Diverse physiological pathways have been shown to regulate longevity, stress resistance, fecundity and feeding rates, and metabolism in Drosophila. Here we tesed physiological traits in flies with Rheb and Myc- Rheb overexpressed in gut progenitor cells, known as enteroblasts (EBs). We found that activation of TOR signaling by overexpression of Rheb in EBs decreases survival and stress resistance. Additionall, we showed that Myc co-expression in EBs reduces fly fecundity and feeding rate. Rheb overexpression enhanced the level of whole body glucose. Higher relative expression of the metabolic genes dilps, akh, tobi and pepck was, however, observed. The role of TOR/Myc in the regulation of genes involved in lipid metabolism and protein synthesis was established. We showed a significant role of TOR/Myc in EBs in the regulation of the JAK/STAT, EGFR and insulin signaling pathways in Drosophila gut. These results highlight the importance of the balance between all different types of cells and confirm previous studies demonstrating that promotion of homeostasis in the intestine of Drosophila may function as a mechanism for the extension of organismal lifespan. Overall, the results demonstrate a role of TOR signaling and its downstream target Myc in EB cells in the regulation of Drosophila physiological processes.
Midgut homeostasis is regulated by multipotent intestinal stem cells (ISCs), which divide and give rise to immature enteroblasts (EBs) or become new stem cells . Enteroblasts can differentiate to enterocytes (ECs) or enteroendocrine (EE) cells. Notch signaling plays an important role in driving EBs to become either ECs or EEs [2,3] (Fig. 1). A recent study summarized findings about maintenance and regulation of ISCs functioning drawing parallels between the fly and mammalian systems . ISCs are diploid, have a small nucleus, and express Delta which is a ligand specific for the Notch receptor. EBs are also diploid with a small nucleus, and express the transcriptional reporter for Notch – Supressor of Hairless (Su(H)) . ECs are polyploid cells with a large nucleus and express the transcription factor Pdm1. EEs are diploid cells with a small nucleus and express the transcription factor Prospero. It was demonstrated, that EEs are generated from Prospero-expressing ISCs, but not from EBs . But another study proposed that EBs give rise to EEs of class II . ECs are involved in nutrient absorption and EEs are important for hormone secretion. Consequently, the functioning of EBs and their differentiation into either ECs or EEs can affect metabolic processes, stress resistance, and aging.
Conserved metabolic pathways are involved in ISC-mediated tissue homeostasis including TOR (target of rapamycin) signaling. TOR is a highly conserved serine/threonine kinase that regulates growth and metabolism in response to nutrient availability, environmental stressors, cellular energy status , and aging . Using an RNAi-based genetic screen, Amcheslavsky and colleagues  identified tuberous sclerosis complex (TSC), a component of TOR signaling, as an important regulator of ISC growth. Kapuria and colleagues  demonstrated that Notch-induced Su(H) activity dowregulates TSC2 expression in EBs. Additionally, it was shown that TSC2 is highly expressed in ISCs  and is downregulated in EBs . A recent study demonstrated that loss of TSC1 and TSC2 function in ISCs lead to the loss of ISCs . Moreover, Rheb overexpression compensates for the effect from TSC1/2 disruption in Drosophila ISCs . A downstream target of TSC1/TSC2 is Rheb, a GTPase that becomes activated in response to TSC2 repression and directly activates TOR kinase. Together, this suggests that TOR signaling is downregulated in ISCs in order to maintain their stemness and is upregulated in EBs, which is a major step for EC differentiation.
D. melanogaster Myc regulates cell growth during development  and together with Rheb possesses oncogenic activity [14,15]. Myc may act independently to regulate ISC growth. Loss of TSC can reduce Myc activity to control excessive ISC growth, and this allows for the restoration of ISC division . In EBs TOR signaling and Myc have a strong impact on cell differentiaion into either EC or EE, which in turn influences metabolic processes, stress resistance, gut integrity, and fly survival.
Our previous study had already demonstrated that both Myc and Rheb expression in stem and progenitor cells have a great impact on Drosophila lifespan, stress resistance and metabolism . The present study aims to show an important role of TOR/Myc signaling axis in EB cells in fruit fly. The study of signaling pathway regulation in ISCs could shed light on potential aging mechanisms. Our study presents novel evidence that TOR and Myc signaling cascades control EB functioning, which is critical for maintaining ISC homeostasis. We used a Su(H) GBE-Gal4 driver to examine the role of ISC progeny – EBs in metabolism and lifespan determination. A UAS-Rheb construct was used to activate the TOR signaling pathway in Drosophila EBs and a UAS-Myc-Rheb construct was used to increase both Rheb and Myc. We found that TOR activation through Rheb overexpression in progenitor stem cells reduced life expectancy, fecundity, and feeding rates but enhanced glucose levels and expression of metabolic genes.
2.1 Fly husbandry and transgenic flies
Before the experiments began, the flies were cultured on standard molasses medium, composed of dry yeast (5%), corn (6.1%), molasses (7.5%), nipagin (0.18%) and propionic acid (0.4%) at 18°C for one year. During the experiments flies were kept at 25°C for two generations on the same diet.
EBs, the immature daughter ISC, can be defined using the Notch signaling reporter Su(H)GBE-LacZ , which is commonly used as an EB marker [2,18]. The Gal4/UAS system was used to overexpress Rheb or both Myc and Rheb proteins in enteroblasts. The EB-specific Gal4 fly line Su(H)GBE-LacZ-Gal4UAS-GFPtub-Gal80ts (Su(H)-Gal4ts) and UAS-Rheb, UAS-Myc-Rheb were backcrossed for eight generations to the w1118 background (Blommington Stock Center). Transgenic flies were obtained from the laboratory of Dr. Bruce Edgar (DKFZ, Heidelberg, Germany). Su(H)-Gal4ts females were mated to males of respective UAS lines. Resultant eggs were allowed to develop at 18°C, because at this temperature Gal80 inhibits the binding of Gal4 protein to UAS. Newly eclosed flies were collected and kept for a three days at 18°C for maiting. Transgenic flies were sexed and shifted to 29°C to induce Su(H)ts, and kept for six days. The final females, which were selected after expression induction, were used for all measurements. All physiological studies were completed at 29°C.
