Insulin/TOR signaling in growth and homeostasis: a view from the fly world

The insulin/TOR pathway is a conserved regulator of cell and organism growth in metazoans. Over the last several years, an array of signaling inputs to this pathway has been defined. However the growth-regulatory outputs are less clear. Drosophila has proven to be a powerful genetic model system in which to study insulin/TOR signaling. This review highlights recent studies in Drosophila that have identified essential outputs and key effectors of the pathway. These include the regulation of ribosome synthesis, mRNA translation, autophagy and endocytosis, through downstream effectors such as Myc, FOXO, HIF1-alpha, TIF-IA, 4EBP and Atg1. This network of outputs and effectors can regulate cell and organismal metabolism, and is essential for the control of tissue growth, responses to starvation and stress, and aging. The mechanisms identified in Drosophila likely operate in most metazoans, and are relevent to our understanding of diseases caused by aberrent insulin/TOR signaling such as cancer, diabetes and obesity.     

Growth-Blocking Peptides As Nutrition-Sensitive Signals for Insulin Secretion and Body Size Regulation

In Drosophila, the fat body, functionally equivalent to the mammalian liver and adipocytes, plays a central role in regulating systemic growth in response to nutrition. The fat body senses intracellular amino acids through Target of Rapamycin (TOR) signaling, and produces an unidentified humoral factor(s) to regulate insulin-like peptide (ILP) synthesis and/or secretion in the insulin-producing cells. Here, we find that two peptides, Growth-Blocking Peptide (GBP1) and CG11395 (GBP2), are produced in the fat body in response to amino acids and TOR signaling. Reducing the expression of GBP1 and GBP2 (GBPs) specifically in the fat body results in smaller body size due to reduced growth rate. In addition, we found that GBPs stimulate ILP secretion from the insulin-producing cells, either directly or indirectly, thereby increasing insulin and insulin-like growth factor signaling activity throughout the body. Our findings fill an important gap in our understanding of how the fat body transmits nutritional information to the insulin producing cells to control body size.     

Drosophila Tribbles Antagonizes Insulin Signaling-Mediated Growth and Metabolism via Interactions with Akt Kinase

Drosophila Tribbles (Trbl) is the founding member of the Trib family of kinase-like docking proteins that modulate cell signaling during proliferation, migration and growth. In a wing misexpression screen for Trbl interacting proteins, we identified the Ser/Thr protein kinase Akt1. Given the central role of Akt1 in insulin signaling, we tested the function of Trbl in larval fat body, a tissue where rapid increases in size are exquisitely sensitive to insulin/insulin-like growth factor levels. Consistent with a role in antagonizing insulin-mediated growth, trbl RNAi knockdown in the fat body increased cell size, advanced the timing of pupation and increased levels of circulating triglyceride. Complementarily, overexpression of Trbl reduced fat body cell size, decreased overall larval size, delayed maturation and lowered levels of triglycerides, while circulating glucose levels increased. The conserved Trbl kinase domain is required for function in vivo and for interaction with Akt in a yeast two-hybrid assay. Consistent with direct regulation of Akt, overexpression of Trbl in the fat body decreased levels of activated Akt (pSer505-Akt) while misexpression of trbl RNAi increased phospho-Akt levels, and neither treatment affected total Akt levels. Trbl misexpression effectively suppressed Akt-mediated wing and muscle cell size increases and reduced phosphorylation of the Akt target FoxO (pSer256-FoxO). Taken together, these data show that Drosophila Trbl has a conserved role to bind Akt and block Akt-mediated insulin signaling, and implicate Trib proteins as novel sites of signaling pathway integration that link nutrient availability with cell growth and proliferation.     

Cyclin G Functions as a Positive Regulator of Growth and Metabolism in Drosophila

In multicellular organisms, growth and proliferation is adjusted to nutritional conditions by a complex signaling network. The Insulin receptor/target of rapamycin (InR/TOR) signaling cascade plays a pivotal role in nutrient dependent growth regulation in Drosophila and mammals alike. Here we identify Cyclin G (CycG) as a regulator of growth and metabolism in Drosophila. CycG mutants have a reduced body size and weight and show signs of starvation accompanied by a disturbed fat metabolism. InR/TOR signaling activity is impaired in cycG mutants, combined with a reduced phosphorylation status of the kinase Akt1 and the downstream factors S6-kinase and eukaryotic translation initiation factor 4E binding protein (4E-BP). Moreover, the expression and accumulation of Drosophila insulin like peptides (dILPs) is disturbed in cycG mutant brains. Using a reporter assay, we show that the activity of one of the first effectors of InR signaling, Phosphoinositide 3-kinase (PI3K92E), is unaffected in cycG mutants. However, the metabolic defects and weight loss in cycG mutants were rescued by overexpression of Akt1 specifically in the fat body and by mutants in widerborst (wdb), the B''-subunit of the phosphatase PP2A, known to downregulate Akt1 by dephosphorylation. Together, our data suggest that CycG acts at the level of Akt1 to regulate growth and metabolism via PP2A in Drosophila.     

