1. Introduction

In Drosophila, females store more fat than males [1–7]. Greater female fat storage has been observed in both mated and unmated females compared with age-matched males [1,3–7]. In flies, as in other animals, triglyceride is the main form of stored fat. Although triglyceride is present in many cell types and organs (e.g., oenocytes, glia, neurons, gut) [5,6,8–14], the majority of triglyceride is stored in an organ called the fat body [3,12,15].

In females, high levels of triglyceride in the fat body play a key role in supporting reproduction and physiology [5,16]. Indeed, triglyceride from the fat body is a key energy source for the developing embryo [17]. Increased fat storage in adult females also supports prolonged survival during nutrient deprivation compared with males [4,6]. Despite these clear benefits of greater fat storage for female fertility, excess fat accumulation in males adversely affects their reproductive output. Specifically, males carrying a mutation that promotes excess triglyceride accumulation show reduced testis size, defects consistent with delays in spermatogenesis, and ultimately a reduction in sperm number [8]. The sex difference in fat storage therefore likely reflects the fact that males and females differ in how much whole-body triglyceride storage supports optimal fertility.

Recent studies have identified genes and pathways that contribute to sex differences in fat storage [5–7,18]. In mated females, the steroid hormone ecdysone acts on neurons to promote food intake, which is associated with increased body fat [5]. In unmated adult females, the insulin/insulin-like growth factor signaling pathway (IIS) plays a key role in maintaining an elevated level of triglyceride storage compared with adult males [18]. Specifically, adult females have higher production of Drosophila insulin-like peptide 3 (Dilp3) and greater insulin sensitivity, leading to higher IIS activity. This elevated IIS activity is important for females to store more triglyceride than males, as adult-specific ablation of the insulin-producing cells reduces body fat in females but not males [18].

In males, body fat is maintained via alternative mechanisms. For example, males show higher expression and activity of two catabolic pathways that promote fat breakdown. One pathway is regulated by triglyceride lipase brummer (bmm), where males show higher bmm mRNA levels compared with females [6]. This elevated bmm expression contributes to the sex difference in fat storage by restricting triglyceride accumulation in males. Similarly, males have higher production and secretion of Adipokinetic hormone (Akh), a key lipolytic hormone in insects [7]. As with bmm, high levels of Akh in males contribute to the sex difference in fat storage by limiting fat accumulation in males [7]. Together, these studies have defined a model of the sex difference in fat storage in which females maintain higher levels of fat storage due to the anabolic action of IIS, whereas males have lower fat storage due to the catabolic effects of bmm and Akh. While some progress has been made in revealing the mechanisms underlying the sex-specific regulation of Akh and IIS [7,18], we do not have a complete understanding of the factors that determine the sex-biased regulation and function of these key metabolic factors.

Recent clues into the regulation of Akh, bmm, and IIS have emerged from studies on peptide hormone function [19–30], as several hormones influence physiology via effects on Akh- and Dilp-producing cells [19–22,24–33]. In particular, recent studies have illuminated an important role for hormones produced by the enteroendocrine (EE) cells of the gut [32,34–36]. Adult Drosophila EE cells produce and release hormones such as Allatostatin A (AstA), Allatostatin C (AstC), neuropeptide F (NPF), tachykinin (Tk), Diuretic hormone 31 (Dh31), Bursicon, CCHamide 1 (CCHa1) and CCHamide 2 (CCHa2), and short neuropeptide F (sNPF) [37–41].

EE cells are identified by expression of the homeodomain protein Prospero in adults [42,43], where EE cells that produce distinct hormones are present in anatomically defined regions of the adult gut [43]. For example, AstA- and Dh31-producing EE cells are located in the posterior midgut, whereas EE cells that produce AstC and Tk are found along the entire length of the midgut [37]. NPF-producing cells are found in the anterior and middle midgut [41]. Supporting a role for EE cell-derived hormones in regulating Akh/IIS, in fed conditions studies show EE cell-derived hormones such as Bursicon inhibit Akh secretion [28], whereas NPF enhances Dilp secretion from the insulin-producing cells [29]. During starvation, AstC promotes Akh release from Akh-producing cells to enable lipid mobilization [22]. While at least two EE cell-derived hormones have been shown to have sex-biased effects on food intake and energy mobilization [22,44], sex differences in the regulation and function of most of these hormones remain unclear. Defining potential differences in EE cells is an important task, as prior studies have revealed profound differences in gut biology between males and females.

