Chronic dysfunction of Stromal interaction molecule by pulsed RNAi induction in fat tissue impairs organismal energy homeostasis in Drosophila

Obesity is a progressive, chronic disease, which can be caused by long-term miscommunication between organs. It remains challenging to understand how chronic dysfunction in a particular tissue remotely impairs other organs to eventually imbalance organismal energy homeostasis. Here we introduce RNAi Pulse Induction (RiPI) mediated by short hairpin RNA (shRiPI) or double-stranded RNA (dsRiPI) to generate chronic, organ-specific gene knockdown in the adult Drosophila fat tissue. We show that organ-restricted RiPI targeting Stromal interaction molecule (Stim), an essential factor of store-operated calcium entry (SOCE), results in progressive fat accumulation in fly adipose tissue. Chronic SOCE-dependent adipose tissue dysfunction manifests in considerable changes of the fat cell transcriptome profile, and in resistance to the glucagon-like Adipokinetic hormone (Akh) signaling. Remotely, the adipose tissue dysfunction promotes hyperphagia likely via increased secretion of Akh from the neuroendocrine system. Collectively, our study presents a novel in vivo paradigm in the fly, which is widely applicable to model and functionally analyze inter-organ communication processes in chronic diseases.

Energy homeostasis is pivotal to life. It is well-established that long-term positive energy balance drives obesity (reviewed in 1,2 ), which is defined as abnormal fat accumulation causative for a number of human diseases 3 .
The prevalence of obesity in children and adults has substantially increased around the world since 1980 4 . Moreover, over the past decades, no country has been successful in combating the obesity pandemic 4 . It is still under debate whether the increased supply of high calorie food 5 combined with sedentary life style 6,7 alone could explain the spread of obesity. But it is clear that the primary driver of obesity is the mismatch of energy intake and expenditure. Specifically, the maintenance of energy balance requires coordination of the major energy handling tissues such as liver (reviewed in 8 ) and adipose tissue (reviewed in 9 ) in mammals. Next to storing energy, adipose tissue also senses the energy status and communicates via adipokines to affect energy intake 10 and expenditure 11 . Therefore, it is vital to understand how energy storage tissue regulates organismal energy balance both in an organ-specific manner and systemically.
Much like mammals, flies also have an energy storage tissue called fat body, which functions similar to liver and white adipose tissue (reviewed in 12 ). Importantly, the majority of extra energy in mammals and flies

Results
Chronic in vivo gene knockdown by RNAi Pulse Induction (RiPI) in adult fly storage tissue. To generate RiPI in adult Drosophila flies (Fig. 1A), we employed the GAL4 system 29 combined with the temperature-sensitive TARGET system 30 to control UAS-RNAi transgenes in a switchable fat body-specific manner (ts-FB-GAL4) 31 . As a proof of concept, we targeted the Adipokinetic hormone receptor (AkhR) gene, a well-characterized key regulator of lipid mobilization in the fat body 30 . Adult flies at the age of six days, which carried the ts-FB-GAL4 and short hairpin (sh) AkhR RNAi transgene (UAS-shAkhRi), were either subjected to a TARGET system-based RiPI (TRiPI) (34)(35)(36)(37)(38)(39)(40)(41)(42)(43)(44)(45)(46)(47)(48) hours at the permissive temperature 29 °C; AkhR-TRiPI On) or kept continuously at the restrictive temperature 18 °C (AkhR-TRiPI Off) (Fig. 1B). To exclude possible confounding effects of the temperature shift per se 32 , we also examined control flies carrying the UAS-shAkhRi transgene only under the two temperature regimes described above (AkhR-TRiPI On control and AkhR-TRiPI Off control) (Fig. 1B). To demonstrate that our conditional expression system is on-off switchable, we took advantage of ts-FB-GAL4 flies carrying a UAS-GFP reporter transgene (see Material & Methods). As expected, GFP mRNA was significantly up-regulated after the induction pulse (day 1) but was at control levels at day 10 after return to the restrictive conditions (Fig. 1C). Similarly, high levels of AkhR siRNA were detected right after the AkhR-TRiPI (21 fold higher than that of AkhR-TRiPI Off flies at day 0). Strikingly, however, AkhR siRNA levels still remained 13-fold higher compared to controls even 10 days after transgene switch-off (Fig. 1D). Consistently, at day 10 after the return to restrictive conditions, AkhR mRNA abundance was just around half compared to control flies; a similar gene knockdown level as observed immediately after the end of TRiPI (Fig. 1E). This chronic AkhR knockdown in the adipose tissue causes persistent body fat increase (Fig. S1A), which is characteristic for AkhR loss-of-function flies 33 . In contrast, we detected no body fat content increase in response to the same temperature shift regimen when ts-FB-GAL4 was absent (Fig. S1A). Importantly, body fat control by tissue-specific, chronic AkhR-TRiPI is not restricted to the UAS-shAkhR transgene but also works in combination with a UAS-long double-stranded (ds)AkhR transgene (Fig. S1B). Collectively, these data suggest that the turnover of shRNA or dsRNA transgene-derived siRNAs targeting AkhR is slow enough to ensure chronic AkhR knockdown for at least three weeks after the induction pulse. This finding opens the possibility to monitor chronic disease progression in response to tissue-specific gene interference in an otherwise undisturbed in vivo setup.
