Cell-autonomous regulation of cell and organ growth in Drosophila by Akt/PKB

Organismal size is determined by a tightly regulated mechanism that coordinates cell growth, cell proliferation and cell death. The Drosophila insulin receptor/Chico/Dp110 pathway regulates cell and organismal size. Here we show that genetic manipulation of the phosphoinositide-3-OH-kinase-dependent serine/threonine protein kinase Akt (protein kinase B) during development of the Drosophila imaginal disc affects cell and organ size in an autonomous manner. Ectopic expression of Akt does not affect cell-fate determination, apoptosis or proliferation rates in imaginal discs. Thus, Akt appears to stimulate intracellular pathways that specifically regulate cell and compartment size independently of cell proliferation in vivo.     

Regulation of pancreatic beta-cell growth and survival by the serine/threonine protein kinase Akt1/PKBalpha

The physiological performance of an organ depends on an interplay between changes in cellular function and organ size, determined by cell growth, proliferation and death. Nowhere is this more evident than in the endocrine pancreas, where disturbances in function or mass result in severe disease. Recently, the insulin signal-transduction pathway has been implicated in both the regulation of hormone secretion from beta cells in mammals as well as the determination of cell and organ size in Drosophila melanogaster. A prominent mediator of the actions of insulin and insulin-like growth factor 1 (IGF-1) is the 3'-phosphoinositide-dependent protein kinase Akt, also known as protein kinase B (PKB). Here we report that overexpression of active Akt1 in the mouse beta cell substantially affects compartment size and function. There was a significant increase in both beta-cell size and total islet mass, accompanied by improved glucose tolerance and complete resistance to experimental diabetes.     

dS6K-regulated cell growth is dPKB/dPI(3)K-independent, but requires dPDK1

Genetic studies in Drosophila melanogaster underscore the importance of the insulin-signalling pathway in controlling cell, organ and animal size. Effectors of this pathway include Chico (the insulin receptor substrate homologue), dPI(3)K, dPKB, dPTEN, and dS6K. Mutations in any of these components have a striking effect on cell size and number, with the exception of dS6K. Mutants in dS6K affect cell size but not cell number, seemingly consistent with arguments that dS6K is a distal effector in the signalling pathway, directly controlled by dTOR, a downstream effector of dPI(3)K and dPKB. Unexpectedly, recent studies showed that dS6K activity is unimpaired in chico-deficient larvae, suggesting that dS6K activation may be mediated through the dPI(3)K docking sites of the Drosophila insulin receptor. Here, we show genetically, pharmacologically and biochemically that dS6K resides on an insulin signalling pathway distinct from that of dPKB, and surprisingly also from that of dPI(3)K. More striking, despite dPKB-dPI(3)K-independence, dS6K activity is dependent on the Drosophila homologue of the phosphoinositide-dependent protein kinase 1, dPDK1, demonstrating that both dPDK1, as well as dTOR, mediated dS6K activation is phosphatidylinositide-3,4,5-trisphosphate (PIP3)-independent.     

Autonomous control of cell and organ size by CHICO, a Drosophila homolog of vertebrate IRS1-4.

The control of growth is fundamental to the developing metazoan. Here, we show that CHICO, a Drosophila homolog of vertebrate IRS1-4, plays an essential role in the control of cell size and growth. Animals mutant for chico are less than half the size of wild-type flies, owing to fewer and smaller cells. In mosaic animals, chico homozygous cells grow slower than their heterozygous siblings, show an autonomous reduction in cell size, and form organs of reduced size. Although chico flies are smaller, they show an almost 2-fold increase in lipid levels. The similarities of the growth defects caused by mutations in chico and the insulin receptor gene in Drosophila and by perturbations of the insulin/IGF1 signaling pathway in vertebrates suggest that this pathway plays a conserved role in the regulation of overall growth by controling cell size, cell number, and metabolism.     

Drosophila PTEN regulates cell growth and proliferation through PI3K-dependent and -independent pathways

The control of cell and organ growth is fundamental to the development of multicellular organisms. Here, we show that dPTEN, a Drosophila homolog of the mammalian PTEN tumor suppressor gene, plays an essential role in the control of cell size, cell number, and organ size. In mosaic animals, dPTEN(-) cells proliferate faster than their heterozygous siblings, show an autonomous increase in cell size, and form organs of increased size, whereas overexpression of dPTEN results in opposite phenotypes. The loss-of-function phenotypes of dPTEN are suppressed by mutations in the PI3K target Dakt1 and the translational initiation factor eif4A, suggesting that dPTEN acts through the PI3K signaling pathway to regulate translation. Although activation of PI3K and Akt has been reported to increase rates of cellular growth but not proliferation, loss of dPTEN stimulates both of these processes, suggesting that PTEN regulates overall growth through PI3K/Akt-dependent and -independent pathways. Furthermore, we show that dPTEN does not play a major role in cell survival during Drosophila development. Our results provide a potential explanation for the high frequency of PTEN mutation in human cancer.     

