The Drosophila insulin receptor InR is similar to its mammalian counterpart in deduced amino acid sequence, subunit structure, and ligand-stimulated protein tyrosine kinase activity. The function of this receptor in D. melanogaster is not yet known. However, a role in development is suggested by the observations that levels of insulin-stimulated kinase activity and expression of InR mRNA are maximal during Drosophila midembryogenesis. In this study, a 2.9-kilobase (kb) cDNA clone corresponding to both the InR tyrosine kinase domain and some of the 3' untranslated sequence was used to determine the tissue distribution of InR mRNA during development. Two principal mRNAs of 11 and 8.6 kb hybridize with the InR cDNA in Northern (RNA) blot analysis. The abundance of the 8.6-kb mRNA increases transiently in early embryos, whereas the 11-kb species is most abundant during midembryogenesis. A similar pattern of expression has been determined by Northern analysis, using an InR genomic clone. In situ hybridization revealed InR transcripts in the ovaries of adult flies, in which the transcripts appear to be synthesized by nurse cells for eventual storage as maternal RNA in the mature oocyte. Throughout embryogenesis, InR transcripts are ubiquitously expressed, although after midembryogenesis, higher levels are detected in the developing nervous system. Nervous system expression remains elevated throughout the larval stages and persists in the adult, in which the cortex of the brain and ganglion cells are among the most prominently labeled tissues. In larvae, the imaginal disc cells exhibit comparatively high levels of InR mRNA expression. The broad distribution of InR mRNA in embryos and imaginal discs is compatible with a role for InR in anabolic processes required for cell growth. The apparently elevated expression of InR mRNA in nervous tissue during mid- and late-embryogenesis coincides with a period of active neurite outgrowth and suggests that dIRH may be involved in this process (Garofalo, 1988).
A monoclonal antibody (Mab E1C) has been generated that recognizes the differentiated nervous system in Drosophila embryos. At the cellular blastoderm stage, Mab E1C behaves as a general ectodermal marker but, in subsequent stages, it also labels the mesoderm. As neurogenesis takes place, staining increases within the neuromeres and is almost exclusively restricted to the nervous tissue by the time neuronal differentiation is completed. In third instar larvae, Mab E1C stains the central nervous system as well as the imaginal discs, which display a staining pattern related to their degree of neuronal differentiation. No labelling can be detected in adult brains or ovaries. Western blots are consistent with this developmental profile and allow the characterization of a major glycoprotein of 135 X 10(3) Mr (135K) that cosediments with a membrane fraction prepared from embryos. Additional glycoproteins (100K and 80K) are extracted from embryo homogenates by immunoaffinity procedures. In larvae, the 100K polypeptide is not detected. The properties of the 135K and 100K components are highly reminiscent of the molecular pattern of the Drosophila insulin receptor homologue. It has been shown that a Mab directed against the human insulin receptor stains the same cells as Mab E1C in imaginal discs and in the CNS. Moreover, this Mab cross-reacts with the 135K and 100K components of the embryonic antigen E1C (Piovant, 1988).
Insulin and insulin-like growth factor (IGF) receptors are members of the tyrosine kinase family of receptors, and are thought to play an important role in the development and differentiation of neurons. The presence of an insulin-like peptide and an insulin receptor (InR) at the body wall neuromuscular junction of developing Drosophila larvae is reported. InR-like immunoreactivity is found in all body wall muscles at the motor nerve branching regions, where it surrounds synaptic boutons. The identity of this immunoreactivity as an InR was confirmed by two additional schemes: in vivo binding of labeled insulin and immunolocalization of phosphotyrosine. Both methods produce staining patterns markedly similar to InR-like immunoreactivity. The presence of an InR in whole larvae was also shown by receptor binding assays. This receptor is more specific for insulin (> 25-fold) than for IGF II, and does not appear to bind IGF I. Among the 30 muscle fibers per hemisegment, insulin-like immunoreactivity is found only on one fiber, and is localized to a subset of morphologically distinct synaptic boutons. Staining in the CNS is limited to several cell bodies in the brain lobes and in a segmental pattern throughout most of the abdominal ganglia, as well as in varicosities along the neuropil areas of the ventral ganglion and brain lobes. Insulin-like peptide and InR are first detected by early larval development, well after neuromuscular transmission begins (Gorczyca, 1993).
The isolation of the Drosophila insulin receptor gene and the recent analysis of loss of function mutants have clearly implicated insulin signaling in embryonic nervous system development. The presence of insulin in the embryo is studied and cellular processes affected by insulin in embryonic neural cells are characterized. A fraction of the cells (7.5%) in the 15-18 h Drosophila embryo contain insulin immunoreactivity. In the embryonic-derived cell line Schneider 1, human insulin is capable of stimulating proliferation and neural differentiation. Thus, the action of insulin on the developing Drosophila nervous system appears to be as pleiotropic as in vertebrates (Pimentel, 1996).
Adult flies contain a specific, high-affinity insulin-binding protein. Insulin-dependent protein tyrosine kinase activity has now been identified in Drosophila. Activity first appears at 6-12 h of embryogenesis, increases during the 12-18-h period and falls to low levels in the adult. 125I-insulin has been cross-linked specifically and with high affinity to a protein (Mr = 135,000) throughout embryogenesis and in the adult. However, during the 6-12- and 12-18-h periods of embryogenesis when insulin-dependent protein tyrosine kinase activity is expressed, another protein (Mr = 100,000) becomes cross-linked to 125I-insulin. Crosslinking to both proteins is competitively inhibited by the addition of 100 nM insulin. It is concluded that the insulin-binding and insulin-dependent protein tyrosine kinase activities of the mammalian insulin receptor are conserved in Drosophila. However, the insulin-dependent protein tyrosine kinase activity of the receptor is detected only during specific times in embryogenesis (Petruzzelli, 1985).
In many metazoans, final adult size depends on the growth rate and the duration of the growth period, two parameters influenced by nutritional cues. In Drosophila, nutrition modifies the timing of development by acting on the prothoracic gland (PG), which secretes the molting hormone ecdysone. When activity of the Target of Rapamycin (TOR), a core component of the nutrient-responsive pathway, is reduced in the PG, the ecdysone peak that marks the end of larval development is abrogated. This extends the duration of growth and increases animal size. Conversely, the developmental delay caused by nutritional restriction is reversed by activating TOR solely in PG cells. Finally, nutrition acts on the PG during a restricted time window near the end of larval development that coincides with the commitment to pupariation. In conclusion, this study shows that the PG uses TOR signaling to couple nutritional input with ecdysone production and developmental timing. Previously studies have shown that the same molecular pathway operates in the fat body (a functional equivalent of vertebrate liver and white fat) to control growth rate, another key parameter in the determination of adult size. Therefore, the TOR pathway takes a central position in transducing the nutritional input into physiological regulations that determine final animal size (Layalle, 2008).
Previous experiments showed that insulin/IGF signaling controls basal levels of ecdysone synthesis in the PG. This, in turn, controls the larval growth rate without modifying the duration of larval growth. These data contrast with the present observations on the role of TOR signaling in the PG and indicate that PG cells discriminate between hormone-mediated activation of InR/PI3K signaling and the nutrient-mediated activation of TOR signaling for the control of ecdysone biosynthesis. Can TOR and InR/PI3K signaling pathways function separately in Drosophila tissues? It has been established both in cultured cells and in vivo that a gain of function for InR/PI3K allows for TORC1 activation through inhibition of TSC2 via direct phosphorylation by AKT/PKB. Such crosstalk between the InR and TOR signaling pathways has important functional implications in cancer cells in which inactivation of the PTEN tumor suppressor leads to an important increase in AKT activity. Nevertheless, the physiological significance of the crosstalk between AKT and TSC2 has been challenged by genetic experiments in Drosophila, leading to the notion that, in the context of specific tissues, TOR and insulin/IGF signaling can be part of distinct physiological regulations for the control of animal growth in vivo. Although not observe in standard conditions, strong InR/PI3K activation in the ring gland shortens larval developmental timing under conditions of food limitation. In light of the present data, this suggests that, in low-food conditions, providing high PI3K activity in PG cells allows for full activation of TOR through the AKT/PKB-mediated inhibitory phosphorylation of TSC2, thus modulating developmental timing. Inversely, a severe downregulation of InR/PI3K signaling in the PG extends larval timing by preventing early larval molts. However, it was observed that strong inhibition of the InR pathway compromises the growth of PG cells, therefore interfering with their capacity to produce normal levels of ecdysone for molting. Overall, previous works as well as the present work highlight the importance of studying signaling networks in the specific contexts (tissue, development) in which these pathways normally operate. This also illustrates that only mild manipulations of these intricate pathways are suitable to unravel the regulatory mechanisms that normally occur within the physiological range of their activities. In conclusion, it is proposed that the insulin/IGF system and TOR provide two separate inputs on PG-dependent ecdysone production: the insulin/IGF system controls baseline ecdysone levels during larval life, and TOR acts upon ecdysone peaks in response to PTTH at the end of larval development (Layalle, 2008).
Important literature describes intrinsic mechanisms controlling a growth threshold for pupariation in insects. After a critical size is attained, the hormonal cascade leading to ecdysone production initiates, and larvae are committed to pupal development, even when subjected to complete starvation. Recent findings in Drosophila by using temperature-sensitive mutants for dInR have revealed that reducing the larval growth rate before the critical size is attained postpones the attainment of this threshold, but has no effect on the final size. Conversely, reducing animals' growth rate after the critical size has been attained leads to strong reduction of the final size. This highlights an important period in the determination of final size, called the terminal growth period (TGP, also called interval to cessation of growth), which spans from the attainment of critical size to the cessation of growth. Due to its exponential rate, growth during that period makes an important contribution to the determination of final size. Interestingly, the duration of the TGP is not affected by the general insulin/IGF system, which explains why reduction of the insulin/IGF system during that period leads to short adults. The present data suggest that the duration of the TGP is an important parameter in the determination of final size that is controlled by TOR. By reducing the level of TOR activity specifically in the PG, neither the growth rate or the critical size for commitment to pupariation is affected. Therefore, the time to attainment of the critical size is not changed. The observation of the developmental transitions in P0206 > TSC1/2 larvae (ectopically expressing TSC1/2) indicate that, indeed, the timing of L1/L2 and L2/L3 molts are not modified. By contrast, the L3/pupa transition is severely delayed, indicating that the interval between attainment of critical size and the termination of growth, i.e., the TGP, is increased. Interestingly, activation of TOR in the PG of fasting larvae leads to a sensible (50%) reduction of the developmental delay induced by low nutrients, whereas it has no effect in normally fed animals. This indicates that the regulation of the TGP by TOR plays an important role in the adaptation mechanisms controlling the duration of larval development under conditions of reduced dietary intake. Other mechanisms, such as the delay to attainment of the critical size due to a reduced growth rate, also contribute to timing of larval development, giving a plausible explanation for the fact that PG-specific TOR activation only partially rescues the increase in larval development timing observed under low-nutrient conditions. Despite characterization in different insect systems, the mechanisms determining the critical size remain to be elucidated. The present study shows that inhibition of TOR signaling in the PG does not modify the minimum size for pupariation. This result is in line with previous findings indicating that nutritional conditions do not modify the critical size in Drosophila. Interestingly, animals depleted of PTTH present an important shift in critical size, indicating that PTTH might participate in setting this parameter. Therefore, mechanisms determining the critical size might reside in the generation or the reception of the PTTH signal, upstream of TOR function in the cascade of events leading to ecdysone production (Layalle, 2008).