For example, males and females differ in overall gut size and shape [45,46] and in the number of intestinal stem cell divisions [45,47–49]. The absorptive lining of the gut also shows sex differences during aging [48,50]. After mating, females show pronounced changes to gut size, function, and gene expression [45,51–54]. While sex differences in intestinal stem cells and enterocytes play a key role in mediating these differences in gut biology, we know less about male-female differences in EE cells. We therefore aimed to perform a detailed characterization of EE cell-derived hormones in males and females, and to determine the contribution of these hormones to physiology in each sex. We reveal profound sex-biased regulation of EE cell-expressed hormones AstC, Tk, and NPF: females show higher mRNA levels of these hormones in the gut than males. For at least two EE cell-derived hormones this sex-biased regulation was physiologically significant, as loss of AstC and Tk in the gut reduced fat storage in females with no effect in males. These female-specific fat storage defects were reproduced by loss of AstC and Tk receptors in neurons and/or neuropeptide-producing cells. While the specific cell type targeted by these EE cell-derived hormones to influence fat storage remains unclear, this reveals a female-specific contribution of EE cell-derived hormones in regulating body fat.

2. Results

2.1. Sex differences in expression of gut-derived peptide hormones

Given that several EE cell-derived hormones such as AstA (FBgn0015591), AstC (FBgn0032336), Tk (FBgn0037976), NPF (FBgn0027109), Dh31 (FBgn0032048) are expressed in cells outside the gut [20,55–69], we used quantitative real-time PCR (qPCR) to assess whole-body mRNA levels of genes encoding EE cell-expressed hormones. In particular, we focused on hormones known to influence whole-body fat metabolism [20,22,29,44,70,71]. In 5-day-old w¹¹¹⁸ unmated adult males and females, we found that whole-body mRNA levels of AstA, AstC, Tk, NPF, and Dh31 were significantly higher in males than in females (Figure 1A-E). To gain further insight into this sex-biased expression, we analyzed mRNA levels of these factors from isolated heads and intestines, as these are the main sites of AstA, AstC, Tk, NPF, and Dh31 production [35,37]. A significant male bias in mRNA levels was found in the head for AstA, AstC, Tk, NPF, and Dh31 (Figure 1F-1J). In contrast, mRNA levels of AstC, Tk, and NPF in isolated intestines showed a strong female bias (Figure 1K-1M). No sex bias in the expression of AstA or Dh31 was observed in the gut (Figure 1N, 1O).

Figure 1

Building on the sex bias in mRNA levels, we next examined mRNA levels of receptors that correspond to EE cell-expressed hormones with sex-biased expression in whole-body, fat body, and head samples. Whole-body mRNA levels of the receptors for AstA (AstA-R2), AstC (AstC-R2), Tk (TkR99D), NPF (NPFR), and Dh31 (Dh31-R) were significantly higher in 5-day-old w¹¹¹⁸ males compared with age-matched females (Figure S1A-E). For most peptides, the male bias was due to a higher mRNA level in the head and not the fat body (Figure S1A-E); however, TkR99D mRNA levels were higher in male fat bodies with no difference in head mRNA levels (Figure S1C). Taken together with our data on peptide mRNA levels, our data suggest sex differences exist in both the expression of EE cell-derived hormones and in the ability of tissues to respond to available peptide.

2.2. Sex determination gene transformer does not regulate sex differences in EE cell-derived peptide mRNA levels

To determine the mechanism by which these differences in mRNA levels are established, we tested a role for sex determination gene transformer (tra). Normally, a functional Tra protein is only expressed in females, where Tra specifies most aspects of female sexual development and behavior [72–75]. Indeed, ectopic Tra expression in males is sufficient to specify many aspects of female sexual development and physiology [7,47,48,73,76,77]. Because tra mRNA is detected in the gut, specifically in ISC and EE cells [47,78], we asked whether broad overexpression of Tra in neurons and/or EE cells contributes to the sex difference in mRNA levels of EE cell-expressed hormones. We found that sex differences in mRNA levels of AstA, AstC, Tk, NPF, and Dh31 were unaffected when we used either voila-GAL4 (Figure 2A-2J) or elav-GAL4 (Figure 2K-2T) to drive Tra expression in these cells. Tra expression in neurons similarly had no effect on mRNA levels of AstC-R2, TkR99D, or Dh31-R in the head (Figure S2A-C). Thus, sex determination tra does not establish sex differences in levels of the mRNAs that encode either EE cell-derived hormones or their receptors.