Neuronal Cbl Controls Biosynthesis of Insulin-Like Peptides in Drosophila melanogaster

The Cbl family proteins function as both E3 ubiquitin ligases and adaptor proteins to regulate various cellular signaling events, including the insulin/insulin-like growth factor 1 (IGF1) and epidermal growth factor (EGF) pathways. These pathways play essential roles in growth, development, metabolism, and survival. Here we show that in Drosophila melanogaster, Drosophila Cbl (dCbl) regulates longevity and carbohydrate metabolism through downregulating the production of Drosophila insulin-like peptides (dILPs) in the brain. We found that dCbl was highly expressed in the brain and knockdown of the expression of dCbl specifically in neurons by RNA interference increased sensitivity to oxidative stress or starvation, decreased carbohydrate levels, and shortened life span. Insulin-producing neuron-specific knockdown of dCbl resulted in similar phenotypes. dCbl deficiency in either the brain or insulin-producing cells upregulated the expression of dilp genes, resulting in elevated activation of the dILP pathway, including phosphorylation of Drosophila Akt and Drosophila extracellular signal-regulated kinase (dERK). Genetic interaction analyses revealed that blocking Drosophila epidermal growth factor receptor (dEGFR)-dERK signaling in pan-neurons or insulin-producing cells by overexpressing a dominant-negative form of dEGFR abolished the effect of dCbl deficiency on the upregulation of dilp genes. Furthermore, knockdown of c-Cbl in INS-1 cells, a rat β-cell line, also increased insulin biosynthesis and glucose-stimulated secretion in an ERK-dependent manner. Collectively, these results suggest that neuronal dCbl regulates life span, stress responses, and metabolism by suppressing dILP production and the EGFR-ERK pathway mediates the dCbl action. Cbl suppression of insulin biosynthesis is evolutionarily conserved, raising the possibility that Cbl may similarly exert its physiological actions through regulating insulin production in β cells.     

Genetic control of size in Drosophila

During the past ten years, significant progress has been made in understanding the basic mechanisms of the development of multicellular organisms. Genetic analysis of the development of Caenorhabditis elegans and Drosophila has unearthed a fruitful number of genes involved in establishing the basic body plan, patterning of limbs, specification of cell fate and regulation of programmed cell death. The genes involved in these developmental processes have been conserved throughout evolution and homologous genes are involved in the patterning of insect and human limbs. Despite these important discoveries, we have learned astonishingly little about one of the most obvious distinctions between animals: their difference in body size. The mass of the smallest mammal, the bumble-bee bat, is 2 g while that of the largest mammal, the blue whale, is 150 t or 150 million grams. Remarkably, even though they are in the same class, body size can vary up to 75-million-fold. Furthermore, this body growth can be finite in the case of most vertebrates or it can occur continuously throughout life, as for trees, molluscs and large crustaceans. Currently, we know comparatively little about the genetic control of body size. In this article we will review recent evidence from vertebrates and particularly from Drosophila that implicates insulin/insulin-like growth factor-I and other growth pathways in the control of cell, organ and body size.     

Insulin reduces apoptosis and increases DNA synthesis and cell size via distinct signalling pathways in Drosophila Kc cells

During development of Drosophila, cell proliferation and size are known to be regulated by insulin. Here we use Drosophila Kc cells to examine the molecular basis for the control of cell growth by insulin. Growing cells in the presence of insulin increased cell number above control levels at 16, 24, 48 and 72 h. We have demonstrated a novel anti-apoptotic effect of insulin (approximately 50%) in these cells, measured by caspase 3-like activity, which contributed to the increase in cell number. The anti-apoptotic effect was observed both in control cells and those in which apoptosis was induced by ultraviolet irradiation. An approximately 2-fold stimulation of bromodeoxyuridine incorporation demonstrated that insulin also increased Kc cell proliferation by stimulating new DNA synthesis. The ability of insulin to increase cell number, stimulate bromodeoxyuridine incorporation and reduce caspase 3-like activity was prevented by PD98059, which inhibits activation of the Drosophila extracellular signal regulated kinase (DERK) pathway, and was unaffected by wortmannin, an inhibitor of Drosophila phosphatidylinositol 3-kinase (DPI3K). Insulin also increased cell size approximately 2-fold and this was prevented by wortmannin and rapamycin, an inhibitor of Drosphilia target of rapamycin (DTOR). In summary, we show that DERK plays an important role in mediating the effect of insulin to reduce apoptosis and increase DNA synthesis whereas the DPI3K/DTOR/Dp70S6 kinase pathway mediates effects of insulin on cell size in Drosophila Kc cells.     

