Development of a silkworms-based evaluation system for the extracts and compounds for their obesity and lipid metabolism improving activity

Development of a silkworms-based evaluation system for the extracts and compounds for their obesity and lipid metabolism improving activity | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Development of a silkworms-based evaluation system for the extracts and compounds for their obesity and lipid metabolism improving activity Mikiyo Wada, Yuki Murata, Hari Prasad Devkota This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4724127/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 23 May, 2025 Read the published version in Scientific Reports → Version 1 posted 10 You are reading this latest preprint version Abstract As lifestyle-related diseases like obesity, dyslipidemia, and non-alcoholic fatty liver disease are increasing globally, the demand for developing therapeutic agents and health foods remains high. However, there is a growing concern worldwide regarding the use of animals for biological testing. Herein, we developed a method using the silkworm, Bombyx mori , to evaluate the effects of compounds on improving obesity and lipid metabolism. A silkworm obesity and lipid metabolism disorder (SOLD) model, fed an 10% glucose diet for 72h showed increased fat body weight and accumulation of neutral fat in the hemolymph and fat body. Administration of fenofibrate reduced neutral fat levels in the hemolymph, and epigallocatechin gallate reduced neutral fat levels in the hemolymph and fat body. Silkworms with improved lipid metabolism, exhibited activation of lipoprotein lipase in muscle tissue, and decreased activities of fatty acid synthase and acetyl-CoA carboxylase, and activation of AMPK in the fat body. Furthermore, enhanced fatty acid beta-oxidation contributed to the promotion of lipolysis. These effects and mechanisms of action observed in silkworms are similar to those found in mammals. These results demonstrate the usefulness of the evaluation system in screening materials for their anti-obesity and lipid metabolism improvement effects using the SOLD model. Biological sciences/Drug discovery Health sciences/Health care hyperlipidemia lipid metabolism obesity animal models silkworm Figures Figure 1 Figure 2 Figure 3 Introduction Today, more than 2.5 billion people worldwide are overweight or have obesity 1 , excessive fat accumulation, posing risk factors for various diseases like type II diabetes, dyslipidemia, cardiovascular disease, and non-alcoholic fatty liver disease (NAFLD). The World Health Organization (WHO) defines people with overweight as a body mass index (BMI) over 25 kg/m² and people with obesity as over 30 kg/m². However, in Asia, individuals with a BMI < 25 face increasing prevalence of hyperglycemia, impaired glucose tolerance, lipid metabolism disorder, and metabolic syndrome due to excessive visceral fat accumulation 2,3 . Most obesity cases are associated with lifestyle disorders, such as overeating and lack of exercise. Moreover, low-income countries witness a rapid rise in people with obesity due to high-carbohydrate diets centered on staple crops 1 . Weight loss can prevent or slow down the progression of obesity-related diseases, thus, improving dietary habits holds potential to reduce health issues and associated medical costs 4 . In recent years, various functional ingredients with anti-obesity and lipid metabolism improvement properties have been discovered in natural products; specifically, there are considerable reports on polyphenols found in plants. Tea polyphenols are known to inhibit lipid absorption, while tea catechins, chlorogenic acid in coffee, capsaicin in capsicum, and curcumin in turmeric inhibit fat accumulation and promote its degradation 5 . While these pharmacological activities have mostly been verified through experiments on mammals, there is a growing movement to abolish animal testing due to animal welfare concerns. Consequently, alternative methods to animal testing are being advocated 6 . The primary alternative method is cell culture systems, but accurately predicting biological reactions involving interactions between tissues and organs is challenging. Insects, however, have attracted attention as pathological models with no animal ethics issues. Insects may appear vastly different from humans, but they possess tissues and organs crucial for glucose and lipid metabolism, with evolutionarily conserved cell signaling pathways. For instance, insects feature a midgut analogous to the mammalian digestive tract, and a fat body analogous to the mammalian liver and adipose tissue. Drosophila melanogaster serves as a representative model organism that demonstrate diet-induced obesity similar to humans when fed high-sugar or high-fat diets, exhibiting characteristics, such as hyperglycemia, increased triglyceride (TG) levels, activation of inflammatory signals, and insulin resistance 7,8,9,10,11,12 . Consequently, Drosophila are utilized as models for