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Equisetin is an anti-obesity candidate through targeting 11β-HSD1

Obesity is increasingly prevalent globally, searching for therapeutic agents acting on adipose tissue is of great importance. Equisetin (EQST), a meroterpenoid isolated from a marine sponge-derived fungus, has been reported to display antibacterial and antiviral activities. Here, we revealed that EQST displayed anti-obesity effects acting on adipose tissue through inhibiting adipogenesis in vitro and attenuating HFD-induced obesity in mice, doing so without affecting food intake, blood pressure or heart rate. We demonstrated that EQST inhibited the enzyme activity of 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1), a therapeutic target of obesity in adipose tissue. Anti-obesity properties of EQST were all offset by applying excessive 11β-HSD1's substrates and 11β-HSD1 inhibition through knockdown in vitro or 11β-HSD1 knockout in vivo. In the 11β-HSD1 bypass model constructed by adding excess 11β-HSD1 products, EQST's anti-obesity effects disappeared. Furthermore, EQST directly bond to 11β-HSD1 protein and presented remarkable better intensity on 11β-HSD1 inhibition and better efficacy on anti-obesity than known 11β-HSD1 inhibitor. Therefore, EQST can be developed into anti-obesity candidate compound, and this study may provide more clues for developing higher effective 11β-HSD1 inhibitors.

Abstract Obesity is increasingly prevalent globally, searching for therapeutic agents acting on adipose tissue is of great importance. Equisetin (EQST), a meroterpenoid isolated from a marine sponge-derived fungus, has been reported to display antibacterial and antiviral activities. Here, we revealed that EQST displayed anti-obesity effects acting on adipose tissue through inhibiting adipogenesis in vitro and attenuating HFD-induced obesity in mice, doing so without affecting food intake, blood pressure or heart rate. We demonstrated that EQST inhibited the enzyme activity of 11b-hydroxysteroid dehydrogenase type 1 (11b-HSD1), a therapeutic target of obesity in adipose tissue. Anti-obesity properties of EQST were all offset by applying excessive 11b-HSD1's substrates and 11b-HSD1 inhibition through knockdown in vitro or 11b-HSD1 knockout in vivo. In the 11b-HSD1 bypass model constructed by adding excess 11b-HSD1 products, EQST's anti-obesity effects disappeared. Furthermore, EQST directly bond to 11b-HSD1 protein and presented remarkable better intensity on 11b-HSD1 inhibition and better efficacy on anti-obesity than known 11b-HSD1 inhibitor. Therefore, EQST can be developed into anti-obesity candidate compound, and this study may provide more clues for developing higher effective 11b-HSD1 inhibitors.

Introduction
Given the known central roles of adipose tissue in obesity, adiposetargeted therapies for obesity are gaining substantial interest 1 . On the one hand, proteins including peroxisome proliferator-activated receptor gamma (PPARg), fatty acid-binding protein (FABP4), fatty acid synthase (FAS), and acetyl-CoA carboxylase (ACC) are now understood to function in adipocyte formation 2 , in inhibiting adipocyte differentiation, and in fat accumulation, and targeted inhibition of the expression and/or activity of these factors has been considered as a potential therapeutic approach for treating obesity 3,4 . On the other hand, promotion of energy expenditure, such as browning in white adipose tissue, has been associated with health benefits for body weight regulation and obesity phenotypes 5 . Therefore, it is of great importance to search for pharmacological molecules capable of regulating lipid metabolism and promoting energy expenditure for combating obesity and the associated diseases 5e8 . However, few anti-obesity products act on adipose tissue directly, so candidate adipose-tissue-targeted anti-obesity agents would be highly welcomed.
11b-hydroxysteroid dehydrogenase type I (11b-HSD1) is an attractive and promising pharmaceutical target for the treatment of obesity and the related diseases 9 due to its roles in adipogenesis 10,11 and energy expenditure 12,13 . 11b-HSD1 converts biologically inactive glucocorticoids (GCs) to their active form 14 , which contribute to enhanced adipogenic differentiation by upregulating adipogenic transcription factors such as PPARg, FABP4, and various C/EBP family members (e.g., C/EBPa, b, and d) 11 . Suppression of 11b-HSD1 expression 15 or activity 16 attenuates the differentiation of adipocytes. Beyond its known roles in adipocyte formation 17 , 11b-HSD1 also inhibits white adipose tissue browning 18,19 , suggesting the potential inhibitory roles of 11b-HSD1 in energy expenditure.
Recent years have seen efforts to develop natural products into 11b-HSD1 inhibitors for treating obesity and/or type 2 diabetes mellitus 20,21 . Glycyrrhetinic acid 22 , carbenoxolone 23 , emodin 24 and other small molecular compounds from natural sources have been developed as 11b-HSD1 inhibitors, but all of them stopped due to poor 11b-HSD1 selectivity and(or) poor anti-obesity efficacy 25e27 , given that no trials of these inhibitors have met primary endpoints for treating obesity, it is clear that the translation of initial findings to clinical impact has proved challenging so far 28 .
In this study, we demonstrated that equisetin (EQST), a natural product 29e31,97 obtained from the marine Fusarium equisetin strain NRRL 5537 32 , is a 11b-HSD1 inhibitor and that confers potent anti-obesity activity in vitro and in vivo by accumulating in adipocyte tissue and through binding to and inhibiting the activity of 11b-HSD1. Our results support the further development of EQST as a drug candidate for the treatment of obesity.