Figure 2

2.3. Gut-derived Tachykinin and Allatostatin C promote female fat storage

Given that EE cell-derived AstC, NPF, and Tk regulate fat storage and phenotypes associated with fat storage (e.g., starvation resistance) in single- and mixed-sex animal groups [22,29,44,71], we wanted to assess whether these hormones contribute to the sex difference in fat storage. We used RNAi to knock down levels of AstC, Tk and NPF with GAL4 drivers targeting these specific EE populations (AstC-GAL4, Tk-GAL4, and NPF-GAL4, respectively). Importantly, GAL4 activity for each driver line was restricted to the gut using R57C10-GAL80, a validated approach to target only the gut cells that produce these hormones [22,44,71]. We found gut-specific loss of AstC (genotype AstC-GAL4>UAS-AstC-RNAi, R57C10-GAL80) caused a significant reduction in body fat in females with no effect in males (Figure 3A). Gut-specific knockdown of Tk (genotype Tk-GAL4>UAS-Tk-RNAi, R57C10-GAL80) similarly showed a trend toward a female-specific decrease in fat storage (GAL4 control p=0.1109; UAS control p=0.0118), with no significant effect on male body fat (Figure 3B). In contrast, gut-specific loss of NPF (NPF-GAL4>UAS-NPF-RNAi, R57C10-GAL80) did not significantly alter whole-body fat storage in either males or females (Figure 3C). Together, these data suggest that the female-biased expression of AstC and Tk in the gut play physiologically significant roles in regulating fat storage in females.

Figure 3

2.4. Allatostatin C receptor and Tachykinin receptor in neurons promote fat storage in females but not males

Neurons and neuropeptide-producing cells are key cell types upon which AstC, Tk, and NPF act to influence physiology [22,29,32,71,79,80]. We therefore predicted that loss of AstC-R2 and TkR99D in these cells would reproduce the reduced fat storage we observed in females with loss of EE cell-derived AstC and Tk. To test this, we used elav-GAL4 to knock down AstC-R2 and TkR99D in post-mitotic neurons and neuropeptide-producing cells. In females, loss of AstC-R2 in neurons and neuropeptide-producing cells caused a significant decrease in body fat, with no effect in males (Figure 4A). This reproduced the body fat phenotype caused by loss of gut AstC. A similar female-specific reduction in fat storage was observed with loss of TkR99D in neurons and neuropeptide-producing cells (Figure 4B), reproducing the fat storage phenotype of females with loss of gut-derived Tk. In line with the lack of body fat effect due to loss of gut-derived NPF, we saw no significant change in fat storage in either males or females with loss of NPFR in neurons and neuropeptide-producing cells (Figure 4C). Together, these data suggest that AstC and Tk may promote whole-body fat storage in females via effects on neurons and neuropeptide-producing cells.

Figure 4

To narrow down the neurons and neuropeptide-producing cells in which AstC-R2 and TkR99D act to mediate their effects on female fat storage, we used cell-type-specific GAL4 drivers to overexpress RNAi transgenes directed at these genes. Given that these gut-derived peptides have been shown to influence metabolic homeostasis and feeding via effects on the insulin-producing cells (IPCs) and the Akh-producing cells (APCs) [19,22,67], we first knocked down AstC-R2 and TkR99D in these cells. We used dilp2-GAL4 to drive expression of UAS-Astc-R2-RNAi and UAS-TkR99D-RNAi in the IPCs, and Akh-GAL4 to drive expression of these transgenes in the APCs. Loss of AstC-R2 in the IPC had no significant effect on fat storage in either males or females compared with sex-matched controls (Figure 4D). IPC-specific loss of TkR99D, on the other hand, caused a significant increase in whole-body fat storage in males (Figure 4E) with no change in females. In the APC, loss of neither receptor altered fat storage in males or females (Figure 4F, 4G). Thus, while Tk normally restricts male body fat via effects in the IPC, AstC and Tk promote female body fat via neurons other than the IPC and APC.

