InteractiveFly: GeneBrief

pumpless : Biological Overview | References

Gene name - pumpless

Synonyms -

Cytological map position - 78C7--8

Function - enzyme

Keywords - behavior, appetite control, fat body

Symbol - ppl

FlyBase ID: FBgn0027945

Genetic map position -

Classification - glycine cleavage system H protein

Cellular location - cytoplasmic

NCBI links: Entrez Gene

ppl orthologs: Biolitmine

pumpless (ppl) plays a role in mediating food intake suppression in response to amino acids, and may be part of a systemic signaling system that regulates growth transition from the larval (feeding) to the pupal (non-feeding) stage of development. It was identified in a genetic screen of larval mutants that were defective in food intake. pumpless larvae initially feed normally upon hatching. However, during late first instar stage, they fail to pump the food from the pharynx into the esophagus and concurrently begin moving away from the food source. Although pumpless larvae do not feed, they do not show the typical physiologic response of starving animals, such as upregulating genes involved in gluconeogenesis or lipid breakdown. Thus the ppl gene appears to function in the fat body in the production of a systemic signal that affects food intake. The pumpless gene is expressed specifically in the fat body and encodes a protein with homology to a vertebrate enzyme involved in glycine catabolism. Feeding wild-type larvae high levels of amino acids can phenocopy the feeding and growth defects of pumpless mutants. The data suggest the existence of an amino acid-dependent signal arising from the fat body that induces cessation of feeding in the larva. This signaling system may also mediate growth transition from larval to pupal stage during Drosophila development (Zinke, 1999).

The foregut may be considered the larval organ of food intake. It consists of the pharynx, the esophagus and the proventriculus, and is innervated by the enteric nervous system. The sequence of food intake in Drosophila larva is highly rhythmic. Food is sucked into the mouth atrium by contraction of the large dorsal pharyngeal muscles, pumped into the esophagus and carried through the proventriculus into the midgut. The assay used for isolating larval feeding mutants is to feed the animals yeast paste containing red dye and to monitor the movement of food along the foregut. With this screen, a number of genes have been identified that are required for proper food intake. These have been placed into two operationally defined classes. One class of mutants shows clear morphological defects in the feeding apparatus. The other class, including ppl, shows no such defects (Zinke, 1999).

In general, mutants that do not feed are expected to have growth defects and an animal may not feed for a variety of reasons. Therefore, it was important to discern whether the feeding defect of ppl mutants was a primary effect, or whether it was a secondary effect due to the general ill-health of the mutant. A major criterion that was used to distinguish between these possibilities was when the feeding phenotype could be first observed relative to the other behavioral abnormalities that might be apparent in the mutants (Zinke, 1999).

The original ppl mutant, ppl-06913, was isolated by screening a collection of P-element-induced lines. In wild-type larvae, the midgut becomes clearly red after a short time of feeding on red yeast; there is no accumulation of food material in the pharynx or the proventriculus. By contrast, ppl-06913 larvae show food accumulation in the pharynx and only very little in the midgut, indicating that the food is not passing into the esophagus. This is not due to a physical blockage in the foregut since cutting the mutant larva in half and gently agitating the head causes the food to flow into the esophagus. Further phenotypic analysis revealed several additional properties of ppl mutants. Initially, the feeding defect is observed starting late first instar stage. In the initial 12 hours after hatching (early first instar), no ppl-06913 mutants were observed with food accumulation in the pharynx. In the next 12 hours (late first instar), several variations on this feeding phenotype were seen, which most likely reflects a time course of the phenotypic progression. Larvae can be seen in which the red food is observed mostly in the midgut and some in the pharynx. The food then accumulates more in the pharynx and less in the midgut. One finally observes mutant larvae in which the food is no longer present in the midgut and are moving about with food only in the pharynx. There is some variation in the size of the ppl-06913 larvae that shows these feeding defects, most likely reflecting differences in the time when the animals lose their ability to pass food into the esophagus. This feeding defect is accompanied by a drastic reduction in growth. The majority of these larvae die within about 3 days after hatching with the food still in the pharynx. About a third of the ppl-06913 with the food accumulated in the pharynx do succeed in swallowing their food, and these invariably grow slightly larger than those that have not swallowed their food; however, these also die as larvae without further growth (Zinke, 1999).