Animal experiments
All animal experiments were approved by the Medical Ethics Committee of Peking Union Medical College, following the National Institutes of Health (NIH) regulations for the care and use of animals in research. 8-Week-old male C57BL/6J mice were purchased from Vital River Laboratories Co., Ltd. (Beijing, China). Animals were randomly assigned to each group (n Z 8) and fed with normal diet (chow) or high-fat diet (HFD). EQST powder were solved with anhydrous alcohol at a concentration of 1 mg/mL and 1% (v/v) Tween-80, and then diluted with distilled water for daily oral gavage. A matched volume of distilled water was given to the control group. Whole-body energy expenditure and respiratory exchange ratio (RER) were monitored by a comprehensive lab animal monitoring system (CLAMS; Columbus, OA, USA). Body-weight was monitored once a week. At the end of the experiment, animals were fasted for 12 h before collecting blood samples, and then culled by cervical dislocation. Livers, epididymal fats, retroperitoneal fats, and subcutaneous fats were collected and weighed separately, some were fixed in 4% (v/v) paraformaldehyde; the rest were snap-frozen using liquid nitrogen for later analyses. Blood samples were stored at 4 C for measurement of serum levels of total cholesterol (TC), triglyceride (TG), low density lipoprotein-cholesterol (LDL-C), high density lipoprotein-cholesterol (HDL-C), leptin, and adiponectin using assay kits (Jian Cheng Biotechnology Company, Nanjing, China).

Preparation for 11b-HSD1 knockout mice
The global 11b-HSD1 knockout mouse model (in a C57BL/6N genetic background) was generated by deleting the genomic DNA fragment from exon 3 of the 11b-HSD1 locus using a CRISPR-Cas9 mediated genome editing system (Cyagen Biosciences Inc., Wuhan, China). The founders were genotyped by PCR, followed by DNA sequencing analysis. Animals were housed at 22e24 C on a 12 h light, 12 h dark cycle, and allowed free access to HFD and drinking water supplemented with corticosterone (100 mg/mL) to establish a 11b-HSD1-bypassed DIO model. PCR primers for the 11b-HSD1 mutation was characterized by PCR with the primers 5 0 -TTCCATGGAGATACTGATAGCCGA-3 0 (forward) and 5 0 -TGGGGTAACTGGGGTTTAAATGAA-3 0 (reverse), yielding a 567-base pair (bp) fragment for the mutant allele, or a 617-bp fragment for the WT allele. For the continuous treatment during the differentiation process, EQST, BVT.2733, or cordycepin were added to the differentiation induction medium, and the final concentration was maintained at 10 mmol/L. For mature adipocytes, 3T3-L1-derived mature adipocytes were exposed to EQST or BVT.2733 (final concentration was maintained at 10 mmol/L) for 24 h.

Histological analysis
Adipose tissues were fixed in 4% paraformaldehyde, and then embedded in paraffin wax. The embedded samples were cut into 4-mm thick slides and mounted on glass slides. The slides were stained with hematoxylin and eosin (H&E).

Oil red O staining
Cells were washed with PBS, and then fixed with 4% paraformaldehyde for 1 h at room temperature, followed by staining with a mixture of 0.5% oil red O dye (resolved in isopropanol and ddH 2 O at a 3:2 ratio) for 20 min. Cells were washed three times with PBS, the images were captured under a microscope. The oil red O dye was resolved with DMSO and subjected for absorbance measurement at 358 nm.