3. Discussion

EE cell-derived hormones regulate body fat in single- and mixed-sex animal groups; however, it has been unclear whether the regulation and function of these peptides differ between the sexes. The goal of our study was to perform a detailed comparison of EE cell-derived hormones between the sexes, and to test if these hormones contribute to the sex difference in fat storage. Our assessment revealed profound female-biased expression of EE cell-expressed hormones within the gut. This differential expression was physiologically significant, as we showed that EE cell-derived Tk and AstC promote female fat storage. Taken together, our data provide additional insight into the mechanism(s) by which unmated female flies achieve higher fat storage than male flies.

While it was not the main goal of our study, our survey of EE cell-expressed hormones in Drosophila revealed that the sex bias in expression was not uniform across tissues. In the gut, mRNA levels of AstC, Tk, and NPF were higher in females than in males. In the brain, mRNA levels of these hormones showed a significant male bias, in line with data from previous reports on Tk [56] and NPF [63]. While it remains unclear whether the tissue-specific sex bias in expression is physiologically significant, peptides derived from the gut and the brain have been shown to mediate distinct effects on physiology and/or behavior. For example, EE cell-derived AstC regulates energy homeostasis and food-seeking behaviors in adult females [22], whereas neuron-derived AstC is involved in regulating locomotion in adult males [59] and the circadian regulation of oogenesis in adult females [69]. Neuron-derived Tk similarly regulates locomotion [64,68], food consumption [67], Dilp secretion [19], and aggression [56], whereas gut-derived Tk regulates intestinal lipogenesis [71] and stem cell divisions in the midgut [81–83]. Supporting a potential sex-specific role for peptides derived from different anatomical sites in regulating physiology, gut-derived AstC stimulates fat breakdown during starvation through the Akh pathway in mated females with no effect in males [22]. Future studies are therefore needed to determine whether there are sex differences in whether the effects of EE cell-derived hormones are primarily mediated by local or systemic mechanisms.

Another important task for future studies will be to elucidate how sex differences in neuropeptide expression are established. The first step in understanding these mechanisms will be to determine which factors specify the sex bias in neuropeptide mRNA levels. Because our data shows that sex determination gene tra does not regulate the sex bias in neuropeptide expression in either the brain or the gut, the role of other factors that influence sexual identity and sexual differentiation must be assessed. One strong candidate is the steroid hormone ecdysone, as virgin females have higher ecdysone titers than males [84–86]. Ecdysone plays a role in regulating sexual differentiation and development [5,87,88], and contributes to male-female differences in multiple aspects of intestinal physiology (e.g., intestinal stem cell proliferation) [45,49] and brain development [89,90]. Another candidate is juvenile hormone, which has been shown to regulate sexual maturation in Drosophila and other insects [91–97]. While it remains unclear whether juvenile hormone titers differ between virgin males and females, juvenile hormone regulates many aspects of gut physiology in mated females (e.g., intestinal lipid accumulation, ISC proliferation) [53,98] and influences brain development [99]. Other than hormones, it is possible that sex determination gene Sex-lethal plays a role in regulating the sex difference in mRNA levels of EE cell-derived hormones, as tra-independent effects of Sex-lethal have been described in the brain [100].

In parallel to identifying the factor(s) responsible for establishing the sex difference in EE cell-expressed hormones, it will be important to reveal the cellular basis for this differential expression. For example, a sex difference in the number of EE cells and neuropeptide-expressing cells in the brain could explain the differences in expression. Supporting this, gut length, overall brain size, and neuron number have been shown to differ between males and females [34,47,101–108]. In the gut, the difference in length is at least partially due to a sex difference in the proliferation of intestinal stem cells [45,47,48], which undergo asymmetric divisions and subsequent differentiation to generate all gut cell types including EE cells [34,42,109–113].