What is the limiting step that is controlled by the TOR sensor during the process of ecdysone production? Results obtained by genetic analysis in vivo are reminiscent of in vitro work on dissected PG in the M. sexta model. In these previous studies, PTTH-induced ecdysone production in the PG was shown to induce the phosphorylation of ribosomal protein S6 and was inhibited by the drug rapamycin, later identified as the specific inhibitor of TOR kinase. Interestingly, rapamycin treatment blocked PTTH-induced, but not db-cAMP-induced, ecdysone production, indicating that the drug does not act by simply inhibiting general protein translation in PG cells, but, rather, by inhibiting a specific step controlling PTTH-dependent ecdysone production. More recently, many studies mostly carried out on large insects have started unraveling the response to PTTH in the PG, leading to ecdysone synthesis. No bona fide PTTH receptor is identified yet, and the previously identified response to PTTH is a rise in cAMP, leading to a cascade of activation of kinases, including PKA, MAPKs, PKC, and S6-kinase. S6-kinase-dependent S6 phosphorylation is currently being considered as a possible bottle-neck in the activation of ecdysone biosynthesis by PTTH. The present genetic analysis of ecdysone production in the Drosophila PG now introduces the TOR pathway, the main activator of S6-kinase, as a key controller of ecdysone production and therefore provides a plausible explanation for the rise of S6-kinase in PG cells following PTTH induction. The phenotypes obtained after TOR inhibition in the PG are remarkably similar to the phenotype obtained after ablation of the PTTH neurons. Moreover, ths study shows here that PTTH expression is not altered upon starvation, and that TOR inhibition in PTTH cells has no effect on the duration of larval development, suggesting that PTTH production is not modified by a nutritional stress. Taken together, these data suggest a model whereby limited nutrients induce a downregulation of TOR signaling in the PG, abolish the capacity of PG cells to respond to PTTH and produce ecdysone, and lead to an extension of the terminal growth period (Layalle, 2008).
In conclusion, this study illustrates how the TOR pathway can be used in a specific endocrine organ to control a limiting step in the biosynthesis of a hormone in order to couple important physiological regulations with environmental factors such as nutrition (Layalle, 2008).
It is important to understand the regulation of stem cell division because defects in this process can cause altered tissue homeostasis or cancer. The cyclin-dependent kinase inhibitor Dacapo (Dap), a p21/p27 homolog, acts downstream of the microRNA (miRNA) pathway to regulate the cell cycle in Drosophila germline stem cells (GSCs). Tissue-extrinsic signals, including insulin, also regulate cell division of GSCs. Intrinsic and extrinsic regulators intersect in GSC division control; the Insulin receptor (InR) pathway regulates Dap levels through miRNAs, thereby controlling GSC division. Using GFP-dap 3'UTR sensors in vivo, this study shows that in GSCs the dap 3'UTR is responsive to Dicer-1, an RNA endonuclease III required for miRNA processing. Furthermore, the dap 3'UTR can be directly targeted by miR-7, miR-278 and miR-309 in luciferase assays. Consistent with this, miR-278 and miR-7 mutant GSCs are partially defective in GSC division and show abnormal cell cycle marker expression, respectively. These data suggest that the GSC cell cycle is regulated via the dap 3'UTR by multiple miRNAs. Furthermore, the GFP-dap 3'UTR sensors respond to InR but not to TGF-beta signaling, suggesting that InR signaling utilizes Dap for GSC cell cycle regulation. The miRNA-based Dap regulation may act downstream of InR signaling; Dcr-1 and Dap are required for nutrition-dependent cell cycle regulation in GSCs and reduction of dap partially rescues the cell cycle defect of InR-deficient GSCs. These data suggest that miRNA- and Dap-based cell cycle regulation in GSCs can be controlled by InR signaling (Yu, 2009).
Previous studies have shown that miRNAs may regulate Dap, thereby controlling the cell division of GSCs. This study shows that the dap 3'UTR directly responds to miRNA activities in GSCs. Using luciferase assays, miR-7, miR-278 and miR-309 were identified as miRNAs that can directly repress Dap through the dap 3'UTR in vitro. Although miR-278 and miR-7 play a role in regulating GSC division and cell cycle marker expression, respectively, neither of these mutants showed as dramatic a defect in the GSC cell cycle as Dcr-1-deficient GSCs. Thus, the dap 3'UTR may serve to integrate the effect of multiple miRNAs during cell cycle regulation. It remains possible that some miRNAs involved in this process remain to be identified. It was further shown that InR signaling controls the dap 3'UTR in GSCs. This led to an exploration of the interaction between InR signaling and miRNA/Dap cell cycle regulation. GSCs deficient for InR or Dcr-1 show similar cell cycle defects. Using starvation to control InR signaling, it was shown that both Dcr-1 and dap are required for proper InR signaling-dependent regulation of GSC division. Further, reduction of dap partially rescues the cell division defect of the InR mutant GSCs, suggesting that InR signaling regulates the cell cycle via Dap. These results suggest that miRNAs and Dap act downstream of InR signaling to regulate GSC division (Yu, 2009).
The data suggest that multiple miRNAs can regulate the 3'UTR of dap: miR-7, miR-278 and miR-309 can regulate the dap 3'UTR directly, whereas bantam and miR-8 may regulate it indirectly, or through cryptic MREs in the dap 3'UTR. Using GFP sensor assays, it was also shown that the dap 3'UTR may be directly regulated by miRNAs in the GSCs in vivo. However, which specific miRNAs control endogenous Dap levels in Drosophila GSCs remains unknown. Mammalian p21cip1 has also been shown to be a direct target for specific miRNAs of the miR-106 family, including miR-290s and miR-372. Further, the mouse miR-290 family has recently been identified as regulating the G1-S transition. In addition, miR-221 and miR-222 have been shown to regulate p27kip1, thereby promoting cell division in different mammalian cancer cell lines. Neither the miR-290 nor miR-220 family is conserved in Drosophila. Together, these results indicate that the CKIs (Dap) might be a common target for miRNAs in regulating the cell cycle in stem cells. However, the specific miRNAs that regulate the CKIs might vary between organisms (Yu, 2009).
This study reveals novel regulatory roles for miR-7 and miR-278 in the GSC cell cycle. miR-7 and miR-278 can directly target Dap. GSCs deficient for miR-278 show a mild but significant reduction in cell proliferation. Ectopic expression of miR-7 in follicle cells reduces the proportion of cells that stain positive for Dap. Furthermore, ablation of miR-7 in GSCs results in a perturbation of the frequency of CycE-positive GSCs. However, the cell division kinetics of miR-7 mutant GSCs is not reduced, by contrast with the dramatic reduction of cell division in Dcr-1-deficient GSCs. It is plausible that miR-7 and miR-278 act in concert with other miRNAs to regulate the level of Dap in GSCs and thereby contribute to cell cycle control in GSCs (Yu, 2009).
The interaction of multiple miRNAs with the dap 3'UTR might integrate information from multiple pathways. Further studies will reveal what regulates miR-7 and miR-278 expression in GSCs and which other miRNAs might act together in Dap regulation. It is known that miR-7 and the transcriptional repressor Yan mutually repress one another in the eye imaginal disk. In this model, Yan prevents transcription of miR-7 until Erk in the Egfr pathway downregulates Yan activity by phosphorylation, thereby permitting expression of miR-7. Conversely, miR-7 can repress the translation of Yan. Thus, a single pulse of Egfr signaling results in stable expression of miR-7 and repression of Yan. Whether similar regulation will be observed between miR-7 and the signaling pathways that regulate GSC division remains to be seen. It has been suggested that miR-7 might regulate downstream targets of Notch, such as Enhancer of split and Bearded. Thus, miR-7 may have a mild repressive effect on multiple targets in GSCs. Further experiments might illuminate this possibility (Yu, 2009).
miR-278, on the other hand, has been implicated in tissue growth and InR signaling. Overexpression of miR-278 promotes tissue growth in eye and wing imaginal discs. Deficiency of miR-278 leads to a reduced fat body, which is similar to the effect of impaired InR signaling in adipose tissue. Interestingly, miR-278 mutants have elevated insulin/Dilp production and a reduction of insulin sensitivity. Furthermore, miR-278 regulates expanded, which may modulate growth factor signaling including InR. Since InR signaling plays important roles in tissue growth and cell cycle control, it will be interesting to further test how miR-278 may regulate InR signaling, and whether InR signaling might regulate miR-278 in a feedback loop in GSCs (Yu, 2009).
Other miRNAs or miRNA-dependent mechanisms might also play roles in Drosophila GSCs. For example, the miRNA bantam is required for GSC maintenance. A recent study has shown that the Trim-NHL-containing protein Mei-P26, which belongs to the same family as Brain tumor (Brat), affects bantam levels and restricts cell growth and proliferation in the GSC lineage (Neumuller, 2008). Interestingly, most miRNAs are upregulated in mei-P26 mutant flies. By contrast, overexpression mei-P26 in bag of marbles (bam) mutants broadly reduces miRNA levels. This suggests that Mei-P26 regulates proliferation and maintenance of GSC lineages via miRNA levels. Since InR signaling cell-autonomously regulates GSC division but not maintenance, the possible interaction between Mei-P26 and InR signaling might be complex (Yu, 2009).
The systemic compensatory effect of insulin secretion in mammals with defective InR signaling is well documented. Insulin levels in mice with liver-specific InR (Insr - Mouse Genome Informatics) knockout are ~20-fold higher than those of control animals owing to the compensatory response of the pancreatic β-cells and impairment of insulin clearance by the liver. Knockout of the neuronal InR also leads to a mild hyperinsulinemia, indicating whole-body insulin resistance. Furthermore, the knockout of components in the InR signaling pathway, such as Akt2 and the regulatory and catalytic subunits of PI3 kinase, also leads to hyperinsulinemia and glucose intolerance. Therefore, a systemic decrease in InR signaling may lead to compensatory responses (Yu, 2009).
To understand the roles of InR signaling in the GSCs while avoiding any systemic compensatory effect the phenotypes of GSC clones were analyzed. Using a panel of cell cycle markers, it was found that InR mutant GSCs show cell cycle defects similar to those of Dcr-1 mutant GSCs: a reduction of cell division rate, an increased frequency of cells staining positive for Dap and CycE, and a decreased frequency of cells staining positive for CycB. Using GFP-dap 3'UTR sensors, it was shown that the dap 3'UTR responds to InR signaling in GSCs, suggesting that InR signaling can regulate Dap expression through the dap 3'UTR. This, together with genetic data indicating that InR/starvation-dependent cell cycle regulation requires Dcr-1 and dap, has led to the proposalthat InR signaling regulates the cell cycle through miRNAs that further regulate Dap levels. Since a reduction in dap only partially rescues the cell cycle defects of InR mutant GSCs, it is possible that InR signaling might also regulate GSC division by additional mechanisms (Yu, 2009).
InR signaling regulates the cell cycle through multiple mechanisms, mainly through the G1-S, but also partly through the G2-M, transition. Recent work has shown a delay in the G2-M transition in GSCs during C. elegans dauer formation. Starvation and InR deficiency may also affect the G2-M checkpoint in Drosophila GSCs (Hsu, 2008). This study has dissected one possible molecular pathway that InR signaling utilizes to regulate the Drosophila GSC G1-S transition and show that InR signaling can control the cell cycle through miRNA-based regulation of Dap (Yu, 2009).
Many studies have connected InR and CKIs to Tor (Target of rapamycin) or Foxo pathways downstream of InR signaling. In S. cerevisiae, the yeast homolog of p21/p27 is upregulated when Tor signaling is inhibited. Foxo, a transcription factor that can be repressed by InR signaling, is known to play important roles in nutrition-dependent cell cycle regulation by upregulating p21 and p27. In C. elegans, starvation causes L1 cell cycle arrest mediated by InR (daf-2) and Foxo (daf-16): InR represses the function of Foxo, thereby downregulating the CKI (cki-1) and upregulating the miRNA lin-4. This study has shown that a miRNA-based regulation of Dap can be coordinated by InR in Drosophila GSCs (Yu, 2009).
Insulin and insulin-like growth factors (Igf1 and Igf2) are known to play important roles in regulating metabolic and developmental processes in many stem cells. In mammals, Igf signaling is required by different stem cell types, including human and mouse ES cells for survival and self-renewal, neural stem cells for expediting the G1-S transition and cell cycle re-entry, and skeletal muscle satellite cells for promoting the G1-S transition via p27kip1 downregulation. This study has dissected the molecular mechanism of the InR pathway in another adult stem cell type, tDrosophila GSCs, showing that InR signaling can regulate stem cell division through miRNA-based downregulation of the G1-S inhibitor Dap. Further studies will reveal whether miRNAs also mediate InR signaling in other stem cell types (Yu, 2009).