2.2 Lifespan and fecundity
To assess lifespan, approximately 75-100 females of each genotype were placed in 1.5L demographic cages. Plastic vials filled with 5 ml of experimental food (5% sucrose, 5% dry yeast, 1.2% agar and 0.18% nipagin) was attached to the cage. Food was changed every other day and dead flies were counted. The experiment was run twice with more than 150 flies tested per genotype.
To determine fecundity, one female and one male were randomly selected and placed into 5 ml vials with 1 ml of experimental food and reproductive rate was measured. The food was changed every day and the number of eggs laid by individual flies were recorded [19,20]. Twenty flies were tested per genotype in two biological replicates.
Food consumption by a single fly was measured using the CAFE assay . Briefly, flies were kept in vials infiltrated by a 5 μl capillary tube filled with food containing 5% yeast extract and 5% sucrose. The capillaries were changed every day and the amount of food eaten was measured over a period of four days. Vials were kept in closed boxes with distilled water to maintain high humidity. There were three control vials without flies to allow correction for any evaporation of food. Ten flies per genotype were tested.
2.4 Malnutrition, starvation and oxidative stress
For the malnutrition experiments, 85-120 flies were kept in groups of 15 in 15 ml vials with 3 ml of medium consisting of 1% sucrose, 1% autolyzed yeasts, or 0.5% of both. 0.5% sucrose was used for starvation assays . Flies involved in oxidative stress resistance experiments were given a 5% sucrose medium supplemented with 20 mM menadione, a polycyclic aromatic ketone that generates intracellular reactive oxygen species (ROS). The vials were changed every 2 days and the number of dead flies recorded at 9 and 12AM and 9 PM. All experiments were run in three repeats.
Flies were decapitated and then centrifuged to extract hemolymph (3000g, 5 min). Resulting hemolymph was used for determination of glucose and trehalose. Pre-weighted bodies were homogenized in 50 mM Na-buffer, centrifuged, and used for determination of glucose and glycogen levels. Measurements were performed using a glucose assay kit (Liquick Cor-Glucose diagnostic kit, Cormay, Poland, Cat. No. 2-203). The glycogen was converted into glucose by amyloglucosidase from Aspergillus niger (25°C, 4 hours). For triglyceride (TAG) determination, flies were weighed, homogenized in 20 mM PBST (phosphate buffered saline containing 0.05% Triton X100), boiled and centrifuged (13000g, 10 min) [21,22]. Resulting supernatants were used for the triglyceride assay with Liquick Cor-TG diagnostic kit (Cormay, Poland, Cat. No. 2-254). Flies of all genotypes were tested in 4-6 independent replicates .
Total whole-body protein content was measured using Coomassie blue dye according to Bradford . Body supernatants were obtained by homogenization in 50 mM potassium phosphate buffer in ratio 1:10 and centrifuged (13000g, 15 min, 4°C). Serum bovine albumin was a standard. Data are expressed as miligrams of protein per gram of wet fly body weight (mg/gww).
2.6 Analysis of gut integrity
Intestinal integrity was used as an aging indicator. We examined flies expressing Rheb and Myc-Rheb in EBs that consumed non-absorbable blue food dye E133 . “Smurf” flies, characterized by the visible blue food dye throughout the body, were considered to have disruption of gut integrity. Fifty females of each genotype were placed into plastic vials with food supplemented with E133 (2.5%) and after 12 hours the number of “smurf” (with dye spread throughout the body) flies was counted. About 200 flies were analysed per genotype.
2.7 Gene expression
Total RNA from heads, whole flies or dissected guts was extracted with the RNeasy Plus Mini Kit (Qiagen) and converted into cDNA with QuantiTect Reverse Transcription Kit (Qiagen). Expression of genes of interest was measured using an ABI Prism 7000 instrument (Applied Biosystems), a SensiFAST SYBR Hi-ROX Kit, and a QuantiTect SYBR Green PCR Kit (Qiagen) under conditions recommended by the manufacturer. mRNA levels for dilp2, dilp3 and dilp 3 were defined in heads; dilp6, akh, tobi, pepck, 4ebp and bmm in whole body; upd2, upd3, soc36, spi, krn, vn, dilp3, pepck and puc in dissected guts. Each analytical and standard reaction was performed in three technical replicates. The levels of transcripts were measured using primer pairs published earlier and shown in table S2 [26,27,28]. The Ct method was used with rp49 as reference gene for heads and whole flies and crq as the reference gene for guts.
2.8 Statistical procedures
To analyze the trends of lifespan in survival curves that were obtained from starvation, malnutrition and oxidative stress resistance assays the log-rank (Mantel-Cox) test was performed. The differences between means were analyzed using ANOVA followed by Newman-Keuls Multiple Comparison Test in Prism GraphPad.
3.1 Lifespan and stress resistance
TOR signaling pathway and its target Myc are involved in regulation of cell growth, ribosome biogenesis and metabolism that, in turn, have impacts on lifespan. Our previous results showed significant impact of TOR/Myc activation in Drosophila EB on fly survival . Mean lifespan of control flies (SuH/+) was about 34 days at 29°C. Overexpression in enteroblasts in Rheb (SuH/rheb) and Myc-Rheb (SuH/Myc-Rheb) shortened mean lifespan by 15% (log-rank test: χ2=18, p<0.0001) and 18% (χ2=36, p < 0.0001), respectively (Fig. 2A and Table S1). Moreover, we conducted two additional controls rheb/+(UAS-Rheb fly line) and myc-rheb/+ (UAS-Myc-Rheb) that were not different from SuH/+. These data indicated that TOR/Myc activation in EB decreased fly survival.
