BACKGROUND
calcium is a key signaling molecule in multi-cellular organisms that regulates a variety of cellular processes  <cit> .
the ip3r  is a ligand gated calcium channel present on the membranes of endoplasmic reticular  stores.
it mediates the release of er calcium upon binding of its cognate ligand ip <dig>  in drosophila there is a single gene, itpr, for the ip3r which is 60% homologous to mammalian ip3r <dig>  <cit> .
previous studies have shown that expression of the drosophila ip3r is widespread in all tissues and cell types examined  <cit> .
however, depending on their allelic strength, itpr mutants exhibit relatively specific metabolic and neuronal phenotypes.
hetero-allelic combinations of strong itpr mutants exhibit metabolic defects, altered feeding and transcriptional changes in metabolic gene pathways during larval stages  <cit> .
itpr
ka1091
and itpr
ug3
are point mutations in the modulatory domain , and in the ligand binding  domain respectively of the ip3r.
these mutants are lethal as homozygotes, while their hetero-allelic combination  is adult viable  <cit> .
recently, we demonstrated the presence of metabolic changes in itpr
ku
adult animals leading to starvation resistance, increased body weight, elevated tags and hyperphagia  <cit> .
in mammals, disorders like type  <dig> diabetes, coronary heart disease, respiratory complications and osteoarthritis are a result of altered fat metabolism  <cit> .
the complexity of these diseases arises in part from regulation of fat metabolism through the interaction of signaling pathways involving multiple tissues and organs.
genetic studies in model organisms help understand aspects of this complexity.
in drosophila, fat metabolism is essential for maintaining energy homeostasis.
nutrient fat in the form of triacylglycerides  is broken down to fatty acids in the mid-gut, absorbed and re-synthesized as tags in the fat bodies  <cit> .
perturbations in fat metabolism can lead to changes in tag levels and consequent obesity  <cit> .
lipids stored in fat body cells are utilized under stress conditions and the storage and mobilization of lipids to target tissues is tightly regulated, based on energy requirements.
this requires communication between the gut, fat body cells and oenocytes, the cells analogous to the mammalian liver in drosophila <cit> .
furthermore, signals from the brain coordinate feeding behavior as well as the utilization of stored tags, finally affecting the body weight of an organism  <cit> .
based on the obese and hyperphagic phenotypes of itpr
ku
it appears that calcium release by the ip3r helps maintain this axis of lipid metabolism and feeding in drosophila.
here we show that peptidergic neurons are an important focus of ip3r function in the context of metabolic control.
RESULTS
the ip3r affects drosophila metabolism through its function in peptidergic neurons
to identify the tissue focus of itpr mutant phenotypes an itpr rnai strain  was used to specifically knockdown the ip3r in all neurons and in the fat body.
pan-neuronal knockdown of the ip3r lead to a significant level of starvation resistance.
in contrast, animals with knock down of the ip3r in the fat body exhibited the same extent of viability post starvation as control animals .
obesity, starvation resistance and hyperphagia in itpr
ka1091/ug3
mutants can be rescued by expression of an itpr
+
cdna in a subset of peptidergic neurons that secrete the insulin-like peptides  amongst other neuropeptides  <cit> .
these cells are marked by the dilp2gal <dig> strain.
to test for necessity of the ip3r in dilp neurons, dsitpr was driven by dilp2gal <dig>  surprisingly, these animals did not exhibit any starvation resistance .
consequently, we tested animals with knockdown of itpr by the dimm gal <dig> that expresses in a larger subset of exclusively peptidergic neurons   <cit> .
this resulted in animals with a significant level of starvation resistance when compared with controls .
in order to test if lack of starvation resistance with ip3r knockdown in dilp <dig> neurons, arose due to insufficient knockdown, we expressed a gfp tagged ip3r transgene  in both dilp <dig> neurons and peptidergic neurons marked by dimmgal <dig>  these strains were tested for the extent of gfp fluorescence in the presence and absence of ip3r knockdown by dsitpr.
gfp expression was abrogated in the dilp neurons with both drivers indicating efficient knock-down .
thus, ip3r knockdown is essential in multiple peptidergic neurons for generating starvation resistance.