Drosophila larval skeletal muscles are single, multinucleated cells of different sizes that undergo tremendous growth within a few days. The mechanisms underlying this growth in concert with overall body growth are unknown. The size of individual muscles correlates with the number of nuclei per muscle cell and with increasing nuclear ploidy during development. Inhibition of Insulin receptor (InR; Insulin-like receptor) signaling in muscles autonomously reduces muscle size and systemically affects the size of other tissues, organs and indeed the entire body, most likely by regulating feeding behavior. In muscles, InR/Tor signaling, Foxo and dMyc (Diminutive) are key regulators of endoreplication, which is necessary but not sufficient to induce growth. Mechanistically, InR/Foxo signaling controls cell cycle progression by modulating dmyc expression and dMyc transcriptional activity. Thus, maximal dMyc transcriptional activity depends on InR to control muscle mass, which in turn induces a systemic behavioral response to allocate body size and proportions (Demontis, 2009).
Therefore, interplay between InR/Tor signaling, Foxo and dMyc activity regulates muscle growth that occurs during Drosophila larval development, in part via the induction of endoreplication. Interestingly, the extent of muscle growth is sensed systemically, regulates feeding behavior and, in turn, influences the size of other tissues and indeed the whole body. Thus, the growth of a single tissue is sensed systemically via modulating a whole-organism behavior (Demontis, 2009).
dMyc, as well as activation of InR signaling, can promote endoreplication in muscles, whereas Foxo and inhibitors of dMyc and of InR/Tor have the opposite effect. dMyc is likely to regulate the expression of genes required for multiple G-S and S-G transitions during endoreplication, similar to vertebrate Myc, which regulates key cell-cycle regulators including cyclin D2, cyclin E, and the cyclin kinase inhibitors p21 and p27 (Cdkn1a and Cdkn1b, respectively). Indeed, aberrant levels of Cyclin E block muscle growth, indicating that proper muscle growth requires tight control of the expression and activity of endoreplication genes. Further, endoreplication is also modulated by Foxo, which is activated in conditions of nutrient starvation, impaired InR/Tor signaling and by other cell stressors. Foxo presumably regulates cell cycle progression at least in part by modulating the expression of evolutionarily conserved Foxo/Myc-target genes, such as dacapo (the Drosophila p21/p27 homolog) and Cyclin E, that regulate the G1-S transition. Interestingly, Foxo and Myc might control different steps in the activation of common target genes (Demontis, 2009).
In addition, it was found that active Foxo can also inhibit dMyc protein activity and regulates dmyc gene expression. Mechanistically, Foxo could influence dMyc activity in several ways. First, it might physically interact with dMyc, although no evidence was found to support this notion. Second, Foxo could regulate the expression of genes that target dMyc for proteasomal degradation, including several ubiquitin E3 ligases that are induced by Foxo during muscle atrophy in mice and humans. However, by analyzing dMyc protein levels by western blot, no significant dMyc protein instability was found upon Foxo overexpression. Third, Foxo might promote the expression of transcriptional regulators that oppose dMyc function, including Mad/Mnt, although no substantial increase in dmnt mRNA levels was detected upon Foxo activation in muscles. Possibly, the expression of other dMyc regulators might be affected by Foxo. Future experiments will be needed to dissect the Foxo-dMyc interaction (Demontis, 2009).
Finally, by manipulating muscle growth and/or endoreplication, it was found that in muscles the ratio of cell size to nuclear size is not constant, and increased nuclear size and DNA content, indicative of ploidy, is necessary but not sufficient to drive growth. Usually, an increase in cell size is matched by an increase in nuclear size, which commonly parallels increases in nuclear ploidy. However, the current findings indicate that in muscles, dMyc-driven variation in nuclear size and ploidy is permissive but not sufficient for substantial growth, even in the presence of increased biogenesis of nucleoli and expression of genes involved in protein translation. This is different from fat body cells, in which dmyc overexpression induces endoreplication and proportional cell growth. Thus, additional instructive signals, possibly modulating protein synthesis, mitochondriogenesis, ribosome biogenesis, sarcomere assembly, and other anabolic responses must be concomitantly received to promote maximal muscle growth. Therefore, increases in cell size and nuclear ploidy are surprisingly uncoupled during muscle growth (Demontis, 2009).
Little is known about the mechanisms that control and coordinate cell, organ and body size, and in particular how muscle growth is matched with the growth of other tissues and of the entire organism. Inhibition of InR/Tor signaling and dMyc activity in muscles impairs, in addition to muscle mass, the size of the entire body and of other internal organs. Similarly, overexpression of Cyclin E in muscles also results in autonomous and systemic growth defects, indicating that, at least in some cases, modulation of muscle growth by means independent from InR signaling can be sensed systemically. In the larva, endoreplicating tissues and organs (gut, salivary glands, epidermis, fat body) are severely affected, whereas non-endoreplicating tissues (brain and imaginal discs) are less affected, indicating distinct tissue responsiveness to this regulation. Similarly, inhibition of Tor signaling in the fat body also primarily affects the size of endoreplicating tissues (Demontis, 2009).
Non-autonomous regulation of tissue size may rely on humoral factors (e.g. hormone-binding proteins, hormones, metabolites) produced by muscles in response to achieving a certain mass. However, alternative models are possible. In particular, decreased and increased larval feeding, respectively, were observed upon inhibition and activation of InR signaling in muscles. This whole-organism behavioral adaptation is possibly due to decreased and increased efficiency of smaller and bigger muscles, respectively, and to regulated expression of neuropeptides that hormonally control feeding behavior. As a consequence of the regulation of feeding behavior, nutrient uptake is decreased and larval growth is blocked in the cells of endoreplicating tissues, which are extremely sensitive to poor nutritional conditions, and to a lesser extent in non-endoreplicating tissues, which are more resistant to limited nutritional supply. In turn, increased or decreased size of non-muscle tissues arise as a consequence of abnormal feeding. Thus, muscle size coordinates with the size of other organs and of the entire body, at least in part via a systemic, behavioral response. Distinct tissues are differently sensitive to this regulation, resulting in altered body proportions (Demontis, 2009).
Understanding the mechanisms regulating muscle mass is of special interest because they underline the etiology of several human diseases. Directly relevant to these studies, both MYC and InR (INSR) signaling have been found to regulate muscle growth and maintenance in humans. Further, muscle atrophy is triggered by FOXO activation in several pathological conditions. In addition, MYC function has been implicated in heart hypertrophy, a process that is conversely regulated by FOXO (Demontis, 2009).
The findings that Foxo functionally antagonizes dMyc during the growth of Drosophila muscles suggest that these factors might also interact similarly in humans. Consistent with this hypothesis, FOXO and MYC regulate, in opposite fashions, the atrophic and hypertrophic programs in human skeletal muscles and cardiomyocytes, and display complementary gene expression and activity in these contexts (Demontis, 2009).
Finally, the finding that during larval development, inhibition of InR signaling in muscles has profound systemic effects might also reflect physiological conditions found in humans. Indeed, defective responsiveness of muscles to Insulin during type II diabetes has autonomous effects on muscle maintenance that are associated with systemic effects on the metabolism of the entire organism, contributing to the improper control of glycemia and the development of metabolic syndrome. This study has identified feeding behavior as part of the systemic response that in Drosophila senses perturbations in muscle mass. These findings might help further elucidate the signals involved in metabolic and growth homeostasis, which may be conserved across evolution (Demontis, 2009).
The insulin-binding and protein tyrosine kinase subunits of the Drosophila melanogaster insulin receptor homolog have been identified and characterized by using antipeptide antibodies elicited to the deduced amino acid sequence of the alpha and beta subunits of the human insulin receptor. In D. melanogaster embryos and cell lines, the insulin receptor contains insulin-binding alpha subunits of 110 or 120 kilodaltons (kDa), a 95-kDa beta subunit that is phosphorylated on tyrosine in response to insulin in intact cells and in vitro, and a 170-kDa protein that may be an incompletely processed receptor. All of the components are synthesized from a proreceptor, joined by disulfide bonds, and exposed on the cell surface. The beta subunit is recognized by an antipeptide antibody elicited to amino acids 1142 to 1162 of the human insulin proreceptor, and the alpha subunit is recognized by an antipeptide antibody elicited to amino acids 702 to 723 of the human proreceptor. Of the polypeptide ligands tested, only insulin reacts with the D. melanogaster receptor. Insulinlike growth factors type I and II, epidermal growth factor, and the silkworm insulinlike prothoracicotropic hormone are unable to stimulate autophosphorylation. Thus despite the evolutionary divergence of vertebrates and invertebrates, the essential features of the structure and intrinsic functions of the insulin receptor have been remarkably conserved (Fernandez-Almonacid, 1987).
The cloning and primary structure of the Drosophila Insulin receptor gene (InR), functional expression of the predicted polypeptide, and the isolation of mutations in the InR locus are reported. These data indicate that the structure and processing of the Drosophila insulin proreceptor are somewhat different from those of the mammalian insulin and IGF 1 receptor precursors. 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 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 which 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 of both types of beta subunits, which in turn 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. Finally, 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, render pleiotropic recessive phenotypes that lead to embryonic lethality. The activity of InR appears to be required in the embryonic epidermis and nervous system among others, since development of the cuticle, as well as the peripheral and central nervous systems are all affected by InR mutations (Fernandez, 1995).
The Insulin-like receptor (InR) gene 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 and identified six independent mutations that led to a loss of expression or function of the receptor protein. 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).
Genetic analyses suggest that the TSC genes act in a parallel pathway that converges on the insulin pathway downstream from Akt. The most convincing evidence for a functional link between the TSC genes and insulin signaling comes from the observation that heterozygosity of TSC1 or TSC2/gigas is sufficient to rescue the lethality of loss-of-function InR mutants. This argues that the TSC genes are intimately linked to insulin signaling, rather than functioning in a totally independent cell-growth pathway. These results suggest that the TSC tumor suppressor genes are novel negative regulators of insulin signaling, and modulating the activities of the TSC genes might provide a potential way to correct insulin signaling defects in certain diseases such as diabetes and obesity (Gao, 2001).
Previous studies have shown that loss of inr or Akt leads to decreased cell size. To investigate the relationship between inr, Akt, and the TSC genes, TSC1;Akt and TSC1;inr double-mutant clones were studied. Cells homozygous for a strong allele of inr, or a null allele of Akt are smaller, and are rarely recovered in adult eye clones because of cell competition during development. However, TSC1;inr or TSC1;Akt1 double-mutant cells showed a similar cell size increase as that observed in TSC1- cells. Furthermore, the competitive disadvantage of inr and Akt mutant cells is also rescued in the TSC1;inr or TSC1;Akt1 double-mutant clones, resulting in larger clones that contained more cells. This result suggests that TSC1 acts genetically downstream from Akt. This observation is compatible with either TSC1 acting molecularly downstream from Akt in the linear InR-PI3K-Akt pathway, or TSC1 acting in a parallel pathway that converges on the insulin pathway downstream from Akt (Gao, 2001).
To distinguish between these two possibilities, cells were generated that were doubly mutant for null alleles of PTEN and TSC1. PTEN is a negative regulator of the InR-PI3K-Akt pathway, and loss of PTEN results in increased Akt activity and cellular growth. It was reasoned that if TSC1 acts downstream from Akt within the InR-PI3K-Akt pathway, it might be expected that PTEN;TSC1 double-mutant cells would show a similar cell-size phenotype to either single mutant. However, if TSC1 acts parallel to the InR-PI3K-Akt the pathway, it might be expected that PTEN;TSC1 double-mutant cells would show additive effects on cell size as compared with each single mutant. PTEN;TSC1 double-mutant photoreceptors are 2.9 times the size of wild-type cells, as compared with 1.9 for PTEN - and 1.8 for TSC1 -. This result strongly suggests that the TSC genes function in a parallel pathway that converges on the insulin pathway at a point downstream from Akt (Gao, 2001).