Next, we targeted by RiPI the ER calcium sensor Stromal interaction molecule (Stim), a key component of the store-operated calcium entry (SOCE). Stim is a broadly expressed, essential gene in flies 28 and mice 26 and accordingly not accessible to conventional long-term knockout mutant analysis in adult animals. We have previously shown that short-term Stim knockdown in the fat body impairs gene function on the mRNA and protein level, which causes excessive lipid accumulation in fly fat cells 15 . Now we subjected six days old adult male flies to an adipose tissue-targeted Stim-TRiPI regimen (Fig. 1B) using a UAS-dsStim RNAi transgene (Stim-RNAi1; Fig. S2A). Consistent with our finding on AkhR, the expression of Stim dsRNA was effectively switched on and off in response to the Stim-TRiPI regimen. Stim dsRNA went high immediately after the induction pulse (day 0) but was back to control levels at day 1 and day 10 after the return to the restrictive temperature ( Fig. 2A). In contrast to the transient dsRNA pulse, the Stim mRNA abundance remained chronically decreased to about 50% at days 1 and 10 (Fig. 2B). In line with the chronic impairment of Stim function, Stim-TRiPI On flies accumulated significant body fat at day 1 after RiPI, and then progressively added fat (doubling at day 10) to reach remarkable 3.5 times the body fat content of Stim-TRiPI Off control flies at day 21 (Fig. 2C). This massive fly obesity development is a universal response to chronic Stim impairment, which is largely independent from fly age and sex. Next to young (Fig. 2C) and older (Fig. S2F) unmated male flies, also virgin female flies (Fig. S2C,G) and mated male flies of different ages (Fig. S2B,H) show significant body fat increase three weeks after Stim-TRiPI. By contrast, continuously mated female flies of different ages were resistant to Stim-TRiPI-dependent obesity (Fig. S2E,I) for currently unknown reasons.
To exclude RNAi off-target effects in the etiology of Stim-TRiPI-dependent obesity, we used a second RNAi transgenic line (Stim-RNAi2), which targets an independent sequence of Stim (Fig. S2A). Consistent with the previous results, the body fat content of Stim-RNAi2 flies increased significantly at day 11 after Stim-TRiPI and remained higher than controls at day 21 (Fig. S3A). Finally, we confirmed the causal role of Stim-TRiPI in fat accumulation by simultaneous expression of Stim-RNAi with either one of the two cDNA-based Stim transgenes: the RNAi-sensitive Stim-RA or the RNAi-resistant Stim-Rm transgene (for details see Material & Methods). Fat body-targeted expression of each of the transgenes causes body fat reduction in flies (Fig. S3B,C), which is characteristic for Stim gain-of-function 15,25 . RiPI on flies, which carry the Stim-dsRNAi and the RNAi-sensitive  29 °C for 38-48hs, starting at day 6 after eclosion of adult flies) of Temperature-sensitive RiPI (TRiPI, based on TARGET system) on target genes, i.e. AkhR. TRiPI On refers to TRiPI activity in response to pulse induction at 29 °C was switched on in adult flies (red bar). TRiPI Off refers to TRiPI activity was at off state in adult flies as they were kept at 18 °C (light red bar). TRiPI On control refers to flies containing the RNAi transgene only, which were also transiently shifted to 29 °C (deep green bar); TRiPI Off control refers to flies of the same genotype compared to TRiPI On control flies, kept constantly at 18 °C (light green). (C) Abdominal GFP mRNA expression of TRiPI flies containing temperature-sensitive GFP transgene displayed substantial higher expression level at day 1 compared to day 10 after Stim-TRiPI On as compared to Stim-TRiPI Off males. (D) High abundance of AkhR RNAi effector (siRNA) produced from the AkhR-shRNAi transgene at day 0 and maintained at significant high levels at day 10 in the abdomen of AkhR-TRiPI On males when compared to AkhR-TRiPI Off males. Relative (Rel.) mRNA or siRNA levels are represented as fold change, which refers to the basis value (=1) obtained from corresponding TRiPI Off. (E) Fly abdominal AkhR mRNA expression level reduced at day 1 and day 10 after AkhR-TRiPI On as compared to AkhR-TRiPI Off males. Relative (Rel.) GFP, siRNA or AkhR RNA levels are represented as fold change, which refers to the basis value (=1) obtained from corresponding TRiPI Off flies. Data are presented as means ± standard deviations from 3-6 replicates. All data were analyzed by the two-tailed unpaired Student's t-test. No *p ≥ 0.05, *p < 0.05, **p < 0.01, ***p < 0.001. www.nature.com/scientificreports www.nature.com/scientificreports/ cDNA-RA transgenes shows progressive fat accumulation as early as day 0 after the pulse induction (Fig. S3D). This fat accumulation profile is consistent with the interpretation that Stim-dsRNA downregulates both, the endogenous and the transgenic overexpression of Stim. In contrast, concomitant pulse induction of Stim-dsRNAi and the RNAi-resistant Stim-Rm transgene rescues the normal body fat phenotype at day 0 but not at day 11 or www.nature.com/scientificreports www.nature.com/scientificreports/ 21 (Fig. S3E). Again, these data are in agreement with the Stim-Rm expression being switched off after RiPI, while the dsRNA-mediated Stim gene knockdown persists.