Nutrient accessibility is an important determinant of organismal lifespan. For this reason we tested fly survival under conditions of malnutrition or complete starvation. Under the condition of low carbohydrate diet (1% sucrose), the survival of both transgenic flies was lower by 11% in SuH/Rheb flies and by 20% in SuH/Myc- Rheb as compared to control group (Fig. 2B and Table S1). Activation of the TOR signaling pathway in enteroblasts by Rheb overexpression had no impact on fly resistance to malnutrition condition when flies are given a low protein diet (1% autolyzed yeast), but Myc-Rheb overexpression shortened lifespan by 17% (χ2=18, p< 0.0001) (Fig. 2C and Table S1). Interestingly, flies exposed to a low carbohydrate, low protein diet (0.5% sucrose and 0.5% autolyzed yeast) Rheb expression decreased the lifespan by 6% (χ2=11, p=0.0007) and Myc co-expression had an even stronger effect where those flies had a 46% reduced lifespan (χ2=78, p < 0.0001) (Fig. 2D and Table S1). Rheb or Myc-Rheb expression in enteroblasts leads to decreased resistance to complete starvation by 15-18% (Fig. 2E and Table S1).
In order to measure oxidative stress susceptibility, flies were fed with menadione in 5% sucrose . There was no significant difference in oxidative stress resistance between transgenic and control flies (Fig. 1F and Table S1). It should be noted that there were no significant differences in stress resistance between two additional control groups.
3.2 Feeding and fecundity
There is a correlation between food consumption, reproduction rate and lifespan in Drosophila. The CAFE assay was used to identify the amount of food consumed by a single fly. Control flies consumed nearly 2 μl of food daily. TOR signaling activation through Rheb over expression did not significantly change the amount of food consumed, but Myc-Rheb expression decreased food intake by 20% (Fig. 3A, p< 0.05). Interestingly, a similar tendency was revealed when fecundity was tested. We found no effect of Rheb expression on fecundity but expression of both Rheb and Myc dramatically decreased reproduction by 30% (Fig. 3B, p< 0.05). There was no significant difference between SuH/+ and Rheb/+ and Myc-Rheb/+ in feeding and fecundity rate. Our data showed that Myc activation in fly EBs affect life-history traits: decreased feeding/fecundity rates.
3.3 Metabolism and gene expression
Glucose, glycogen and TAGs are parameters extensively used as measures of carbohydrate and fat metabolism . Glucose, glycogen and TAG levels were measured in flies of all genotypes. Our results showed that activation of TOR signaling through Rheb overexpression in enteroblasts only increased the level of body glucose, but did not change total hemolymph glucose, glycogen, triglyceride or protein levels. Body glucose levels were 32% higher in Rheb-expressing flies compared to the averaged control. Myc-Rheb co-expressing flies had 1.5- fold higher body glucose compared to the control (p< 0.05) (Fig. 4A). Interestingly, there was 15% higher body glucose level in SuH/Myc-Rheb flies as compared to SuH/Rheb (p < 0.05). The level of glycogen in control flies was about 60 mg/gww. None of the expressed constructs in EBs had an impact on glycogen levels in EBs (Fig. 4C). Moreover, hemolymph glucose concentration, triglyceride content and protein levels were not affected by TOR activation and Myc co-expression (Fig. 4B, D, and E).
To better understand the molecular mechanisms underlying the changes in metabolism resulting from Rheb and Myc-Rheb overexpression in EBs we evaluated gene expression by in Myc-Rheb-expressing flies quantitative realtime reverse transcriptase PCR (qRT-PCR). The transcript levels of important longevity and metabolism genes were measured in Rheb and Myc-Rheb expressing flies. TOR activation through Rheb overexpression in Drosophila enteroblasts increased the expression of dilp2, dilp3 in fly heads and dilp6 in bodies. However, only dilp2 and 3 transcript levels, measured in heads, were significantly upregulated in flies expresing Myc-Rheb (Fig. 5A, B, D, p < 0.05). Additionally, there was a 3.5-4-fold increase in akh transcript levels when Rheb was overexpressed in enteroblasts (p <0.05). However, Myc-Rheb expression also enhanced akh expression (Fig. 5E). There was a 2-fold increase in tobi mRNA levels in the flies that expressed Rheb in progenitor cells (p<0.05), but Myc-Rheb expression had no impact on tobi levels (Fig. 5F). The relative expression of brummer (bmm), encoding a TAG lipase that regulates fat metabolism, was higher by 50% in flies that expressed Rheb and increased by 45% in Myc-Rheb-expressing flies (Fig. 5I, p<0.05). However, there was no significant difference in bmm transcript level comparing Rheb and Myc-Rheb expressing flies. Both genetic manipulations caused a nearly 4-fold increase in pepck transcript level compared to the control (Fig. 5G, p<0.05). Additionally, expression of 4ebp was higher by 50% compared to control when Rheb was overexpressed in enteroblasts (p < 0.05). Myc co-expression caused 5-fold higher transcript level of 4ebp gene (Fig. 5H, p<0.05).
3.4 Gut-specific effects of Rheb and Myc-Rheb overexpression
A recent study showed that intestinal barrier dysfunction is an important factor in pathophysiology of aging, because loss of intestinal integrity is associated with altered metabolic signaling pathways and leads to organismal death . Thus, we supplemented the fly food with an unabsorbable blue food dye for the detection of “smurf” flies . We monitored this phenotype every 5 days and by experimental day 30 about 5-10% of smurf flies were observed per genotype. There was no difference between genotypes (not shown). However, the expression of gut-specific genes was highly affected by Rheb and Myc-Rheb overexpression in EBs. The transcript levels of upd2 and upd3, which encode ligands for JAK/STAT signaling, were higher by 4-fold when Rheb was overexpressed in EBs (Fig. 6A and B, p<0.05). The transcript level of soc36, a target for JAK/STAT signaling, increased 1.5-fold in flies overexpressing Rheb (p<0.05), but Myc co-expression decreased soc36 expression by 75% (Fig. 6C, p<0.05).