Insulin receptor substrate and p55 orthologous adaptor proteins function in the Caenorhabditis elegans daf-2/insulin-like signaling pathway

An insulin-like signaling pathway regulates development and lifespan in Caenorhabditis elegans. Genetic screens that identified many components of the C. elegans insulin pathway did not identify homologs of insulin receptor substrates or the phosphoinositide 3-kinase (PI3K) adaptor/regulatory subunit, which are both required for signaling by mammalian insulin/insulin-like growth factor I pathways. The C. elegans genome contains one homolog of each protein. The C. elegans versions of insulin receptor substrate (IST-1) and PI3K p50/p55 (AAP-1) share moderate sequence similarity with their vertebrate and Drosophila counterparts. Genetic experiments show that ist-1 and aap-1 potentiate C. elegans insulin-like signaling, although they are not required for signaling in the pathway under most conditions. Worms lacking AAP-1 activity because of the mutation aap-1(m889) constitutively arrest development at the dauer larval stage when raised at high temperatures. aap-1 mutants also live longer than wild-type animals, a phenotype observed in other C. elegans mutants with defects in DAF-2 signaling. Interestingly, IST-1 appears to be required for signaling through a pathway that may act in parallel to AGE-1/PI3K.     

A genetic screen for dominant modifiers of a small-wing phenotype in Drosophila melanogaster identifies proteins involved in splicing and translation

Studies in the fly, Drosophila melanogaster, have revealed that several signaling pathways are important for the regulation of growth. Among these, the insulin receptor/phosphoinositide 3-kinase (PI3K) pathway is remarkable in that it affects growth and final size without disturbing pattern formation. We have used a small-wing phenotype, generated by misexpression of kinase-dead PI3K, to screen for novel mutations that specifically disrupt organ growth in vivo. We identified several complementation groups that dominantly enhance this small-wing phenotype. Meiotic recombination in conjunction with visible markers and single-nucleotide polymorphisms (SNPs) was used to map five enhancers to single genes. Two of these, nucampholin and prp8, encode pre-mRNA splicing factors. The three other enhancers encode factors required for mRNA translation: pixie encodes the Drosophila ortholog of yeast RLI1, and RpL5 and RpL38 encode proteins of the large ribosomal subunit. Interestingly, mutations in several other ribosomal protein-encoding genes also enhance the small-wing phenotype used in the original screen. Our work has therefore identified mutations in five previously uncharacterized Drosophila genes and provides in vivo evidence that normal organ growth requires optimal regulation of both pre-mRNA splicing and mRNA translation.     

Akt/protein kinase B promotes organ growth in transgenic mice

One of the least-understood areas in biology is the determination of the size of animals and their organs. In Drosophila, components of the insulin receptor phosphoinositide 3-kinase (PI3K) pathway determine body, organ, and cell size. Several biochemical studies have suggested that Akt/protein kinase B is one of the important downstream targets of PI3K. To examine the role of Akt in the regulation of organ size in mammals, we have generated and characterized transgenic mice expressing constitutively active Akt (caAkt) or kinase-deficient Akt (kdAkt) specifically in the heart. The heart weight of caAkt transgenic mice was increased 2.0-fold compared with that of nontransgenic mice. The increase in heart size was associated with a comparable increase in myocyte cell size in caAkt mice. The kdAkt mutant protein attenuated the constitutively active PI3K-induced overgrowth of the heart, and the caAkt mutant protein circumvented cardiac growth retardation induced by a kinase-deficient PI3K mutant protein. Rapamycin attenuated caAkt-induced overgrowth of the heart, suggesting that the mammalian target of rapamycin (mTOR) or effectors of mTOR mediated caAkt-induced heart growth. In conclusion, Akt is sufficient to induce a marked increase in heart size and is likely to be one of the effectors of the PI3K pathway in mediating heart growth.     