To test the general applicability of the RiPI approach for chronic disease modeling, we tested a TARGET-independent switchable gene expression system. Accordingly, we combined Stim-dsRNA with the drug (Mifepristone)-inducible geneSWITCH (GS) system 34 to achieve switchable ubiquitous expression control using dautherlessGS (daGS 35 ) (Fig. 2D). As in the case of Stim-TRiPI, drug-dependent RNAi pulse induction (DRiPI) of Stim caused a significant transient induction of Stim-RNAi just after RiPI (day 0), which returned to control levels at day 4 and following ( Fig. 2E). Comparing to fat body-targeted Stim-TRiPI, ubiquitous Stim-DRiPI also reduced Stim mRNA to about 40% of the abundance in control flies at day 8 (Fig. 2F). Consequently, this chronic Stim knockdown caused progressive body fat accumulation (Fig. 2G). Notably, this obesity is not only independent from age and sex of the flies but also from food composition. While the absolute fly body fat content varies widely in response to dietary composition, the relative body fat content of Stim-DRiPI flies is consistently at least doubled compared to controls on rich and poor food (Fig. S3F).
Collectively, our data present RiPI as a versatile tool to monitor chronic disease progression caused by long-term tissue-specific gene impairment. Specifically, our findings establish Stim-RiPI as potent method to study the pathophysiological consequences of chronic SOCE impairment in the fly adipose tissue.

Chronic adipose tissue impairment of Stromal interaction molecule causes metabolic disease in flies.
After having demonstrated that Stim-RiPI flies suffer from excessive fat accumulation, we asked whether these flies also display other phenotypes characteristic for mammalian obesity such as increased girth, elevated body weight, and reduced physical fitness. While Stim-TRiPI On and Stim-TRiPI Off flies are indistinguishable at day 1 ( Fig. 3A), abdominal girth of day 21 Stim-TRiPI On flies is visibly inflated compared to Stim-TRiPI Off flies (Fig. 3B). Consistently, Stim-TRiPI On flies gained about 22% body weight in three weeks to become 18% heavier than age-matched Stim-TRiPI Off flies (Fig. 3C) and a dry weight gain of Stim-TRiPI On flies is also observed (Fig. S3G). Consistent with inflated abdominal girth, and increased body weight, thin layer chromatography (TLC) data confirmed that the body TAG content of day 21 Stim-TRiPI On flies was 90% higher compared with day 21 Stim-TRiPI Off flies (Fig. 3D,E). Fly fat body tissue is functionally similar to mammalian adipose tissue and liver, which is a major organ for fat and glycogen reserve 12  Consistent with their increased fat reserves, obese Stim-TRiPI flies are more starvation resistant compared with control flies (Fig. S4A). Noteworthy, substantial post mortem lipid stores suggest a lipid mobilization impairment in Stim-TRiPI flies (Fig. S4B). Much in contrast to fat, the glycogen storage under ad libitum feeding and the glycogen mobilization upon starvation is comparable between Stim-TRiPI On and Stim-TRiPI Off control flies (Fig. S4C). By contrast, we found that day 10 Stim-DRiPI On flies displayed substantially higher level (around 1.9 fold) of circulating sugar levels compared to the value of day 10 Stim-TRiPI Off control flies (Fig. S4D). These results demonstrate that the metabolic dysregulation of body energy stores includes not only a defect in TAG mobilization but also hyperglycemia, which likely contributes to or at least correlates with the obesity phenotype.
We asked next, whether Stim-TRiPI-dependent obesity in flies correlates with physical fitness impairment. Indeed, the climbing ability of day 24 Stim-TRiPI flies was reduced by 40% compared to controls (Fig. S4D). Also, the median lifespan of Stim-DRiPI flies was significantly reduced by about 21% compared to corresponding control flies (Fig. S4E), which is reminiscent of the increased mortality correlated with human obesity 4 . Collectively, these data show that the Stim-TRiPI model specifically affects the storage lipid metabolism and recapitulates hallmarks of mammalian obesity.
