GYY4137 Promotes Mice Feeding Behavior via Arcuate Nucleus Sulfur-Sulfhydrylation and AMPK Activation

Hydrogen sulfide (H2S) is an endogenous gaseous molecule and plays important biological and neurochemical roles in many processes such as the neural activity and immunity. The arcuate nucleus (ARC) of hypothalamus is a control center for appetite and energy metabolism. AMPK is a gage kinase in the monitoring of energy status and regulation of energy metabolism, and it can be activated by H2S via CaMKKβ/AMPK pathway. But the role of H2S in ARC and appetite has not been reported. Here we studied the orexigenic effect of H2S and the mechanisms by means of GYY4137, a water soluble and slow-releasing donor of H2S, and protein sulfur-sulfhydrylation analysis. We demonstrated that GYY4137-derived H2S increased food intake of mice, augmented the production of neuropeptide Y (NPY), and elevated the protein sulfur-sulfhydrylation level and the activation of AMPK and CaMKKβ in ARC. Blocking sulfur-sulfhydrylation with DTT eliminated GYY4137-induced activation of AMPK and CaMKKβ. DTT and preventing AMPK activation in ARC with Compound C and Ara-A could both attenuate the orexigenic effect of GYY4137. These findings suggest that H2S enhances appetite through protein sulfur-sulfhydrylation and the activation of AMPK and NPY function in ARC.


INTRODUCTION
Hydrogen sulfide (H 2 S) is produced endogenously in mammals by metabolism of sulfurcontaining amino acids under the catalysis of special enzymes (Predmore et al., 2012;Sheibani et al., 2017) and has been regarded as the third bio-active gas or gaseous signaling molecule by accumulated evidence (Gadalla and Snyder, 2010;Predmore et al., 2012;Yu et al., 2017). H 2 S plays important physiological and pathological roles in many biological processes and acts as a critically functional regulator in several brain regions, such as the hippocampus and cerebral cortex (Giuliani et al., 2013;Yakovlev et al., 2017;Zhu et al., 2017). The arcuate nucleus (ARC) of hypothalamus is an important control center for appetite and energy metabolism (Joly-Amado et al., 2014;Reis et al., 2015). The effect of H 2 S on ARC and feeding behavior has not been explored.
Adenosine 5 -monophosphate (AMP)-activated protein kinase (AMPK) is an important modulator and gage molecule in energy metabolism and food intake (Oh et al., 2016;Dong et al., 2017). By sensing the available level of energetic substrates, AMPK determines the activation or suppression of orexigenic neurons such as neuropeptide Y (NPY) neurons and controls the production of NPY in ARC, the appetite regulation center in hypothalamus, and thus regulates feeding behavior (Blankenship et al., 2016;Carneiro et al., 2016). Ca 2+ /calmodulin-dependent protein kinase β (CaMKKβ) is a vital upstream activator of AMPK (Anderson et al., 2008), and it has also been reported that AMPK-mediated feedback phosphorylation of CaMKKβ regulates the CaMKKβ/AMPK signaling cascade and may be physiologically important for intracellular maintenance of Ca 2+ -dependent AMPK activation by CaMKKβ (Nakanishi et al., 2017).
As an endogenous signaling molecule, H 2 S exerts pivotal functions in many aspects, including immunity, metabolism, cardiovascular, and neural activity (Gadalla and Snyder, 2010;Gong et al., 2010;Predmore et al., 2012;Weber et al., 2016). Studies have reported that H 2 S elevates intracellular calcium level through glutamate receptor (Lee et al., 2006;Zhang and Bian, 2014) and increases AMPK activity (Lee et al., 2012;Zhou et al., 2014). By promoting AMPK activation, H 2 S exerts various physiological functions such as antiinflammatory and cytoprotective effects. It is reported that CaMKKβ-dependent activation of AMPK is critical to the effects of H 2 S on neuroinflammation suppression and insulin sensitivity enhancement Chen et al., 2017;Wang et al., 2017). However, there is no report about the role of H 2 S-induced activation of AMPK in feeding behavior.
In the present study, we reported the orexigenic effect of GYY4137, a water soluble and slow-releasing donor of H 2 S, on mice and the underlying mechanism. We found that this effect of H 2 S is mediated by the sulfur-sulfhydrylation of ARC proteins, including AMPK and CaMKKβ, which subsequently increases the activation of AMPK and NPY function in ARC and food intake of mice.