In the course of these studies, a striking genetic interaction between the TSC genes and inr mutations was observed. Flies homozygous for a strong loss-of-function inr allele, are larval lethal. However, homozygous inr mutant flies that are heterozygous for TSC1 can survive to adults, suggesting that a mere 50% reduction in the dosage of the TSC1 gene can rescue the developmental arrest of an inr mutant (Gao, 2001).
Similarly, heterozygosity of TSC2 is sufficient to rescue the lethality of another inr mutant. Flies carrying the allelic combination inr353/inrl(3)05545 are 100% lethal. However, approximately 39% of inr353/inrl(3)05545 flies that are heterozygous for TSC2 can survive to adults. Taken together, these results provide convincing in vivo evidence that the TSC genes are negative regulators of insulin signaling in development (Gao, 2001).
The Drosophila gene insulin-like receptor (InR) is homologous to mammalian insulin receptors as well as to C. elegans daf-2, a signal transducer regulating worm dauer formation and adult longevity. A heteroallelic, hypomorphic genotype of mutant InR is described that yields dwarf females with up to an 85% extension of adult longevity and dwarf males with reduced late age-specific mortality. Treatment of the long-lived InR dwarfs with a juvenile hormone analog restores life expectancy toward that of wild-type controls. It is concluded that juvenile hormone deficiency, which results from InR signal pathway mutation, is sufficient to extend life-span, and that in flies, insulin-like ligands nonautonomously mediate aging through retardation of growth or activation of specific endocrine tissue (Tatar, 2001).
Molecular similarity between fly InR and worm daf-2 suggests that mutants of InR in flies should affect adult life-span, as do mutants of daf-2 in worms. InR and daf-2 are members of the insulin receptor family with homology to mammalian insulin and insulin-like growth factor-1 (IGF-1) receptors. Worms carrying temperature-sensitive mutations in daf-2 form dauers at high temperatures, but at lower temperatures develop directly into adults with extended longevity and resistance to exogenous stress. Genotypes homozygous for mutant InR have been reported to be lethal, but several heteroallelic combinations of InR alleles produce viable, dwarf adults that are slow to develop: InREC34/InRE19 and InRGC25/InRE19, and InRE19/InRp5545. In addition, InRE19/InRE19 is viable and dwarf, once crossed into a new isogenic background. Dwarf females eclose with extremely immature ovaries, and the egg chambers of young adults remain previtellogenic (Tatar, 2001).
Measurement of INR kinase activity indicates that the InRp5545 and InRE19 alleles both confer loss of INR function. Basal activity of heterozygotes +/InRE19 and +/InRp5545 was 45% of that of the wild type. Insulin stimulation increases kinase activity of INR from +/+ and +/InRE19 flies by 60%, but only by 26% from +/InRp5545 flies. Basal kinase activity of INR from InRE19/InRE19 and from InRp5545/InRE19 flies is 14% and 11% of that of the wild type, respectively; neither type is stimulated by insulin. InRp5545 is a P-element insertion in exon-1; the molecular lesion of InRE19 has yet to be identified, but it does not appear to occur in the known coding region of the gene (Tatar, 2001).
Life tables of InR mutant adults were compared to concurrent cohorts of a wild-type coisogenic strain. Dwarfs of InREC34/InRE19 and InRGC25/InRE19 are short-lived. Dwarf InRE19/InRE19 and nondwarf +/InRp5545 have moderately reduced survival; nondwarf +/InRE19 individuals are normal. In contrast, females of InRp5545/InRE19 are 85% longer lived than wild-type controls and overall present reduced age-specific mortality. The life-span of female D. melanogaster is also extended by mutation of the insulin receptor substrate homolog chico. Survivorship among male InR genotypes follows the pattern observed for females. Relative to the wild type, InRp5545/InRE19 males exhibit high mortality as early adults, but because of reduced mortality at late ages, dwarf life expectancy at 10 days is 43% greater than that of controls. It is likely that not all InR alleles increase longevity because the gene is highly pleiotropic, with some alleles producing developmental defects that carry over to the adult stage, which counterbalance positive effects of the allele upon aging (Tatar, 2001).
The fact that InR mutants are nonvitellogenic suggests a plausible mechanism for the extended longevity of InRp5545/InRE19 flies. Drosophila overwinter as adults in a reproductive diapause where egg development is arrested at previtellogenic stages. In many insects, including Drosophila, reproductive diapause is proximally controlled through down-regulation of juvenile hormone (JH) synthesis by the corpora allata (CA). Ovaries of InR dwarf females morphologically resemble ovaries of diapause wild-type flies, and exogenous application of the JH analog methoprene to dwarf females initiates vitellogenesis. Females of InRE19/InRE19 respond to a single treatment of methoprene in a dose-dependent manner, but females of InRp5545/InRE19 require continuous exposure to hormone to induce any vitellogenesis. Direct assay of adult JH synthesis verified that CA activity is reduced in InR dwarfs to about 23% of the wild-type level. Because reduced JH synthesis is seen in InRE19/InRE19 flies, which exhibit normal life-span, as well as in long-lived InRp5545/InRE19 flies, the simple lack of JH may not be enough to extend longevity (Tatar, 2001).
Loss of corpora allata JH accounts for dwarf infertility. Mutation of InR may increase longevity because infertility reduces allocation of metabolic resources to reproduction and frees resources for somatic maintenance or because reduced JH in mutant flies induces specific physiological mechanisms of somatic persistence normally expressed during adult reproductive diapause. Adult D. melanogaster in reproductive diapause age at negligible rates and are stress resistant; these traits are reversed by treatment with methoprene. Extended survival is characteristic of adult reproductive diapause in acridid grasshoppers and in the monarch butterfly, and surgical ablation of the corpora allata to eliminate adult JH synthesis induces both diapause and increased longevity. Consistent with the notion that reduced JH synthesis can directly extend life-span, InR dwarf flies show somatic physiological changes: (1) triglycerides are elevated fourfold (F = 32.2, P < 0.001), as observed in diapause D. triauraria and in dwarf D. melanogaster mutant for chico, and (2) Cu/Zn-superoxide dismutase concentration is increased twofold, as is characteristic of long-lived mutants of C. elegans. Measured in young adults, no difference in mass-specific metabolic rate is detected. It is suggested that infertility need not be the direct cause of slowed aging in InR mutants; JH may simply control both fertility and life-span (Tatar, 2001).
To test directly whether JH modulates survival in InRp5545/InRE19 female dwarfs, a test was made of whether treatment with methoprene restores wild-type longevity to these mutants, even if it does not fully restore fertility. In concurrent trials of dwarf and wild-type flies, survival of methoprene-treated InRp5545/InRE19 females is reduced toward the level observed in coisogenic controls. This rescue is physiological rather than toxicological because, in wild-type controls, methoprene produces no significant change relative to ethanol-treated flies (Tatar, 2001).
The InR pathway may alter endocrine function in two ways. Adult CA is derived from neurosecretory tissue of the larval ring gland. Adult dwarf CA may be immature upon metamorphosis as a result of cell autonomous effects of InR upon the development of neuroendocrine cells. A second way InR may alter endocrine function is that JH secretion by CA may be impaired by reduced neuropeptide transmission in the adult brain, due to a reduction of INR function in brain areas where it is normally expressed (Tatar, 2001).
In C. elegans, the insulin/IGF-1 pathway influences dauer formation, fertility, and aging in part through nonautonomous, secondary signaling; sterility is not required for extended longevity in C. elegans because some long-lived daf-2 are fully fertile. For Drosophila, InR affects neurosecretory tissue specialized for secretion of juvenile hormone. Therefore, mutations in the insulin signaling pathway in flies autonomously affect cell proliferation, growth, and body size, but nonautonomously affect diapause, reproduction, and life-span through effects upon specific neuroendocrine cells. Deficiency in a juvenoid-like hormone signal in worms and in flies may extend longevity because its absence leads to the inappropriate expression of parallel physiological programs normally reserved for dauer or diapause (Tatar, 2001).
This invertebrate model may have parallels with mammalian aging. Ames and Snell mice are mutant for the genes Prop-1 or Pit-1, respectively, and are defective for pituitary development. Consequently, they are deficient in growth hormone, prolactin, and thyroid-stimulating hormone, leading to hypothyroidism and presumably reduced synthesis of thyroxin, a retinoid hormone with potential functional similarity to JH. These mice are phenotypically dwarf, mildly obese, and long-lived. A remarkably similar phenotype is observed in mice lacking insulin receptor function in the central nervous system or those lacking the chico homolog, IRS-2, in all tissues: increased fat mass and infertility with accompanying neuroendocrine deficiency. Although effects on life-span in these mice remain to be determined, the concordance of phenotypes suggests that insulin signaling may be central to a common mechanism that exists across taxa for the neuroendocrine regulation of metabolism and the reproductive state, and their associated consequences upon aging (Tatar, 2001).
The Drosophila gene chico encodes an insulin receptor substrate that functions in an insulin/insulin-like growth factor (IGF) signaling pathway. In the nematode C. elegans, insulin/IGF signaling regulates adult longevity. Mutation of chico extends fruit fly median life-span by up to 48% in homozygotes and 36% in heterozygotes. Extension of life-span is not a result of impaired oogenesis in chico females, nor is it consistently correlated with increased stress resistance. The dwarf phenotype of chico homozygotes was also unnecessary for extension of life-span. The role of insulin/IGF signaling in regulating animal aging is therefore evolutionarily conserved (Clancy, 2001).
In Drosophila, the insulin/IGF receptor INR, the insulin receptor substrate Chico, the phosphatidylinositol 3-kinase (PI3K) Dp110/p60, and the PI3K target protein kinase B (PKB, also known as DAkt1) form a signaling pathway that regulates growth and size. The effects on aging of hypomorphic mutations in Inr (equivalent to daf-2) and PKB, and null mutations in chico and the catalytic (Dp110, equivalent to age-1) and adapter (p60) PI3K subunits were examined. All mutants were tested as heterozygotes. chico1 and PKB3 homozygotes and InrGC25/InrE19 transheterozygotes, which form viable dwarf adults, were also examined. The remaining mutations were homozygous lethal (Clancy, 2001).
Most mutants tested had normal or significantly decreased life-span. For example, PKB3 homozygotes and InrGC25/InrE19 flies are short-lived. By contrast, chico1 extends life-span. Homozygous chico1 females exhibit an increase of median and maximum life-span of up to 48% and 41%, respectively. chico1 heterozygotes also exhibit increases in median life-span of up to 36% and 13% in females and males, respectively. Homozygous males, however, are slightly short-lived (Clancy, 2001).
Of the mutations tested, only chico1 increases life-span. This may be because the effect of reduced IIS on life-span depends on the degree to which signaling is reduced. Unlike the other null mutations in IIS genes tested, chico1 is not homozygous lethal, presumably because the INR receptor can signal to PI3K directly, as well as indirectly via Chico. Thus, chico1 mutants may be long-lived because of the relatively mild reduction in pathway activity that they bring about. Notably, severe IIS mutations in C. elegans can cause premature mortality in some adults, although the maximum life-span of populations is invariably increased. This is probably why InrGC25/InrE19 flies are short-lived: Demographic analysis indicates that a reduction in the age-specific mortality rate acceleration occurs, whose effect on survival is masked by an elevated rate of age-independent mortality. Furthermore, a different heteroallelic Drosophila Inr mutant to that tested here exhibits an 85% increase in female life-span. By contrast, in short-lived PKB3 populations, no reduction in mortality rate acceleration is seen. This raises the possibility that a second pathway downstream of chico might regulate aging in Drosophila. Interestingly, Chico contains potential binding sites for the Drk/Grb2 docking protein, consistent with signaling via Ras/mitogen-activated protein kinase (Clancy, 2001). 30 March 2008PDK1 regulates growth through Akt and S6K in Drosophila
The insulin/insulin-like growth factor-1 signaling pathway promotes growth in invertebrates and vertebrates by increasing the levels of phosphatidylinositol 3,4,5-triphosphate through the activation of p110 phosphatidylinositol 3-kinase. Two key effectors of this pathway are the phosphoinositide-dependent protein kinase 1 (PDK1) and Akt/PKB. Although genetic analysis in C. elegans has implicated Akt as the only relevant PDK1 substrate, cell culture studies have suggested that PDK1 has additional targets. In Drosophila, dPDK1 (FlyBase name: Protein kinase 61C) controls cellular and organism growth by activating Akt1 and S6 kinase, dS6K. Furthermore, dPDK1 genetically interacts with dRSK but not with dPKN (FlyBase name: Protein kinase related to protein kinase N), encoding two substrates of PDK1 in vitro. Thus, the results suggest that dPDK1 is required for dRSK but not dPKN activation and that it regulates insulin-mediated growth through two main effector branches, dAkt and dS6K (Rintelen, 2001).