Next, we asked how long-term knockdown of Stim changes systemic energy homeostasis. Consistent with previous acute RNAi expression studies 15 , we observed that Stim-TRiPI On flies increased food intake by 90% immediately after pulse induction (Fig. 4A) and these flies stayed hyperphagic the following 10 days (Fig. 4B). Similarly, food intake was increased by around 50% in Stim-DRiPI On flies for eight days using this alternative RNAi induction system (Fig. S5A). These data suggest that hyperphagia contributes to extra fat accumulation. In line with this hypothesis are the results from restrictive pair-feeding experiments. In this paradigm, Stim-TRiPI On flies were offered slightly less than the food amount consumed by ad libitum fed Stim-TRIP Off flies to largely match the dietary intake of experimental and control flies. Under pair-feeding, the body fat content of Stim-TRiPI On flies increased by moderate 127% during the first ten days, and reach 1.85-fold the value of age-matched Stim-TRiPI Off flies (Fig. 4C). The corresponding body fat values increase under ad libitum fed conditions are 343% during the first ten days (with a 30% increase already at day 1 after pulse induction) to reach 2.57-fold the value of Stim-TRiPI Off flies (Fig. 4C). To assess the contribution of hyperphagia-dependent lipogenesis we used dietary 14 C glucose pulse-labeling to compare de novo lipid synthesis between Stim-DRiPI On and Off flies (Fig. S5B) as glucose intake is known to drive lipogenesis 36 . Indeed, the 24 h 14 C incorporation into neutral lipids of Stim-DRiPI On flies was almost five times higher compared to Stim-DRiPI Off flies (Fig. S5C), which substantially exceeds the corresponding incorporation into polar lipids (79%; Fig. S5D) or the food intake increase. These results suggest that in addition to hyperphagia, hyperactivity of TAG biosynthesis and/or reduced energy expenditure causes the excessive fat accumulation. Therefore, we used the respirometry-based metabolic rate estimation in fly 37 to detect a 29% decrease in CO 2 production at noon and a 17% decrease in the evening in the of Stim-DRiPI On flies compared to the corresponding control flies (Fig. S5E). Collectively, these data www.nature.com/scientificreports www.nature.com/scientificreports/ demonstrate that hyperphagia, increased lipogenesis, and reduced energy expenditure contribute to fly obesity in response to chronic Stim knockdown by unknown molecular mechanisms.

Adipose tissue lipolysis dysfunction, Adipokinetic hormone resistance, and insulin signaling impairment caused by chronic impairment of Stromal interaction molecule.
To address the tissue-autonomous mechanisms of chronic Stim knockdown, we performed differential transcriptome profiling by comparative RNAseq analysis on dissected fat body tissues of day 10 Stim-TRiPI On and Off flies ( Fig. S6A-C). Unsurprisingly, gene ontology analysis revealed a complex regulatory response to chronic SOCE impairment suggesting the down-regulation of various metabolic processes (Fig. 5A). A group of significantly down-regulated genes comprises aralar1, Mdh1, Mdh2, Got1, and Got2 (Fig. 5B,C), all of which encode enzymes involved in NADH shuttling between mitochondria and cytoplasm (Fig. S6D). To understand the correlation between Akh signaling and genes involved in NADH shuttling, we co-analyzed the RNAseq dataset of fly larval fat body samples in response to Akh overexpression vs. control condition 38 and our RNAseq dataset of adult fly fat body tissue under Stim-TRiPI On vs. Off condition. Intriguingly, 47 genes were up-regulated in larval fly fat body overexpressing Akh but down-regulated in Stim-TRiPI On adult fly fat body tissue: four of them are Mdh1, Got1, Got2 and aralar1, which could be considered as Akh signaling reporter genes (Fig. S7A). Moreover, Mdh1 mRNA is down-regulated as early as day 1 in Stim-TRiPI On flies (Fig. 5D) but not in Stim-TRiPI On control flies (Fig. S7B). Another gene contrarily expressed under the described conditions is Gprk2 (Fig. S7A), which is directly correlated with cAMP levels in fly tissue 39 . Consistently, both RNAseq and RT-qPCR show that the AkhR mRNA level is up-regulated in the fat body of Stim-TRiPI On flies, however not the AkhR downstream signaling gene tobi 40 (Fig. 5E). Collectively, these finding suggest a differential impairment of Akh signaling in the fat body of obese Stim-TRiPI flies.
Previous research showed that Akh/AkhR signaling is causative for lipid mobilization 33,41 . Consistently, RNAseq indicates the transcriptional down-regulation of the paralogous lipase genes, doppelgänger of brummer (dob) and brummer (bmm), which encodes the fly homolog of mammalian adipose triglyceride lipase (ATGL) 42 in response to chronic Stim impairment (Fig. S8A). Moreover, this analysis suggests a coordinated down-regulation of mitochondrial and peroxisomal β-oxidation pathways in obese Stim-TRiPI On flies (Fig. S8A). We confirmed the transcriptional down-regulation of lipid catabolic pathway genes by selected RT-qPCR analyses of bmm, carnitine O-palmitoyltransferase (CPT1), dACADVL, and dATP5B (Fig. S8A,C). In contrast, midway (mdy), which encodes the homolog of the key human lipid synthesis enzyme diacylglycerol acyltransferase 1 (DGAT1), was  www.nature.com/scientificreports www.nature.com/scientificreports/ only significantly up-regulated at day 1 but not at day 10 in Stim-TRiPI On fat body (Fig. S8D). Similarly, other key genes involved in lipid biosynthesis such as dFAS (fatty acid synthase gene), dACS (Acetyl Coenzyme A synthetase gene), dLipin (Mg 2 + -dependent PA phosphatase gene) were not significantly up-regulated at day 10 in Stim-TRiPI On fat body (Fig. S8D,E). Collectively, these data suggest that a partial Akh resistance as well as impairment of lipolysis and mitochondrial function mediate obesity progression in the fat body in response to chronic Stim knockdown.