Animals
Male C57BL/6J mice (aged 8-10 weeks) were used for the experiments in this study. Mice were singly housed in standard cages with a small PVC pipe for environmental enrichment. The colony room was maintained at constant temperature (22 ± 1 • C) and humidity (60-70%) on a controlled 12-h light/dark cycle (lights on at 20:00). All experimental manipulations and tests were carried out during the dark cycle. Laboratory standard food for mice and water were available ad libitum in home cages. All experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the U.S. National Institutes of Health. The adult and neonatal mice were obtained from Laboratory Animal Center of Anhui Medical University. The use of animals for all experimental procedures was approved by the Animal Welfare Committee of Anhui Medical University.

Feeding Experiment
We investigated the effect of GYY4137, a water soluble and slow-releasing donor of H 2 S (Szabo and Papapetropoulos, 2017) on feeding behavior of mice. The mice were divided into five dosage groups and their food intake was detected (measured by weighing the consumption of food for mice) at 2 h after the intraperitoneal (i.p.) injection of GYY4137 (10% in saline, w/v). Then, under the treatment of GYY4137 at a final concentration of 150.4 mg/kg, the cumulative food consumptions of mice were measured at 1, 2, 4, and 8 h after drug administration. To explore the blocking effect of DTT, Compound C, and Ara-A on the orexigenic effect of GYY4137, mice were treated with the blocking drugs through intra-lateral ventricle (i.c.v.) injection 10 min before the administration of GYY4137. The mice in the control groups were all administrated with 0.9% saline, vehicle of the drugs. Food consumptions of mice treated with GYY4137 (100 nmol per mouse) via i.c.v. injection was also measured at 2 h after the administration. As for the detection and comparison of the S-sulfhydrylation levels in the ARC of the normal feeding and food restricted mice, the mice in food restricted group were fasted for 12 h, and then the ARC tissues were isolated for S-sulfhydrylation assay.

Surgical Procedure and Microinjection
The cannula was implanted according to stereotaxic mouse brain atlas (Paxinos and Franklin, 2012) with some modifications. Mice were anesthetized with chloral hydrate (350 mg/kg, i.p.) and placed in a stereotaxic apparatus (Stoelting) with the bregma and posterior on the same level. The body temperature was maintained at 37.0 • C by an electric incandescent lamp. A small hole was drilled into the bone to insert a cannula into the lateral ventricle (LV, 0.7 mm posterior to the bregma, 1.0 mm lateral to the midline, 2.2 mm vertical from the cranial theca). A stainless steel cannula (length 6 mm, OD 0.5 mm) was implanted into the place according to the coordinates located above. The cannula was fixed to the skull with the aid of dental acrylic resin and dental cement. Cannula placement was confirmed by analyzing the brain slices of mice cannula-implanted under magnifying glass after perfusion, fixation, and slicing (thickness 50 µm) of the brain. The experiments began after a 5-day recovery period from surgery. The drugs were injected into the lateral ventricle with a microsyringe (2 µL) connected by a PE-10 polyethylene tubing (10 cm) to a needle (OD 0.2 mm), which was introduced into the lateral ventricle through the cannula fixed to the head of mouse. The injection volume was set to 2 µL within a period of 2 min.

Preparation of ARC Neurons
Primary cultures of hypothalamic ARC neurons were prepared as previously described with some modifications (Chen et al., 2012;Wu et al., 2013;Palomba et al., 2015). Briefly, C57BL/6J mice, 1-2 days postnatal, were humanely killed by decapitation. The ARC tissue was quickly dissected and transferred to PBS-buffered solution containing 135 mM NaCl, 5 mM KCl, 1 mM CaCl2, 1 mM MgCl2, pH 7.3, and finely chopped. Then the ARC tissues were treated with 0.125% trypsin in PBS-balanced solution for 20 min at 37 • C and gently triturated using flame-polished Pasteur pipettes. Cell suspension was centrifuged for 7 min at 1000 g, and then the cell pellets were resuspended in the Dulbecco's modified Eagle's medium (DMEM) and F-12 supplement (1:1) with 10% fetal bovine serum before plating onto glass-bottomed dishes (MatTek) coated with poly-L-lysine (20 µg/mL for 1-2 h) and kept at 37 • C in 5% CO 2 incubator. After overnight incubation in DMEM, the medium was changed to neurobasal medium (Gibco) containing 15 mmol/L glucose supplemented with 2% B27, 2 mM glutamine, 10 µg/mL penicillin, and 10 µg/mL streptomycin. The ARC neurons were fed with fresh medium every 48 h. Microscopically, glial cells were not apparent in cultured cells employing this protocol. The neurons were maintained for 6-8 days in primary culture until used for subsequent cell experiments.