Intracellular triglyceride (TG) quantification
Cells were scraped and re-suspended in 1% TritonX-100 and heated to 80e100 C for 5 min to solubilize the intracellular triglycerides. The triglyceride content was measured using a triglyceride quantification assay kit (BioVision, Inc., Milpitas, CA, USA) according to the manufacturer's instructions.

11b-HSD1 and 11b-HSD2 enzyme activity assays
The enzyme activity of 11b-HSD1 in cells was evaluated according to its capability to convert cortisone to cortisol, and the enzyme activity of 11b-HSD2 in cells was evaluated according to its capability to convert corticosterone to 11-dehydrocorticosterone 33,34 . 3T3-L1 preadipocytes were seeded into 6-well culture plates and induced into mature adipocyte as above. 3T3-L1-derived mature adipocyte were exposed to 160 nmol/L cortisone or 160 nmol/L corticosterone for 24 h. The reaction mixtures were collected and analyzed for cortisol levels using an ELISA kit (R&D Systems, Minneapolis, MN, USA), according to the manufacturer's instructions.

Quantitative real time-PCR analysis
Total RNA was isolated from cells using TRIzol Reagent (Ambion, Austin, TX, USA) and was reverse-transcribed into cDNA using EasyScript One-Step gDNA Removal and cDNA Synthesis SuperMix (TransGen Biotech, Beijing, China). Gene transcript levels of Pparg, Ucp-1, Fabp4, Fas, Acc-1, Srebp1, Perilipin, Hsl, Lpl was quantified using the TransStart Top Green qPCR SuperMix (TransGen Biotech, Beijing, China) according to the manufacturer's protocol. Glyceraldehyde-3-phosphate dehydrogenase (Gapdh) was used as housekeeping control. Primers used for quantitative PCR are listed in Supporting Information Table S1.

Multiple reaction monitoring (MRM)
MRM analysis was performed on a 6495 Triple-Quadrupole Mass Spectrometer coupled with an Agilent 1260 Infinity HPLC system (Agilent, Santa Clara, CA, USA). Twenty micrograms of trypticdigested peptides from each sample were loaded onto a reversephase analytical column (Agilent ZORBAX SB-C18, 3.5 mm, 15 cm in length, 500 mm inner diameter). The aforementioned mass spectrometer was used to measure m/z values and signal intensities for peptides sequentially eluted from the analytical column. MRM-MS analysis was performed in positive ion mode, with the ion spray capillary voltage and nozzle voltage set at 3000 and 1200 V, respectively. All raw MRM-MS data were processed using Skyline platform. All integrated peaks were manually inspected to confirm correct peak detection and accurate integration. The MRM acquisition method was initially built with eight to fourteen precursor/fragment ion pairs (transitions) for each light and heavy peptide. Reverse response curves for examining linearity were generated from MRM-MS data from each serially diluted heavy peptide spiked into 25 mg of tryptic-digested sample. Eight concentration points (approximately 1.56e200 fmol/mL) were used, and a tryptic-digested pooled sample (without heavy peptides added) was used as a blank to estimate background levels.
Triplicate MRM-MS analyses were performed for each group.

Reverse docking
The two-dimensional molecular structure of EQST (Fig. 1A)  Generally, if the affinity value is less than À6 kcal/mol, the small molecule can be considered as a potential ligand of the receptor 35 .
The receptors with a docking score less than À9 kcal/mol are considered as the potential target proteins of EQST. Obesityrelated proteins with a relevance score higher than 10 were retrieved from the GeneCards database (https://www.genecards. org/) by searching the term of "obesity". The two sets of the proteins were subjected to Venn analysis using the Venny 2.1.0 online tool to identify candidate obesity-related targets of EQST.