concurrently, when an itpr
+
cdna was over-expressed in either all neurons or peptidergic neurons in the background of itpr
ku
, the rescued animals displayed a viability profile similar to that of controls .
next we measured the body weights of animals with pan-neuronal and peptidergic knockdown of the ip3r.
similar to what has been observed for itpr
ku
these knockdown animals showed significantly higher body weights after feeding for 144 hours.
body weights remained significantly elevated post-starvation for 72 hours.
knockdown of itpr exclusively in the fat body however, fails to elicit the above phenotype.
additionally, the elevated body weight of itpr
ku
mutants  was rescued by over-expression of itpr
+
in peptidergic neurons .
pan-neuronal knock down of the ip3r also lead to elevated levels of tags in the abdomen at 144 hrs after feeding.
moreover, in flies with itpr knockdown using pan-neuronal and peptidergic gal4s the tag levels are high even after starvation for 72 hrs.
the fat body knockdown of itpr animals also have slightly but significantly higher tag levels post starvation .
this result agrees with the partial rescue of the elevated tags of itpr
ku
by over expression of itpr
+
exclusively in the fat body observed previously  <cit> .
on the other hand, over expression of itpr
+
in the peptidergic neurons alone completely rescues the elevated tags post feeding and starvation .
these data suggest that itpr function in peptidergic neurons is both necessary and sufficient for maintaining the level of stored tags in fat body cells of adult drosophila.
ip3r function in peptidergic neurons regulates food intake
obesity of itpr
ku
derives in part from excess feeding or hyperphagia  <cit> .
feeding behavior in drosophila is regulated by neurons which innervate and control satiety signals in the gut  <cit> .
the presence of peptidergic neuronal cell bodies on the proventriculus, the insect stomach, has been reported previously  <cit> .
similarly, we observed that both pan-neuronal gal <dig> and the peptidergic gal <dig> strains  used in our studies express in neurons that innervate the gut, with cell bodies on the proventriculus .
to test if excess feeding observed in itpr
ku
flies has a neuronal focus, animals with either pan-neuronal knockdown of the ip3r or with knockdown in peptidergic domains were tested for the quantity of food ingested within a fixed time period by including an edible red dye in their food.
a significantly higher quantity of red dye was observed in abdomens of animals with either pan-neuronal or peptidergic neuron knockdown of the ip3r .
oil red o staining demonstrated elevated tags in the guts of flies with ip3r knockdown indicating greater ingestion and digestion of lipids .
next we tested if expression of itpr
+
in peptidergic neurons rescued the excess feeding observed in itpr
ku
<cit> .
itpr+ expression in peptidergic neurons of itpr
ku
rescued hyperphagia as evident from the significantly reduced level of red dye in the abdomens of rescued animals compared with control animals .
ip3r function in peptidergic neurons affects transcription of a gut lipase
for obese and hyperphagic animals, excess storage of tags is likely to be accompanied by altered levels of lipid metabolizing enzymes in the gut.
magro, encoded by cg <dig>  is the drosophila homolog of mammalian gastric lipase, lipa and is expressed in the anterior part of the gut and released into the intestine to digest tags.
levels of magro have been shown to modulate tag levels as knocking down magro shows reduction in tag levels  <cit> .
itpr
ku
mutants have elevated levels of tags as well as elevated levels of magro <cit> .
since peptidergic knockdown of itpr phenocopies the itpr
ku
mutant, transcript levels of the gastric lipase magro were measured from isolated guts of appropriate genotypes .
magro expression in guts from flies with knockdown of the ip3r in peptidergic neurons appears significantly elevated.
moreover, expression of itpr
+
in peptidergic neurons of itpr
ku
animals could rescue the elevated levels of magro transcripts .
thus, ip3r mediated signals in peptidergic neurons appear to regulate magro transcription in the gut in a manner that is not yet understood.
discussion and
CONCLUSIONS
insulin-like peptides , which are secreted by a subset of the medial neurosecretory cells in the brain , regulate lipid homeostasis in the fat body cells of adult drosophila <cit> .
the obese phenotype observed in adult itpr mutants suggested a role for ip <dig> mediated calcium signaling in modulating ilp release and secretion.