Immunofluorescence Assay
After treatment, the mouse was anesthetized with chloral hydrate and continuously perfused through the left ventricle with 4% polyformaldehyde for 0.5-1 h. Then the whole brain was isolated and immersed into 4% polyformaldehyde for 24 h. The brain was sliced using oscillating slicer (Leica) and the brain slices were rinsed with 10 mM PBS. Then the slices were treated by permeabilization with 10 mM PBS containing 0.3% Triton X-100 (v/v) for 30 min, and then blocked with 3% BSA-PBS (w/v) for 1 h, and incubated with 1:50 anti-c-Fos and 1:50 anti-NeuN antibodies in 10 mM PBS containing 0.3% Triton X-100 and 1% BSA and 2% goat serum (v/v) overnight at 4 • C. After washes with PBS, slices were incubated with 1:100 Rhodamine-conjugated goat anti-rabbit and FITC-conjugated goat anti-rat secondary antibodies in 10 mM PBS containing 0.3% Triton X-100 and 1% BSA and 2% goat serum for 1 h at room temperature. Finally, the slices were mounted on glass slides with 30% glycerin, and visualized and captured with a fluorescent microscopy (Olympus BX43, Japan) for the expression and co-localization of c-Fos and neuronal marker NeuN. The fluorescence quantification software is Scion Image 4.0 (Scion Inc., Fredrick, MD, United States). We took the c-Fos/NeuN ratio of their fluorescence intensity of each single cell as data to make comparison between the vehicle and GYY-treated groups.

Measurement of NPY Content
The procedure for NPY content detection was executed as our previous study (Wu et al., 2013) with minor modification. After treatment for 1 h, mice were sacrificed by decapitation under 10% chloral hydrate anesthesia, and the entire ARC tissue was isolated from the brain. To minimize the deviation of operationinduced stimulation to the mice, we gave the control group with equal volume of saline injections and get the sample preparations for detection both at 1 h after treatment. Isolated tissues were washed twice with ice-cold PBS and then lyzed on ice in extraction buffer containing 50 mM Tris base (pH 7.4), 100 mM NaCl, 1% NP-40 (v/v), 10 mM EDTA, 20 mM NaF, 1 mM phenylmethylsulfonyl fluoride, 3 mM Na 3 VO 4 , and protease inhibitors. The homogenates were centrifuged at 12,000 g for 15 min at 4 • C. Supernatant was separated and stored at −80 • C until use. Protein concentration was determined using the BCA protein assay kit (Pierce Biotechnology, Inc.), and NPY content was measured by a commercial enzyme-linked immunosorbent assay kit (R&D Systems, Minneapolis, MN, United States) according to the manufacturer's protocol. NPY content was expressed as picogramas per microgram of protein.

Western Blot Assay
Western blot assay was processed as our previous studies with minor modifications (Li et al., 2016;Hou et al., 2017). The sample preparations for western blotting were lyzed solutions of the whole cells or tissue which include nuclear and cytosolic fractions. ARC tissues isolated from the brain of mice after treatment were homogenized rapidly in icecold lysis buffer containing protease and phosphatase inhibitors (as above) and centrifuged for 15 min (12,000 g, 4 • C). The supernatant fraction was collected and stored at −80 • C for western blot assay. Protein concentration was examined by BCA assay and 30 µg of protein sample was loaded in each lane for comparison, with GAPDH as loading control. Following SDS-PAGE electrophoresis, the proteins were transferred to polyvinylidenedifluoride membrane. After blocking with 5% BSA in Tris-buffered saline containing 0.1% Tween-20 (v/v) for 1 h at room temperature, transferred membranes were incubated overnight at 4 • C with appropriately diluted primary antibodies against each protein below: anti-c-Fos (1:1000), anti-NPY (1:800), anti-phospho-AMPKα (Thr172; 1:500), anti-AMPKα (1:1000), anti-phospho-CaMKKβ (Ser458/495; 1:500), anti-CaMKKβ (1:1000), and anti-GAPDH (1:4000). Membranes were then incubated with HRP-conjugated secondary antibodies (anti-rabbit 1:2000, anti-rat 1:3000) for 1 h at room temperature before signals were visualized using the SuperSignal West Pico ECL kit (Thermo Scientific, Rockford, IL, United States). Images were captured with Micro Chemi (DNR Bio-imaging systems, Israel) and the optical density of the bands was determined using Scion Image software (Fredrick, MD, United States). All assays were performed at least three times. Results are presented as percentage of control after normalization (% control) in each blot to correct the variations between blots.

