Bombesin receptor subtype‐3‐expressing neurons regulate energy homeostasis through a novel neuronal pathway in the hypothalamus

Abstract Objectives Bombesin receptor subtype‐3 (BRS‐3) has been suggested to play a potential role in energy homeostasis. However, the physiological mechanism of BRS‐3 on energy homeostasis remains unknown. Thus, we investigated the BRS‐3‐mediated neuronal pathway involved in food intake and energy expenditure. Materials and Methods Expression of BRS‐3 in the rat brain was histologically examined. The BRS‐3 neurons activated by refeeding‐induced satiety or a BRS‐3 agonist were identified by c‐Fos immunostaining. We also analyzed expression changes in feeding‐relating peptides in the brain of fasted rats administered with the BRS‐3 agonist. Results In the paraventricular hypothalamic nucleus (PVH), dorsomedial hypothalamic nucleus (DMH), and medial preoptic area (MPA), strong c‐Fos induction was observed in the BRS‐3 neurons especially in PVH after refeeding. However, the BRS‐3 neurons in the PVH did not express feeding‐regulating peptides, while the BRS‐3 agonist administration induced c‐Fos expression in the DMH and MPA, which were not refeeding‐sensitive, as well as in the PVH. The BRS‐3 agonist administration changed the Pomc and Cart mRNA level in several brain regions of fasted rats. Conclusion These results suggest that BRS‐3 neurons in the PVH are a novel functional subdivision in the PVH that regulates feeding behavior. As the MPA and DMH are reportedly involved in thermoregulation and energy metabolism, the BRS‐3 neurons in the MPA/DMH might mediate the energy expenditure control. POMC and CART may contribute to BRS‐3 neuron‐mediated energy homeostasis regulation. In summary, BRS‐3‐expressing neurons could regulate energy homeostasis through a novel neuronal pathway.


| INTRODUCTION
The central nervous system (CNS) plays an important role in energy homeostasis. The disruption of this system leads to the dysregulation of feeding behavior and energy metabolism, which is associated with obesity. Obesity contributes to the development of a large number of diseases, including diabetes mellitus, hypertension, coronary heart disease, and certain types of cancer and hence remains an important social problem (Guh et al., 2009;Pi-Sunyer, 2009). Although several anti-obesity agents are in the market, their efficacy is limited (Butsch, 2015). Therefore, research on CNS-acting drugs with regulatory effects on both feeding behavior and peripheral energy metabolism could represent a potential strategy for developing potent anti-obesity drugs.
Bombesin-like peptides are known to serve as neuromodulators in the CNS. Bombesin was originally isolated from the skin of frogs as a 14-amino acid peptide with smooth muscle contraction activity; thereafter, several bombesin-like peptides were identified from various species. There are two such peptides in mammals, gastrinreleasing peptide (GRP) and neuromedin B (NMB), which bind to the G-protein-coupled receptors (GPCR) GRP receptor (GRPR) and NMB receptor (NMBR), respectively (Corjay et al., 1991;Ramos-Álvarez et al., 2015;Whitley, Moore, Giraud, & Shulkes, 1999). Bombesin receptor subtype-3 (BRS-3) was identified as the third member of this GPCR subfamily, based on sequence similarity, and was found to be expressed in the CNS (Fathi et al., 1993). However, despite the sequence similarity, BRS-3 does not have a high affinity with any known bombesin-like peptides, and its natural ligand remains unknown (Ramos-Álvarez et al., 2015). Although the physiological function of BRS-3 remains unclear, BRS-3-deficient (Brs-3 -/Y ) mice are known to develop obesity and have a reduced metabolic rate and increased food intake (FI) (Guan et al., 2010;Ladenheim et al., 2008;Ohki-Hamazaki et al., 1997;Yamada, Santo-Yamada, Wada, & Wada, 2002). Several nonpeptide BRS-3 agonists with an antiobesity effect in animals have recently been developed (Chobanian et al., 2012;Guan et al., 2010Guan et al., , 2011Lateef, Abreu-Vieira, Xiao, & Reitman, 2014;Matsufuji et al., 2015;Metzger et al., 2010;Sebhat et al., 2011). In particular, Guan et al. reported that Bag-1, a potent and selective small-molecule BRS-3 agonist, increased the fasting metabolic rate and body temperature and reduced the FI and body weight (BW) in mice. These results suggest that BRS-3 might serve as an attractive drug target for treating obesity .
Feeding and energy metabolism are regulated by the neural network of central and peripheral nervous systems, in which the hypothalamus plays a central role. The neurons in the hypothalamus receive inputs from the periphery through the brain stem and from the midbrain and cortex, which subsequently integrate the information associated with the energy status (Waterson & Horvath, 2015).
Recently, we reported a novel selective nonpeptide BRS-3 agonist, compound-A (Nio et al., 2017). Compound-A shows clear anorectic effects and enhanced energy expenditure in rats, suggesting that this compound is an useful tool compound to assess the function of BRS-3 neurons. Thus, in this study, we examined the hypothalamic neural network underlying the BRS-3-expressing neuron-mediated energy homeostasis regulation using compound-A.