however, significant differences were observed between the phenotypes of itpr mutant animals rescued by expression of an itpr
+
cdna as compared with rescue by over-expression of drosophila ilp <dig>  suggesting that ip3r mutants affect a broader axis of neurohormonal control than the one defined by insulin signaling  <cit> .
ip3-mediated calcium signaling and its modulation of the neurohormonal axis, leading to obesity, have now been investigated in greater detail.
the ilp secreting medial neurosecretory cells also secrete a number of other neuropeptides, which regulate stress and metabolism in drosophila <cit> .
while it is possible that these neuropeptides, in addition to the ilps, contribute to the itpr mutant phenotype of obesity this seems unlikely because knockdown of the ip3r in neurons which secrete ilps and additional neuropeptides  had no effect on starvation resistance and obesity in itpr mutants.
the peptidergic neurons, defined by dimmgal <dig>  include the dilp neurons plus other neurosecretory cells in the brain  <cit> .
because knockdown of the ip3r in peptidergic neurons phenocopied the itpr mutant, and restoring itpr function specifically in peptidergic neurons rescued mutant phenotypes, we conclude that ip3r function and ca2+ release affects lipid metabolism primarily through regulation of neuropeptide secretion.
the composite phenotypes of hyperphagia and obesity in ip3r mutants and peptidergic knockdown animals suggest a role for these neuropeptides in regulation of feeding and tag storage and utilisation in the fatbody.
our data support the idea that feeding and lipid mobilisation are regulated by non-overlapping sets of neurosecretory cells, possibly comprising the mnscs which regulate tag utilisation in the fatbody, and other neuropeptide secreting cells such as the neurons of the stomatogastric system, which regulate satiety and feeding.
moreover, it is likely that feedback mechanisms exist between these two groups of neurons such that knockdown of the ip3r in one set can be compensated by the other, as in the case of knockdown in the dilp neurons.
a recent study revealed the role of drosulfakinin , a neuropeptide expressed in the dilp neurons in feeding and satiety  <cit> .
the target cells of dsk are not known and in the light of our observations, we hypothesize that these might be peptidergic neurons of the stomatogastric nervous system.
indeed, axonal projections from the mnscs have been shown to target the proventriculus and midgut in both larval and adult animals  <cit> .
thus, when ip3r knockdown affects both sets of neurons, as in dimmgal <dig> knockdown and in itpr mutants, it is likely that such feedback mechanisms are abrogated.
as a consequence despite the existence of sufficient tag stores in the fatbody the animals continue to feed excessively, leading to a further increase in tag deposits and obesity.
the altered neurohormonal axis created by knockdown of itpr in peptidergic neurons, and in itpr
ku
mutants, leads to non-cell autonomous effects such as up-regulation of the gastric lipase cg <dig>  which has been previously reported to be expressed mainly in the proventriculus and then delivered to the intestinal lumen  <cit> .
our data support the idea that magro transcription is regulated by neuropeptides released from the stomatogastric nervous system .
stimuli received by the sns are likely to derive from elevated feeding and body tag levels.
a similar neurohormonal gut brain axis is known to exist in mammals in which the vagus nerve which innervates the gastro-intestinal tract plays a role in regulating feeding, satiety and nutrient absorption  <cit>  and responds to the orexin neuropeptides  <cit> .
a better understanding of signaling mechanisms that regulate interactions between these axes is likely to help in devising new therapeutic measures for human obesity.