Sulfhydrylation Detection
For the measurement of the sulfhydrylation levels of protein samples, the isolated tissue was homogenated in ice-cold lysis solution without EDTA. After centrifuged for 15 min (12,000 g, 4 • C), the supernatant fraction was isolated and aliquoted into the 96-well plate. Equal volume of DTNB [5,5 -Dithio bis-(2nitrobenzoic acid)] reaction buffer (10 mM MgCl 2 , 30 mM KCl, 25 mM Tris-HCl, 4 mM DTNB, pH 8.0) was also added to the wells and the reaction proceeded at 37 • C in dark place for 15 min with moderate shaking. Then the absorbance of the wells was determined by a microplate reader at 415 and 595 nm. After deducting the blank control value, the absorbance value is directly proportional with the sulfydrylation level of the detected samples in a linear range.

Sulfur-Sulfhydrylation Measurement
Protein sulfur-sulfhydrylation measurement was carried out according to previous studies (Paul and Snyder, 2012;Li et al., 2016;Saha et al., 2016) with some modifications. After treatment and dissection, the ARC tissues were collected and homogenated in special lysis solution prepared with HEN buffer (250 mM HEPES, 1 mM EDTA, 0.1 mM neocuproine; the pH was regulated to 7.7 with NaOH). The tissue homogenate was centrifuged (12,000 g, 4 • C, 15 min) and 400 µL of supernatant was transferred into a boiling tube. Then, 1600 µL of selective sulfhydryl sealing agent (MMTS) solution [20 mM MMTS in HENS buffer (HEN buffer:25% sodium dodecyl sulfonatein H 2 O = 9:1)] was added into the boiling tube to selectively seal the free sulfhydryl of the total proteins. After 50 • C water bath with slight shaking for 20 min, 100 µL of biotin-HPDP diluent (4 mM biotin-HPDP in DMSO) was then added into the boiling tube under darkness. Then the biotinylation reaction proceeded at 37 • C with slight shaking in dark place for 3 h. After that, the boiling tube was filled with −20 • C prefrozen acetone, and then kept stewing in −20 • C refrigerator for 30 min. Then the boiling tube was immediately centrifuged for 20 min (12,000 g, 4 • C), and the acetone was poured and volatilized out. Finally, 100 µL of HEN buffer was used to redissolve the precipitation. Then the sulfur-sulfhydryl biotinylated protein sample was quantitatively determined, and then inactivated with non-reducing protein loading buffer. After SDS-PAGE electrophoresis, the proteins were transferred to polyvinylidenedifluoride membrane and blocked with 5% BSA for 1 h at room temperature. At last, the transferred membrane was incubated with HRP-conjugated streptavidin for 2 h at room temperature before signals were visualized using the ECL kit. As for the sulfur-sulfhydrylation detection of AMPKα and CaMKKβ, only a fraction of the total lysates was determined and loaded as references, and most of the protein samples were selectively biotinylated as above and precipitated by streptavidin-agarose beads for sulfursulfhydrylation proteins. Then the biotinylated proteins were eluted and detected by primary antibodies for western blot assay.

Statistical Analysis
All data analysis was performed using SPSS 18.0 software (SPSS Inc., United States) and values were expressed as the mean ± SEM. Repeated measures analysis of variance (ANOVA) was used for the comparison of food intake between control and GYY groups at different time levels. One-way ANOVA and LSD post hoc test were used for comparisons between two or more groups. All tests for comparisons were set at α = 0.05 by two-side, and differences at the P < 0.05 level were considered statistically significant.