The relative expression of EGFR signaling genes spi (Spitz), krn (Keren), vn (Vein) were measured. Relative gene expression of spi increased when TOR signaling was activated through Rheb overexpression in EBs (Fig. 6D, p<0.05). However, no significant difference in krn transcript levels were observed when Rheb and Myc-Rheb were expressed (Fig. 6E). Expression of vn markedly increased (5-fold) in Rheb-expressing flies (Fig. 6F, p<0.05).
Drosophila intestine expresses two insulin-like peptides, Dilp3 and Dilp7. Dilp3 is expressed in the midgut and foregut muscles  and acts directly to induce proliferation and midgut growth via asymmetric and symmetric division . We observed 12-fold higher dilp3 transcript levels in Rheb-expressing flies and an increase of about 4-fold when Rheb and Myc were co-expressed (Fig. 6G, p<0.05). Next, we examined the ability of the TOR/Myc pathway to modulate gluconeogenesis in fly gut cells. Fig. 5H demonstrates 3.5-fold higher pepck transcript levels in Rheb-Myc-expressing flies (p<0.05). Given the clear role of puc (puckered) in controlling tissue homeostasis , we examined relative puc expression in fly gut to see if this might also be correlated with organismal survival. Rheb overexpression in EB led to a 4.5-fold increase in puc transcript levels in Drosophila gut cells (Fig 5I, p<0.05).
The TOR pathway is an important regulatory system for stem cell protection, maintenance and proliferation . It is a key nutrient sensing pathway that can increase lifespan if inhibited . However our study showed that TOR signaling activation through Rheb overexpression in EBs shortens fly lifespan. The inhibition of TOR in EBs is sufficient to change their commitment from an EC fate to an EE fate . TOR activation in intestinal stem cell progeny favors the differentiation to ECs , and this differentiation event may be critical for shortening organismal lifespan. Consequently, an imbalance between ECs and EEs decreases fly lifespan. Intestinal stem cell proliferation increases during aging, which causes an accumulation of ISC progenitor cells that have impaired terminal differentiation pathways . Our previous study showed that the lifespan of Drosophila was decreased when Rheb was overexpressed in ISCs and lifespan was even shorter when Myc was coexpressed . Present results propose a critical role for TOR/Myc signaling pathway in progenitor stem cells, EBs, which in turn can have a strong impact on Drosophila stress resistance and lifespan.
We also found that food availability significantly impacts fly survival. Flies that expressed Rheb and Myc-Rheb in EBs were less resistant to starvation and malnutrition. But our previous results showed that low carbohydrate or low protein diets increase lifespan of Rheb and Myc-Rheb-expressing flies . Choi and colleagues  demonstrated that starvation caused a delay in EC growth and a prolonged contact between ISCs and their progenitors. Drosophila adults that fed on a protein-poor diet had an increased number of stem cell progeny and a slower proliferation rate, indicating that nutrition influences ISC proliferation . Similarly, Choi and colleagues observed a strong reduction in EC reduplication in flies fed a low protein diet . Flies with defects in ISC growth had a thinner gut epithelium and were more sensitive to food quality.
Starvation and oxidative stress resistance are mediated through 4E-BP, a downstream target of TOR . Furthermore, Tettweiler and colleagues demonstrated that 4E-BP has significant impact on lifespan in that 4ebp overexpression is a protection against starvation and oxidative stress . In response to stressful agents such as pathogens and ROS-inducing compounds, ISCs increase proliferative rates, which allows for the repair of damaged cells . This fly phenotype closely resembles the aging phenotype. Treatment with stressful agents, such as paraquat exposure which induces oxidative stress, has been shown to increase ISC number and activity in the guts of aged flies . Our data demonstrated that menadione exposure shortened the lifespan of all experimental flies. Furthermore, TOR/Myc activation in EBs had no impact on organismal oxidative stress response. However, the lower resistance to oxidative stress when TOR/Myc was activated in ISCs , suggesting a critical role of midgut stem cells, but not their progenitors, in the regulation of stress resistance.
Of significance, this work also uncovered the role of TOR/Myc in Drosophila EBs on food consumption and its impact on fecundity rates. The conserved TOR signaling pathway plays a central role in the regulation of nutrient intake and maintaining nutrient balance . Balance in food consumption is important for organismal growth, development, and survival. Feeding rate and egg production are interrelated biological processes in Drosophila. Feeding rate is controlled by a molecular pathway which regulates egg production through an ejaculate “sex peptide” . Previous studies have shown that increases in female feeding correlated with higher reproductive rates reflected by higher egg production . Our results demonstrated that TOR activation through Rheb overexpression together with Myc co-expression decreased the amount of food consumed by a single fly. A similar effect was observed when Myc-Rheb was overexpressed in ISCs . Simultaneously, this group of transgenic flies showed lower fecundity. These data indicate that Myc, as a transcription factor, in Drosophila enteroblasts may play an important role in linking nutrition and reproduction. Moreover, Su(H)-driver which is expressed in EBs has an impact on the expression of sex determinants, which may have significant impact on fecundity rate.
