscular dystrophies and neuronal abnormalities caused by defects in this complex. Using a fluorescence polarization assay, a high conservation in Dg-Dys interaction between human and Drosophila is demonstrated. Genetic and RNAi-induced perturbations of Dg and Dys in Drosophila cause cell polarity and muscular dystrophy phenotypes: decreased mobility, age-dependent muscle degeneration and defective photoreceptor path-finding. Dg and Dys are required in targeting glial cells and neurons for correct neuronal migration. Importantly, Dg interacts with insulin receptor and Nck/Dock SH2/SH3-adaptor molecule in photoreceptor path-finding. This is the first demonstration of a genetic interaction between Dg and InR (Shcherbata, 2007).
The Dg-Dys binding interface is highly conserved in humans and Drosophila. Both proteins are required for oocyte cellular polarity and interact in this process. Futhermore, mutants of both Dg and Dys genes show symptoms observed in muscular dystrophy. Reduction of Dg and Dys proteins results in age-dependent mobility defects. Eliminating Dg and Dys specifically in mesoderm derived tissues reveals that these proteins are required for muscle maintenance in adult flies: age-dependent muscle degeneration was observed in mutant tissues. Dg-Dys complex is also required for neuron path-finding and has both cell autonomous and non-cell autonomous functions for this process. Further, in neuronal path-finding process Dg interacts with InR and an SH2/SH3-domain adapter molecule Nck/Dock (Shcherbata, 2007).
Animal models have been used efficiently in muscular dystrophy studies. Some of the models are naturally occurring mutations (mdx-mouse, muscular dystrophy dog, cat and hamster), others have been generated by gene targeting. However, the regulation and the control of Dg-Dys complex are not understood, and no successful therapeutics exist yet for muscular dystrophies. Recently developed models for muscular dystrophy exist in C. elegans and zebrafish. In C. elegans Dys mutant, the transporter snf-6 that normally participates in eliminating acetylcholine from the cholinergic synapses, is not properly localized, resulting in an increased acetylcholine concentration at the neuromuscular junction and muscle wasting (Kim, 2004). The function of Dys in neuromuscular junctions has been addressed in Drosophila. These results bring up the possibility that muscular dystrophies in humans might also at least partly be attributed to the altered kinetics of acetylcholine transmission through neuromuscular junctions (Shcherbata, 2007).
Drosophila acts as a remarkably good model for age-dependent progression of muscular dystrophy. Dg and Dys reduction in Drosophila show age-dependent muscle degeneration and lack of climbing ability. It is tempting to speculate that the common denominator between different defects observed in Dg-Dys mutants in Drosophila and C. elegans is defective cellular polarity. The defects observed in C. elegans could be due to a defect in polarization of a cell, which will generate a neuromuscular junction that leads to miss-targeted snf-6. Similarly, Drosophila Dg-Dys complex is required for cellular polarity in the oocyte. In addition, neural defects observed are plausibly due to polarity defects in the growing axon (Shcherbata, 2007).
Similar to neuronal defects observed in human muscular dystrophy patients, neuronal defects were also found in Drosophila Dg and Dys mutant brains. In vertebrate brains, Dg affects neuronal migration (Montanaro, 2003; Qu, 2004) possibly through interaction of neurons with their glial guides. The neuronal migration and process outgrowth have been shown to require supportive input from glial cells and involve the formation of adhesion junctions along the length of the soma. Also, the outgrowth of the leading process involves rapid extension and contraction over the length of the glial fiber. Disruption of the cytoskeletal organization within the neuron, either of actin filaments, has been shown to inhibit glial-mediated neuronal migration. The glial function in this process is less well studied (Shcherbata, 2007).