To understand why Stim-DRiPI On flies have higher circulating sugar levels, we checked the expression of the Hex-C gene, a homolog of mammalian Glucokinase (GCK) likely involved in glucose clearance by fly fat body 43 and insulin signaling 44 . Stim-TRiPI On flies show around 35% reduced expression of the Hex-C gene at day 10 ( Fig. S9A), which might account for the hyperglycemia. Since insulin-deficient flies display hyperglycemia 45 and insulin signaling in the fat body promotes circulating sugar clearance 46 , we checked peripheral insulin signaling in Stim-TRiPI On flies. The insulin signaling target gene d4EBP's expression was not significantly up-regulated at day 1, and mildly but significantly up-reguated by around 25% at day 10 ( Fig. S9B), which is repressed by the insulin signaling through active phosphorylated Akt 47 . Besides, although insulin signaling was shown to promote fly fat cell proliferation and fat accumulation 48 , we did not find a measurable increase of new DNA synthesis indicated by EdU staining in Stim-TRiPI On fly fat body tissues (Fig. S9C). Consistently, the phosphorylated Akt (at Ser505 43 but not Thr342 49 ) level, the downstream readout of insulin signaling 50 , shows a reduced trend by 37% in Stim-DRiPI On peripheral tissues (Figs S9D, S10A, S11A,B,C). Moreover, loss-of-function of the three central insulin peptide genes dIlp2,3,5, the master regulators of sugar homeostasis 51,52 , did not prevent the extra fat accumulation of Stim-DRiPI On flies (Fig. S9E). Consistent with our previous finding 15 , insulin producing cells (IPCs) of Stim-TRiPI On flies also show reduced staining intensity of dIlp-2 by around 30% (Fig. S9F,G), indicative of increased dIlp-2 secretion 53 . In line with this, RNAseq and RT-qPCR analysis identified the up-regulation of insulin secretion promoting gene CCHamide-2 (CCHa2) 54 by around 67% and dawdle (daw) 55 by around 35%, but down-regulation of insulin signaling inhibiting gene Ecdysone-inducible gene L2 (ImpL2) 56 by around 85% and insulin secretion suppression gene Limostatin (Lst) 57 by around 70% at the mRNA level (Fig. S10A). To measure circulating dIlp-2 directly, we combined the Stim-DRiPI system with dIlp-2HF (epitope-tagged dIlp-2 gene) 52

and found no significant difference on dIlp-2HF levels between obese Stim-DRiPI On and Stim-DRiPI
Off flies (Fig. S10D). While the contradictory findings on circulating dilp levels need future research attention, the results collectively show that insulin signaling is impaired in the fat body tissues of obese Stim-RiPI flies and partially dispensible for long-term body fat storage increase.

Systemic Adipokinetic hormone signaling controls hyperphagia in response to chronic
Stromal interaction molecule dysfunction in the fat body. Adipokinetic hormone signaling does not only promote lipid catabolism in the fat body, but also regulates food intake with ectopic Akh expression causing hyperphagia 58,59 . Therefore, we asked if Stim dysfunction of fat body tissue promotes food intake via Akh signaling. We first quantified the Akh mRNA levels of day 1 and day 10 Stim-TRiPI On flies to find no expression differences compared to control flies (Fig. 6A). Since Akh signaling is controlled by Akh peptide secretion from the neuroendocrine corpora cardiaca (CC) cells 60 , reduced Akh immunostaining intensity in CC cells is used as a proxy for increased secretion of the hormone 60,61 . Therefore, we assessed Akh peptide levels by comparative immunohistochemistry on dissected CC cells of day 10 Stim-TRiPI On/Off flies using antibodies directed against Akh and the synaptic cytoskeletal protein Bruchpilot (served as neuronal marker for signal normalization) (Fig. 6B,C). Normalized Akh staining intensity in CC cells of Stim-TRiPI On flies was significantly reduced by around 27% compared to Stim-TRiPI Off flies (Fig. 6B,C) suggesting increased Akh secretion in response to chronic SOCE impairment in the fat body. To directly address the role of Akh in obese flies subject to chronic Stim fat body dysfunction, we compared the body fat content and the food intake of Akh heterozygous and homozygous mutant flies subjected to Stim-DRiPI On/Off (Fig. 6D,E). Loss-of-Akh completely suppressed obesity development (Fig. 6D) and hyperphagia (Fig. 6E) in Stim-DRiPI On flies.