 calcium is a key signaling molecule in multi-cellular organisms that regulates a variety of cellular processes. the ip3r is a ligand gated calcium channel present on the membranes of endoplasmic reticular stores. it mediates the release of er calcium upon binding of its cognate ligand ip in drosophila there is a single gene, itpr, for the ip3r which is 60% homologous to mammalian ip3r. previous studies have shown that expression of the drosophila ip3r is widespread in all tissues and cell types examined. however, depending on their allelic strength, itpr mutants exhibit relatively specific metabolic and neuronal phenotypes. hetero-allelic combinations of strong itpr mutants exhibit metabolic defects, altered feeding and transcriptional changes in metabolic gene pathways during larval stages. itpr ka1091 and itpr ug3 are point mutations in the modulatory domain, and in the ligand binding domain respectively of the ip3r. these mutants are lethal as homozygotes, while their hetero-allelic combination is adult viable. recently, we demonstrated the presence of metabolic changes in itpr ku adult animals leading to starvation resistance, increased body weight, elevated tags and hyperphagia. in mammals, disorders like type diabetes, coronary heart disease, respiratory complications and osteoarthritis are a result of altered fat metabolism. the complexity of these diseases arises in part from regulation of fat metabolism through the interaction of signaling pathways involving multiple tissues and organs. genetic studies in model organisms help understand aspects of this complexity. in drosophila, fat metabolism is essential for maintaining energy homeostasis. nutrient fat in the form of triacylglycerides is broken down to fatty acids in the mid-gut, absorbed and re-synthesized as tags in the fat bodies. perturbations in fat metabolism can lead to changes in tag levels and consequent obesity. lipids stored in fat body cells are utilized under stress conditions and the storage and mobilization of lipids to target tissues is tightly regulated, based on energy requirements. this requires communication between the gut, fat body cells and oenocytes, the cells analogous to the mammalian liver in drosophila. furthermore, signals from the brain coordinate feeding behavior as well as the utilization of stored tags, finally affecting the body weight of an organism. based on the obese and hyperphagic phenotypes of itpr ku it appears that calcium release by the ip3r helps maintain this axis of lipid metabolism and feeding in drosophila. here we show that peptidergic neurons are an important focus of ip3r function in the context of metabolic control.  the ip3r affects drosophila metabolism through its function in peptidergic neurons to identify the tissue focus of itpr mutant phenotypes an itpr rnai strain was used to specifically knockdown the ip3r in all neurons and in the fat body. pan-neuronal knockdown of the ip3r lead to a significant level of starvation resistance. in contrast, animals with knock down of the ip3r in the fat body exhibited the same extent of viability post starvation as control animals. obesity, starvation resistance and hyperphagia in itpr ka1091/ug3 mutants can be rescued by expression of an itpr + cdna in a subset of peptidergic neurons that secrete the insulin-like peptides amongst other neuropeptides. these cells are marked by the dilp2gal strain. to test for necessity of the ip3r in dilp neurons, dsitpr was driven by dilp2gal surprisingly, these animals did not exhibit any starvation resistance. consequently, we tested animals with knockdown of itpr by the dimm gal that expresses in a larger subset of exclusively peptidergic neurons. this resulted in animals with a significant level of starvation resistance when compared with controls. in order to test if lack of starvation resistance with ip3r knockdown in dilp neurons, arose due to insufficient knockdown, we expressed a gfp tagged ip3r transgene in both dilp neurons and peptidergic neurons marked by dimmgal these strains were tested for the extent of gfp fluorescence in the presence and absence of ip3r knockdown by dsitpr. gfp expression was abrogated in the dilp neurons with both drivers indicating efficient knock-down. thus, ip3r knockdown is essential in multiple peptidergic neurons for generating starvation resistance. concurrently, when an itpr + cdna was over-expressed in either all neurons or peptidergic neurons in the background of itpr ku, the rescued animals displayed a viability profile similar to that of controls. next we measured the body weights of animals with pan-neuronal and peptidergic knockdown of the ip3r. similar to what has been observed for itpr ku these knockdown animals showed significantly higher body weights after feeding for 144 hours. body weights remained significantly elevated post-starvation for 72 hours. knockdown of itpr exclusively in the fat body however, fails to elicit the above phenotype. additionally, the elevated body weight of itpr ku mutants was rescued by over-expression of itpr + in peptidergic neurons. pan-neuronal knock down of the ip3r also lead to elevated levels of tags in the abdomen