obesity and fatty liver disease to evaluate the effects of plant extracts and lactic acid bacteria 13,14,15,16 . However, their extremely small body size poses challenges in measuring food intake and collecting tissues. In mammals, the liver, muscle, and adipose tissue play crucial roles in lipid metabolism, underscoring the importance of assessing metabolic changes in each tissue in an insect model. Moreover, since food intake significantly influences fat accumulation and degradation, precise quantification becomes imperative. The silkworm, Bombyx mori , has attracted attention as an insect model due to its larger body size. Fifth instar silkworms are large, measuring about 4–6 cm, facilitating the easy collection of hemolymph, fat bodies, and muscle. Moreover, these silkworms, daily, consume food equivalent to their own body weight, experiencing significant growth from 1 g after molting to 5 g within three days during which fat accumulates in the fat body. This makes silkworms ideal for verifying the effects of diet-induced inhibition of fat accumulation and degradation, a process that typically spans 10–25 days in Drosophila, within a few days. Furthermore, when fed an artificial diet containing (10GD) for 18 h, silkworms display symptoms similar to human lipid metabolism disorders, including hyperglycemia, increased fat body weight, and increased TG levels in fat bodies and hemolymph 17 . Despite these similarities, currently, there are no established evaluation systems for lipid metabolism enhancers utilizing silkworms. Additionally, Drosophila fed a high-fat diet supplemented with fenofibrate (FBT), a drug used to treat hyperlipidemia, exhibit lower TG levels in their bodies 16 , through the precise mechanism remains unclear. In this study, we established a silkworm with obesity and lipid metabolism disorder (SOLD) model by feeding the worms with a 10GD diet for 72 h. Then, we evaluated the usefulness of the model using the therapeutic effect of FBT and epigallocatechin gallate (EGCG), a functional anti-obesity ingredient. Result Silkworms develop obesity and dyslipidemia when fed a 10% glucose diet (10GD) Silkworms were divided into groups fed either a normal diet (ND) or a 10GD on the first day of the fifth instar (Fig. 1 A). The 10GD group showed decreased food intake and body weight compared to the ND group (Fig. 1 B and C). Fat body weight was higher in the 10GD group (Fig. 1 D and E) showing a time-dependent increase, peaking at 72 h. Furthermore, levels of neutral fat in both the fat body (Fig. 1 F) and hemolymph (Fig. 1 G), as well as hemolymph sugar levels (Fig. 1 H) and bombyxin, a silkworm insulin-like peptide (Fig. 1 I), were significantly increased in silkworms fed with 10GD for 72 h compared to the ND group. These findings suggest that silkworms reared on a 10GD for 72 h develop a phenotype similar to obesity and lipid metabolism disorders observed in mammals. Figure 1 . Evaluation of the effects of hyperlipidemia drugs and anti-obesity functional ingredients on lipid metabolism in the silkworm obesity and lipid metabolism disorder (SOLD) model To assess the efficacy of the SOLD model, silkworms were fed with 10GD supplemented with FBT, EGCG and AMP-activated protein kinase (AMPK) activator (AICAR), a target similar to EGCG, and metformin (MT) and pioglitazone (PG), both antidiabetic drugs that have no effect on improving lipid metabolism, for 72 h (Fig. 2 A). In the SOLD silkworms, food intake and body weight decreased (Supplementary Fig. 1A and B), while fat body weight, NF levels in hemolymph and fat bodies, and hemolymph sugar levels significantly increased compared to the ND group (Fig. 2 B–D). In the FBT group, the hemolymph NF levels decreased in a concentration-dependent manner compared to the untreated group (Fig. 2 D). The EGCG group exhibited a concentration-dependent decrease in NF levels in the fat bodies and hemolymph compared to the untreated group (Fig. 2 C and D). Moreover, hemolymph sugar levels tended to decrease for both compounds compared to the untreated group (Supplementary Fig. 1C). The AICAR group showed a significant decrease in NF levels in fat bodies (Fig. 2 C). Additionally, both the EGCG and AICAR groups showed a trend toward lower fat body weight (Fig. 2 B). However, no significant improvement was observed in the MT and PG groups. These results indicate that the effects of FBT and EGCG on the SOLD model resemble their effects on mammals. Mechanism of action underlying the effects of FBT and EGCG on the SOLD model To clarify the mechanism of action of FBT and EGCG on the SOLD model, we evaluated the activation status of enzymes involved in lipid synthesis signaling in fat bodies using Western blotting (Fig. 2 E). Compared to the ND group, the SOLD model showed increased activities of fatty acid synthase (FAS) activities (Fig. 2 F), along with decreased levels of the phosphorylated acetyl-CoA carboxylase (p-ACC) (Fig. 2 G), and phosphorylated AMPK (p-AMPK) (Fig. 2 H). The FBT group displayed a significant