The pronounced effect of loss of dPDK1 function on head size suggests that it is a dominant constituent in the dInr pathway. To test this possibility, the ability of complete and partial loss-of-function alleles of dPDK1 to reverse phenotypes caused by either overexpression of dInr or by mutations in dPTEN, the 3-phosphatidylinositide phosphatase, was evaluated. Overexpression of a wild-type dInr cDNA under the control of GMR-Gal4 leads to a marked increase in eye size and a slightly rough eye surface, an effect dominantly suppressed by removing one copy of dPDK1. Further reduction of dPDK1 function by the dPDK11/4 heteroallelic combination reduces the eye to almost wild-type size, suggesting that the amount of dPDK1 protein is rate-limiting for the dInr overgrowth phenotype. Null mutations in dPTEN cause lethality, and removal of dPTEN function in clones stimulates cell autonomous growth, suggesting that increased levels of PIP3 promote growth and are the likely cause of lethality. Thus, if dPDK1 is an essential target of PIP3, mutations in dPDK1 may suppress the dPTEN phenotype. Surprisingly, some dPTEN/dPDK1 double mutant flies survive to adulthood, indicating that the presumed PIP3-induced lethality is primarily caused by the hyperactivation of dPDK1 or of one of its targets (Rintelen, 2001).
These results show that dPDK1 is an essential component in the insulin signaling pathway in the control of cell growth and body size through its two substrates, dAkt and dS6K. These results are distinct from the genetic evidence in C. elegans where Akt is the primary target of PDK1 in dauer formation. Because mutations in the insulin signaling pathway do not show an autonomous alteration of cell size in C. elegans, the regulation of the rate of protein synthesis through S6K does not seem to be a primary target of this pathway. However, the fact that dPDK1 may yet have additional substrates is suggested by the genetic interaction with dRSK gain-of-function mutations and because viable dPDK1 males are almost completely sterile. Although mutations in components of the insulin signaling pathway such as dInr, chico, Dp110/PI(3)K, and dAkt cause female sterility, male sterility is not observed. Further genetic dissection of dPDK1 function is required to determine the role of dPDK1 in male fertility. These findings in Drosophila are consistent with the absence of insulin growth factor-1-induced activation of S6K, Akt, and RSK in mammalian PDK1-/- embryonic stem cells, and therefore provide evidence for the functional conservation of branch points in kinase networks during evolution (Rintelen, 2001).
Insulin/IGF signaling during development controls growth and size, possibly by coordinating the activities of the Ras and PI 3-kinase signaling pathways. In vertebrates, the IR and IGFR act through IRS1-IRS4 proteins, which are multifunctional adaptors that link insulin and IGF signaling to the Ras/MAPK and phosphoinositide 3'-kinase (PI 3-kinase) signaling pathways. The pleckstrin homology domain (PH) and phosphotyrosine binding domain (PTB) of the IRS proteins are believed to mediate binding to phosphoinositol phosphates and the juxtamembrane NPXY motif of IR/IGFR, respectively. Grb2 (Drosophila homolog Drk) is an adaptor protein containing SH2 and SH3 domains. It has been suggested that Grb2 may, via its binding to IRS, link insulin/IGF to the Ras/MAPK pathway and thereby control proliferation. The Drosophila homolog of the SH2 domain containing p85 PI 3-kinase adaptor subunit, p60, binds Chico/IRS and thereby recruits the p110 catalytic subunit of PI 3-kinase [which converts phosphoinositol(4,5)P2 (PtdIns(4,5)P2) into phosphoinositol(3,4,5)P3 (PtdIns(3,4,5)P3)] to the plasma membrane. The p110 PI 3-kinase belongs to the class I PI 3-kinases implicated in the metabolic effects of insulin. The classical effectors that mediate the biological outcomes of insulin and IGF downstream of IRS have been divided into two functional branches: the Ras/MAPK proliferation pathway, and the PI 3-kinase metabolic, growth and survival pathway (Oldham, 2002).
To analyze the role of the different domains of Chico/IRS under physiological conditions, a panel of effector site mutants was created in a genomic rescue construct for chico that disrupts the PH or the PTB domains or the putative binding sites of Drk/Grb2 and p60. The constructs include the cis-regulatory sequences that permit expression of chico in its normal spatial and temporal pattern. The wild-type chico construct fully restores the defects of chico homozygous null mutants. In this manner, the effector site mutants were assayed for the ability to rescue the three different phenotypes associated with complete loss of Chico function: body size reduction, female sterility and lipid alterations. The Drk/Grb2 consensus binding site mutant is able to fully rescue the reduced weight to the same extent as the wild-type rescue construct. Therefore, the presence of a functional Drk binding site in Chico and thus the link to the activation of the Ras/MAPK kinase pathway is not required for its wild-type function. In contrast, the PH and PTB domain mutants and the double p60 PI 3-kinase binding site mutant were unable to rescue the reduced body weight. The latter result is surprising because InR contains additional functional PI 3-kinase binding sites in its C-terminal tail, an extension shared only with the C. elegans InR homolog, Daf-2, and not the mammalian IR or IGFR. This suggests that the presence of additional p60 binding sites in the InR C-terminal tail is not sufficient in vivo to mediate wild-type levels of growth and proliferation in the absence of the Chico p60 PI 3-kinase binding sites and that the InR C-terminal tail may contribute only low levels of PI 3-kinase signaling. Although the PTB domain mutant fails to restore normal body weight, it rescues the female sterility associated with the loss of Chico function. With the exception of the full rescue of the lipid accumulation observed in Drk/Grb2 mutant, all the other effectors only partially restore the change in lipid accumulation (Oldham, 2002).
To test whether increasing PtdInsP3 levels in an InR or PI 3-kinase p110 mutant background is sufficient to restore growth, the function of a negative regulator of the insulin pathway was eliminated. The 3'-phosphoinositol-specific lipid phosphatase, PTEN acts as a negative regulator of the PI 3-kinase pathway by converting PtdInsP3 generated by PI 3-kinase into PtdInsP2. Used were a null (Pten2L117) and a hypomorphic (Pten2L100) allele of Pten, identified in a screen for genes involved in growth control. As shown by HPLC analysis of the phospholipids in extracts of Pten mutant larvae, the loss of PTEN function results in a 2-fold increase in PtdInsP3 levels. This is consistent with the increase in PtdInsP3 seen in Pten-deleted murine fibroblasts. One prominent biological effect of these increased PtdInsP3 levels in Drosophila is a substantial increase in size in both larvae and pupae. To test whether loss of PTEN function, and consequently increased PtdInsP3 levels, is sufficient to restore growth or viability in InR null mutants, InR and Pten double mutants were generated by creating mosaic animals using the eyeless-Flipase (eyFlp) tissue-specific recombination system. In such animals, the head consists of homozygous mutant tissue, whereas the rest of the body is heterozygous for the same mutation. While loss of PTEN function (Pten2L117) in the head results in a fly with a disproportionately larger head (with more and larger cells), loss of InR function (InR327) results in flies with smaller heads (pinhead) compared to the wild type. Heads doubly mutant for Pten2L117and InR327, however, are almost the size of heads singly mutant for Pten2L117. Also, two different lethal heteroallelic InR combinations (InR304/InR327 or InR304/InR25), which arrest at the second larval instar stage, develop to the pupal stage (15%-17% of 33% expected) and even to pharate adults in the presence of reduced PTEN levels (Pten2L117/Pten2L100). These results demonstrate that complete loss of PTEN function can largely substitute for InR-mediated growth and proliferation in the absence of InR function and that the Ras/MAPK pathway plays little or no role in the InR mediated control of cell growth. This notion is further supported by the observation that complete loss of InR function in the compound eye does not result in a loss of photoreceptors, a hallmark of loss of Ras pathway function (Oldham, 2002).
The rescue of lethal, null InR mutant combinations to near viability by reducing PTEN activity strengthens the argument that a PtdInsP3-dependent signaling pathway is the primary effector for InR-derived growth and proliferation. In support of this observation, PI 3-kinase and Akt have been isolated as retroviral oncogenes, suggesting that activation of PI 3-kinase and Akt is sufficient to mediate growth, proliferation, and oncogenesis in vertebrate systems. In Drosophila and mammals, overexpression of PI 3-kinase causes increased growth; but this is not sufficient for proliferation as is the removal of Pten. From this premise, it has been proposed that PI 3-kinase and PTEN regulate similar yet distinct pathways. Alternatively, it is possible that they do function uniquely in the same pathway and that the difference may be due to altered location and function because of overexpression, or to differential feedback of PI 3-kinase versus PTEN. For example, since PI 3-kinase has been shown to act as a serine/threonine protein kinase on IRS, this may have a negative feedback effect on the insulin pathway that might not be evident in Pten loss-of-function mutations. Nevertheless, PI 3-kinase is absolutely critical in controlling size because using an allelic series of PI 3-kinase mutants in combination with the ey-Flp sytem resulted in a range of different head sizes. Furthermore, expressing an activated and dominant-negative form of PI 3-kinase in Drosophila imaginal discs or the heart of the mouse also leads to a corresponding increase or decrease in cell and organ size. Thus, the PI 3-kinase/PTEN cycle can be considered a dedicated growth rheostat, and the InR pathway is an evolutionary conserved module for regulating the range of growth and size (Oldham, 2002).
Loss of PTEN function results in a metabolically similar phenotype as loss of murine PTP1B (Ptpn1), an IR-specific tyrosine phosphatase, in that hyperactivation of the IR pathway causes resistance to high-fat-diet-induced obesity because of increased basal metabolism. These metabolic lipid effects have likely been conserved during evolution because the increased lipid levels in chico mutants are reminiscent of the enhanced lipid content in Irs2 deleted and NIRKO mice (Oldham, 2002).
Collectively, these data firmly establish Drosophila as a valid model organism for the study of metabolic diseases like diabetes and obesity as well as for the study of growth disorders like cancer. Pten mutant flies are larger in size due to increased cell size and number, but have a corresponding decrease in energy stores, a situation completely opposite that of mutations in positive components of the insulin signaling pathway like InR, chico, PI 3-kinase, and dAkt. These large viable Pten mutants show that a reduction of PTEN function is sufficient for increased organism size. This fact suggests that the four-fold size difference between viable InR and Pten mutants can simply be controlled by the amount of PtdInsP3 and this phenomenon may possibly be extended to vertebrate size regulation. Thus, in Drosophila, the InR/PI 3-kinase/PTEN pathway combines both metabolism and growth control into one pathway that later diverged into two separate, yet interacting systems in mammals (Oldham, 2002).
Insulin-IGF receptor (InR) signaling has a conserved role in regulating lifespan, but little is known about the genetic control of declining organ function. This study describes progressive changes of heart function in aging fruit flies: from one to seven weeks of a fly's age, the resting heart rate decreases and the rate of stress-induced heart failure increases. These age-related changes are minimized or absent in long-lived flies when systemic levels of insulin-like peptides are reduced and by mutations of the only receptor, InR, or its substrate, Chico. Moreover, interfering with InR signaling exclusively in the heart, by overexpressing the phosphatase PTEN or the forkhead transcription factor FOXO, prevents the decline in cardiac performance with age. Thus, insulin-IGF signaling influences age-dependent organ physiology and senescence directly and autonomously, in addition to its systemic effect on lifespan. The aging fly heart is a model for studying the genetics of age-sensitive organ-specific pathology (Wessells, 2004).