at 144 hrs after feeding. moreover, in flies with itpr knockdown using pan-neuronal and peptidergic gal4s the tag levels are high even after starvation for 72 hrs. the fat body knockdown of itpr animals also have slightly but significantly higher tag levels post starvation. this result agrees with the partial rescue of the elevated tags of itpr ku by over expression of itpr + exclusively in the fat body observed previously. on the other hand, over expression of itpr + in the peptidergic neurons alone completely rescues the elevated tags post feeding and starvation. these data suggest that itpr function in peptidergic neurons is both necessary and sufficient for maintaining the level of stored tags in fat body cells of adult drosophila. ip3r function in peptidergic neurons regulates food intake obesity of itpr ku derives in part from excess feeding or hyperphagia. feeding behavior in drosophila is regulated by neurons which innervate and control satiety signals in the gut. the presence of peptidergic neuronal cell bodies on the proventriculus, the insect stomach, has been reported previously. similarly, we observed that both pan-neuronal gal and the peptidergic gal strains used in our studies express in neurons that innervate the gut, with cell bodies on the proventriculus. to test if excess feeding observed in itpr ku flies has a neuronal focus, animals with either pan-neuronal knockdown of the ip3r or with knockdown in peptidergic domains were tested for the quantity of food ingested within a fixed time period by including an edible red dye in their food. a significantly higher quantity of red dye was observed in abdomens of animals with either pan-neuronal or peptidergic neuron knockdown of the ip3r. oil red o staining demonstrated elevated tags in the guts of flies with ip3r knockdown indicating greater ingestion and digestion of lipids. next we tested if expression of itpr + in peptidergic neurons rescued the excess feeding observed in itpr ku. itpr+ expression in peptidergic neurons of itpr ku rescued hyperphagia as evident from the significantly reduced level of red dye in the abdomens of rescued animals compared with control animals. ip3r function in peptidergic neurons affects transcription of a gut lipase for obese and hyperphagic animals, excess storage of tags is likely to be accompanied by altered levels of lipid metabolizing enzymes in the gut. magro, encoded by cg is the drosophila homolog of mammalian gastric lipase, lipa and is expressed in the anterior part of the gut and released into the intestine to digest tags. levels of magro have been shown to modulate tag levels as knocking down magro shows reduction in tag levels. itpr ku mutants have elevated levels of tags as well as elevated levels of magro. since peptidergic knockdown of itpr phenocopies the itpr ku mutant, transcript levels of the gastric lipase magro were measured from isolated guts of appropriate genotypes. magro expression in guts from flies with knockdown of the ip3r in peptidergic neurons appears significantly elevated. moreover, expression of itpr + in peptidergic neurons of itpr ku animals could rescue the elevated levels of magro transcripts. thus, ip3r mediated signals in peptidergic neurons appear to regulate magro transcription in the gut in a manner that is not yet understood. discussion and  insulin-like peptides, which are secreted by a subset of the medial neurosecretory cells in the brain, regulate lipid homeostasis in the fat body cells of adult drosophila. the obese phenotype observed in adult itpr mutants suggested a role for ip mediated calcium signaling in modulating ilp release and secretion. however, significant differences were observed between the phenotypes of itpr mutant animals rescued by expression of an itpr + cdna as compared with rescue by over-expression of drosophila ilp suggesting that ip3r mutants affect a broader axis of neurohormonal control than the one defined by insulin signaling. ip3-mediated calcium signaling and its modulation of the neurohormonal axis, leading to obesity, have now been investigated in greater detail. the ilp secreting medial neurosecretory cells also secrete a number of other neuropeptides, which regulate stress and metabolism in drosophila. while it is possible that these neuropeptides, in addition to the ilps, contribute to the itpr mutant phenotype of obesity this seems unlikely because knockdown of the ip3r in neurons which secrete ilps and additional neuropeptides had no effect on starvation resistance and obesity in itpr mutants. the peptidergic neurons, defined by dimmgal include the dilp neurons plus other neurosecretory cells in the brain. because knockdown of the ip3r in peptidergic neurons phenocopied the itpr mutant, and restoring itpr function specifically in peptidergic neurons rescued mutant phenotypes, we conclude that ip3r function and ca2+ release affects lipid metabolism primarily through regulation of neuropeptide secretion. the composite phenotypes of hyperphagia and obesity in ip3r mutants and peptidergic knockdown animals suggest a role for these neuropeptides in regulation of feeding and tag storage and utilisation in the