哺乳动物不育系20样激酶MST1

哺乳动物不育系20样激酶1(mammalian sterile 20-like kinase 1,Mst1)基因是果蝇Hippo基因在哺乳动物中的同源基因,编码丝氨酸/苏氨酸激酶,主要参与细胞生长、增殖、凋亡以及器官大小等的调控。最近的研究表明,Mst1基因具有抑癌作用,其功能的缺失与肿瘤的发生密切相关。本文就MST1的结构与功能、促凋亡作用机制及其在医学研究中的应用作一简要综述。     

Drosophila short neuropeptide F signalling regulates growth by ERK-mediated insulin signalling

Insulin and insulin growth factor have central roles in growth, metabolism and ageing of animals, including Drosophila melanogaster. In Drosophila, insulin-like peptides (Dilps) are produced by specialized neurons in the brain. Here we show that Drosophila short neuropeptide F (sNPF), an orthologue of mammalian neuropeptide Y (NPY), and sNPF receptor sNPFR1 regulate expression of Dilps. Body size was increased by overexpression of sNPF or sNPFR1. The fat body of sNPF mutant Drosophila had downregulated Akt, nuclear localized FOXO, upregulated translational inhibitor 4E-BP and reduced cell size. Circulating levels of glucose were elevated and lifespan was also extended in sNPF mutants. We show that these effects are mediated through activation of extracellular signal-related kinases (ERK) in insulin-producing cells of larvae and adults. Insulin expression was also increased in an ERK-dependent manner in cultured Drosophila central nervous system (CNS) cells and in rat pancreatic cells treated with sNPF or NPY peptide, respectively. Drosophila sNPF and the evolutionarily conserved mammalian NPY seem to regulate ERK-mediated insulin expression and thus to systemically modulate growth, metabolism and lifespan.     

Controlling the size of organs and organisms

A key difference between yeast and metazoans is the need of the latter to regulate cell proliferation and growth to create organs (and organisms) of reproducible size and shape. Great progress has been made in understanding how growth, cell size and the cell cycle are controlled in metazoans. Recent work has shown that disruption of conserved components of the insulin and Tor kinase pathways can alter organ size, indicating that the normal functioning of these pathways is essential for organ size control. However, disruption of genes that regulate patterning and of genes that control cell adhesion and cell polarity has a much more dramatic effect on final organ size than does manipulation of the cell cycle or of basal growth control mechanisms. These data point to an 'organ-size checkpoint' that regulates cell division, cell growth and apoptosis. Recent data suggests that cell competition may play an important role in implementing the organ-size checkpoint.     

Insulin receptor-mediated organ overgrowth in <Emphasis Type="Italic">Drosophila</Emphasis> is not restricted by body size

Recent studies have demonstrated that insulin receptor (Inr) signalling plays an important role in determining organ size and maintaining proportionality in normal animals. However, it is unclear whether the activity of this pathway in a developing organ is invariably a dominant determinant of its mass or whether size can be restricted by other non-autonomous growth regulatory mechanisms if a tissue starts to outgrow the rest of the body. To test this in Drosophila, we induced excess Inr-dependent growth by removal of the Inr signalling antagonist, DPTEN, in the eyes of flies with dramatically different body sizes. Although our data suggest a very limited level of growth competition between organs in animals with giant eyes, there do not appear to be mechanisms by which neighbouring structures substantially inhibit eye overgrowth, even when the resulting organ is many times enlarged relative to the rest of the animal. Overall, our results support a simple model in which organs are normally maintained in proportion to each other, primarily because they all respond similarly to levels of insulin-like factors and other growth regulators. Given the evolutionarily conserved role of insulin receptor signalling in growth control, our data may also help to explain why this pathway is so frequently hyperactivated in mammalian tumours.     

The functions of insulin signaling: size isn't everything,even in Drosophila

Mammalian insulin and insulin-like growth factors (IGFs) signal through several receptors with different ligand specificities to regulate metabolism and growth. This regulation is defective in diabetes and in a wide variety of human tumors. Recent analysis in Drosophila melanogaster has revealed that insulin-like molecules (known as DILPs in flies) also control growth and metabolism, but probably do so by signaling through a single insulin receptor (InR). The intracellular signaling molecules regulated by this receptor are highly evolutionarily conserved. Work in flies has helped to dissect the network of InR-regulated intracellular signaling pathways and identify some of the critical players in these pathways and in interacting signaling cascades. Surprisingly, these studies have shown that DILPs control tissue and body growth primarily by regulating cell growth and cell size. Changes in cell growth produced by these molecules may subsequently modulate the rate of cell proliferation in a cell type-specific fashion. At least part of this growth effect is mediated by two small groups of neurons in the Drosophila brain, which secrete DILPs into the circulatory system at levels that are modulated by nutrition. This signaling center is also involved in DILP-dependent control of the fly's rate of development, fertility, and life span. These surprisingly diverse functions of InR signaling, which appear to be conserved in all higher animals, reflect a central role for this pathway in coordinating development, physiology, and properly proportioned growth of the organism in response to its nutritional state. Studies in flies are providing important new insights into the biology of this system, and the identification of novel components in the InR-regulated signaling cascade is already beginning to inform the development of new therapeutic strategies for insulin-linked diseases in the clinic.     