Based on complementation analysis, several other alleles of ppl were identified (Russell, 1996). ppl-00217 shows a significant reduction in growth, with the body size being approximately the same as the bigger class of ppl-06913 mutants. ppl-00217 therefore is most likely a weaker allele than ppl-06913. ppl-78Cb3 and ppl-78Cb1 show a slight difference in the feeding defect. Both can feed upon hatching. They then gradually stop feeding, as seen by the disappearence of food in the midgut; however they do not show food accumulation in the pharynx. These variations in the phenotype may reflect differences in the strength of the alleles and the exact point in the deglutition cycle at which food intake is halted. Analysis has focused on ppl-06913, as this represents a null allele (Zinke, 1999).

In addition to the cessation of food intake, ppl larvae display another interesting food response. Wild-type first instar larvae feed continuously and essentially remain buried in the food the entire time, rarely straying far from the food source; when these are deprived of food for a while and then placed near a food source, they will quickly move towards it. By contrast, ppl mutants move away from the food source and start wandering about; some leave the food, wander about, then return again, and this cycle can be repeated. The movement of the body per se is indistinguishable from wild type, and the mutants move about with the same rigor, respond to touch, and can right themselves rapidly when turned upside down, as in wild-type animals (Zinke, 1999).

The progression of the ppl mutant phenotype can be summarized as follows. There is at the beginning no discernable defect in feeding, and it is impossible to distinguish the wild type from mutants shortly after hatching. During late first instar, the feeding phenotype is apparent as seen by the accumulation of food in the pharynx. As the food intake defect becomes more prominent, the mutant larvae begin leaving the food and start wandering about. These observations indicate that the food intake defect in ppl mutants precedes the appearance of general lethargic characteristics and suggest a primary defect in the feeding response (Zinke, 1999).

These observations suggested that ppl larvae, which are essentially not feeding, are not showing a typical starvation response. To further investigate this, molecular markers were sought that would be regulated by starvation. Two such markers, phosphoenolpyruvate carboxykinase (Pepck) and lipase 3 (Lip3) were found. The Drosophila Pepck gene encodes an enzyme that is highly homologous to mammalian PEPCK, which catalyzes the rate-limiting step of gluconeogenesis. It is known that the PEPCK gene in mammals is regulated at the transcriptional level by nutritional signals. The Drosophila Lip3 gene shows significant homology to the acid hydrolase lipases, which is a member of a larger class of lipases that are involved in lipid metabolism. It was of interest to determine how these genes would be regulated upon starvation. Both genes show a constant level of expression during second instar larvae, with both increasing slightly at the end of second instar; upon starvation, however, both genes are highly upregulated. It was then asked how these genes would be regulated in ppl larvae. Neither of these genes are upregulated. Taken together, these data indicate that the lack of food intake and growth in ppl larvae is not accompanied by a normal physiologic response to starvation (Zinke, 1999).

In situ hybridization shows that ppl is expressed exclusively in the fat body of developing embryos and larvae. The fat body is the major organ of metabolic regulation and energy storage in insects and can be viewed as performing the combined function of the liver and adipocytes of mammals. ppl expression level increases during the first and second instars, but decreases near the end of third instar. Since many genes are downregulated during starvation, and since ppl larvae are not feeding, there was the possibility that the inability to detect ppl transcripts was due to its downregulation. Although ppl is indeed downregulated upon starvation of wild-type larvae, significant ppl expression could still be detected (Zinke, 1999).

Since ppl is expressed specifically in the fat body, ppl mutants were examined for defects in this organ. The morphology of the fat body is highly dependent on the nutritional state of the larva. In normal well-fed larva, the fat body is enriched in lipids, as seen by Sudan Black histochemical staining. When larvae are starved, lipid becomes undetectable in the fat body. In ppl mutant larvae, one can still detect lipids in the fat body. It should be noted that, since the mutant larvae are not feeding, they have less fat than wild-type larvae of the same age. In ppl larvae that have grown slightly bigger because they were successful in swallowing food, there is a corresponding increase in the lipid staining of the fat body. The breakdown of lipids can be prevented by addition of sugar, and since larvae grown on sugar do not grow in size, these were also used to compare with ppl larvae. These larvae show staining in the fat body comparable to that found in some of the ppl mutants. Furthermore, when ppl mutants are completely starved for food by removing them to a wet filter paper, they eliminate their lipid contents. It should be noted that despite the breakdown of lipids during starvation, the fat body itself remains intact as can be seen by staining of the fat body nuclei. These results indicate that ppl larvae still have fat bodies, and can accumulate and break down lipids depending on nutritional conditions (Zinke, 1999).