Molecular docking simulation of EQST to 11b-HSD1
The molecular docking simulation was conducted using Molecular Operating Environment (MOE; Chemical Computing Group Inc., Montreal, Canada) v2018.01. The 2D structure of EQST was drawn in ChemBioDraw 2014 and was converted to its 3D conformations through energy minimization. The 3D structure of the protein 11b-HSD1 was retrieved from the RCSB Protein Data Bank (https://www.rcsb.org/; PDB code 3D5Q). The protein structure was prepared by using the QuickPrep module. The protonation states and the orientations of the hydrogens were optimized by LigX at a pH 7 at 300 K. The docking process was performed using an AMBER10/EHT force field, along with an internal dielectric constant of 1 and an external dielectric constant of 80, as well as an implicit solvation model of reaction field (R-field). The Triangle Matcher algorithm was used for the initial placement of 1000 returned conformations, and the top 100 conformations ranked by the London dG scoring function were further refined through energy minimization, followed by rescoring using Generalized Born/Volume Integral (GB/VI) solvation energy. The induced-fit protocol was applied for energy minimization: the ligand was fully flexible during the conformation sampling process, and the side chains of the receptor were also allowed to move. Finally, 20 top-ranked poses were retained, and the most representative pose was retrieved by visual inspection for further analysis (See detailed methods in Supporting Information).

Cellular thermal shift assays (CETSA)
3T3-L1-derived adipocytes were collected and freezeethawed three times using liquid nitrogen. The lysates were divided into three aliquots, with one aliquot being incubated with 20 mmol/L EQST, the second with 20 mmol/L BVT.2733, and the third with DMSO as a negative control. After 1 h at room temperature, the samples were heated individually at different temperatures (40e72 C) for 3 min, followed by cooling for 3 min on ice before centrifuging at 11,000 Â g for 10 min at 4 C (Laborzentrifugen, Sigma, Osterode am Harz, Germany). The supernatant was then analyzed by Western blotting.

Statistical analyses
Statistical analyses were performed with GraphPad Prism (version 8.4.3). Independent Student's t-tests were used to compare the means of numerical variables. Data are presented as the mean AE standard error of mean (SEM). Statistical significance was defined as P values < 0.05. Experiments involving measurements of obesity indexes were performed in a blinded fashion with randomly grouped individuals. Imaging and histology were also performed and analyzed in a blinded fashion prior, to the decoding of the sample identities.

EQST inhibited terminal differentiation of preadipocytes
A screen of marine natural product compounds showed that equisetin (EQST, Fig. 1A) exhibited apparently strong lipidlowering activity in 3T3-L1 preadipocyte cells. We first evaluated the cytotoxicity of EQST, CCK-8 assay result demonstrated that EQST did not show cytotoxicity to 3T3-L1 preadipocyte cells (Supporting Information Fig. S1). To investigate the lipidlowering effects of EQST, we continuously exposed differentiating 3T3-L1 preadipocytes to different doses of EQST, in experiments which used a reported inhibitor of adipogenesis 36 as a positive control (cordycepin, CPN). Quantification at the end point of the 3T3-L1 preadipocyte differentiation procedure showed that both total lipid content and TG content were significantly decreased in the cells given EQST (1 and 10 mmol/L) compared to untreated model cells (Fig. 1BeD). Moreover, we found that EQST dose-dependently suppressed both the mRNA and protein abundances of known pan-adipocyte markers including PPARg and FABP4 37 ( Fig. 1E and G). Genes that function in lipogenesis (Srebp1, Acc, Fas) 38 and in lipid droplet formation (Fsp27 and Plin5) 39,40 were also down-regulated in response to EQST (Fig. 1F).
In addition, we found that EQST dramatically increased the expression of UCP1 in 3T3-L1-derived mature adipocytes (Fig. 1E and G). UCP1 is a mitochondrial protein that functions in thermogenesis, specifically by uncoupling oxidative phosphorylation to enable non-shivering thermogenesis in brown and beige adipocytes 41e43 . These observations together indicate that EQST can somehow suppress the expression of key adipogenic factors while also promoting the expression of a gene for increased thermogenic energy expenditure.