In the brain, males and females differ in the number of neurons found within many identified clusters [101,102,104–108,114], including the cells that produce NPF [63] and Tk [56]. Differences in neuron number have been primarily attributed to sex-specific programmed cell death [107,114–117]; however, sex differences in neuroblast cell death and/or proliferation may also play a role [106,118–120].

Beyond the effects of cell number, sex differences in EE cell-derived hormone mRNA levels may also be due to differential gene and/or protein expression of these factors, which have been reported for other peptide hormones [7,18,63]. Because sex differences in the activity of peptide hormone-producing cells have also been previously described [7], it is clear that a detailed examination of sex differences in EE cells, and more generally in neuropeptide-producing cells, is needed to gain a comprehensive picture of how these cells differ between males and females. Benefits of such a detailed study include gaining insight into potential mechanisms underlying sex differences in other aspects of physiology and behavior. For example, EE cells regulate ISC homeostasis [81,121], and EE cell-derived hormones act locally and systemically to regulate appetite, food ingestion, food digestion, gut motility, and immune responses [35,80,122,123]. Importantly, male-female differences in many of these phenotypes have been reported [51,52,124–127].

Overall, our findings identify EE cell-derived hormones AstC and Tk as important factors that promote higher fat storage in Drosophila adult virgin females. This builds on a recent paper identifying a key role for IIS in promoting higher levels of fat storage in unmated females but not males [18], advancing knowledge of the factors that establish an optimal level of stored fat in each sex.

4. Materials and methods

4.1. Fly strains

The following fly strains from the Bloomington Drosophila Stock Center were used: w¹¹¹⁸ (#3605), voila-GAL4 (#80572), dilp2-GAL4 (IPCs) (#37516), UAS-tra^F (#4590), UAS-NPF-RNAi (#27237), UAS-TkR99D-RNAi (#27513), UAS-AstC-RNAi (#25868), UAS-NPFR-RNAi (#25939), UAS-AstC-R2-RNAi (#36888), UAS-Tk-RNAi (#25800), elav-GAL4 (#458). We obtained R57C10-GAL80; NPF-GAL4, R57C10-GAL80; Tk-GAL4 and R57C10-GAL80; AstC-GAL4 as kind gifts from Dr. Kim Rewitz at the University of Copenhagen, and Akh-GAL4 was a kind gift from Dr. Mike Gordon at The University of British Columbia.

4.2. Fly husbandry

Fly media was prepared with the following ingredients: 20.5 g/L sucrose, 70.9 g/L D-glucose, 48.5 g/L cornmeal, 45.3 g/L yeast, 4.55 g/L agar, 0.5 g/L CaCl₂•2H₂O, 0.5 g/L MgSO₄•7H₂O, 11.77 mL/L acid mix (propionic acid/phosphoric acid). For all experiments, we allowed female flies to lay eggs on grape juice agar plates for 12 hr. At 24 hr after egg laying, 50 larvae were picked into vials containing 10 mL of food and reared at 22°C. Males and females were distinguished by the presence of sex combs in the late pupal period and placed into single-sex vials to eclose. After eclosion, adult flies were maintained at a density of twenty flies per vial in single-sex groups. Unless otherwise stated, all experiments used 5-to 7-day-old unmated flies.

4.3. Adult weight

Groups of 10 flies were placed in pre-weighed 1.5 ml microcentrifuge tubes (Diamed Lab Supplies, DIATEC610-2550) and weighed on an analytical balance (Mettler-Toledo, ME104).

4.4. Whole-body triglyceride measurements

One biological replicate consisted of five flies. Flies were collected in a 1.5 ml tube and homogenized in 350 μl of 0.1% Tween (Amresco, 0777-1L) in 1X phosphate-buffered saline (PBS; Sigma-Aldrich, P5493) using 50 μl of glass beads (Sigma-Aldrich, Z250473) that were agitated at 8 m/s for 5 s (OMNI International BeadRuptor 24).

Triglyceride concentration was measured using the Stanbio Triglyceride Liquid Reagent (FT7610, BD386a/d, BD386b) according to the manufacturer’s instructions and as described previously [13,18] with minor modifications. Briefly, 10 μl of either homogenate or triglyceride standard (FT7610) was added to 190 μl of activated triglyceride reagent (Enzymatic Triglyceride Reagent, BD386a/d; Triglyceride Activator, Cat. No. BD386b) in a 96-well plate. After a 15 min incubation at room temperature, the absorbance was read at 540 nm (Thermo Scientific – Multiskan FC Microplate Photometer).