reduction in FAS and an upward trend in p-AMPK and p-ACC compared to the untreated group. Similarly, the EGCG group showed significantly lower FAS levels and higher p-AMPK and p-ACC levels compared to the untreated group (p < 0.01, p < 0.05). These compounds act similarly to their effects in mammals. The AICAR group showed a decreasing trend in FAS and a significant increase in p-AMPK and p-ACC. While the MT group showed a significant decrease in FAS and an increase in p-AMPK, the PG group did not significantly differ from the untreated group. Figure 2 . LPL activity In mammals, FBT activates LPL promotes TG degradation in the blood, EGCG decreases LPL activation in adipose tissue18 21 , and green tea, rich in EGCG, increases LPL activation in muscle 19 . In the SOLD model, LPL activity showed no significant differences in either fat body (Fig. 3 A) or muscle (Fig. 3 B) compared to the ND group. However, in the FBT group, there was an increasing trend in muscle LPL activity. The EGCG group showed a significant decrease in fat body LPL activity (p < 0.05) and a significant increase in muscle LPL activity compared to the ND group (p < 0.01). Furthermore, the AICAR group showed a significant decrease in fat body LPL and a tendency toward increased muscle LPL. Conversely, there were no significant changes observed in the MT and PG groups compared to the untreated group. These results suggest that FBT and EGCG regulate AMPK activation and LPL activity, indicating their action on LPL activity in the silkworm mirrors their effects on mammalian LPL. Fatty acid oxidation activity FBT and EGCG are known to promote the β-oxidation of fatty acids in mammals. To determine whether this effect is present in the SOLD model, we measured the RQ as an indicator of fatty acid oxidation. Silkworms were initially fed with 10 GD for 48 h to induce obesity and dyslipidemia. Subsequently, from 48–75 h, they were fed with ND mixed with FBT, EGCG, or AICAR. During this period, O 2 consumption and CO 2 emissions were monitored using a metabolic measurement system (MK-5000RQ/MS) designed for small animals (Fig. 3 C). RQ was calculated by dividing CO 2 emissions by O 2 consumption (RQ = VCO 2 /VO 2 ). Data collection was paused for the first 3 h after switching to the ND to allow the silkworms to acclimate to the measurement environment. Data were then collected continuously for 24 h from 51th to 75th h. RQ values represent the average of each 8-h period divided into 3 segments over the 24-h periods. In the SOLD model, RQ decreased over time from the start of measurements. In the non-treated group, it remained above 1 at 17–24 h. Conversely, in the FBT, EGCG, and AICAR groups, the RQ at 17–24 h was below 1 (Fig. 3 D). The RQ at 17–24 h was lowest in the EGCG and AICAR groups, which aligns with the observation that both groups exhibited the lowest NF levels in hemolymph and fat bodies (Fig. 3 E–G). Figure 3 . Discussion Establishment of the SOLD model In this study, we aimed to construct an evaluation system for screening compounds that could improve obesity and lipid metabolism disorders, using the silkworm model. Insects possess fat bodies that serve analogous functions of mammalian adipose tissue and liver. Sugars ingested are metabolized into trehalose within the fat body, stored in the hemolymph, or converted into TG and glycogen, which accumulate in the fat body 20 . Silkworms fed with 10GD showed a progressive increase in fat body weight over time (Fig. 1 D), alongside elevated levels of hemolymph bombyxin, suggesting enhanced trehalose uptake into fat bodies and increased fat synthesis. Moreover, the 10GD group exhibited higher levels of hemolymph sugar and NF in fat body and hemolymph (Fig. 1 F–H). In humans, obesity is characterized by excessive body fat accumulation, typically with a BMI of ≥ 25 1 , yet there is a growing population of individuals with high visceral fat levels despite a low BMI 21 . Silkworms fed with 10GD showed reduced overall body weight but increased fat body weight compared to those on a ND, suggesting that the SOLD model bears similarity to visceral fat obesity in human. The pathways for fatty acid biosynthetic are shared between insects and mammals. In mice with high-sugar or high-fat diets induced obesity, there is an increase in the activity of key enzymes for fatty acid synthesis, such as FAS and ACC. Conversely, the activity of AMPK, a signaling protein that inhibits FAS and ACC, decreases 22,23,24,25,26,27 . In the SOLD model, compared to the ND group, there was an increase in FAS expression and a decrease in the inactive form of ACC (p-ACC), while the activated form of AMPK (p-AMPK), responsible for phosphorylating ACC, decreased (Fig. 2 F–H). These results suggest that the SOLD model induces diet-induced obesity through mechanisms similar to those observed in mammals. Usefulness of the SOLD model as an animal model for obesity To assess the utility of the SOLD model, we evaluated the effects of FBT, a drug for hyperlipidemia, and EGCG, known