In addition to the important role of TOR in regulating growth and aging, it also regulates various aspects of cellular metabolism. In particular, we were interested in how TOR and Myc activation in EBs affect energy metabolism. We observed higher levels of whole body glucose and akh transcript levels in Rheb and Myc-Rheb expressing flies. Previous studies have demonstrated a hypothetical link between glucose levels and glucagonlike peptide AKH levels [41,42]. DILPs are important insulin signaling molecules that display antagonistic properties to AKH in regulation of Drosophila metabolism . Insulin signaling pathways regulate ISC proliferation in Drosophila  and DILP3 in particular is known to promote ISC differentiation. Activation of TOR signaling pathways through Rheb overexpression in EBs lead to higher dilp2,dilp3, dilp6 transcript levels in fly heads. Previous studies have shown that insulin-producing cells sense nutritional signals for DILP production in Drosophila [43,44]. Additionally, Okamoto and Nishmura  illustrated the existence of a feedback mechanism between DILPs and FOXO. Fly intestine dilp3 transcript levels increased 12-fold and 4-fold, respectively, in response to Rheb and Myc-Rheb expression. According to these results, a possible feedback mechanism may exist between DILP3 and Rheb in fly guts. Specifically, Rheb overexpression in EBs controls metabolic processes in Drosophila gut by regulating dilp3 expression. Elevated dilp3 levels in the gut may be important for maintaining tissue homeostasis. Moreover, DILPs and AKH have an impact on tobi gene expression which was elevated in Rheb expressing flies . This increase suggests that TOR activated flies have more insulin signaling in their peripheral tissues compared to control flies. A possible reason why insulin and AKH regulatory signals increased simultaneously could be that transgenic flies have a faster metabolism or that these outputs are regulated by peptide synthesis or release. Drosophila PEPCK is involved in gluconeogenesis and glycerogenesis . Interestingly, elevated pepck levels correspond with elevated whole body glucose levels in Rheb and Myc-Rheb expressing flies. Although there are many studies to support this theory [47,48], some evidence suggests that fluctuating levels of pepck do not affect levels of plasma glucose in mice . Future studies directly assessing the effect of changes in pepck expression on glucose levels are warranted. Only Myc-Rheb expression in EBs enhance pepck expression in Drosophila gut cells, suggesting that Myc may directly regulate gluconeogenesis and glycerogenesis. Previous study also demonstrated the involvement of TOR/Myc in ISCs in regulation of metabolities content .
Finally, we were interested in the role of TOR and Myc activation in the regulation of lipid metabolism. Brummer lipase (bmm) is responsible for TAG mobilization from fly fat bodies . TOR signaling activation through Rheb overexpression as well as Rheb-Myc expression were found to lead to higher bmm expression, but bmm expression was not different between Rheb and Myc-Rheb expressing flies. These results suggest that glucose metabolism, insulin signaling, and TAG mobilization are dependent on TOR activation in EBs. We also observed elevated 4ebp transcript levels in flies that expressed Rheb, and a much more dramatic increase in flies that co-expressed Myc transcription factor. It was shown that 4E-BP is activated during starvation and oxidative stress conditions, but its absence impairs fly survival . The study of Tahmasebi and colleagues  showed that 4E-BP has a complex impact on the reprogramming process of embryonic fibroblasts into pluripotent stem cells in mice. Moreover, enhanced reprogramming is a result of increased Myc and Sox2 translation . Myc can be considered a downstream target of TOR because its activity is in part controlled by other downstream TOR targets such as S6K [51,52]. TOR and Myc are involved in ribosome biogenesis and protein synthesis . Therefore, the observation of elevated 4ebp expression in Rheb- and Myc-Rheb-expressing flies prompted the analysis of total protein levels in transgenic Drosophila lines. Interestingly, neither Rheb nor Myc-Rheb overexpression in EBs influenced protein level in fly body. But we expected that increased 4ebp expression would increase whole body protein levels because activated (phosphorylated) 4EBP allows translation to proceed. However, we have also found in our study that TOR activation through Rheb overexpression may reduce feeding or appetite and does not help survival when flies are given low-nutrient diets. Perhaps the increase in expression of 4ebp during TOR activation is not in response to TOR activation alone, but the other phenotypes it induces. For future studies, it would be interesting to analyze relative levels of phospho-4EBP and unphosphorylated 4EBP to determine which processes are actually occurring. Another possible explanation of our observation that total protein levels did not differ between experimental fly lines is the existence of compensatory mechanism of protein synthesis by 4ebp overexpression in response to TOR signaling activation, but in a TOR-independent manner. However, these suggestions need additional experiments to make more specific conclusions.
We were interested whether death of Rheb and Myc-Rheb-expressing flies is the result of disruption of gut integrity. Smurf assays did not help us to confirm or deny this theory. Aging is associated with reduced cellular regeneration and defects in tissue homeostasis. We found no difference in the number of transgenic “Smurf” flies compared to the control group, indicating that the unabsorbable blue dye did not penetrate the intestinal wall in either of the experimental fly groups. Similar trends were obtained when we expressed Rheb and Myc-Rheb in ISCs . Therefore, we were interested in observing the effects of TOR and Myc activation on the molecular pathways that control EB viability and proliferation. Intestinal stem cell homeostasis is regulated by Notch, JAK/STAT, EGF, Hippo, insulin, and Wnt signaling pathways . Consequently, the transcript levels of genes that encode ligands for EGFR (spi, krn, vn), JAK/STAT (upd2, upd3, and downstream gene soc36), and insulin signaling (dilp3) were measured in Drosophila gut. The JAK/STAT signaling pathway is highly conserved from flies to mammals and plays essential roles during development. It also serves as a regulator of stem cell differentiation. Signaling ligands for this pathway, Upd2 and 3, activate the JAK/STAT pathway activity in ISCs . We found higher Upd2 and Upd3 transcript levels when Rheb is overexpressed in Drosophila EBs, indicating that the JAK/STAT pathway is activated. Recent data revealed that these cytokines are controlled by dTOR expression, indicating TOR as a possible regulator of JAK/STAT . We also show that Rheb-expressing flies have an elevated transcription of soc36, a JAK/STAT target gene. Thus, our findings suggest that TOR signaling activation in EBs leads to increased JAK/STAT activity. We recently showed that Rheb overexpression in Drosophila ISCs enhanced the relative expression of upd2 and soc36 in fly guts . Moreover, the study of Wang and Huang indicated that JAK/STAT acts downstream of TOR signaling by inhibiting Upd secretion . But Myc is post-transcriptionally upregulated upon JAK/STAT activation  (Fig. 1A). Inhibition of JAK/STAT signaling promotes gut homeostasis and extends lifespan . Interestingly, soc36 acts as a pathway repressor via feedback inhibition. Moreover, soc36 suppresses EGFR signaling  and can interact with multiple signaling pathways to maintain appropriate stem cell functionality.