Since Stim-TRiPI On flies have higher circulating sugar levels than control flies, the increased Akh secretion cannot be triggered by lower circulating sugar levels as reported previously 60 . Thus, we propose that increased Akh secretion in Stim-RiPI On flies might be regulated by unknown fat body tissue secreted factors. RNAseq analysis on Stim-TRiPI fat body tissues identified 16 differentially expressed genes encoding proteins belong to "secreted" group (supplementary data file 2). Interestingly, Lst, CCHa2, daw, Est-6, and PGRP-SD were also transcriptionally regulated in fly larval fat body overexpressing Akh (Fig. S10B), which qualifies them as fat body-derived signal candidates to regulate Akh secretion. Collectively, these results support the model that the chronic impairment of Stim activity in the fat body remotely triggers Akh secretion from the CC cells by currently unknown mechanisms, which in turn stimulates food intake and thereby contributes to obesity progression. the malate-aspartate shuttle genes only Mdh1 is an early response gene to Stim-TRiPI On as revealed by comparison to the Stim-TRiPI Off flies. (E) RT-qPCR identifies transcriptional up-regulation of the AkhR gene but no change on tobi mRNA level. Data are presented as means ± standard deviations from 3-6 replicates. All qRT-PCR data were analyzed by the two-tailed unpaired Student's t-test. No *p ≥ 0.05, *p < 0.05, **p < 0.01, ***p < 0.001. (2019) 9:6989 | https://doi.org/10.1038/s41598-019-43327-y www.nature.com/scientificreports www.nature.com/scientificreports/ . Data are presented as means ± standard deviations. All data were analyzed by the two-tailed unpaired Student's t-test. No *p ≥ 0.05, *p < 0.05, **p < 0.01, ***p < 0.001. www.nature.com/scientificreports www.nature.com/scientificreports/

Discussion
Chronic knockdown of target genes by RNAi Pulse Induction (RiPI). Here we present in vivo evidence for chronic targeted gene knockdown following RiPI in the adult Drosophila fat body. We found that a short pulse induction of shRNA targeting the AkhR gene generates persisting siRNAs, which causes significant down-regulation of AkhR for at least 10 days. Persistence of RNAi has been associated with RNA-dependent RNA polymerase (RdRP)-mediated siRNA amplification in C. elegans 62,63 and in human cells 64 . In Drosophila, however, it remains controversial whether the genome encodes a functional RdRP 65,66 . Therefore, slow degradation of the transgene-derived siRNAs might confer the chronic gene knockdown. In fact, RNAi effector double-stranded siRNAs (21nt and 24nt) are more stable than the 18nt double-stranded RNAs in the human cytosolic extract 67 . Moreover, in human HEK293T cells, the anti-sense strand of siRNA is more resistant to intracellular nucleases compared to the sense strand of the siRNA duplex, which is likely due to the incorporation of anti-sense siR-NAs into the activated RNA induced silencing complex (RISC) 68,69 . Therefore, the involvement of RISC might allow the slow degradation of siRNAs in adult Drosophila fat body cells. The slow decline of the siRNA level is apparently sufficient to chronically knockdown the endogenous gene expression of AkhR and Stim, which causes progressive body fat increase. This mode of action is further supported by the fact that pulsed overexpression of RNAi resistant Stim-mRNA only transiently rescues the fat content increase due to Stim-TRiPI. Consistently, long-term gene silencing (at least 11 days) is also observed in adult flies after injection of low concentrations of dsRNAs 70 . Similarly, in an EGFP-transgenic mouse model, the inhibition of the reporter expression lasts as long as two months after siRNA injection 71 . In summary, we show here that in vivo RiPI generates long-lasting RNAi, which allows chronic knockdown of target genes in a tissue-specific manner. We propose that RiPI is a versatile tool to study causative relationships and temporal sequences in inter-organ communication processes. A Drosophila obesity model triggered by chronic Stromal interaction molecule dysfunction in the fly adipose tissue. Using RiPI, we established a Drosophila obesity model based on chronic, adipose tissue-directed knockdown of Stim, which shares remarkable similarity to characteristics of human obesity. First, the visibly enlarged abdomen of the obese flies corresponds to increased waist circumference, which gains importance as meaningful parameter to assess android adiposity 72 . Similarly, body fat accumulation causes significant weight gain, another readout to quantify obesity in rodents 73 and human 74 . Second, the excessive fat accumulation correlates with climbing deficits of the obese flies, with physical fitness reduction being another hallmark of human adiposity 75 . Moreover, obese Stim-TRiPI flies have reduced life span, which is reminiscent of the higher mortality rates in human obesity patients 76 . Third, we demonstrate that early-onset hyperphagia drives the positive energy balance in Stim-TRiPI flies. Consistently, increased food intake is the major driver of human obesity 5 . Hyperphagia is linked to increased dietary glucose conversion into storage fat in obese Stim-TRiPI flies. Notably, increased food intake and elevated glucose conversion into storage lipids has also been reported after silencing obesity blocking neurons in the fly central brain 77 . With hyperphagia being an important contributor, obesity development in Stim-RiPI flies is not monocausal. It is noteworthy that the rise in fat storage in Stim-DRiPI substantially exceeds the food intake increase. Moreover, matching the food intake of Stim-TRiPI On and Off flies still results in body fat accumulation. Importantly, there is a significantly reduced metabolic rate of Stim-DRiPI flies. Finally, the observed hyperglycemia at day 10, physical fitness reduction at day 24 and shortened life span of Stim-TRiPI On flies are associated with obesity development, similar to type 2 diabetes (T2D) 78 , exercise intolerance 79 and mortality 80 , which are also highly correlated with human obesity. In summary, chronic knockdown of Stim in the adult fat body causes fly obesity by a number of physiological factors culminating in organismal energy imbalance similar to mammalian adiposity.