Drosophila photoreceptor path-finding provides an excellent system for genetic dissection of neuronal outgrowth and target recognition. During the formation of the nervous system, newly born neurons send out axons to find their targets. Each axon is led by a growth cone that responds to extracellular axon guidance cues and chooses between different extracellular substrates upon which to migrate. Recent work has also identified a variety of intracellular signaling pathways by which these cues induce cytoskeletal rearrangements, but the proteins connecting signals from cell surface receptors to actin cytoskeleton have not been clearly determined. Dg is a good candidate for linking receptor signaling to the remodeling of the actin cytoskeleton and thereby polarizing the growth cone. Perturbation of Dg-Dys complex causes phenotypes that resemble Nck/Dock-Pak-Trio axon path-finding phenotypes, suggesting that Dg may be one of the key players in Nck/Dock signaling pathway for axon guidance and target recognition in Drosophila (Shcherbata, 2007).
Interestingly, Insulin receptor-tyrosine kinase (InR) mutants also show similar phenotypes to those of Nck/Dock signaling in photoreceptor axon path-finding and these two proteins show genetic and biochemical interactions. These data have led to speculations of mammalian InR acting in conjunction with Nck/Dock pathway in learning, memory and eating behavior. The current data now add Dg-Dys complex to this pathway; similar to what is seen in the case of Dg and Dys photoreceptor mutants, InR mutants show no obvious defects in patterning of the photoreceptors. However, the guidance of photoreceptor cell axons from the retina to the brain is aberrant. Furthermore, genetic and biochemical evidence suggests that InR function in axon guidance involves the Dock-Pak pathway rather than the PI3K-Akt/PKB pathway. Independently, biochemical interaction between Nck/Dock and Dg has been reported supporting the hypothesis that InR, Dg and Nck/Dock interaction regulates Dg-Dys complex. Furthermore, Dg interacts genetically with InR and Dock in photoreceptor axon path-finding. Since Dys interacts with Dg but not with InR and Dock, it is tempting to speculate that Dg can selectively interact with either Dys or InR and Dock. One possibility is that the tyrosine kinase activity of InR could regulate the Dg-Dys interaction by tyrosine phosphorylation in the Dg-Dys binding interphase. This tyrosine phosphorylation could prohibit the Dg-Dys interaction and thereby result in rearrangements in the actin cytoskeleton. Alternatively, other components observed in Dg-Dys complex might be involved in this regulation. However, it is also possible that potential polarity defects in the Dg mutant axons result in defective InR membrane localization. Interestingly, in another cell type, the Drosophila oocyte, InR, Dg and Dys also show similar phenotypes. In addition, insulin-like growth factors (IGF) and InR are important in maintaining muscle mass in vertebrates. Further connection of InR to Dg-Dys complex comes from experiments showing that muscle specific expression of IGF counters muscle decline in mdx-mice. The work presented in this study is the first demonstration of genetic interaction between Dg and InR. Future biochemical studies should unravel the molecular mechanism of this interaction (Shcherbata, 2007).
Dg-Dys complex is required both in neural and in targeting glial cells for correct neuronal axon path-finding in Drosophila brain. These data reveal that Dg-Dys complex also has a non-cell autonomous effect on axon path-finding and suggest that Dg-Dys-controlled ECM both from neuron and glial cells regulate neuronal axon path-finding. Further experiments are required to reveal whether long-range Laminin fibers are involved in this process, as has been shown in epithelial planar polarity, or whether glial processes are observed in close proximity to the neural growth cone. Interestingly, similar phenotypes are observed with Integrin mutants, suggesting that, as in planar polarity, Integrin and Dg-Dys complex might act in concert to regulate the process of ECM organization that will regulate the cytoskeleton of the cells involved (Shcherbata, 2007).
Taken together, the phenotypes caused by Drosophila Dg and Dys mutations are remarkably similar to phenotypes observed in human muscular dystrophy patients, and therefore suggest that functional dissection of Dg-Dys complex in Drosophila should provide new insights into the origin and potential treatment of these fatal neuromuscular diseases. As a proof of principle, using Drosophila as a model, InR has now been identified as a signaling pathway that genetically interacts with Dg. Future studies are directed to unravel the molecular mechanism of Dg and InR-Dock interactions in invertebrates as well as vertebrates (Shcherbata, 2007).
Home page: The Interactive Fly © 1997 Thomas B. Brody, Ph.D.
The Interactive Fly resides on the
Society for Developmental Biology's Web server.