| Animals
F344/Jcl rats (male, 5-week-old) were purchased from CLEA Japan (Tokyo, Japan). They were fed on a high-fat diet (HFD; D12451: Research Diets, NJ, USA) from the age of 5 weeks to achieve dietinduced obesity (DIO). Male Sprague Dawley (SD) rats were also purchased from CLEA Japan. Melanin-concentrating hormone receptor-1-deficient (Mchr1 −/− ) mice were originally established through the targeted disruption of exon 2 of the Mchr1 gene and backcrossed to a C57BL/6J background for four times with a speed congenic system. All animals were maintained at an appropriate temperature (23-25°C) under a 12-hr light and dark cycle (7:00-19:00 for rats, 7:30-19:30 for Mchr-1 −/− mice). All the animal experiments were conducted in compliance with a protocol that was reviewed by the Institutional Animal Care and Use Committee of Takeda Pharmaceutical Company Limited.

| In vitro agonistic activity
With regard to functional assays, the agonist-induced mobilization of intracellular Ca 2+ was measured in CHO-K1 cells that overexpressed BRS-3 using an aequorin bioluminescence assay (duplicate experiments).

| Pharmacokinetic parameters of compound-A in SD rats
To determine the pharmacokinetic parameters of compound-A, male 8-week-old SD rats (n = 3) were orally administered certain compounds (1 mg/kg BW/5 ml, suspended in 0.5% methylcellulose aqueous solution) via cassette dosing. The plasma compound concentrations were measured using liquid chromatography-tandem mass spectrometry.

| Measurement of FI and BW after compound-A administration in SD or DIO-F344 rats
Male SD rats (10-week-old) were divided into five groups (4-5 rats in each group) based on the nocturnal FI during the previous night and the BW on the morning of the drug administration. The SD rats were orally administered with vehicle (0.5% methylcellulose solution), compound-C (3, 10, and 30 mg/kg), or sibutramine (10 mg/kg) at 1-2 hr prior to the onset of the dark phase. FI was measured 24 hr after drug administration, whereas BW was measured 24 hr after drug administration. Two weeks before the study, DIO-F344 rats were fed on a powdery HFD and had been acclimatized to oral dosing. The DIO-F344 rats (male, 47-, 52-or 53-week-old) were divided into four or five groups (4-8 rats in each group) based on the nocturnal FI during the previous night and the BW on the morning of the drug administration. The DIO-F344 rats were orally administered with vehicle (0.5% methylcellulose solution), compound-A or compound-C (3, 10, and 30 mg/kg), or sibutramine (1 mg/kg) at 1-2 hr prior to the onset of the dark phase. FI was measured 4, 16, and 24 hr after drug administration, whereas BW was measured 24 hr after drug administration.  underwent fasting for 16 hr and were then housed individually in the metabolic chamber of an Oxymax system (Columbus Instructions, OH, USA) for acclimatization to the chamber. The rats were divided into three groups according to the BW and EE at approximately 10 a.m. for 1 hr. At 11:00 a.m., the rats were orally administered with vehicle (0.5% methylcellulose solution), compound-A (30 mg/kg), or CL316,243

| Measurement of EE after compound-A administration in fasted DIO-F344 rats
(2 mg/kg), and the heat production and respiratory exchange ratio (RER) were measured over 5 hr with 10-min interval. Data were averaged in every 30 min.