Since ppl encodes an enzyme that may be involved in glycine catabolism, it was asked whether feeding high levels of glycine to normal larvae would also lead to similar growth and feeding defects as ppl mutants. Therefore, wild-type larvae food mixed with different concentrations of glycine was fed. At 0.1 M added glycine, no effect on growth was observed, whereas 0.4 M glycine led to significant growth retardation. Larvae fed high glycine diet also pupariate at significantly later times; this also depends on the concentration of glycine. Eclosion is also delayed corresponding to the delay in pupariation, indicating that the retardation of growth occurs during the larval stage. These effects are completely reversible, since taking the animals out of high glycine food into normal food results in resumption of the normal growth rate. These results indicated that high levels of dietary glycine can significantly reduce growth (Zinke, 1999).

Pepck and Lip3 are upregulated in starved wild-type larvae, whereas no upregulation is observed in ppl mutants. It was therefore asked whether glycine or other nutrient components would affect the transcription of these genes. When wild-type larvae are raised in saline solution plus sugar, Lip3 is downregulated completely. Here however, Pepck expression is downregulated but not to the same extent as that of Lip3. Adding glycine plus sugar results in a greater suppression than sugar alone, restoring Pepck expression almost to the basal level. Glycine alone has a similar effect as sugar alone. This indicates that Lip3 expression is completely dependent on sugar, whereas Pepck expression is dependent on both sugar and glycine (Zinke, 1999).

Whether the growth-retarding effect could be brought about by other amino acids was then examined. Lysine has an even greater effect on growth retardation than glycine, whereas serine, alanine and proline have less of an effect. Finally, it was asked whether the specific food intake defect of ppl-06913 larvae could be phenotyped in addition to the growth defects. Of the five amino acids tested, one of these, lysine, could phenocopy the characteristic accumulation of food in the pharynx. The fact that the specific pharynx phenotype could be phenocopied with lysine but not with glycine may be due to the fact that exogenously feeding high amino acids leads to similar but not identical outcomes as endogeneously preventing amino acid breakdown. In this respect, it is not known know if, in ppl mutants or in wild-type animals fed high amounts of amino acids, there is a steady-state accumulation of glycine or other amino acids in the body; it could also be that they are rapidly converted to another metabolite. A detailed biochemical characterization of ppl mutants, such as measuring the amino acid levels in the larvae, should provide insights into this issue. Taken together, these data indicate that, although the ppl mutation appears to be affecting glycine metabolism, the ability to depress growth and food intake is not glycine specific but is shared by different amino acids (Zinke, 1999).

The path from intake of nutrients to alterations in feeding response requires the coordinate functioning of many organ systems. The fact that ppl is exclusively expressed in the fat body suggests that the fat body may be an important relay point in conveying nutritional signals that regulate food intake. What other organs might be involved? It is helpful to address this issue in light of classical studies performed on various insects. These studies have provided a conceptual framework in which interacting central and peripheral organ systems communicate through humoral factors to regulate growth and development: neurosecretory cells of the brain send signals to the endocrine organs, causing them to release hormones that act on target tissues. The key point for the current analysis is that the activity of the neurosecretory cells in the brain, and thus the triggering of the relay system, is dependent on nutritional cues. For example, it has been shown in the blood-feeding bug Rhodnius that molting occurs at a specific time after a blood meal and that only one meal is needed for each stage. If the brain is removed, molting takes place only when a certain time has passed since the feeding. Interestingly, a fat body derived mitogenic signal that is dependent on amino acids controls neuroblast proliferation in the brain (Britton, 1998). Thus, the effect of amino acids on food intake that was observed in this study may also be mediated through the brain (Zinke, 1999).

These findings can be seen in the context of molecular studies in rodents on feeding and body weight regulation. The product of the mouse obese gene, leptin, is secreted by the adipocytes and acts on the brain as an afferent signal to regulate food intake. Although there is no evidence that a leptin homolog exists in Drosophila, the underlying logic in the interplay of central and peripheral organs in the production and relay of nutrient-dependent signals may share similarities. In this respect, it has been shown that extirpation of the ring gland (the master neuroendocrine organ of Diptera insects) in Drosophila hydei results in increased size of the fat body. This result is strikingly reminiscent of studies in mammals in which destruction of specific part of the hypothalamus leads to obesity (Zinke, 1999).