During normal tissue homeostasis in the Drosophila midgut JAK/STAT signaling acts as a part of a greater regulatory network. It acts together with EGFR and Wnt signaling. This coordination is necessary for ISC maintenance and promotes differentiation of EBs. Specifically, EGFR signaling is required for ISC proliferation induced by JAK/STAT , and both JAK/STAT and EGFR are required for the regeneration of midgut epithelium . McNeill and colleagues  illustrated the existence of crosstalk between TOR and EGFR signaling. Moreover, TOR controls the expression of several EGFR components, such as argos, rho, and pntP2 . We found elevated levels of vn transcripts in Rheb-expressing flies suggesting that vein is regulated by TOR signaling. Our study also revealed increased spi transcript levels when Myc-Rheb is overexpressed in Drosophila EBs, indicating that Myc takes part in the regulation of spi expression. Together, the data suggests that TOR/Myc activation influences EGFR activity in fly EBs.
Puckered (puc) is also known to be involved in maintaining the balance between cell differentiation and proliferation . It plays an important role in controlling cell viability and may act as a tumor suppressor gene. We found that TOR activation through Rheb overexpression enhanced the relative expression of puc. This data is interesting since Rheb is known to have oncogenic properties [14,15]. Therefore, a balance between Rheb and puc expression may be critical for fly survival, and TOR/Myc signaling is likely to be a key regulator of ISC proliferation and differentiation. Together, our data concerning several key metabolic pathways suggests that TOR/Myc activation has an important role in maintaining cell viability in the fly intestine.
Stem cells play a critical role in maintaining tissue homeostasis, which is necessary for organismal survival. Moreover, the TOR/Myc signaling pathway has been shown to be a conserved regulator of longevity. Here, we specifically focus on how activation of TOR and Myc signaling in progenitors cells regulate different aspects of fly metabolism, stress resistance, and aging. Our results demonstrate a significant decrease in lifespan and stress resistance when TOR is activated in Drosophila EBs. Myc overexpression leads to lower fecundity and feeding levels. Finally, we show that TOR/Myc signaling controls fly metabolism by differentially regulating the expression of key metabolic genes. Our results highlight the importance of TOR/Myc signaling in the regulation of various physiological processes in Drosophila progenitor cells. Identification of changes in the expression of specific genes encoding signal transduction proteins involved in proliferation and differentiation suggest that TOR/Myc activation is critical for maintaining Drosophila gut homeostasis.
We would like to thank Semanyuk U. for technical assistance.
Funding: The research received partial support from a discovery grant from the Natural Sciences and Engineering Research Council of Canada (#6793) to KBS.
Conflict of interest: Authors state no conflict of interest.
 Zeng X., Chauhan C., Hou S.X., Characterization of midgut stem cell- and enteroblast-specific Gal4 lines in Drosophila, Genesis, 2010, 48, 607-611 Search in Google Scholar
 Ohlstein B., Spradling A., Multipotent Drosophila intestinal stem cells specify daughter cell fates by differential notch signaling, Science, 2007, 315, 988-992 Search in Google Scholar
 Perdigoto C.N., Schweisguth F., Bardin A.J., Distinct levels of Notch activity for commitment and terminal differentiation of stem cells in the adult fly intestine, Development, 2011, 138(21), 4585-4595 Search in Google Scholar
 Li H., Jasper H., Gastrointestinal stem cells in health and disease: from flies to humans, Dis Model Mech, 2016, 9(5), 487-99 Search in Google Scholar
 Hou S.X., Intestinal stem cell asymmetric division in the Drosophila posterior midgut, Cell Physiol., 2010, 224, 581-584 Search in Google Scholar
 Biteau B., Jasper H., Slit/Robo signaling regulates cell fate decisions in the intestinal stem cell lineage of Drosophila, Cell Rep, 2014, 7, 1867-1875 Search in Google Scholar
 Beehler-Evans R., Micchelli C.A., Generation of enteroendocrine cell diversity in midgut stem cell lineages, Development, 2015, 142, 654-664 Search in Google Scholar
 Wullschleger S., Loewith R., Hall M.N., TOR signaling in growth and metabolism, Cell, 2006, 124(3), 471-484 Search in Google Scholar
 Lushchak O., Strilbytska O., Piskovatska V., Storey K.B., Koliada A., Vaiserman A., The role of the tor pathway in mediating the link between nutrition and longevity, Mech Ageing Dev, 2017, 164, 127-138 Search in Google Scholar
 Amcheslavsky A., Ito N., Jiang J., Ip Y.T., Tuberous sclerosis complex and Myc coordinate the growth and division of Drosophila intestinal stem cells, J Cell Biol, 2011, 193(4), 695-710 Search in Google Scholar
 Kapuria S., Karpac J., Biteau B., Hwangbo D., Jasper H., Notch-mediated suppression of TSC2 expression regulates cell differentiation in the Drosophila intestinal stem cell lineage, PLoS Genet, 2012, 8(11), e1003045 Search in Google Scholar
 Quan Z., Sun P., Lin G., Xi R., TSC1/2 regulates intestinal stem cell maintenance and lineage differentiation through Rheb-TORC1-S6K but independently of nutritional status or Notch regulation, J Cell Sci, 2013, 126(17), 3884-92 Search in Google Scholar