Chronic knockdown of Stromal interaction molecule causes adipose tissue dysfunction. Our study
highlights the critical roles played by Stim in interaction with Akh/AkhR signaling and insulin signaling in the fly fat body tissue. Reduced expression of Mdh1 and Gprk2 suggests impaired Akh/AkhR signaling in the fat body of Stim-TRiPI flies. Mammalian MDH1 has been linked to glycolysis in cells with mitochondrial dysfunction 81 , obese Stim-TRiPI On flies display normal glycogen storage and mobilization during starvation. Similar findings are also observed in Akh A , Akh AP , and AkhR 1 mutant fly larvae 82 and adult flies, albeit their capability to mobilize glycogen is weakly impaired 41 . A possible explanation is that flies employ corazonin, a starvation-responsive pathway complementary to Akh, to utilize glycogen 83 . In addition to storage glycogen, the reduced expression of genes involved in lipolysis predicts an impairment of starvation-induced storage lipid mobilization. Indeed, obese Stim-TRiPI flies display an abnormal lipid mobilization profile under starvation and die with residual fat resources. Similarly, impaired lipid mobilization is also observed in flies with loss-of-function mutation in the TAG lipase gene bmm 42 or in flies lacking either InsP3R 84 or AkhR 33 . Consistently, loss-of-function of STIM1/2 in mammalian cells, also impairs lipolysis via down-regulation of cAMP 26 . Moreover, decreased catecholamine-stimulated lipolysis has been identified in human obese individuals 85 . Collectively, our results show that fat body tissue of obese Stim-RiPI On flies is resistant in response to Akh signaling, which drives the obesity development.
Moreover, our study supports the possibility to model T2D in adult flies. Obese Stim-TRiPI flies show reduced expression of the glucose clearance gene Hex-C, whose mammalian homolog was also suppressed in T2D patients 86 . Besides, we also provide evidences to support that obese Stim-TRiPI flies have hyperglycemia, impairment of insulin signaling in fat body tissue, and larger lipid droplets. Similar features were also described in fly larvae reared on high sugar diet 36,87 , which resemble mammalian insulin resistance 88 . Regarding unchanged circulating dIlp-2 level in obese Stim-DRiPI flies, insulin-like peptide secretion might be interfered by the knockdown of Stim in the insulin producing cells of Stim-DRiPI flies mediated by ubiquitous driver daGS, more investigation on circulating insulin levels of obese Stim-DRiPI flies by specific driver need to be done in future. Interestingly, the indicators of insulin signaling impairment mentioned above occur at later stage of Stim-RiPI obesity development, and accordingly are possibly the consequence of Stim-TRiPI On mediated-fat gain, which also supports the concept that obesity compromises insulin signaling.
www.nature.com/scientificreports www.nature.com/scientificreports/ Metabolic fat body dysfunction drives organismal energy imbalance. Apart from the specific role of the fat body in storage lipid handling and glucose clearance, we show that chronic knockdown of Stim in this organ remotely promotes Akh secretion from the fly CC neuroendocrine cells, which leads to hyperphagia. Our RNAseq and gene expression analysis indicate a list of genes encoding candidate hormone or secreted proteins. Among them, CCHa2, daw, and Lst has been shown to function as hormones to regulate insulin-like peptide secretion 54,55,57 . In addition, CCHa2, daw, Lst are also regulated by Akh overexpression in opposite direction. Whether differential expression of these genes mentioned above mediate the (mis)communication between the fat body and the CC cells is currently unknown. Nevertheless, the communication between the fat body and the CC cells is essential for the food intake increase as well as further obesity development induced by long-term knockdown of Stim. Interestingly, a study provided evidence that muscle tissue in flies communicates with the CC cells to control Akh secretion via the myokine Unpaired2 (Upd2) 89 . Upd2 had been previously shown to act as adipokine, which signals the fed state from the fat body. Unlike mammalian leptin, Upd2 remotely acts on insulin-producing cells in the central brain to regulate insulin secretion but not food intake 90 . Recently, Akh mRNA expression was shown to be regulated by a gut-neuronal relay via midgut-secreted peptide Buriscon α in response to nutrients 91 . Given the fact that the transcription of Akh is unaffected in Stim-RiPI On flies, identification of the adipokine, which regulates the Akh release directly or indirectly to affect food intake in the Stim-RiPI fly obesity model requires future research efforts.