EQST inhibited HFD-induced obesity and enhanced energy expenditure in HFD-induced obese mice
We next investigated the potential in vivo anti-obesity effect(s) of EQST. C57BL/6J mice were given a high-fat diet (HFD) for 10 weeks to establish a classic diet-induced obese (DIO) model, after which mice were given EQST daily (i.g.) for 6 weeks (n Z 8).
Orlistat, an approved drug for long-term treatment of obesity 44 , was used as a positive control. At Week 16, the EQST-treated mice all weighed significantly less than the vehicle control group, and our testing of three EQST concentrations (20,40, and 80 mg/kg) showed that this effect on weight gain was in dose-dependent manner (Fig. 2AeD). It should be noted that EQST treatment did not affect food intake (Supporting Information Fig. S2), heart rate, or blood pressure (Supporting Information Table S2), which led us to speculate that EQST may act directly on adipose tissue. Pursuing this, experiments using SpragueeDawley (SD) rats to determine the tissue distribution of EQST showed that a 10 mg/kg dose of EQST given to rats by oral administration for 7 consecutive days led to EQST accumulation of 0.38 AE 0.13 mg/g in adipose tissue, a significantly higher level than detected in lung (0.26 AE 0.08 mg/g) or skin (0.24 AE 0.05 mg/g) (Supporting Information Table S3). The detected EQST in adipose tissue supports the plausibility that the observed anti-adipogenesis effects could result from the EQST's lipid-lowering effects in adipocytes.
We also found that the EQST-treated mice had significantly reduced body fat compared to the vehicle control DIO animals (Fig. 2E), and a closer examination revealed significant reductions in the weight of subcutaneous, epididymal, and retroperitoneal white adipose tissue (sWAT, eWAT, and rWAT; Fig. 2F). Potentially helping to explain their retarded body weight gain, the EQST-treated mice showed significant reduced content for blood lipids including TG, TC, and LDL (Fig. 2G). H&E staining showed that EQST treatment significantly reduced the sizes of individual adipocytes within both eWAT and sWAT in DIO (Fig. 2H). Finally, EQST reduced the expression levels of PPARg and FABP4 in subcutaneous fat (Supporting Information Fig. S3C) and visceral fat (Supporting Information Fig. S4) extracted from the DIO mice. These data from HFD-induced obese mice demonstrate that EQST acts to retard body weight gain and show that EQST affects adipose tissues by suppressing lipid accumulation.
We also monitored oxygen uptake (VO 2 ) kinetics for the DIO mice and found that mice treated with EQST had significantly higher whole-body energy expenditure ( Fig. 3A and B, and Fig. S3A) than vehicle control mice. Moreover, mice in EQST group had a lower respiratory quotient (RQ, VCO 2 /VO 2 ) than did control mice, suggesting that EQST treatment may induce a fuel switch toward fatty acid oxidation (Fig. S3B). Immunoblotting result showed that EQ treatment significantly increased the expression of UCP1 in sWAT of DIO mice (Fig. S3C). We also extracted proteins from sWAT of the DIO mice and performed multiple reaction monitoring (MRM), and MRM-based quantitation showed that EQST treatment significantly increased the sWAT accumulation of proteins known to function in fatty acid oxidation, lipid metabolism (e.g., CPT2, ETFDH, HADH, HADHA, HADHB 45 ), the electron transport chain (e.g., UQCRC1, SDHA 46 ), and the TCA cycle (e.g., SDHA, ACO2 47 ) (Fig. 3C). These in vivo results suggest that EQST can both inhibit adipogenesis and induce elevated energy expenditure, apparently by ameliorating obesity-linked metabolic impairment.