The Temporal Requirements for Insulin Signaling During Development in Drosophila

Recent studies have indicated that the insulin-signaling pathway controls body and organ size in Drosophila, and most metazoans, by signaling nutritional conditions to the growing organs. The temporal requirements for insulin signaling during development are, however, unknown. Using a temperature-sensitive insulin receptor (Inr) mutation in Drosophila, we show that the developmental requirements for Inr activity are organ specific and vary in time. Early in development, before larvae reach the “critical size” (the size at which they commit to metamorphosis and can complete development without further feeding), Inr activity influences total development time but not final body and organ size. After critical size, Inr activity no longer affects total development time but does influence final body and organ size. Final body size is affected by Inr activity from critical size until pupariation, whereas final organ size is sensitive to Inr activity from critical size until early pupal development. In addition, different organs show different sensitivities to changes in Inr activity for different periods of development, implicating the insulin pathway in the control of organ allometry. The reduction in Inr activity is accompanied by a two-fold increase in free-sugar levels, similar to the effect of reduced insulin signaling in mammals. Finally, we find that varying the magnitude of Inr activity has different effects on cell size and cell number in the fly wing, providing a potential linkage between the mode of action of insulin signaling and the distinct downstream controls of cell size and number. We present a model that incorporates the effects of the insulin-signaling pathway into the Drosophila life cycle. We hypothesize that the insulin-signaling pathway controls such diverse effects as total developmental time, total body size and organ size through its effects on the rate of cell growth, and proliferation in different organs.     

The temporal requirements for insulin signaling during development in Drosophila

Recent studies have indicated that the insulin-signaling pathway controls body and organ size in Drosophila, and most metazoans, by signaling nutritional conditions to the growing organs. The temporal requirements for insulin signaling during development are, however, unknown. Using a temperature-sensitive insulin receptor (Inr) mutation in Drosophila, we show that the developmental requirements for Inr activity are organ specific and vary in time. Early in development, before larvae reach the “critical size” (the size at which they commit to metamorphosis and can complete development without further feeding), Inr activity influences total development time but not final body and organ size. After critical size, Inr activity no longer affects total development time but does influence final body and organ size. Final body size is affected by Inr activity from critical size until pupariation, whereas final organ size is sensitive to Inr activity from critical size until early pupal development. In addition, different organs show different sensitivities to changes in Inr activity for different periods of development, implicating the insulin pathway in the control of organ allometry. The reduction in Inr activity is accompanied by a two-fold increase in free-sugar levels, similar to the effect of reduced insulin signaling in mammals. Finally, we find that varying the magnitude of Inr activity has different effects on cell size and cell number in the fly wing, providing a potential linkage between the mode of action of insulin signaling and the distinct downstream controls of cell size and number. We present a model that incorporates the effects of the insulin-signaling pathway into the Drosophila life cycle. We hypothesize that the insulin-signaling pathway controls such diverse effects as total developmental time, total body size and organ size through its effects on the rate of cell growth, and proliferation in different organs.     

A gain-of-function screen identifies wdb and lkb1 as lifespan-extending genes in Drosophila

The insulin/insulin-like growth factor (IGF) and the target of rapamycin (TOR) signaling pathways are known to regulate lifespan in diverse organisms. However, only a limited number of genes involved in these pathways have been examined regarding their effects on lifespan. Through a gain-of-function screen in Drosophila, we found that overexpression of the wdb gene encoding a regulatory subunit of PP2A, and overexpression of the lkb1 gene encoding a serine/threonine kinase, reduced organ size and extended lifespan. Overexpression of wdb also reduced the level of phosphorylated AKT, while overexpression of lkb1 increased the level of phosphorylated AMPK and decreased the level of phosphorylated S6K. Taken together, our results suggest that wdb- and lkb1-dependent lifespan extension is mediated by downregulation of S6K, a downstream component of the insulin/IGF and TOR signaling pathways.