GYY4137 Has Orexigenic Effect on Mice With Dosage and Time Dependence
In order to explore the effect of GYY4137-dervied H 2 S on feeding behavior of mice and find the appropriate administration dosage, we divided the experimental animals homogenously into five groups (vehicle, 37.6, 75.2, 150.4, and 300.8 mg/kg) and tested their food consumption in 2 h after the application of GYY4137 or vehicle (saline) via i.p. injection. We found that GYY4137 augmented food intake in most of the dosage groups (37.6, 75.2, and 150.4 mg/kg), but with 150.4 mg/kg being the effective dosage [n = 14 mice for each group; F (4,65) = 3.873, P = 0.009; vehicle, 0.392 ± 0.032; 37.6 mg/kg, 0.441 ± 0.030, P = 0.424 vs vehicle; 75.2 mg/kg, 0.495 ± 0.045, P = 0.081 vs vehicle; 150.4 mg/kg, 0.589 ± 0.057, P = 0.003 vs vehicle; 300.8 mg/kg, 0.398 ± 0.039, P = 0.926 vs vehicle; Figure 1A]. Then we asked how long time the orexigenic effect of GYY4137 could maintain. We measured the cumulative food consumption after GYY4137 (150.4 mg/kg) administration by four time point (1, 2, 4, and 8 h after application), and the result suggested that GYY4137 promotes feeding behavior of mice for hours and it significantly increased food intake in 2 and 4 h after the application  Data are expressed as means ± SEM. One-way ANOVA and post hoc tests and repeated measures ANOVA were used. * P < 0.05, * * P < 0.01 vs vehicle.
P < 0.001; Supplementary Figure S1]. These results indicate that S-sulfhydrylation may mediate the effects of GYY4137 on ARC proteins and it may also play important role in the regulation of food intake.

DISCUSSION
In the present study, we found that GYY4137-derived H 2 S has orexigenic effect on mice and the possible underlying mechanisms. Our investigation showed that GYY4137 could promote mice food intake, augment the production of NPY and simultaneously, enhance the sulfur-sulfhydrylation and activation of AMPK and CaMKKβ in ARC. Blocking sulfursulfhydrylation with DTT eliminated GYY4137-induced activation of AMPK and CaMKKβ. DTT and preventing AMPK activation in ARC with Compound C amd Ara-A, could both attenuate the orexigenic effect of GYY4137. We used GYY4137 as a H 2 S donor to investigate the possible effect of H 2 S on ARC and food intake of mice. We found that GYY4137/H 2 S increased food consumption within the physiological concentration range of H 2 S (50-200 µM, with GYY4137 at 100-800 µM). GYY4137 is a water soluble, slow, and stable releasing H 2 S donor. For example, with a single dose of GYY4137 at 100 µM, the concentration of GYY4137-derived H 2 S can keep at 20 µM for about 1-5 h in vivo (Rose et al., 2015). The optimal observation time for GYY action is within 1-5 h after treatment. Since repeated administration via i.p. injection may induce adverse stimulation to mice, we used a single dose treatment and tested the food consumption of mice during the 1-8 h period. In our study, the orexigenic effect of a single dose of GYY4137 maintained for 4 h, and at 8 h after GYY4137 administration the orexigenic effect disappeared. Thus, the timedependence of the orexigenic effect of GYY4137 is probably associated with its chemical and H 2 S-releasing property. Besides, the highest dose of GYY in our study is 300.8 mg/kg, which is a relatively safe dose lower than the toxic amount but could also induce some unexpected neuroexcitable effects such as increased locomotive activity, epilepsy, hypertension, and pain sensitization (Feng et al., 2013;Kuksis et al., 2014;Luo et al., 2014), and that may affect the feeding behavior of the mice under this treatment level.
Modification of substrate proteins by sulfur-sulfhydrylation is a principal action mode of H 2 S (Mustafa et al., 2011;Paul and Snyder, 2012). We hypothesized that sulfur-sulfhydrylation of certain target proteins in ARC is the foundation of the orexigenic effect of H 2 S. DTT is a blocker of S-sulfhydrylation, which is commonly used in blocking the S-sulfhydrylation and functions of H 2 S (Mustafa et al., 2011;Paul and Snyder, 2012;Sen, 2017). Our results showed that GYY4137/H 2 S significantly increased the proteins sulfur-sulfhydryl level, which could be blocked by DTT. This result confirmed that DTT could be used as an efficient sulfur-sulfhydrylation blocker. AMPK in ARC plays critical roles in the control of appetite and feeding behavior (Oh et al., 2016;Dong et al., 2017). Our experiments found that DTT attenuated the activation of AMPK and CaMKKβ induced by GYY4137, and DTT and AMPK inhibitors both successfully attenuated the orexigenic effect of GYY4137. So we deemed protein sulfur-sulfhydrylation substantially contributory to H 2 S-induced CaMKKβ/AMPK activation and food intake. The sulfur-sulfhydrylation effect of H 2 S can be quickly abolished by the breakage of sulfur-sulfhydryl bond, which is a brittle and reversible chemical bond (Mustafa et al., 2011;Paul and Snyder, 2012). We found that the enhanced sulfur-sulfhydrylation in ARC caused by GYY4137 was almost completely blocked by the administration of DTT, which meat DTT given through i.c.v. injection is efficient in controlling sulfur-sulfhydrylation of ARC proteins. Feeding study also showed that DTT significantly attenuated the orexigenic effect of GYY4137 on mice, and thus demonstrated that GYY4137-derived H 2 S stimulates feeding behavior via signaling regulation involving protein sulfursulfhydrylation.