EQST targets 11b-HSD1
We next attempted to identify potential targets of EQST to help explore the mechanism(s) underlying EQST's anti-obesity effects. Briefly (Fig. 4A), we used a reverse docking method to examine EQST's apparent capacity to interact with proteins having solved structures in the PDB database: 40 candidate target proteins had binding propensity (PLB) scores higher than 9 (docking score less than À9 kcal/mol). The GeneCards database (https://www. genecards.org/) lists a total of 466 putative obesity-related proteins, and a Venn analysis indicated that 8 of these overlapped with our reverse docking candidates, among which 11b-HSD1 had the highest GeneCards relevance score for the obesity phenotype (Supporting Information Table S4).
The full name of 11b-HSD1 is 11b-hydroxysteroid dehydrogenase I; this enzyme functions in lipid metabolism and adipogenesis, specifically (in humans) by mediating the conversion of the inactive corticosteroid cortisone into its active form (cortisol). (F) qPCR analysis of lipogenesis genes (Srebp1, Acc, Fas) and lipid droplet formation genes (Fsp27, Plin5) normalized by Gapdh, n Z 3. (G) Immunoblotting against the PPARg, FABP4 and UCP1 proteins, n Z 3. All data are presented as mean AE SD of at least three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.005 vs. Model group. P value was assessed by two-tailed Student's t-test. CPN: Cordycepin group.
Note that inhibition of 11b-HSD1 has been reported as beneficial for treating obesity 48 . We assayed potential inhibitory impacts of EQST on 11b-HSD1 in experiments using 3T3-L1-derived mature adipocytes, EQST did not affect 11b-HSD1 protein abundance (Supporting Information Fig. S5), and we found that EQST inhibited the enzyme activity of 11b-HSD1 at a dose of 1 and 10 mmol/L (Fig. 4B). Human-liver-derived HepG2 cells express little or no 11b-HSD1 49 , so we constructed a human cell model based on exogenous expression of 11b-HSD1 in HepG2 cells. Treatment of 11b-HSD1 expressing (or empty vector) HepG2 cells with EQST (10 mmol/L) showed that EQST does inhibit 11b-HSD1 enzyme activity in human cells (Fig. 4C).
In mice, 11b-HSD1 transforms the inactive substrate 11-dehydrocortisone (11-DHC) into the active product corticosterone (CORT) 48 . Excessive 11-DHC levels promote the expression of 11b-HSD1 in vitro 50 , while excessive CORT inhibits the expression of 11b-HSD1 in vitro 51 ; these regulatory outcomes are based on substrates or products binding at the 11b-HSD1 active site 52 . We exposed 3T3-L1 preadipocytes that had been cultured with or without 40 nmol/L 11-DHC to EQST and  Z 100 mm). Data are presented as mean AE SD, n Z 8. **P < 0.01, ***P < 0.001 compared to Chow group. # P < 0.05, ## P < 0.01, ### P < 0.001 compared to HFD group. P value was assessed by twotailed Student's t-test. ORL: Orlistat group. examined total lipid and TG content: the presence of an inhibitory 11-DHC concentration blocked any EQST-induced alteration of lipid content and of TG content (Fig. 4D and E). We then exposed 3T3-L1 preadipocytes that had been cultured with or without 40 nmol/L CORT to EQST and examined both total lipid and TG levels. Our findings that the presence of an inhibitory CORT concentration blocked any EQST-induced alteration of lipid or TG content supports direct involvement of 11b-HSD1 in EQST's antiobesity effects (Supporting Information Fig. S6A and S6B); that is EQST's capacity to inhibit 11b-HSD1's transformation of 11-DHC to CORT can explain the reduction of lipid accumulation we observed earlier in 3T3-L1 preadipocytes. We also investigated the 11b-HSD1 inhibition effects of EQST in human primary adipocytes, in experiments using the previously reported human 11b-HSD1 inhibitor 53 as a positive control (PF-915275). Human primary adipocytes were exposed to 10 mmol/L EQST or 10 mmol/L PF-915275 for 24 h. Compared to untreated model cells, the total lipid content was significantly decreased in the cells given EQST (10 mmol/L) and PF-915275 (10 mmol/L) (Supporting Information Fig. S7).

11b-HSD1 is required for EQST's in vivo anti-obesity effects
Building from findings with cell cultures, we also generated homozygous 11b-HSD1 gene knockout C57BL/6J mice. Compared with the wild-type mice, knockout of 11b-HSD1 decreased the body weight of mice under 4 weeks HFD-feeding, whereas EQST's anti-obesity effects disappeared in 11b-HSD1 knockout mice (Supporting Information Fig. S9). To further explore the relationship between EQST and 11b-HSD1, we performed a rescue experiment. For 4 weeks, we fed 11b-HSD1 knockout mice a HFD supplemented with excess corticosterone (100 mg/mL) and prophylactically co-administered with EQST (80 mg/kg) or positive control (orlistat, 80 mg/kg) to establish a 11b-HSD1bypassed DIO model (n Z 6).
With these 11b-HSD1 knockout mice, there was no difference in body weight between the vehicle and EQST-treated groups, although there was an obvious anti-obesity effect from the Orlistat treatment (Fig. 5AeC). We also found that the weights of different fat deposits in the 11b-HSD1 knockout mice ( Fig. 5D and E) and the serum levels of biochemical indicators (TG, TC, LDL, HDL, and FFA; Fig. 5F) were effectively improved by orlistat treatment; no such effects resulted from EQST treatment of these animals. These results suggest that EQST cannot inhibit the obesity induced by excessive exogenous glucocorticoids in 11b-HSD1 knockout mice, which confirmed that 11b-HSD1 is required for the EQST's anti-obesity effects (Figs. 4 and 5).
We then treated differentiating 3T3-L1 preadipocytes with EQST or BVT.2733 and found that EQST was more effective in reducing lipid content than BVT.2733 at the same dose of 10 mmol/L (Fig. 6EeG). Further, we constructed a prophylactical drug delivery model to compare their anti-obesity in vivo efficacy. We fed C57BL/6J mice a HFD and co-administered 40 mg/kg EQST or 40 mg/kg BVT.2733 for 4 weeks. The body weights and body fat percentages of the EQST-treated animals were significantly lower than in the vehicle-treated DIO mice ( Fig. 6HeJ and Supporting Information Fig. S14A). EQST also outperformed BVT.2733 regarding serum lipid profiles (Fig.  S14B). These data indicate that EQST exerts more potent antiobesity effects than the known anti-obesity agent and 11b-HSD1 inhibitor BVT.2733.