Stem cells reside in specialized niches that provide signals required for their maintenance and division. Tissue-extrinsic signals can also modify stem cell activity, although this is poorly understood. This study reports that neural-derived Drosophila insulin-like peptides (DILPs) directly regulate germline stem cell division rate, demonstrating that signals mediating the ovarian response to nutritional input can modify stem cell activity in a niche-independent manner. A crucial direct role is demonstrated for DILPs in controlling germline cyst growth and vitellogenesis (LaFever, 2005).
Germline and somatic stem cells support oogenesis throughout adult life in Drosophila. Germline stem cells (GSCs) reside within a specialized niche where they are exposed to a unique combination of signals required for stem cell function. However, GSCs are also controlled by tissue-extrinsic signals, such as Drosophila insulin-like peptides (DILPs), which mediate the ovarian response to nutrition. On a protein-rich diet, germline and somatic stem cells have high division rates, and their progeny exhibit high division and development rates. On a protein-poor diet or under reduced insulin signaling, rates of division and development are reduced, and progression through vitellogenesis is blocked. It remains unclear, however, how DILPs control the response of GSCs in coordination with their differentiating progeny and with follicle cells (LaFever, 2005).
In adult females, DILPs are produced in two clusters of medial neurosecretory cells in the brain, and stage 10 follicle cells express dilp5 mRNA. Ablation of brain DILP-producing cells results in reduced egg production rates and a partial block in vitellogenesis. To examine the role of the brain DILP-producing cells in previtellogenic stages, they were ablated and follicle cell proliferation rates were measured. Females missing brain DILP-producing cells (ablated) have a severely impaired ability to up-regulate follicle cell proliferation in response to a protein-rich diet. The rate of germline development is reduced in coordination with follicle cell divisions because no abnormalities are observed in previtellogenic egg chambers. Ablation of DILP-producing cells reduces the size of eclosed adults and delays development. Ablated females in which these developmental defects are rescued by an hs-dilp2 transgene expressed during larval stages show a reduced follicle cell proliferation rate, comparable to that of nonrescued, ablated females. Thus, the impaired response to a protein-rich diet is not a secondary consequence of the developmental defects. Moreover, the 2.3-fold delay caused by ablation of brain DILP-producing cells is very similar to that caused by blocking reception of DILP signals by the germ line. This indicates that the brain is the major source of DILPs that determine the rate of egg chamber development with little, if any, contribution from dilp5-expressing follicle cells (LaFever, 2005).
To examine whether DILPs control the rate of germline development directly or indirectly, germline cysts unable to respond to DILPs were created by inducing Drosophila insulin receptor (dinr) mutant clones using the flipase (FLP)/FLP-recognition target (FRT) technique. Germline cysts homozygous for dinr339, a genetic null allele, had normal morphology and correct cell number; however, 83% of these cysts were developmentally delayed, showing markedly decreased size relative to neighboring wild-type egg chambers. Further quantification of these data showed a 2.4-fold delay in the development of dinr339 cysts. Similar results were obtained for germline cysts homozygous for dinrE19 and dinr353, which are viable hypomorphic alleles; 78% and 64% of dinrE19 and dinr353 cysts, respectively, were developmentally delayed. These results reveal that dinr function is required cell autonomously for a normal rate of germline cyst development. Thus, the rate of cyst development is regulated by a DILP signal that is received directly (LaFever, 2005).
Progression through vitellogenesis requires DILP signaling; however, it has been unclear whether this role is direct. Reduced juvenile hormone levels are present in homozygous viable dinr mutants, and the block in yolk uptake in these mutants can be partially bypassed by treatment with methoprene, a juvenile hormone analog, suggesting an indirect role for DILPs in promoting vitellogenesis. To specifically address whether direct activation of germline cysts by DILPs is required for vitellogenesis, mosaic ovarioles were examined in which the entire germ line was homozygous dinr mutant for the ability of their egg chambers to undergo vitellogenesis. All egg chambers containing dinr339 or dinrE19 homozygous mutant cysts failed to progress through vitellogenesis and degenerated. In the case of dinr353, the allele with the higher level of dinr activity, only one out of six egg chambers containing homozygous mutant cysts failed to undergo vitellogenesis. These results suggest that the level of insulin signaling within the germ line controls vitellogenesis, revealing a direct role for DILPs in this process. Moreover, complete loss of dinr function in the germ line results in a complete block in vitellogenesis, whereas this block is partial upon ablation of brain DILP-producing cells. Thus, DILP5 expressed in stage 10 follicle cells likely signals in combination with brain DILPs to regulate vitellogenesis (LaFever, 2005).
It was next asked whether DILPs control GSC division rate directly by binding to receptors on their surface (a cell-autonomous requirement for dinr in GSCs) or indirectly by regulating signals produced by niche cells (a non-cell-autonomous requirement). dinr mosaic ovarioles containing one wild-type and one mutant GSC were examined and the number of wild-type versus mutant cystoblasts and cysts present in their germaria was counted. Because each cystoblast or cyst corresponds to one GSC division, the ratio of mutant to wild-type cystoblasts and cysts is a measure of their relative division rates. For dinr339 homozygous mutant GSCs, a relative division rate of 0.31 was found, whereas, for wild-type GSCs, it was 0.90. Similarly, the relative division rates of dinr353 and dinrE19 GSCs were 0.55 and 0.65, respectively. Thus, dinr homozygous mutant GSCs divide more slowly than wild-type GSCs, and GSC division rate appears sensitive to the level of dinr activity. These results demonstrate that GSCs directly receive the DILP signal to regulate their division rate without mediation by the stem cell niche (LaFever, 2005).
Germline and somatic cells respond to nutritional status in a coordinated manner; however, it is unclear whether somatic cells receive the DILP signal directly (a cell-autonomous role of dinr in follicle cell proliferation) as does the germ line, or indirectly through secondary signals (a non-cell-autonomous role). The percentages of dinr mutant and control follicle cells were measured in mosaic ovarioles carrying one wild-type and one dinr mutant somatic stem cell. If follicle cells receive the DILP signal directly, the reduced level of insulin signaling in dinr mutant follicle cells should result in lower rates of proliferation (i.e., fewer mutant than control follicle cells should be observed), whereas if they receive the signal indirectly, the proliferation rates should be similar. In dinrE19 mosaic ovarioles, 51% of follicle cells were wild-type and 49% were mutant, indicating similar proliferation rates. dinr mutant follicle cells appeared to enter the endoreplicative cycle normally, but pycnotic (degenerating) nuclei and cell death were observed within dinrE19 and dinr339 mutant follicle cell clones starting at stage 8. These results reveal that although a reduction in dinr activity delays germline cyst development cell autonomously, it does not cause a cell-autonomous reduction in follicle cell proliferation rate. Furthermore, in ovarioles carrying a fully dinr mutant germ line, excess follicle cells were not observed, showing that proliferation of surrounding wild-type follicle cells remains coordinated with germline growth. These results suggest that follicle cells respond indirectly to increased DILP levels through a secondary signal from the germ line. Similar degrees of coordination between germ line and soma have been observed in the presence of developmentally delayed dMyc mutant germline clones (LaFever, 2005).
These data demonstrate that tissue-extrinsic DILP signals can directly modify GSC proliferative activity, acting in parallel to signals from their niche. Evidence is provided that, in addition to its previously reported indirect roles in Drosophila and mammals through secondary hormonal signals, insulin signaling plays a crucial direct role during Drosophila oogenesis in regulating not only GSC division rate but also germline cyst development rate and progression through vitellogenesis. Insulin may, therefore, have important direct roles in mammalian oogenesis. Finally, the data suggest that the coordinated response of germline and somatic cells to nutrition involves communication between these tissues. These results have broad significance, in light of the long-known effects of nutrition on human fertility and of the high degree of conservation of insulin signaling functions (LaFever, 2005).
Stem cell maintenance depends on local signals provided by specialized microenvironments, or niches, in which they reside. The potential role of systemic factors in stem cell maintenance, however, has remained largely unexplored. This study shows that insulin signaling integrates the effects of diet and age on germline stem cell (GSC) maintenance through the dual regulation of cap cell number (via Notch signaling) and cap cell-GSC interaction (via E-cadherin) and that the normal process of GSC and niche cell loss that occurs with age can be suppressed by increased levels of insulin-like peptides. These results underscore the importance of systemic factors for the regulation of stem cell niches and, thereby, of stem cell numbers (Hsu, 2009).
The stem cell microenvironment (niche) controls stem cells, and niche aging leads to stem cell decline. The Drosophila germline stem cell (GSC) niche includes terminal filament cells, cap cells, and escort stem cells, and GSC fate and activity require direct contact with cap cells and exposure to niche-derived signals. GSCs also respond to systemic signals, such as Drosophila insulin-like peptides (DILPs), which directly modulate their proliferation. Increased age leads to decreased niche size and signaling and GSC loss. The molecular basis for age-dependent changes in the niche, however, remains poorly understood (Hsu, 2009).
Because diet influences aging, its effects on GSC maintenance were examined, exploiting the fact that GSCs can be unambiguously identified by their anteriorly anchored fusome (a membranous cytoskeletal structure) and by their juxtaposition to cap cells. A decrease was observed in GSC numbers in well-fed females over time. In females on a poor diet, however, the rate of GSC loss was significantly increased (Hsu, 2009).
Insulin secretion and signaling respond to diet and diminish in aging humans. Using a phosphoinositide 3-kinase reporter, reduced insulin signaling was found in older ovaries. To address if GSC maintenance requires insulin signaling, GSC numbers were measured in Drosophila insulin receptor (dinr) mutants. The dinr339/dinrE19 females contain slightly fewer GSCs at eclosion and lose them significantly faster than controls. GSC death was not observed in dinr339/dinrE19 or control germaria, suggesting that GSC loss results from differentiation (Hsu, 2009).
The chico1 homozygotes, which lack insulin receptor substrate, a major insulin pathway component, also show increased GSC loss. Thus, insulin signaling controls GSC maintenance. Next, whether DILP expression in germarial somatic cells could counteract the wild-type age-dependent GSC loss was tested. The c587-GAL4 driver was used to express a UAS-dilp2 transgene, encoding the DILP most closely related to human insulin, and thereby increase the local levels of insulin-like signals. GSC loss on rich and poor diets was significantly suppressed by DILP2 overexpression, although this was less pronounced in 4-week-old females on a poor diet. The less effective rescue on a poor diet could potentially be attributable to lower expression of the c587-GAL4 driver, to the actions of additional diet-dependent signals, or to a combination thereof. Nevertheless, these results suggest that the normal GSC loss observed in wild-type females as their age increases results largely from reduced insulin signaling (Hsu, 2009).
DILPs control GSC division directly, leading to a cell-autonomous dinr requirement. It was therefore asked whether dinr is required within GSCs for their maintenance. In genetic mosaics, homozygous dinr339 or dinrE19 GSCs are not lost at a higher rate than control GSCs, demonstrating that DILPs do not promote GSC maintenance directly (Hsu, 2009).
It was next hypothesized that insulin signaling may regulate GSC fate via the niche. Indeed, expression of wild-type dinr in somatic cells of dinr339/dinrE19 germaria rescued GSC loss. To examine dinr339/dinrE19 niche structure, terminal filament and cap cells were counted. Terminal filament cell numbers in dinr339/dinrE19 and control females are similar. In contrast, dinr339/dinrE19 females eclose with fewer cap cells and also lose them faster over time, suggesting that insulin signaling controls cap cell number during development and adulthood. Moreover, DILP2 overexpression suppresses the wild-type age-dependent cap cell number decrease. It is concluded that DILPs control GSC niche size and that the reduced cap cell numbers observed with increased female age at least in part reflect low insulin signaling levels (Hsu, 2009).
It was next asked whether DILPs control cap cell number directly. In mosaic germaria containing β-gal-negative dinr339 or control cap cells, the distribution (and average number) of β-gal-negative cap cells was indistinguishable, indicating that dinr does not control cap cell number cell autonomously. It is possible that a second cell type, such as terminal filament cells, produces an intermediate factor; alternatively, cap cells themselves may control their own maintenance via paracrine signaling (Hsu, 2009).