| Effect of compound-A on FI and BW in
Mchr-1 −/− mice Male Mchr-1 −/− mice and age-matched male wild-type mice (38-week-old) were individually housed and fed on a HFD (D-12451) from 6 weeks of age and were acclimatized to oral dosing for 1 week.
They were divided into three groups according to the nocturnal FI of the previous day and the BW on the morning of the day of drug administration. They were orally administered vehicle (0.5% methylcellulose solution), compound-A (100 mg/kg), or sibutramine (10 mg/kg).
The FI and BW were measured at 16 and 24 hr after drug administration, respectively.

| Histochemistry
To determine the expression of BRS-3 mRNA and feeding-related neuropeptides, brain samples from adult male SD rats (7-week-old) were examined. To identify the c-Fos-immunoreactive (ir) neurons after refeeding, brain samples were collected from adult male SD rats (11-week-old) following 48-hr fasting or 2-hr refeeding after 48-hr fasting. To indicate the c-Fos-ir neurons following compound-A administration, the DIO-F344 rats (male, 43-week-old) were orally administered vehicle (0.5% methylcellulose solution) or compound-A (30 mg/kg) at 9:00 a.m. in an ad libitum condition, and the brains were sampled 2 hr after dosing.
For single c-Fos immunohistochemistry (IHC), single in situ hybridization (ISH), and double staining for ISH and IHC, the animals were anesthetized and perfused with saline, followed by 4% paraformaldehyde, via the left cardiac ventricle, prior to brain sampling. After the brains were cryoprotected with 30% sucrose, frozen coronal sections (40 μm) were prepared with a freezing cryostat and used for staining. For double ISH, the animals were anesthetized, and the removed brains were frozen using O.C.T. compound (Sakura Finetek Japan, Tokyo, Japan) in liquid nitrogen. Coronal fresh-frozen sections were cut to 16 μm and used for staining.  After processing, the sections were mounted and then examined via light microscopy or confocal laser microscopy.

| Antibodies
Primary antibodies used in this study are listed in Table 1. We used two different anti-c-Fos antibodies, Santa Cruz Biotechnology sc-52 and sc-253. sc-52 recognized the predicted molecular weight of 62 kDa on Western blots (manufacturer's datasheet), and it has been validated for detection of nuclear-localized c-Fos proteins (Brown, Gentry, & Rowland, 1998). sc-253 was used for immunostaining after ISH reaction. This antibody recognized the predicted molecular weight of 62 kDa on Western blots (manufacturer's datasheet) and it has been validated for detection of nuclear-localized c-Fos proteins (Konsman & Blomqvist, 2005). For the marker of oxytocin-containing neuron, anti-oxytocin antibody (Calbiochem, PC226L) was used.

Staining is completely eliminated by pretreatment of antibody with
oxytocin, but no staining is detected by pre-absorption of antibody with vasopressin (manufacturer's datasheet). This antibody has been validated for detection of cell bodies and fibers in oxytocin mRNA localized nucleus, such as PVH and supraoptic nucleus (Luo, Kaur, & Ling, 2002). For the marker of AVP-containing neuron, anti-AVP antibody (Phoenix, H-065-07) was used. This antibody does not react with oxytocin (manufacturer's datasheet) and it has been validated for detection of cell bodies and fibers in AVP-mRNA localized nucleus, such as PVH and supraoptic nucleus (Yi et al., 2008).
After sections at both end of ROI were removed, two to four sections in each animal were selected and the 0.58-or 0.29-mm 2 images, including the unilateral nucleus, were acquired using Nikon ECLIPSE E800 (two images were obtained from one section). The  c-Fos-positive cells in the BRS-3 mRNA-positive neurons were calculated from the acquired 0.29-mm 2 images; two to four sections