fatbody. our data support the idea that feeding and lipid mobilisation are regulated by non-overlapping sets of neurosecretory cells, possibly comprising the mnscs which regulate tag utilisation in the fatbody, and other neuropeptide secreting cells such as the neurons of the stomatogastric system, which regulate satiety and feeding. moreover, it is likely that feedback mechanisms exist between these two groups of neurons such that knockdown of the ip3r in one set can be compensated by the other, as in the case of knockdown in the dilp neurons. a recent study revealed the role of drosulfakinin, a neuropeptide expressed in the dilp neurons in feeding and satiety. the target cells of dsk are not known and in the light of our observations, we hypothesize that these might be peptidergic neurons of the stomatogastric nervous system. indeed, axonal projections from the mnscs have been shown to target the proventriculus and midgut in both larval and adult animals. thus, when ip3r knockdown affects both sets of neurons, as in dimmgal knockdown and in itpr mutants, it is likely that such feedback mechanisms are abrogated. as a consequence despite the existence of sufficient tag stores in the fatbody the animals continue to feed excessively, leading to a further increase in tag deposits and obesity. the altered neurohormonal axis created by knockdown of itpr in peptidergic neurons, and in itpr ku mutants, leads to non-cell autonomous effects such as up-regulation of the gastric lipase cg which has been previously reported to be expressed mainly in the proventriculus and then delivered to the intestinal lumen. our data support the idea that magro transcription is regulated by neuropeptides released from the stomatogastric nervous system. stimuli received by the sns are likely to derive from elevated feeding and body tag levels. a similar neurohormonal gut brain axis is known to exist in mammals in which the vagus nerve which innervates the gastro-intestinal tract plays a role in regulating feeding, satiety and nutrient absorption and responds to the orexin neuropeptides. a better understanding of signaling mechanisms that regulate interactions between these axes is likely to help in devising new therapeutic measures for human obesity. 
we show that knockdown of the ip3r, either in all neurons or in peptidergic neurons alone, mimics known itpr mutant phenotypes. thus, the focus of observed itpr mutant phenotypes of starvation resistance, increased body weight, elevated lipid storage and hyperphagia derive primarily from peptidergic neurons. the inositol, trisphosphate receptor is a ligand gated calcium channel present on the membranes of endoplasmic reticular stores. while the phenotype manifests in cells of the fat body, genetic studies suggest dysregulation of a neurohormonal axis. the present study shows that itpr function in peptidergic neurons is not only necessary but also sufficient for maintaining normal lipid metabolism in drosophila. the peptidergic neuron domain includes, but is not restricted to, the medial neurosecretory cells as well as the stomatogastric nervous system. our results suggest that intracellular calcium signaling in peptidergic neurons affects lipid metabolism by both cell autonomous and non-autonomous mechanisms. cdna in the same set of peptidergic neurons rescues metabolic defects of itpr transcript levels of a gene encoding a gastric lipase cg, which is known to regulate triacylglyceride storage, can be regulated by itpr knockdown and over-expression in peptidergic neurons.
 intracellular calcium signaling regulates a variety of cellular and physiological processes. the inositol, trisphosphate receptor is a ligand gated calcium channel present on the membranes of endoplasmic reticular stores. in previous work we have shown that drosophila mutants for the ip3r become unnaturally obese as adults with excessive storage of lipids on a normal diet. while the phenotype manifests in cells of the fat body, genetic studies suggest dysregulation of a neurohormonal axis.  we show that knockdown of the ip3r, either in all neurons or in peptidergic neurons alone, mimics known itpr mutant phenotypes. the peptidergic neuron domain includes, but is not restricted to, the medial neurosecretory cells as well as the stomatogastric nervous system. conversely, expression of an itpr + cdna in the same set of peptidergic neurons rescues metabolic defects of itpr ku mutants. transcript levels of a gene encoding a gastric lipase cg, which is known to regulate triacylglyceride storage, can be regulated by itpr knockdown and over-expression in peptidergic neurons. thus, the focus of observed itpr mutant phenotypes of starvation resistance, increased body weight, elevated lipid storage and hyperphagia derive primarily from peptidergic neurons.  the present study shows that itpr function in peptidergic neurons is not only necessary but also sufficient for maintaining normal lipid metabolism in drosophila. our results suggest that intracellular calcium signaling in peptidergic neurons affects lipid metabolism by both cell autonomous and non-autonomous mechanisms. calciumlipid homeostasishyperphagiamagro 