Notch signaling controls cap cell number during niche formation and in adults. Notch hyperactivation during development forms ectopic cap cells, leading to excess GSCs. Conversely, defective Notch signaling reduces niche size and GSC number. Notch activation is strongly detected in larval terminal filament and cap cells and is also detected in adult cap cells. Notch signaling was examined in dinr mutants using the E(spl)mβ-CD2 reporter. Every control germarium had strong CD2 expression in both terminal filament and cap cells. In contrast, CD2 levels were severely reduced in dinr339/dinrE19 germaria, indicating that insulin signaling controls Notch activation in the niche (Hsu, 2009).
It was next asked if the reduced cap cell number in dinr mutants was attributable to impaired Notch signaling. Weak hypomorphic dinrE19/dinr353 females have no reduction in GSC or cap cell number. Similarly, Notch heterozygotes (half the Notch dosage) have normal GSC and cap cell numbers. In contrast, dinrE19/dinr353 females heterozygous for Notch have significantly reduced GSC and cap cell numbers. A decrease in small cap cell number has been reported for Notch heterozygotes; this discrepancy may reflect slightly reduced insulin signaling in the latter study attributable to diet (Hsu, 2009).
To determine if Notch signaling is sufficient to rescue dinr defects, an activated form of Notch was expressed in the somatic cells of dinr339/dinrE19 germaria, and the GSC and cap cell loss phenotypes were rescued. These results and the genetic interaction between dinr and Notch are consistent with the insulin pathway acting upstream or in parallel to Notch. Nevertheless, the reduced Notch reporter levels in dinr mutants favor the model that insulin signaling leads to Notch activation, thereby controlling cap cell number and, indirectly, GSC maintenance (Hsu, 2009).
GSCs and terminal filament cells express the Delta ligand for Notch, and removal of Delta function from GSCs has been reported to affect niche activity. It was reasoned that dinr could be required in GSCs, terminal filament cells, the cap cell population, or a combination thereof to control Delta production and Notch activation. dinr mosaic germaria were examined in which all GSCs were dinr339 homozygous, and the number of cap cells in those germaria was indistinguishable from control numbers, suggesting that dinr is not required in GSCs for Notch signaling. DILPs may instead regulate Delta within terminal filament or cap cells or, alternatively, act via other intermediate signals to regulate Notch activation within the niche (Hsu, 2009).
Cap cell and GSC numbers correlate. Indeed, in germaria containing control β-gal-negative cap cells (control C1), total cap cell and GSC numbers are roughly proportional. Remarkably, despite similar cap cell numbers, a significant fraction of germaria in which dinr mutant cap cells are present contains fewer GSCs relative to control C1 or C2 (i.e., germaria without cap cell clones from dinr mosaics). Thus, although dinr does not control cap cell number per se autonomously, it is required within cap cells either for the optimal production and/or secretion of a GSC maintenance factor(s) or to promote GSC attachment (Hsu, 2009).
Niche-derived bone morphogenetic protein (BMP) signals directly stimulate GSCs to repress differentiation. To test if insulin signaling controls BMP pathway activation in GSCs, the Dad-lacZ reporter was used. Dad-lacZ levels in dinr339/dinrE19 and control females are indistinguishable, showing that dinr does not control BMP signaling. Insulin signaling in cap cells must therefore control another GSC maintenance signal and/or the cap cell-GSC association (Hsu, 2009).
To investigate if dinr controls the physical interaction between cap cells and GSCs, the percentage of dinr339 versus control cap cells directly contacting GSCs was measure in mosaic germaria. Indeed, 21% of dinr339 cap cells contact GSCs, compared with 50% of control cap cells, indicating that dinr339 cap cells have significantly reduced attachment to GSCs. These results suggest that insulin signaling in cap cells controls their association with GSCs. Alternatively, insulin signaling may regulate the production of a short-range GSC maintenance signal, such that only GSCs in contact with dinr mutant cap cells are affected (Hsu, 2009).
E-cadherin-mediated adhesion between cap cells and GSCs is required for retaining GSCs in the niche. Therefore E-cadherin levels were measured at the GSC-cap cell junction. In controls, it was found that E-cadherin levels vary with changes in the fusome, a membranous cytoskeletal structure. When the fusome is round, its predominant morphology, there is a higher intensity of E-cadherin at the junction, although when the fusome is elongated, the intensity is lower. The intensity of E-cadherin at the junction of cap cells with GSCs displaying elongated fusomes in dinr339/dinrE19 mutants is similar to that of control. In contrast, the round fusome GSC-cap cell junctions contain significantly lower E-cadherin levels in dinr mutants than in controls. These results suggest that insulin signaling influences E-cadherin levels at the GSC-cap cell junction and may explain the age-dependent E-cadherin reduction that contributes to GSC loss (Hsu, 2009).
These studies demonstrate that systemic insulin-like signals integrate inputs from diet and age to regulate GSC maintenance via the niche. Specifically, it is proposed that DILPs control cap cell number via Notch and also E-cadherin- mediated GSC retention within the niche. Because diet and insulin signaling control GSC proliferation, it is also likely that the proliferation decline reported in older females results from reduced insulin signaling. These results also provide insights into recent findings that systemic factors from young mice can restore Notch activation and skeletal muscle progenitor proliferation and regenerative capacity to old mice in heterochronic parabiotic pairings. Finally, the results are intriguing in light of the well-established connection between low insulin signaling, restricted diet, and extended lifespan and of studies in C. elegans suggesting that GSCs may have a negative effect on longevity. It is conceivable that excessive stem cell activity in general is deleterious and that slight decreases in stem cell number or activity with age as a result of reduced insulin signaling may actually promote longevity (Hsu, 2009).
Alcedo, J. and Kenyon, C. (2004). Regulation of C. elegans longevity by specific gustatory and olfactory neurons. Neuron 41: 45-55. 14715134
Ament, S. A., Corona, M., Pollock, H. S. and Robinson, G. E. (2008). Insulin signaling is involved in the regulation of worker division of labor in honey bee colonies. Proc. Natl. Acad. Sci. 105(11) :4226-31. PubMed Citation: 18337502
Araki, E., et al. (1994). Alternative pathway of insulin signalling in mice with targeted disruption of the IRS-1 gene. Nature 372: 186-190. 7526222
Bateman, J. M. and McNeill, H. (2004). Temporal control of differentiation by the Insulin receptor/Tor pathway in Drosophila. Cell 119: 87-96. 15454083
Baugh, L. R. and Sternberg, P. W. (2006). DAF-16/FOXO regulates transcription of cki-1/Cip/Kip and repression of lin-4 during C. elegans L1 arrest. Curr. Biol. 16(8): 780-5. 16631585
Belgacem, Y. H. and Martin, J. R. (2006). Disruption of insulin pathways alters trehalose level and abolishes sexual dimorphism in locomotor activity in Drosophila. J. Neurobiol. 66(1): 19-32. PubMed Citation: 16187303
Britton, J. S. and Edgar, B. A. (1998). Environmental control of the cell cycle in Drosophila: nutrition activates mitotic and endoreplicative cells by distinct mechanisms. Development 125(11): 2149-58. 9570778
Britton, J. S., et al. (2002). Drosophila's insulin/pi3-kinase pathway coordinates cellular metabolism with nutritional conditions. Dev. Cell 2: 239-249. 11832249
Brogiolo, W., et al. (2001). An evolutionarily conserved function of the Drosophila insulin receptor and insulin-like peptides in growth control. Current Biology 11: 213-221. 11250149
Butler, A. A. and Roith, D. L. (2001). Control of growth by the somatropic axis: Growth hormone and the insulin-like growth factors have related and independent roles. Annu. Rev. Physiol. 63: 141-164. 11181952
Chen, C., Jack, J. and Garofalo, R. S. (1996). The Drosophila insulin receptor is required for normal growth. Endocrinology 137(3): 846-856. 8603594
Chiu, S. L., Chen, C. M. and Cline, H. T. (2008). Insulin receptor signaling regulates synapse number, dendritic plasticity, and circuit function in vivo. Neuron 58(5): 708-19. PubMed Citation: 18549783
Clancy, D. J., et al. (2001). Extension of life-span by loss of Chico, a Drosophila Insulin receptor substrate protein. Science 292: 104-6. 11292874
Corona, M., et al. (2007). Vitellogenin, juvenile hormone, insulin signaling, and queen honey bee longevity. Proc. Natl. Acad. Sci. 104: 7128-7133. PubMed Citation: 17438290
Danielsen, A. G., et al. (1995). Activation of protein kinase Ca inhibits signaling by members of the insulin receptor family. J. Biol. Chem. 270(37): 21600-21605. 7545165
Demontis, F. and Perrimon, N. (2009). Integration of Insulin receptor/Foxo signaling and dMyc activity during muscle growth regulates body size in Drosophila. Development 136(6): 983-93. PubMed Citation: 19211682
Dennis, P., et al. (2001). Mammalian TOR: a homeostatic ATP sensor. Science 294: 1102-1105. 11691993
Fernandez, R., et al. (1995). The Drosophila insulin receptor homolog: a gene essential for embryonic development encodes two receptor isoforms with different signaling potential. EMBO J. 14(14): 3373-3384. 7628438
Fernandez-Almonacid, R. and Rosen, O. M. (1987). Structure and ligand specificity of the Drosophila melanogaster insulin receptor. Molec. Cell. Biol. 7: 2718-2727. 3118188
Fernandez, A. M., et al. (2001). Functional inactivation of the IGF-I and insulin receptors in skeletal muscle causes type 2 diabetes. Genes Dev. 15: 1926-1934. 11485987
Gao, X. and Pan, D. (2001). TSC1 and TSC2 tumor suppressors antagonize insulin signaling in cell growth. Genes Dev. 15: 1383-1392. 11390358
Garofalo, R. S. and Rosen, O. M. (1988). Tissue localization of Drosophila melanogaster insulin receptor transcripts during development. Molec. Cell. Biol. 8: 1638-1647. 2454394
Gorczyca, M., Augart, C. and Budnik, V. (1993). Insulin-like receptor and insulin-like peptide are localized at neuromuscular junctions in Drosophila. J. Neurosci. 13(9): 3692-3704. 8366341
Hetru, C., et al. (1991). Isolation and structural characterization of an insulin-related molecule, a predominant neuropeptide from Locusta migratoria Eur. J. Biochem. 201: 495-499
Hopper, N. A. (2006). The adaptor protein soc-1/Gab1 modifies growth factor receptor output in C. elegans. Genetics 173(1): 163-75. 16547100
Hsu, H. J., LaFever, L. and Drummond-Barbosa, D. (2008). Diet controls normal and tumorous germline stem cells via insulin-dependent and -independent mechanisms in Drosophila. Dev. Biol. 313: 700-712. PubMed Citation: 18068153
Hsu, H. J. and Drummond-Barbosa, D. (2009). Insulin levels control female germline stem cell maintenance via the niche in Drosophila. Proc. Natl. Acad. Sci. 106(4): 1117-21. PubMed Citation: 19136634