 Wu D.C., Johnston L.A., Control of wing size and proportions by Drosophila myc, Genetics, 2010, 184(1), 199-211 Search in Google Scholar
 Mavrakis K.J., Zhu H., Silva R.L., Mills J.R., Teruya-Feldstein J., Lowe S.W., et al., Tumorigenic activity and therapeutic inhibition of Rheb GTPase, Genes Dev, 2008, 22(16), 2178-2188 Search in Google Scholar
 Johnston L.A., Prober D.A., Edgar B.A., Eisenman R.N., Gallant P., Drosophila myc regulates cellular growth during development, Cell, 1999, 98, 779-790 Search in Google Scholar
 Strilbytska O., Semaniuk U., Storey K., Edgar B., Lushchak O., Activation of TOR/MYC signaling axis in intestinal stem and progenitor cells affects longevity, stress resistance and metabolism in Drosophila, Comp Biochem Physiol B Biochem Mol Biol, 2017, 203, 92-99 Search in Google Scholar
 Furriols M., Bray S., A model Notch response element detects Suppressor of Hairless-dependent molecular switch, Curr Biol, 2001, 11(1), 60-64 Search in Google Scholar
 Micchelli C.A., Perrimon N., Evidence that stem cells reside in the adult Drosophila midgut epithelium, Nature, 2006, 439(7075), 475-479 Search in Google Scholar
 Lushchak O.V., Gospodaryov D.V., Rovenko B.M., Glovyak A.D., Yurkevych I.S., Klyuba V.P., et al., Balance between macronutrients affects lifespan and functional senescence in fruit fly Drosophila melanogaster, J Gerontol A Biol Sci Med Sci, 2012, 67(2), 118-25 Search in Google Scholar
 Lushchak O.V., Gospodaryov D.V., Rovenko B.M., Yurkevych I.S., Perkhulyn N.V., Lushchak V.I., Specific dietary carbohydrates differentially influence the life span and fecundity of Drosophila melanogaster, J Gerontol A Biol Sci Med Sci, 2014, 69(1), 3-12 Search in Google Scholar
 Rovenko B.M., Perkhulyn N.V., Gospodaryov D.V., Sanz A., Lushchak O.V., Lushchak V.I., High consumption of fructose rather than glucose promotes a diet-induced obese phenotype in Drosophila melanogaster, Comp Biochem Physiol A Mol Integr Physiol, 2015a, 180, 75-85 Search in Google Scholar
 Rovenko B.M., Kubrak O.I., Gospodaryov D.V., Perkhulyn N.V., Yurkevych I.S., Sanz A., et al., High sucrose consumption promotes obesity whereas its low consumption induces oxidative stress in Drosophila melanogaster, J Insect Physiol, 2015b, 79, 42-54 Search in Google Scholar
 Rovenko B.M., Perkhulyn N.V., Lushchak O.V., Storey J.M., Storey K.B., Lushchak V.I. Molybdate partly mimics insulinpromoted metabolic effects in Drosophila melanogaster, Comp Biochem Physiol C Toxicol Pharmacol, 2014, 165, 76-82 Search in Google Scholar
 Bradford M.M., A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Analytical Biochemistry, 1976, 72, 248-254 Search in Google Scholar
 Rera M., Bahadorani S., Cho J., Koehler C.L., Ulgherait M., Hur J.H., et al., Modulation of longevity and tissue homeostasis by the Drosophila PGC-1 homolog, Cell Metab, 2011, 14(5), 623-634 Search in Google Scholar
 Kapan N., Lushchak O.V., Luo J., Nässel D.R., Identified peptidergic neurons in the Drosophila brain regulate insulin-producing cells, stress responses and metabolism by coexpressed short neuropeptide F and corazonin, Cell Mol Life Sci, 2012, 69(23), 4051-4066 Search in Google Scholar
 Luo J., Lushchak O.V., Goergen P., Williams M.J., Nässel D.R., Drosophila insulin-producing cells are differentially modulated by serotonin and octopamine receptors and affect social behavior, PLoS One, 2014, 9(6), e99732 Search in Google Scholar
 Lushchak O.V., Carlsson M.A., Nässel D.R., Food odors trigger an endocrine response that affects food ingestion and metabolism, Cell Mol Life Sci, 2015, 72(16), 3143-3155 Search in Google Scholar
 Tennessen J.M., Barry W., Cox J., Thummel C.S., Methods for studying metabolism in Drosophila, Methods, 2014, 68(1), 105-115 Search in Google Scholar
 Rera M., Clark R.I., Walker D.W., Intestinal barrier dysfunction links metabolic and inflammatory markers of aging to death in Drosophila, Proc Natl Acad Sci USA, 2012, 109(52), 21528-21533 Search in Google Scholar
 Veenstra J.A., Agricola H.J., Sellami A., Regulatory peptides in fruit fly midgut, Cell Tissue Res, 2008, 334, 499-516 Search in Google Scholar
 O’Brien L.E., Soliman S.S., Li X., Bilder D., Altered modes of stem cell division drive adaptive intestinal growth, Cell, 2011, 147, 603-614 Search in Google Scholar
 Bjedov I., Toivonen J.M., Kerr F., Slack C., Jacobson J., Foley A., Partridge L., Mechanisms of life span extension by rapamycin in the fruit fly Drosophila melanogaster, Cell Metab, 2010,11(1), 35-46 Search in Google Scholar
 Wang L., Jones D.L., The effects of aging on stem cell behavior in Drosophila, Exp Gerontol, 2011, 46, 340-344 Search in Google Scholar
 Choi N.H., Lucchetta E., Ohlstein B., Nonautonomous regulation of Drosophila midgut stem cell proliferation by the insulin-signaling pathway, Proc Natl Acad Sci USA, 2011, 108, 18702-18707 Search in Google Scholar
 Tettweiler G., Miron M., Jenkins M., Sonenberg N., Lasko P.F., Starvation and oxidative stress resistance in Drosophila are mediated through the eIF4E-binding protein, d4E-BP, Genes Dev, 2005, 19(16), 1840-1843 Search in Google Scholar
 Amcheslavsky A., Jiang J., Ip Y.T., Tissue damage-induced intestinal stem cell division in Drosophila, Cell Stem Cell, 2009, 4(1), 49-61 Search in Google Scholar
 Biteau B., Hochmuth C.E., Jasper H., JNK activity in somatic stem cells causes loss of tissue homeostasis in the aging Drosophila gut, Cell Stem Cell, 2008, 3(4), 442-455 Search in Google Scholar