Discussion
Natural products represent a rich source of diverse molecules for drug development. EQST is a meroterpenoid which was first isolated from a marine sponge-derived fungus for its antibacterial activity 29 . EQST can also be used as an HIV-1 integrase inhibitor 61,62 . Here, we demonstrate that the marine natural product EQST is an 11b-HSD1 inhibitor that confers potent anti-obesity activity by acting on adipose tissue. eWAT and sWAT are the main adipose tissues which undergo adipocyte hypertrophy in the early stages of HFD exposure 63 ; over-loaded lipids distributed in the blood can also lead to ectopic fat deposition 1 , which leads to metabolic syndrome in obese individuals 64 . Reducing body weight and improving fat distribution are regarded as highly relevant aspects for developing anti-obesity drugs 65 . In our study, EQST administration effectively inhibited adipogenesis in vitro and suppressed adipocyte size of eWAT and sWAT in mice, indicating that EQST reduced fat weight by alleviating adipose tissue expansion through suppressing adipocyte hypertrophy. Moreover, EQST accumulated in adipose tissue and did not affect food intake, blood pressure or heart rate, indicating that EQST exerts its antiobesity effects on adipose tissue directly, rather than acting on the CNS or on the cardiovascular system. Elevated TCA cycle activity has been linked to increased expression of UCP1 and to the reduction of adipocyte size in brown adipose tissue (BAT), eWAT, and sWAT; this can help promote WAT browning and prevent lipid deposition to the liver and kidneys 66 . Knockout or downregulation of genes function in lipid droplets formation also help increase UCP1 expression and induces the appearance of brown fat-like phenotype, which promote WAT browning 67,68 . EQST significantly inhibited lipid droplets formation genes (Fsp27 and Plin5) expression in vitro, EQST treatment also significantly increased UCP1 expression and induced the expression of markers associated with fat burning and elevated TCA cycle activity in sWAT of HFDinduced DIO mice, indicating that EQST promotes energy expenditure in sWAT via promoting WAT browning. Increased expression of PPARg is known to promote the polarization of adipose tissue macrophages (ATM) 69 , and our observation of EQST-induced PPARg repression hints that EQST may exert some anti-inflammatory effects on adipose tissues. More studies will be needed to assess potential impacts of EQST on regulating adipose tissue macrophages. There is a close relationship between UCP1, BAT, and WAT browning 70 , and it should be informative to conduct additional experiments to explore EQST's impacts for enhancing BAT function and/or promoting WAT browning. For example, primary adipose tissue could be isolated and treated with EQST, followed by analysis seeking phenotypes related to beige adipocytes, beige-progenitor cells 70 , and possibly even mitochondrial dynamics 71 .
11b-HSD1 plays a role in metabolic disease by producing locally excessive active glucocorticoids (GCs) in adipose tissue. The metabolism of GCs in obese individuals is unbalanced, resulting from the local upregulation of the expression and activity of 11b-HSD1 in WAT of obese individuals 14,72e75 , 11b-HSD1 is also upregulated during the differentiation process of 3T3-L1 preadipocytes 56,57 . This upregulation results in elevated tissue GCs levels, and excess GCs in adipose tissue have been shown to cause adiposity and insulin resistance 76 specifically by inducing PPARg-dependent adipogenesis 77 . Given that the effects of GCs in tissue are known to depend on the enzymatic activity of 11b-HSD1 48,76 , the inhibition of 11b-HSD1 has been considered a potential anti-obesity therapeutic strategy 78e84 . We found that EQST could inhibit the 11b-HSD1's promotion of lipogenesis induced by excess substrate 11-DHC. And EQST-induced downregulation of genes integral to adipogenesis and lipid biosynthesis all disappeared in the 11b-HSD1-knockdown cells we examined. Knockout of 11b-HSD1 has been proved to be beneficial to metabolic diseases 78e80,85 , whereas enhanced metabolic adaptability of EQST observed in wild-type mice disappeared in 11b-HSD1-KO obese mice supplemented with excessive GC, confirming that 