| Quantitative reverse transcriptase (RT)-PCR of peptides in the brain regions of DIO-F344 rats after compound-A administration
DIO-F344 rats (male, 53-week-old) were orally administered vehicle (0.5% methylcellulose solution) or compound-A (30 mg/kg) at 9:00 a.m. following fasting for 16 hr or under free access food and water conditions. At 1 hr after dosing, the rats were anesthetized, and the samples including ARC, PVH, lateral hypothalamic area (LHA), or nucleus tractus solitarius (NTS) were dissected. In detail, 1 mm of brain slices was prepared using Brain Matrix (Muromachi Kicai, BS-Z 2000C, Tokyo, Japan), and then, the area including ROI was roughly dissected by razors. Total RNA was extracted using ISOGEN (Nippon Gene, Tokyo, Japan), and then, cDNA was prepared from total RNA by oligo (dT) primer using SuperScript II reverse transcriptase (Invitrogen).
Expression analysis (TaqMan RT-PCR) was performed by ABI Prism 7900HT (Applied Biosystems, MA, USA) using TaqMan ® Gene Expression Master Mix (Applied Biosystems), with the primers and probes listed in Table 2. For rat cyclophilin, predesigned primers and probe (Rn00452692_m1; Assays-on-Demand, Applied Biosystems) were used. The mRNA expression in each gene was normalized to the cyclophilin mRNA expression using ddCT method.

| Statistical analysis
Data involving more than two groups were assessed by one-way analysis of variance (ANOVA) followed by Williams test. Differences T A B L E 2 TaqMan probe and primer sequences used for the expression analysis between two groups were assessed using Student's t test or Aspin-Welch's t test. In Figure 2, statistical differences were analyzed with Student's t test or Aspin-Welch test, followed by Bonferroni's correction, for 9-time point comparisons.

| Profile of the BRS-3 agonist compounds
Compound-A is an active conformer (tR2(IC)) of BRS-3 agonist as previously reported (Nio et al., 2017). Compound-A had agonistic activity with an EC 50 value of 100 nM (95% confidence interval: 59-172 nM) as per the aequorin assay (Ca 2+ ) against rat BRS-3, but did not show agonistic action at 10 uM to human GRPR and NMBR. Compound-C is the racemate of compound-A (Nio et al., 2017) and had agonistic activity with an EC 50 value of 130 nM against rat BRS-3 (Ca 2+ ). The pharmacokinetic profile of compound-A (1 mg/kg, po) in SD rats was determined and the maximum plasma concentration (Cmax), time at which the Cmax was observed (Tmax), and bioavailability (BA) were found to be 69.1 ng/ml, 0.5 hr, and 21.7%, respectively. Our previous study revealed that the compound-C can pass the blood-brain barrier, suggesting that compound-A could pass the blood-brain barrier (Nio et al., 2017).

| Anti-obesity effect of single oral administration of compound-A in DIO-F344 rats
We examined the effect of compound-A and compound-C on the

| Distribution of BRS-3 mRNA in the adult rat brain
To demonstrate the localization of BRS-3 mRNA within the CNS, we performed an ISH study in adult SD rat brains. ISH was performed using a rat BRS-3 cRNA probe in coronal sections and showed specific signals for the antisense probe on cell cytoplasm in a wide range of CNS regions (Figure 3)  (Liu et al., 2002;Zhang et al., 2013). The data are summarized in Table 3.

| Localization of feeding-relating neuropeptides in BRS-3 mRNA-positive neurons
Neurons in the PVH and ARC secrete several neuropeptides that regulate feeding behavior, such as CRH, oxytocin, AVP, NPY, and α-MSH processed from POMC. We performed double staining of BRS-3 mRNA with these peptides in adult SD rats to demonstrate their colocalization. Oxytocin and AVP were detected using IHC, whereas CRH, NPY, and POMC were examined using ISH. In the PVH,

| Induction of c-Fos proteins in the BRS-3 mRNA-positive neurons following refeeding
Refeeding-induced satiety is considered to activate anorexigenic neurons. To clarify the BRS-3-expressing neurons involved in feeding suppression, adult SD rats were subjected to a 48-hr fasting; at 2 hr after refeeding, the distribution of c-Fos-ir neurons in the hypothalamus was evaluated using IHC. In our experimental condi-

| Induction of c-Fos proteins in BRS-3 mRNApositive neurons following single oral administration of compound-A in DIO-F344 rats
To determine the neurons activated by BRS-3 agonists, 30 mg/kg of compound-A was orally administrated to DIO-F344 rats, and the distribution of c-Fos-ir neurons was examined by IHC after 2 hr. The  T A B L E 3 Distribution of BRS-3 mRNA in the rat central nervous system of c-Fos in the BRS-3 neurons was slightly, but significantly, increased after compound-A administration (Figure 8j-l).