Although ppl mutants are not feeding, they do not display the typical response characteristics of wild-type animals that have been deprived of food. When wild-type larvae are starved, specific behavioral and physiologic responses are invoked, including the transcriptional upregulation of Pepck and Lip3. This is accompanied by a drastic reduction in the size of the fat body since the stored lipid is broken down. In addition, when larvae that have not been fed are now presented with a food source, these animals will move towards the food source and remain there. ppl mutants, although they do not feed, are not growing and die about the same time as starved larvae, do not show this response. Pepck and Lip3 genes are not upregulated; the fat body still stores lipids, and the mutant larvae wander away from the food source. This indicates that the starvation response is suppressed in ppl mutants. Although it is not known how this is effected, the wandering movement that ppl larvae display provides a hint, in that the behavior is reminiscent of wild-type larvae at late third instar larvae. Wild-type larvae essentially feed continuously up until the late third instar stage. A short time before pupariation, however, in a process that is dependent on the hormone ecdysone, they leave the food and start wandering about the surrounding in what is termed the 'wandering' stage. During this period, they empty their gut and do not feed, and the lipids in their fat body are not broken down. These observations reveal that ppl mutants, as first instar larvae, share several characteristics of non-feeding third instar wild-type animals and suggest that some aspects of the ppl mutant phenotype may be due in part to a premature activation of a developmental program that is normally activated shortly prior to pupariation. Therefore, ppl gene may be involved in mediating the normal sequence of events, including those that are responsive to ecdysone, that lead to pupariation (Zinke, 1999).

Under optimal conditions, Drosophila larvae pupariate within a narrow range with respect to both time and body size. However, restricting food intake during the larval stage can greatly alter the timing of pupariation as well as the size of the pupa and adult. In addition, removal of the corpora allata (a major endocrine organ which, in Drosophila, is part of the ring gland) during earlier larval stages in a variety of insects leads to premature pupariation and, correspondingly, very small pupae and adults. These classical studies suggest that systemic signals can regulate body size in response to nutrient availability. Interestingly, heteroallelic combinations of insulin receptor mutants, as well as mutants of the chico gene, which encodes a homolog of a vertebrate insulin receptor substrate, show eclosion delay and have small body size. Furthermore, the fat body produces growth factors that act with insulin to promote proliferation of imaginal disc cells (Kawamura, 1999). Since ppl gene appears to function in the fat body in the production of a systemic signal that affects food intake and timing of pupariation, it is possible that ppl interacts with genes in the insulin signaling system to regulate body size (Zinke, 1999).

As protein biosynthesis drives growth, it is not surprising that amino acids regulate nutrient intake in a variety of organisms. In yeast, amino acids have been shown to control diverse cellular processes that have direct connection to nutrient availability, such as autophagy, pseudohyphal formation and cell growth. In Dictyostelium, ammonia, which is a common catabolic product of all amino acids, is known to be a signaling molecule that controls food response behavior and development. In hydra, which possess a nerve net that is concentrated around the mouth, specific feeding response can be elicited by the amino acid tyrosine. Amino acids have also been implicated in appetite suppression. It has been shown in rats, for example, that direct injection of amino acids into the hypothalamus reduced food intake. In Drosophila larvae, which are continuous feeders and may not be subject to short term, periodic feeding controls, amino acids could act as a nutrient signal for progressing to the non-feeding pupal stage. Since amino acids have such diverse biochemical roles in the body, it is not known which pathway might be involved in regulating feeding response (Zinke, 1999).

However, these considerations bring to mind the discussion on the origin of hormones and intercellular signaling by Tomkins (1975), who suggested that neurotransmitters, which are in many cases amino acid derivatives or are themselves amino acids, may have initially evolved for transducing information related to amino acid accumulation (Zinke, 1999).


Search PubMed for articles about Drosophila pumpless

Britton, J. and Edgar, B. (1998). Environmental control of the cell cycle in Drosophila: nutrition activates mitotic and endoreplicative cells by distinct mechanisms. Development 125: 2149-2158. 9570778

Fujiwara, K., et al. (1991). The primary structure of human H-protein of the glycine cleavage system deduced by cDNA cloning. Biochem. Biophys. Res. Commun. 176: 711-716.

Kawamura, K., et al. (1999). A new family of growth factors produced by the fat body and active on Drosophila imaginal disc cells. Development 126, 211-219. 9847235

Mathews, C. and van Holde, K., editors. Biochemistry. Redwood City: The Benjamin/Cummings Publications, 1990.

Russell, S. R., et al. (1996). The Drosophila Eip78C gene is not vital but has a role in regulating chromosome puffs. Genetics 144: 159-170. 8878682

Tomkins, G. (1975). The metabolic code. Science 189: 760-763

Yamamoto, M., et al. (1991). The glycine cleavage system: occurrence of two types of chicken H-protein mRNAs presumably formed by the alternative use of the polyadenylation consensus sequences in a single exon. J. Biol. Chem. 266: 3317-3322. 1993703

Zinke, I., et al. (1999). Suppression of food intake and growth by amino acids in Drosophila: the role of pumpless, a fat body expressed gene with homology to vertebrate glycine cleavage system. Development 126: 5275-5284. 10556053

Biological Overview

date revised: 22 March 2001

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