Drosophila Adiponectin Receptor in Insulin Producing Cells Regulates Glucose and Lipid Metabolism by Controlling Insulin Secretion

Adipokines secreted from adipose tissue are key regulators of metabolism in animals. Adiponectin, one of the adipokines, modulates pancreatic beta cell function to maintain energy homeostasis. Recently, significant conservation between Drosophila melanogaster and mammalian metabolism has been discovered. Drosophila insulin like peptides (Dilps) regulate energy metabolism similarly to mammalian insulin. However, in Drosophila, the regulatory mechanism of insulin producing cells (IPCs) by adipokine signaling is largely unknown. Here, we describe the discovery of the Drosophila adiponectin receptor and its function in IPCs. Drosophila adiponectin receptor (dAdipoR) has high homology with the human adiponectin receptor 1. The dAdipoR antibody staining revealed that dAdipoR was expressed in IPCs of larval and adult brains. IPC- specific dAdipoR inhibition (Dilp2>dAdipoR-Ri) showed the increased sugar level in the hemolymph and the elevated triglyceride level in whole body. Dilps mRNA levels in the Dilp2>dAdipoR-Ri flies were similar with those of controls. However, in the Dilp2>dAdipoR-Ri flies, Dilp2 protein was accumulated in IPCs, the level of circulating Dilp2 was decreased, and insulin signaling was reduced in the fat body. In ex vivo fly brain culture with the human adiponectin, Dilp2 was secreted from IPCs. These results indicate that adiponectin receptor in insulin producing cells regulates insulin secretion and controls glucose and lipid metabolism in Drosophila melanogaster. This study demonstrates a new adipokine signaling in Drosophila and provides insights for the mammalian adiponectin receptor function in pancreatic beta cells, which could be useful for therapeutic application.     

p53- and ERK7-Dependent Ribosome Surveillance Response Regulates Drosophila Insulin-Like Peptide Secretion

Insulin-like signalling is a conserved mechanism that coordinates animal growth and metabolism with nutrient status. In Drosophila, insulin-producing median neurosecretory cells (IPCs) regulate larval growth by secreting insulin-like peptides (dILPs) in a diet-dependent manner. Previous studies have shown that nutrition affects dILP secretion through humoral signals derived from the fat body. Here we uncover a novel mechanism that operates cell autonomously in the IPCs to regulate dILP secretion. We observed that impairment of ribosome biogenesis specifically in the IPCs strongly inhibits dILP secretion, which consequently leads to reduced body size and a delay in larval development. This response is dependent on p53, a known surveillance factor for ribosome biogenesis. A downstream effector of this growth inhibitory response is an atypical MAP kinase ERK7 (ERK8/MAPK15), which is upregulated in the IPCs following impaired ribosome biogenesis as well as starvation. We show that ERK7 is sufficient and essential to inhibit dILP secretion upon impaired ribosome biogenesis, and it acts epistatically to p53. Moreover, we provide evidence that p53 and ERK7 contribute to the inhibition of dILP secretion upon starvation. Thus, we conclude that a cell autonomous ribosome surveillance response, which leads to upregulation of ERK7, inhibits dILP secretion to impede tissue growth under limiting dietary conditions.     

Lint,a transmembrane serine protease,regulates growth and metabolism in Drosophila

Insulin signaling in Drosophila has a significant role in regulating growth, metabolism, fecundity, stress response, and longevity. The molecular mechanism by which insulin signaling regulates these vital processes is dependent on the nutrient status and oxygen availability of the organism. In a genetic screen to identify novel genes that regulate Drosophila insulin signaling, we discovered lumens interrupted (lint), a gene that has previously been shown to act in tracheal development. The knockdown of lint gene expression using a Dilp2Gal4 driver which expresses in the neuronal insulin producing cells (IPCs), led to defects in systemic insulin signaling, metabolic status and growth. However, our analysis of lint knockdown phenotypes revealed that downregulation of lint in the trachea and not IPCs was responsible for the growth phenotypes, as the Gal4 driver is also expressed in the tracheal system. We found various tracheal terminal branch defects, including reduction in the length as well as number of branches in the lint knockdown background. Our study reveals that substantial effects of lint downregulation arose because of tracheal defects, which induced tissue hypoxia, altered systemic insulin/TOR signaling, and resulted in effects on developmental growth regulation.     