| Anorexigenic effect of BRS-3 agonist in Mchr1 −/− mice
A previous study showed that expression of MCH and its receptor mRNA was increased in the hypothalamus of BRS-3 -/Y mice (Maekawa, Quah, Tanaka, & Ohki-Hamazaki, 2004). Thus, the antiobesity effect of BRS-3 agonist was considered to be mediated via the MCH pathway. As we have shown the anti-obesity effect of compound-A in mice (Nio et al., 2017), compound-A can be used for mouse studies. Then, we attempted to validate this hypothesis using HFD-fed Mchr1 −/− mice and HFD-fed wild-type mice. In addition to sibutramine (10 mg/kg), single oral administration of compound-A (100 mg/kg) significantly decreased the FI and BW in both wild-type mice and Mchr1−/− mice (Figure 9a-d).

| Effect of compound-A on feeding-relating peptide gene expression in the brain of DIO-F344 rats
To clarify the pathophysiological role of BRS-3 on the FI in the rat brain, we measured the gene expression of feeding-relating peptides Pomc, Cart, Npy, Agrp, Hcrt, Pmch, Crh, Oxt, Avp, and Bdnf (listed in Table 2) in the ARC, PVH, LHA, and NTS after compound-A administration. As compound-C did not decrease the FI and BW in normal chow-fed SD rats (Figure 1a