Iiboshi, Y., et al. (1999). Amino acid-dependent control of p70(s6k). Involvement of tRNA aminoacylation in the regulation. J. Biol. Chem. 274: 1092-1099. 9873056
Ikeya, T., et al. (2002). Nutrient-dependent expression of Insulin-like Peptides from neuroendocrine cells in the CNS contributes to growth regulation in Drosophila. Curr. Biol. 12: 1293-1300. 12176357
Kawakami, A., et al. (1989). Structure and organization of four clustered genes that encode bombyxin, an insulin-related brain secretory peptide of the silkmoth Bombyx mori Proc. Natl. Acad. Sci. 86: 6843-6847. 2674935
Kim, H., Rogers, M. J., Richmond, J. E. and McIntire, S. L. (2004). SNF-6 is an acetylcholine transporter interacting with the dystrophin complex in Caenorhabditis elegans. Nature 430: 891-896. Medline abstract: 15318222
Kleijn, M. and Proud, C. G. (2000). Glucose and amino acids modulate translation factor activation by growth factors in PC12 cells. Biochem. J. 347: 399-406. 10749669
Lee, R. Y., Hench, J. and Ruvkun, G. (2001). Regulation of C. elegans DAF-16 and its human ortholog FKHRL1 by the daf-2 insulin-like signaling pathway. Curr. Biol. 11(24):1950-7. 11747821
Lee, S. S., Kennedy, S., Tolonen, A. C. and Ruvkun, G. (2003). DAF-16 target genes that control C. elegans life-span and metabolism. Science 300(5619): 644-7. 12690206
LaFever. L. and Drummond-Barbosa, D. (2005). Direct control of germline stem cell division and cyst growth by neural insulin in Drosophila. Science 309: 1071-1073. Medline abstract: 16099985
Leibiger, B., et al. (2001). Selective insulin signaling through a and b insulin receptors regulates transcription of insulin and glucokinase genes in pancreatic cells. Molec. Cell 7: 559-570. 11463381
Lin, K., Dorman, J.B., Rodan, A., and Kenyon, C. (1997). daf-16: An HNF-3/forkhead family member that can function to double the life-span of Caenorhabditis elegans. Science 278: 1319-1322. 9360933
Lin, K., Hsin, H., Libina, N. and Kenyon, C. (2001). Regulation of the Caenorhabditis elegans longevity protein DAF-16 by insulin/IGF-1 and germline signaling. Nat. Genet. 28(2): 139-45. 11381260
Layalle, S., Arquier, N., Léopold, P. (2008). The TOR pathway couples nutrition and developmental timing in Drosophila. Dev. Cell 15(4): 568-77. PubMed Citation: 18854141
Marin-Hincapie, M. and Garofalo, R. S. (1995). Drosophila insulin receptor: Lectin-binding properties and a role for oxidation-reduction of receptor thiols in activation. Endocrinology 136(6): 2357-2366. 7750456
Marin-Hincapie, M. and Garofalo, R. S. (1999). The carboxyl terminal extension of the Drosophila insulin receptor homologue binds IRS-1 and influences cell survival. J. Biol. Chem. 274(35): 24987-24994. 10455177
Marr, M. T., D'Alessio, J. A., Puig, O. and Tjian, R. (2007). IRES-mediated functional coupling of transcription and translation amplifies insulin receptor feedback. Genes Dev. 21(2): 175-83. Medline abstract: 17234883
Masumura, M., Ishizaki, H., Nagata, K., Kataoka, H., Suzuki, A. and Mizoguchi, A. (1997). Glucose stimulates the release of bombyxin, an insulin-related peptide of the silkworm Bombyx mori. Comp. Biochem. Physiol. Part B Biochem. Mol. Biol. 118: 349-357. 9440228
Masumura, M., et al. (2000). Glucose stimulates the release of bombyxin, an insulin-related peptide of the silkworm Bombyx mori. Gen. Comp. Endocrinol. 118: 393-399. 10843790
Montanaro, F. and Carbonetto, S. (2003). Targeting dystroglycan in the brain. Neuron 37: 193-196. Medline abstract: 12546815
Murakami, S. and Johnson, T. E. (2001). The OLD-1 positive regulator of longevity and stress resistance is under DAF-16 regulation in Caenorhabditis elegans. Curr. Biol. 11(19): 1517-23. 11591319
Neumuller, R. A., Betschinger, J., Fischer, A., Bushati, N., Poernbacher, I., Mechtler, K., Cohen, S. M. and Knoblich, J. A. (2008). Mei-P26 regulates microRNAs and cell growth in the Drosophila ovarian stem cell lineage. Nature 454: 241-245. PubMed Citation: 18528333
Nishida, Y., et al. (1986). Cloning of a Drosophila cDNA encoding a polypeptide similar to the human insulin receptor precursor. Biochem. biophys. Res. Commun. 141: 474-481. 3099787
Ogg, S., Paradis, S., Gottlieb, S., Patterson, G. I., Lee, L., Tissenbaum, H. A. and Ruvkun, G. (1997). The Fork head transcription factor DAF-16 transduces insulin-like metabolic and longevity signals in C. elegans. Nature 389: 994-999. 9353126
Oldham, S., et al. (2000). Genetic and biochemical characterization of dTOR, the Drosophila homolog of the target of rapamycin. Genes Dev. 14: 2689-2694. 11069885
Oldham, S., et al. (2002). The Drosophila insulin/IGF receptor controls growth and size by modulating PtdInsP3 levels. Development 129: 4103-4109. 12163412
Ookuma, S., Fukuda, M. and Nishida, E. (2003). Identification of a DAF-16 transcriptional target gene, scl-1, that regulates longevity and stress resistance in Caenorhabditis elegans. Curr. Biol. 13(5): 427-31. 1262019
Piovant, M., and Lena, P. (1988). Membrane glycoproteins immunologically related to the human insulin receptor are associated with presumptive neuronal territories and developing neurones in Drosophila melanogaster. Development. 103(1): 145-56. 3143540
Petruzzelli, L., et al. (1985). Acquisition of insulin-dependent protein tyrosine kinase activity during Drosophila embryogenesis. J. Biol. Chem. 260(30): 16072-5. 3934169
Petruzzelli, L., et al. (1986). Isolation of a Drosophila genomic sequence homologous to the kinase domain of the human insulin receptor and detection of the phosphorylated Drosophila receptor with an anti-peptide antibody. Proc. Natl. Acad. Sci. 83(13): 4710-4. 3014506
Piekny, A. J., Wissmann, A. and Mains, P. E. (2000). Embryonic morphogenesis in Caenorhabditis elegans integrates the activity of LET-502 Rho-binding kinase, MEL-11 myosin phosphatase, DAF-2 insulin receptor and FEM-2 PP2c phosphatase. Genetics 156(4): 1671-89. 11102366
Pimentel, B., et al. (1996). Insulin acts as an embryonic growth factor for Drosophila neural cells. Biochem. biophys. Res. Commun. 226(3): 855-861. 8831701
Poltilove, R. M. K., et al. (2000). Characterization of Drosophila insulin receptor substrate. J. Biol. Chem. 275(30): 23346-23354. 10801879
Puig, O., Marr, M. T., Ruhf, R. L. and Tjian, R. (2003). Control of cell number by Drosophila FOXO: downstream and feedback regulation of the insulin receptor pathway. Genes and Development 17: 2006-2020. 12893776
Puig, O. and Tjian, R. (2005). Transcriptional feedback control of insulin receptor by dFOXO/FOXO1. Genes Dev. 19(20): 2435-46. 16230533
Qu, Q. and Smith, F. I. (2004). Alpha-dystroglycan interactions affect cerebellar granule neuron migration. J. Neurosci. Res. 76: 771-782. Medline abstract: 15160389
Raught, B., Gingras, A. C. and Sonenberg, N. (2001). The target of rapamycin (TOR) proteins. Proc. Natl. Acad. Sci. 98: 7037-7044. 11416184
Richard-Parpaillon, L., et al. (2002). The IGF pathway regulates head formation by inhibiting Wnt signaling in Xenopus. Dev. Biol. 244: 407-417. 11944947
Rintelen, F., Stocker, H., Thomas, G. and Hafen, E. (2001). PDK1 regulates growth through Akt and S6K in Drosophila. Proc. Natl. Acad. Sci. 98: 15020-15025. 11752451
Ruan, Y., et al. (1995). The Drosophila insulin receptor contains a novel carboxyl-terminal extension likely to play an important role in signal transduction. J. Biol. Chem. 270(9): 4236-4243. 7876183
Rulifson, E. J., Kim, S. K. and Nusse, R. (2002). Ablation of insulin-producing neurons in flies: Growth and diabetic phenotypes. Science 296: 1118-1120. 12004130
Samuelson, A. V., Carr, C. E. and Ruvkun, G. (2007). Gene activities that mediate increased life span of C. elegans insulin-like signaling mutants. Genes Dev. 21(22): 2976-94. PubMed citation: 18006689
Schmelzle, T. and Hall, M. N. (2000). TOR, a central controller of cell growth. Cell 103: 253-262. 11057898
Shcherbata, H. R., et al. (2007). Dissecting muscle and neuronal disorders in a Drosophila model of muscular dystrophy. EMBO J. 26(2): 481-93. Medline abstract: 17215867
Sim, C. and Denlinger, D. L. (2008). Insulin signaling and FOXO regulate the overwintering diapause of the mosquito Culex pipiens. Proc. Natl. Acad. Sci. 105(18): 6777-81. PubMed Citation: 18448677
Smit, A. B., et al. (1988). Growth-controlling molluscan neurons produce the precursor of an insulin-related peptide. Nature 331: 535-538
Tamemoto, H., et al. (1994). Insulin resistance and growth retardation in mice lacking insulin receptor substrate-1. Nature 372(6502): 182-6. 7969452
Tatar, M., et al. (2001). A mutant Drosophila insulin receptor homolog that extends life-span and impairs neuroendocrine function. Science 292: 107-10. 11292875
Tiessen, J., Underwood, L. and Ketelslegers, J. (1999) Regulation of insulin growth factor-1 in starvation and injury. Nutr. Rev. 57: 167-176. 10439629
Tissenbaum, H. A. and Guarente, L. (2001). Increased dosage of a sir-2 gene extends lifespan in Caenorhabditis elegans. Nature 410(6825): 227-30. 11242085
Tu, M. P., Yin, C. M. and Tatar, M. (2005). Mutations in insulin signaling pathway alter juvenile hormone synthesis in Drosophila melanogaster. Gen. Comp. Endocrinol. 142: 347-356. PubMed Citation: 15935161
Wessells, R. J., Fitzgerald, E., Cypser, J. R., Tatar, M. and Bodmer, R. (2004). Insulin regulation of heart function in aging fruit flies. Nat. Genet. 36(12): 1275-81. 15565107
Williams, K. D., Busto, M., Suster, M. L., So, A. K., Ben-Shahar, Y., Leevers, S. J. and Sokolowski, M. B. (2006). Natural variation in Drosophila melanogaster diapause due to the insulin-regulated PI3-kinase. Proc. Natl. Acad. Sci. 103(43): 15911-5. PubMed Citation: 17043223
Withers, D. J., et al. (1998). Disruption of IRS-2 causes type 2 diabetes in mice. Nature 391: 900-904. 9495343
Wu, Q., Zhang, Y., Xu, J. and Shen, P. (2005). Regulation of hunger-driven behaviors by neural ribosomal S6 kinase in Drosophila. Proc. Natl. Acad. Sci. 102(37): 13289-94. 16150727
Xu, G. G. and Rothenberg, P. L. (2001). Insulin receptor signaling in the beta-cell influences insulin gene expression and insulin content: evidence for autocrine beta-cell regulation. Diabetes 47: 1243-1252. 9703324
Yamaguchi, T., Fernandez, R. and Roth, R.A. (1995). Comparison of the signaling abilities of the Drosophila and human insulin receptors in mammalian cells. Biochemistry 34(15): 4962-4968. 7711018
Yeh, T. C., et al. (1996). Characterization and cloning of a 58/53-kDa substrate of the insulin receptor tyrosine kinase. J. Biol. Chem. 271(6): 2921-2928. 8621681
Yenush, L., et al. (1996). The Drosophila insulin receptor activates multiple signaling pathways but requires insulin receptor substrate proteins for DNA synthesis. Molec. Cell. Biol. 16(5): 2509-2517. 8628319
Yu, H. and Larsen, P. L. (2001). DAF-16-dependent and independent expression targets of DAF-2 insulin receptor-like pathway in Caenorhabditis elegans include FKBPs. J. Mol. Biol. 314(5): 1017-28. 11743719
Yu, J. Y., et al. (2009). Dicer-1-dependent Dacapo suppression acts downstream of Insulin receptor in regulating cell division of Drosophila germline stem cells. Development 136(9): 1497-507. PubMed Citation: 19336466
Zhang, H., et al. (2000). Regulation of cellular growth by the Drosophila target of rapamycin dTOR. Genes Dev. 14: 2712-2724. 11069888
date revised: 10 December 2009
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