4.5. RNA extraction, cDNA synthesis, and Quantitative real-time PCR (qPCR)

One biological replicate consisted of 3-5 adult fly guts, or 10 adult fly heads, or 5 whole-body adult flies. Samples were homogenized in 500 μl Trizol (Thermo Fisher Scientific; 15596018). Chloroform was added to Trizol to separate the mixture into aqueous and organic phases, and isopropanol was added to the aqueous phase (in a fresh tube) to precipitate the RNA. RNA was resuspended in either 20-25 μl (for guts and heads) or 200 μl (for whole-body) of molecular biology grade water (Corning, 46-000-CV). RNA was stored at -80°C until use. Each experiment contained 5-10 biological replicates per sex and per genotype; each experiment was repeated twice.

For genomic DNA elimination and cDNA synthesis, an equal amount of RNA per reaction was DNase-treated and reverse transcribed according to manufacturer’s instructions using the QuantiTect Reverse Transcription Kit (Qiagen, 205314). Relative mRNA transcript levels were quantified using qPCR as described previously [6]. Data were normalized to the average fold change of Actin5C and β-tubulin. For a full primer list, refer to Document S1.

4.6. Statistical analysis

Statistical analyses and data presentation were completed using GraphPad Prism 9 (GraphPad Software, San Diego, CA, USA). All data were tested for normality using the Shapiro-Wilk test. Normally-distributed data were subjected to parametric tests as appropriate, including Student’s t-test and two-way ANOVA followed by Bonferroni post-hoc test. For non-normally distributed data, we used the Mann-Whitney test. For two-way ANOVA involving data that do not satisfy the normality assumption, aligned rank transformation was first applied using the art() function from the ARTool R package [128]. Then, ANOVA was performed on the transformed data with the base R anova() function. Finally, the art.con() function from the ARTool package was used to extract the main as well as the interaction effects. Default parameters were used in each step of the analysis. For all statistical analyses, differences were considered significant if p<0.05.

Data availability

All data generated or analyzed during this study are included in the manuscript and supporting files.

Acknowledgements

The authors thank FlyBase, which is supported by a grant from the National Human Genome Research Institute at the U.S. National Institutes of Health (U41 HG000739) and by the British Medical Research Council (MR/N030117/1). Stocks obtained from the Bloomington Drosophila Stock Center (NIHP40OD018537) were used in this study. The authors thank the TRiP at Harvard Medical School (NIH/NIGMS R01-GM084947) for providing transgenic RNAi fly stocks and/or plasmid vectors used in this study. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. We thank members of the Rideout lab for valuable feedback. We acknowledge that our research takes place on the traditional, ancestral, and unceded territory of the Musqueam people; a privilege for which we are grateful.

Additional information

CRediT authorship contribution statement

Puja Biswas

Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Visualization, Writing – original draft.

Elizabeth J. Rideout

Conceptualization, Funding acquisition, Methodology, Resources, Supervision, Writing – original draft, Writing – review & editing.

Funding

This study was supported by operating grants to EJR from the Canadian Institutes for Health Research (PJT-153072 and PJT-183786), CIHR Sex and Gender Science Chair program (GS4-171365), Michael Smith Foundation for Health Research (16876), and the Canada Foundation for Innovation (JELF-34879). PB was supported by a 4-year CELL fellowship from UBC.

Funding

Canadian Institutes of Health Research (CIHR) (PJT-153072)

• Elizabeth Rideout

Canadian Institutes of Health Research (CIHR) (PJT-183786)

• Elizabeth Rideout

Canadian Institutes of Health Research (CIHR) (GS4-171365)

• Elizabeth Rideout

Michael Smith Health Research BC (MSFHR) (16876)

• Elizabeth Rideout

Canada Foundation for Innovation (CFI) (JELF-34879)

• Elizabeth Rideout

Additional files

Document S1. Supplemental Figures S1-S4.

Table S1. Excel file containing raw data with calculations.

Table S2. Excel file containing statistics for all data.