| DISCUSSION
In this study, we examined the hypothalamic neuronal pathway underlying energy homeostasis regulation by BRS-3-expressing neurons using compound-A as a tool compound. Compound-A is a novel orally available, selective, small-molecule BRS-3 agonist that we have recently reported (Nio et al., 2017). The in vivo specificity of compound-A to BRS-3 has been shown using BRS-3-deficient mice (Nio et al., 2017). In addition to our previous study, this study showed that the single oral administration of compound-A reduced the FI and BW in DIO-F344 rats and increased the EE in fasted DIO-F344 rats (Figures 1   and 2). Thus, this compound can be used for analyzing the mechanism of BRS-3-expressing neuron-mediated energy homeostasis under single oral administration.
The PVH plays an important role in the regulation of numerous physiological processes, including feeding and energy metabolism (Seoane-Collazo et al., 2015;van Swieten et al., 2014) and is roughly divided into two parts: PVHm and PVHp. The neurons in the PVHm mainly contain oxytocin and AVP, whereas those in the PVHp mainly contain CRH and TRH; these peptides suppress feeding behavior.
TRH neurons in PVH are also reported to have orexigenic function . Previous reports have indicated that refeeding mainly activates the AVP-positive neurons in the PVHm and CRHpositive neurons in the PVHp (Timofeeva et al., 2005). However, we found that the BRS-3-expressing neurons in the PVH did not contain CRH, oxytocin, or AVP ( Figure 4). Although this is different from the previous reports that only a few CRH-and oxytocin-positive neurons overlap with BRS-3 (Bagnol & Grottick, 2008;Zhang et al., 2013), it is  (Figure 4), consistent with previous studies (Zhang et al., 2013).
In our experimental condition, refeeding did not induce c-Fos expression in the ARC ( Figure 5) and compound-A administration induced only slight c-Fos induction in BRS-3-expressing neurons ( Figure 8). As previous study showed that refeeding significantly leads to c-Fos induction in ARC (Wu et al., 2014), the lack of c-Fos induction in this study was due to the difference of experimental condition. In addition, it would be supposed that limited c-Fos induction by compound-A might be due to potent inhibitory regulation to these neurons. Hence, the involvement F I G U R E 1 0 Effect of compound-A on feeding-relating peptide gene expression in the brain of DIO-F344 rats. DIO-F344 rats under ad libitum (Ad lib) conditions were fasted for 16 hr (Fast) and then orally administered with vehicle or compound-A (30 mg/kg). At 1 hr after dosing, the brain samples were dissected and mRNA level listed in Table 2 was measured. (a-k) The expression levels of Crh (a), Oxt (b), and Avp (c) in the PVH, Pomc (d), Cart (e), Npy (f), and Agrp (g) in ARC, Pmch (h), Hcrt (i), and Cart (j) in the LHA, and Cart (k) mRNA in the NTS were measured via quantitative RT-PCR. The mRNA expression of each gene was normalized to that of cyclophilin. Results are presented as mean values ± standard deviation (n = 5-6). § p < .05 versus vehicle (Ad lib), *p < .05 versus vehicle (Fast) (Student's t test) of these BRS-3-expressing neurons in energy homeostasis remains unknown. Nonetheless, we found that the expression of Pomc, Cart, and Npy mRNA is increased in the ARC at 1 hr after compound-A administration ( Figure 9). This suggests that there are indirect pathways to activate these neurons using BRS-3 agonists. Although compound-A did not elicit significant c-Fos induction, the percentage of c-Fos in the BRS-3 neurons was slightly, but significantly, increased in the ARC ( Figure 8) (Kong et al., 2012). Moreover, Bagnol et al. reported that approximately 80% of the BRS-3-expressing neurons in the posterior ARC are GABAergic (Bagnol & Grottick, 2008). Thus, it is likely that BRS-3 neurons in the ARC may be involved in EE enhancement.
Although we focused on hypothalamic regulation by the BRS-3 agonist, there are several extrahypothalamic nuclei where BRS-3 is strongly expressed, such as in the LPB and MHb (Figure 3). LPB reportedly works as a hub that integrates signals from several brain regions to modulate feeding and BW (Wu, Boyle, & Palmiter, 2009;Wu, Clark, & Palmiter, 2012). LPB also reportedly mediates the Bagnol. et al. has reported that no BRS-3 mRNA expression is observed in the LHA MCH neurons (Bagnol & Grottick, 2008), and thus, it is likely that MCH pathway is not important for BRS-3-mediated energy regulation. These results strongly support our hypothesis that the well-known feeding-related peptides are not the primary regulators involved in the anti-obesity effect mediated by the BRS-3-expressing neurons. On the other hand, we found a significant change in the expression of Pomc mRNA in the ARC and Cart mRNA in the ARC, LHA, and NTS as a result of compound-A administration ( Figure 10).
Hence, these peptides could lead to at least partial energy regulation by BRS-3, whereas we observed a compound-A-induced increase of Npy mRNA, a potent orexigenic peptide, in ARC ( Figure 10). As it is clear that BRS-3 agonist finally exerts anti-obesity effects, this NPY increase might be a compensatory reaction. Regarding this controversy, further investigation is needed.
This study has certain limitations. As only acute responses in the CNS were examined, we cannot exclude the possibility that the chronic responses of BRS-3-expressing neurons associated with energy homeostasis would differ from our results. Moreover, even in the hypothalamus, there are areas that have not been examined, such as BRS-3-expressing neurons in the SCN. SCN may be involved in the BRS-3 agonist-mediated anti-obesity effect via circadian rhythm regulation (Nio et al., 2017). The histological assessment in this study was conducted under nonbiased condition, although the quantification method does not fully satisfy the criteria of "Stereological methods" stated in Schmitz & Hof, 2005. In detail, the ROIs in this study were not identified with the Nissl staining of adjacent sections and were not fully covered with the regularly spaced series of sections. Strict stereological method should have been applied; however, the ROIs in this study were reliably identified using the standard cytoarchitectonic and anatomical landmarks as reported in previous studies (Göktalay & Millington, 2016;Ryan et al., 2014), and the procedures including the section selection were conducted under blinded conditions.
In conclusion, the present study supposed that BRS-3-expressing neurons regulate energy homeostasis through a novel neuronal pathway in the hypothalamus. Our findings suggested that BRS-3 could be used as a marker of a novel neuronal population associated with energy homeostasis. Still, our hypothesis should be confirmed through more direct methodology, such as optogenetics and DREADD (Designer Receptors Exclusively Activated by Designer Drugs) in the future research. At present, no high-affinity endogenous ligand of BRS-3 has been identified, except for that of the BRS-3 phylogenetic subgroup in Drosophila (Ida et al., 2012;Ikeda et al., 2015;Sano et al., 2015).
Further study of the BRS-3-related neural pathway, including the deorphanization of BRS-3, is needed to determine the mechanism underlying energy homeostasis and to evaluate BRS-3 as a potential therapeutic target.

ACKNOWLEDGMENTS
We would like to thank Drs. Shoki Okuda, Yoshiyuki Tsujihata, Masakuni Noda, Tsuyoshi Maekawa, and Yukio Yamada for their advice and comments for this study. This research did not receive any specific grant from funding agencies in the public, commercial, or notfor-profit sectors.

CONFLICT OF INTERESTS
The authors declare no competing financial interests.