AMPK is a critical gage molecule in the monitoring of energy status and controlling of energy metabolism (Kohno et al., 2011;Oh et al., 2016;Dong et al., 2017). In the ARC region, AMPK mainly determines the activity of glucose sensitive neurons and regulates the secretion of orexigenic neuropeptide such as NPY, which is the most abundant neuropeptide in brain and an important appetite-promoting hormone (Kohno et al., 2011;Blankenship et al., 2016;Carneiro et al., 2016). Calcium signal also plays pivotal roles in the production of NPY and energy metabolism (Anderson et al., 2008;Wu et al., 2013). Accumulated evidence suggests that H 2 S is an important functional regulator of several ion channels (Peers et al., 2012;Zhang and Bian, 2014) and influences the intracellular calcium signals (Lee et al., 2006;Tang et al., 2008;Marino et al., 2016). Ca 2+ is an important activator of CaMKKβ/AMPK signaling cascade (Wu et al., 2013;Nakanishi et al., 2017), and H 2 S activates CaMKKβ/AMPK signaling in many kinds of cells (Lee et al., 2012;Zhou et al., 2014). Therefore, we suppose that H 2 S augments NPY content and food intake via increasing intracellular calcium level and activating CaMKKβ/AMPK signaling in ARC orexigenic neurons, though the enhancement of calcium signals in orexigenic neurons needs to be proved in further study.
From this study, we can also draw out some other questions for the future investigations. First, is inhaling a proper dose of H 2 S gas capable of inducing the orexigenic effect? Second, whether endogenous H 2 S and its sulfur-sulfhydrylation function are involved in the regulation of food intake? It has been reported that fasting or diet can increase the endogenous production of H 2 S (Hine and Mitchell, 2015;Nakano et al., 2015). So we can suppose that endogenous H 2 S, to some extent, may be implicated in refeeding increase induced by energy stress such as starvation or fasting, and perhaps protein sulfursulfhydrylation is involved in the process of the orexigenic effect. Third, since H 2 S influences intracellular Ca 2+ level in many cells and calcium signal is a critical activating factor for hypothalamic CaMKKβ/AMPK (Lee et al., 2012;Wu et al., 2013;Zhou et al., 2014;Chen et al., 2017), which is involved in the regulation of energy balance (Anderson et al., 2008), is calcium signal also contributory for the enhanced activation of CaMKKβ/AMPK in ARC and the increased food intake caused by exogenous H 2 S? Therefore, whether H 2 S may activate AMPK through augmenting intracellular calcium signal in ARC, and whether sulfur-sulfydrylation has substantial contribution to this process still need further research. What is more, unbiased identification of the abundant S-sulfhydrated proteins in the GYY4137-treated arcuate lysate by bidirectional electrophoresis and bio-mass spectrometry would represent an important future direction and will help in finding special sites for S-sulfhydrylation in individual and specific proteins.

CONCLUSION
In summary, we confirmed the orexigenic effect of H 2 S by means of GYY4137 administration and its possible mechanism via sulfur-sulfhydrylation of certain functional proteins. GYY4137derived H 2 S increases the activation of energy metabolic gage AMPK and its upstream kinase CaMKKβ via sulfursulfhydrylation, further enhances the activity of the orexigenic neurons in ARC including NPY neurons, and subsequently promotes appetite. This study provides foundation for the investigation of the roles of endogenous H 2 S in the hypothalamus and energy metabolism which influences food intake, and the function of protein sulfur-sulfhydrylation on the production of appetite-regulating neuropeptides including NPY. Besides, our investigation also suggests that H 2 S-derived modulating agents may be of value in the design and development of therapeutic medications for diseases such as anorexia nervosa.

AUTHOR CONTRIBUTIONS
JZ designed and substantially performed the experiments, analyzed the data, and wrote the paper. X-HL, BG, and L-YD helped in biochemical detections and provided expert advice. KD, J-JF, and A-QS helped in animal feeding and experiments. W-NW designed and oversaw the experiments, analyzed and interpreted the data, and co-wrote and developed the manuscript.