Body size regulation by maturation steroid hormones: a <Emphasis Type="Italic">Drosophila</Emphasis> perspective

The mechanism that determines the specific body size of an animal is a fundamental biological question that remains largely unanswered. This aspect is now beginning to be understood in insect models, particularly in Drosophila melanogaster, with studies highlighting the importance of nutrient-responsive growth signaling pathways involving insulin/insulin-like growth factor signaling (IIS) and target of rapamycin (TOR) (IIS/TOR). These pathways operate in animals, from insects to mammals, adjusting the growth rate in response to the nutritional condition of the organism. Organismal growth is closely coupled with the process of developmental maturation mediated by maturation steroid hormones, which is influenced greatly by environmental and nutritional conditions. Recent Drosophila studies have been revealing the mechanisms responsible for this phenomenon. In this review, I summarize some important findings about the steroid hormone regulation of Drosophila body growth, calling attention to the influence of developmental nutritional conditions on animal size determination.     

Local requirement of the Drosophila insulin binding protein imp-L2 in coordinating developmental progression with nutritional conditions

In Drosophila, growth takes place during the larval stages until the formation of the pupa. Starvation delays pupariation to allow prolonged feeding, ensuring that the animal reaches an appropriate size to form a fertile adult. Pupariation is induced by a peak of the steroid hormone ecdysone produced by the prothoracic gland (PG) after larvae have reached a certain body mass. Local downregulation of the insulin/insulin-like growth factor signaling (IIS) activity in the PG interferes with ecdysone production, indicating that IIS activity in the PG couples the nutritional state to development. However, the underlying mechanism is not well understood. In this study we show that the secreted Imaginal morphogenesis protein-Late 2 (Imp-L2), a growth inhibitor in Drosophila, is involved in this process. Imp-L2 inhibits the activity of the Drosophila insulin-like peptides by direct binding and is expressed by specific cells in the brain, the ring gland, the gut and the fat body. We demonstrate that Imp-L2 is required to regulate and adapt developmental timing to nutritional conditions by regulating IIS activity in the PG. Increasing Imp-L2 expression at its endogenous sites using an Imp-L2-Gal4 driver delays pupariation, while Imp-L2 mutants exhibit a slight acceleration of development. These effects are strongly enhanced by starvation and are accompanied by massive alterations of ecdysone production resulting most likely from increased Imp-L2 production by neurons directly contacting the PG and not from elevated Imp-L2 levels in the hemolymph. Taken together our results suggest that Imp-L2-expressing neurons sense the nutritional state of Drosophila larvae and coordinate dietary information and ecdysone production to adjust developmental timing under starvation conditions.     

Role of Fat Body Lipogenesis in Protection against the Effects of Caloric Overload in Drosophila

The Drosophila fat body is a liver- and adipose-like tissue that stores fat and serves as a detoxifying and immune responsive organ. We have previously shown that a high sugar diet leads to elevated hemolymph glucose and systemic insulin resistance in developing larvae and adults. Here, we used stable isotope tracer feeding to demonstrate that rearing larvae on high sugar diets impaired the synthesis of esterified fatty acids from dietary glucose. Fat body lipid profiling revealed changes in both carbon chain length and degree of unsaturation of fatty acid substituents, particularly in stored triglycerides. We tested the role of the fat body in larval tolerance of caloric excess. Our experiments demonstrated that lipogenesis was necessary for animals to tolerate high sugar feeding as tissue-specific loss of orthologs of carbohydrate response element-binding protein or stearoyl-CoA desaturase 1 resulted in lethality on high sugar diets. By contrast, increasing the fat content of the fat body by knockdown of king-tubby was associated with reduced hyperglycemia and improved growth and tolerance of high sugar diets. Our work supports a critical role for the fat body and the Drosophila carbohydrate response element-binding protein ortholog in metabolic homeostasis in Drosophila.