11b-HSD1 is required for the anti-obesity effect of EQST. GCs and 11b-HSD1 also regulate brown adipocyte function. GCs transcriptionally upregulating miR-19b, suppress the expression of beta-1-adrenergic receptors (ADRB1) 19 , which mediates adrenergic stimulation of UCP1 86 . GCs also transcriptionally upregulate miR-27b and suppress the expression of zinc finger transcriptional co-regulator PR domain-containing protein 16 (PRDM16) 18 , which plays role in the beige transdifferentiation from white adipocyte and the brown preadipocyte differentiation 70,87 . Moreover, in hypertrophic adipocytes, increased 11b-HSD1 expression contributes to mitochondrial dysfunction with reduced fatty acid oxidation (FAO) and aerobic respiration, thus inhibiting energy expenditure 13 . Reducing GCs content by inhibiting the expression of 11b-HSD1 has been proved to promote browning by upregulating UCP1 and other BAT-specific proteins 88,89 . The observed abolition of EQST-enhanced UCP1 expression after 11b-HSD1-knockdown suggested that EQST upregulates UCP1 in an 11b-HSD1-dependent manner, yet more studies will be required to determine whether EQST and 11b-HSD1 influence WAT browning and if so to characterize their underlying mechanisms.
We also found that EQST displayed a higher-affinity 11b-HSD1 inhibition and better anti-obesity efficacy than the known 11b-HSD1 inhibitor BVT.2733, which has been reported to improve metabolism disorders 54,88,90e93 and to target adipose tissue 54,91 , but was not appropriate for further development because of the low 11b-HSD1 inhibition potent in human 26 .
Structurally, EQST was predicted to form hydrogen bond interactions with 11b-HSD1's Ser170 and Tyr183 residues; both Ser170 and Tyr183 are known to be key active site residues for cortisone binding with 11b-HSD1 52 and the formation of H-bond interactions with Ser170 and Tyr183 has been previously proposed as a possible approach for enhancing the activity of 11b-HSD1 inhibitors 94 . BVT.2733 was predicted to form hydrogen bond interactions with 11b-HSD1 residues Ser170 and Tyr177; Tyr177 has been proposed to play a role in 11b-HSD1's substrate and inhibitor binding but unlikely a hydrogen bond donor 95 , this may give reason to the BVT.2733's weaker combination with 11b-HSD1 and weaker 11b-HSD1 inhibitory effect even if BVT.2733 was inserted deeper in the binding pocket of 11b-HSD1 than EQST (Supporting Information Fig. S8). 11b-HSD2, another isozyme of 11b-HSD, functions in the inactivation of active GCs 48 . Non-selective 11b-HSD2 inhibition may increase active GCs content and lead to long-term side effects because of the reduction of GCs inactivation in tissues 59,60 . Therefore, high selective inhibition over 11b-HSD1 is required for the development of 11b-HSD1 inhibitors 58,59 . The observed unchanged 11b-HSD2 activity after EQST treatment in vitro suggested that EQST displayed a better-selectivity 11b-HSD1 inhibition. These results may help to explain EQST's anti-obesity effects.
EQST exhibits favorable medicinal potential since that EQST achieve antibacterial effect by inhibiting de novo synthesis of fatty acids 96 and inhibiting quorum sensing 30 , and external application of EQST can effectively promote wound healing by inhibiting bacteria 31 . Our results also demonstrate that EQST confers potent anti-obesity activity. EQST can also be used to synthesize cancer migration inhibitors fusarin A by chemical synthesis 97 . Thus, the development of EQST may provide more clues for drug development. With the establishment of chemical synthesis of EQST 98 and the discovery of key biosynthetic enzymes of EQST 99 , more resources for EQST-related research would be provided, which will gradually promote the development of EQST and more marine natural products.

Conclusions
Our results establish (i) that EQST inhibited HFD-induced obesity in mice by acting on adipose tissue; (ii) that EQST targets and blocks the enzymatic activity of 11b-HSD1; and (iii) that EQST outperformed the 11b-HSD1 inhibitor and anti-obesity agent BVT.2733. Consequently, EQST is a favorable potential candidate for the development of anti-obesity treatments. Our research may provide more information for the development of novel 11b-HSD1 inhibitors.