 Vargas M.A., Luo N., Yamaguchi A., Kapahi P., A role for S6 kinase and serotonin in postmating dietary switch and balance of nutrients in D. melanogaster, Curr Biol, 2010, 20(11), 1006-1011 Search in Google Scholar
 Barnes A.I., Wigby S., Boone J.M., Partridge L., Chapman T., Feeding, fecundity and lifespan in female Drosophila melanogaster, Proc Biol Sci, 2008, 275(1643), 1675-1683 Search in Google Scholar
 Bharucha K.N., Tarr P., Zipursky S.L., A glucagon-like endocrine pathway in Drosophila modulates both lipid and carbohydrate homeostasis, J Exp Biol, 2008, 211, 3103-3110 Search in Google Scholar
 Kim S.K., Rulifson E.J., Conserved mechanisms of glucose sensing and regulation by Drosophila corpora cardiaca cells, Nature, 2004, 431(7006), 316-320 Search in Google Scholar
 Okamoto N., Nishimura T., Signaling from glia and cholinergic neurons controls nutrient-dependent production of an insulin-like peptide for Drosophila body growth, Dev Cell, 2005, 35(3), 295-310 Search in Google Scholar
 Nässel D.R., Kubrak O.I., Liu Y., Luo J., Lushchak O.V., Factors that regulate insulin producing cells and their output in Drosophila, Front Physiol, 2013, 4, 252 Search in Google Scholar
 Buch S., Melcher C., Bauer M., Katzenberger J., Pankratz M.J., Opposing effects of dietary protein and sugar regulate a transcriptional target of Drosophila insulin-like peptide signaling, Cell Metab, 2008, 7, 321-332 Search in Google Scholar
 Okamura T., Shimizu H., Nagao T., Ueda R., Ishii T., ATF-2 regulates fat metabolism in Drosophila, Mol Biol Cell, 2007, 18(4), 1519-1529 Search in Google Scholar
 She P., Burgess S.C., Shiota, M., Flakoll P., Donahue E.P., Malloy C.R., et al., Mechanisms by which liver-specific PEPCK knockout mice preserve euglycemia during starvation, Diabetes, 2003, 52(7), 1649-1654 Search in Google Scholar
 Yang J., Kalhan S.C., Hanson R.W., What is the metabolic role of phosphoenolpyruvate carboxykinase? J Biol Chem, 2009, 284, 27025-27029 Search in Google Scholar
 Grönke S., Mildner A., Fellert S., Tennagels N., Petry S., Müller G., et al., Brummer lipase is an evolutionary conserved fat storage regulator in Drosophila, Cell Metab, 2005, 1(5), 323-330 Search in Google Scholar
 Tahmasebi S., Alain T., Rajasekhar V.K., Zhang J.P., Prager-Khoutorsky M., Khoutorsky A., et al., Multifaceted regulation of somatic cell reprogramming by mRNA translational control, Cell Stem Cell, 2014, 14(5), 606-616 Search in Google Scholar
 Parisi F., Riccardo S., Daniel M., Saqcena M., Kundu N., Pession A., et al., Drosophila insulin and target of rapamycin (TOR) pathways regulate GSK3 beta acitivty to control Myc stability and determine Myc expression in vivo, BMC Biol, 2011, 9, 65 Search in Google Scholar
 Zhang H.H., Lipovsky A.I., Dibble C.C., Sahin M., Manning B.D., S6K1 regulates GSK3 under conditions of mTOR-dependent feedback inhibition of Akt, Mol Cell, 2006, 24(2), 185-197 Search in Google Scholar
 van Riggelen J., Yetil A., Felsher D.W., MYC as a regulator of ribosome biogenesis and protein synthesis, Nat Rev Cancer, 2010, 10(4), 301-309 Search in Google Scholar
 Jiang H., Edgar B.A., Intestinal stem cells in the adult Drosophila midgut, Exp Cell Res, 2011, 317(19), 2780-2788 Search in Google Scholar
 Bauzek N., JAK/STAT signaling in stem cells and their niches in Drosophila, JAKSTAT, 2013, 2(3), e25686 Search in Google Scholar
 Wang W., Li Y., Zhou, L., Yue H., Luo H., Role of JAK/STAT signaling in neuroepithelial stem cell maintenance and proliferation in the Drosophila optic lobe, Biochem Biophys Res Commun, 2011, 410, 714-720 Search in Google Scholar
 Li H., Qi Y., Jasper H., Preventing age-related decline of gut compartmentalization limits microbiota dysbiosis and extends lifespan, Cell Host Microbe, 2016, 19(2), 240-253. Search in Google Scholar
 Ren F., Shi Q., Chen Y., Jiang A., Ip T., Jiang H., et al., Drosophila Myc integrates multiple signaling pathways to regulate intestinal stem cell proliferation during midgut regeneration, Cell Research, 2013, 23, 1133-1146 Search in Google Scholar
 Bina S., Wright V.M., Fisher K.H., Milo M., Zeidler M.P., Transcriptional targets of Drosophila JAK/STAT pathway signalling as effectors of haematopoietic tumour formation, EMBO Rep, 2010, 11(3), 201-207 Search in Google Scholar
 Jiang H., Patel P.H., Kohlmaier A., Grenley M.O., McEwen D.G., Edgar B.A., Cytokine/Jak/Stat signaling mediates regeneration and homeostasis in the Drosophila midgut, Cell, 2009, 137, 1343-1355 Search in Google Scholar
 McNeill H., Craig G.M., Bateman J.M., Regulation of neurogenesis and epidermal growth factor receptor signaling by the insulin receptor/target of rapamycin pathway in Drosophila, Genetics, 2008, 179, 843-853 Search in Google Scholar
 McEwen D.G., Peifer M., Puckered, a Drosophila MAPK phosphatase, ensures cell viability by antagonizing JNK-induced apoptosis, Development, 2005, 132(17), 3935-3946 Search in Google Scholar
target of rapamycin
Janus kinase and signal transducer and activator of transcription
epidermal growth factor receptor
intestinal stem cell
suppressor of hairless
tuberous sclerosis complex
capillary feeding assay
reactive oxygen species
Drosophila insulin-like peptide
target of brain insulin
4E binding protein
© 2017 Olha M. Strilbytska et al.
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