In conclusion, our work introduces RNAi Pulse Induction as a novel in vivo paradigm for chronic, tissue-specific gene interference. RiPI makes essential genes accessible to long-term functional analysis in the adult fly, as exemplified here by establishing a Drosophila obesity model caused by chronic knockdown of Stim in the adult fat body. Moreover, this study reveals, that the fat body integrates the tissue-autonomous and the systemic branches of Akh signalling: by regulation of lipid mobilization via SOCE in the fat body, and possibly by remote-control of Akh secretion from the CC cells. Recently, the evolutionarily conserved role of SOCE in controlling energy metabolism has attracted the interest of mammalian studies 26,27 . While Akh is structurally not conserved to humans, there is a growing number of remotely-controlled orexigenic peptide hormones in mammals with asprosin being one of the latest additions 92,93 . Collectively, our findings in the fly add further evidence to the existence of conserved regulatory principles in animal energy homeostasis control emanating from SOCE signalling in fat storage tissues.

Material and Methods
Fly stocks and husbandry. The following Drosophila fly stocks were used in this study: StimRNAi1 94 , ts-FB-  Table S1. Quantification of body fat, glycogen, and protein levels. Unless stated differently, three to six replicates of each condition (typically five adult male flies per replicate) were collected. A coupled-colorimetric assay for TAG equivalents was carried out as previously described 97 . Glycogen level was determined with method described in Gáliková et al. 41 . Protein level for normalization of body fat and glycogen content was quantified with Bicinchoninic acid assay (BCA) as described in Gáliková et al. 58 .
Fly body weight measurement. Three to six replicates of five adult males each were transferred into a 1.5 mL Eppendorf tube and the total weight determined using a Sartorius Microbalance MC5 (Sartorius AG, Göttingen, Germany). For dry weight, the open tubes containing flies were incubated at 65 °C for 24 hours. The empty tube weights were previously determined by averaging the values of three different measurements and subtracted from the total weight to determine the wet and dry weight of the flies. The experiment was repeated twice.
Food intake and physiological assays. To quantify adult fly food intake, a capillary feeding (CAFE) assay was performed according to Ja et al. 98 with slight modifications 31 . At least 36 flies per condition were assayed for food intake analysis. The following physiological assays were carried out based on previously described methods with minor modifications: starvation resistance assay 42 , startle-induced climbing assay 41 , and metabolic rate assay 37 ; for further details see supplementary information.
Ex vivo adult fly fat body tissue imaging, lipid and cell area quantification. Three to five flies per condition were analyzed. The experiment was done as described in Baumbach et al. 25 . Further details refer to supplementary information.
Thin layer chromatography (TLC). Four replicates (five adult males per replicates) were collected for TLC, which was done as described in Baumbach et al. 25 with minor modifications. Experiments were repeated twice. Details are described in supplementary information.
www.nature.com/scientificreports www.nature.com/scientificreports/ RT-qPCR. Three to six independent biological replicates were used. RT-SYBR qPCR was done as described in Beller et al. 31 . RT-Taqman qPCR followed the instructions of the kit manufacturer. Details including primer sequences on quantitative RT-qPCR are described in supplementary information.

RNAseq analysis of adult fly fat body tissues.
Abdominal fat body tissues of adult male flies at day 10 and 11 after Stim-TRiPI On/Off induction were dissected in cold PBS buffer, snap frozen in liquid nitrogen, and immediately stored at −80 °C. Dissected fat body tissues of about 20 flies were pooled as one biological replicate per condition of each Stim-TRiPI On/Off experiment. Three independent biological replicates of fat body tissues were used for total RNA isolation using a commercial RNA extraction kit (Norgen BIOTEK, Cat. #: 36200). Typical RNA samples contained at least 1 μg of RNA with concentrations around 50 ng/μL. RNA sample quality analysis, library preparation, sequencing, sequence data trimming, mapping, and reads counting were performed by the Max Planck-Genome-centre Cologne, Germany (http://mpgc.mpipz.mpg.de). Further details on RNAseq quality control, gene differential expression analysis and gene ontology analysis are described in supplementary information.
Western blot analysis. Three independent biological replicates were used for the experiments. Akh and dIlp-2 immunostaining and confocal imaging. Akh and dIlp-2 immunohistochemistry was performed as described 25 with modifications described in supplementary information.
Circulating sugar level measurement. Circulating sugar level of adult flies was determined as described 41 with modifications described in supplementary information.
Statistical analysis. Unless stated differently, data were analysed by two-tailed unpaired Student's t-tests using Microsoft Excel 2011. Statistical significance of the differences between samples is represented by asterisk: *p < 0.05, **p < 0.01, ***p < 0.001. All error bars represent standard deviations of the mean.