for its ability to inhibit fat accumulation in humans. FBT exhibits various lipid-improving effects, including lowering blood TG levels and increasing blood HDL cholesterol, attributed to the activation of peroxisome proliferator-activated receptor (PPAR)-α. PPARα is strongly expressed in tissues with active β-oxidation, such as liver, kidney, skeletal muscle, and heart, in humans 28 . Its activation increases the production and activity of LPL in muscle and adipose tissue by decreasing the production of ApoC III, thus, facilitating TG degradation in blood and free fatty acid uptake in tissues 29 . Furthermore, PPARα activation enhances β-oxidation and suppressing TG synthesis by activating AMPK in the liver and muscle 30 . In the SOLD model, administration of FBT led to a concentration-dependent decrease in hemolymph NF levels (Fig. 2 D) and a decrease in FAS alongside an increase in p-AMPK and p-ACC in fat bodies (Fig. 2 F–H). Furthermore, the FBT-treated group showed an increasing trend in muscle LPL activity (Fig. 3 B) and a decreasing trend in RQ (Fig. 3 D) compared to the untreated group, suggesting an increase in β-oxidation. The effects and mechanisms of action of FBT observed in the SOLD model closely resembled those seen in mammals. EGCG, a polyphenol abundant in green tea, is recognized for its ability to lower body weight and prevent obesity, diabetes, and cardiovascular diseases 31 . In mice with diet-induced obesity, EGCG decrease fat accumulation, blood TG, cholesterol, liver TG, blood glucose levels and the intestinal absorption of lipids 32,33,34 . EGCG primary mechanisms involve inhibiting fatty acid synthesis by activating AMPK in the liver, skeletal muscle and adipose tissue, as well as enhancing β-oxidation 35,36,37 ; additional effects include decreased LPL activity in adipose tissue 18 , increased expression of PPARα in muscle 38 and a decrease in RQ 39 [ 41 ]. Additionally, green tea increases LPL activity in muscle tissue 19 . In the SOLD model, administration of EGCG led to a reduction in NF levels in the fat body and hemolymph (Fig. 2 C and D). Moreover, the EGCG-treated group showed increased p-AMPK and p-ACC in the fat body (Fig. 2 G and H), decreased fat body LPL activity (Fig. 3 A), increased muscle LPL activity (Fig. 3 B), and decreased RQ (Fig. 3 D). The effects of EGCG on lipid metabolism in the SOLD model were consistent with those in the mammalian model with obesity. In this study, the effects of FBT and EGCG on the SOLD model demonstrated increased muscle LPL activity and decreased RQ. High RQ and low RQ indicate fat synthesis and β-oxidation, respectively 40 . Our results suggest that FBT and EGCG influenced myocytes LPL in silkworm, promoting the uptake of hemolymph NF and fat oxidation in muscle tissue. In the flying insect, LPL is found in the fat bodies, muscle and ovaries 41,42 . Diglycerides (DG), degraded by fat body lipase, bind to lipophorin, an insect lipoprotein in hemolymph and are uptake into the muscle cells via myocyte membrane LPL for ATP production 43,44 . Our report is the first to suggest that in the silkworm, LPL is expressed in muscle and involved in ATP production. In the SOLD model, the effective dose of FBT (10 mg/kg/day) for improving lipid metabolism was approximately one-tenth of that in mice (100 mg/kg/day 45 ) and 3–5 times that administered in humans (1.8–2.7 mg/kg/day). The effective dose of EGCG in the SOLD model (100–200 mg/kg/day) was about twice that used in mice (50–100 mg/kg/day 36 ) and approximately 10 times that used in humans (10 mg/kg/day). Despite these differences, the effective doses of both compounds in the SOLD model were comparable to those in mice or humans, suggesting their potential utility as alternative models. Furthermore, FBT and EGCG not only suppress fat accumulation by activating AMPK in the liver but also prevent the onset of NAFLD in mice 46, 47 . These effects were observed in the fat body of silkworms, suggesting that the SOLD model may offer a means to evaluate improved effects on NAFLD. The SOLD model offers a means to evaluate lipid metabolism with a small sample, within a short timeframe, and at a low cost, and without encountering animal ethics issues. These results indicate the utility of the SOLD model for screening therapeutic drugs and functional food ingredients aimed at improving obesity and lipid metabolism disorders. Materials and Methods Chemicals Trehalase from porcine kidney and palmitoyl coenzyme A lithium salt was purchased from Sigma Aldrich Japan, Co, LLC. (Tokya, Japan). AICAR (S7863) was purchased from Selleck Chemicals (Houston TX, USA). (−)-Epigallocatechin gallate hydrate, pioglitazone hydrochloride, fenofibrate were obtained from Tokyo Chemical Industry, Co. (Tokyo, Japan). D-(+)-glucose were purchased from Nacalai Tesque, Inc. (Kyoto, Japan). Metformin hydrochloride, trehalose dihydrate, oil Red O, 4%-paraformaldehyde phosphate buffer solution, heparin sodium, 4-methylumbelliferyl oleate and 2-propanol were obtained from FUJIFILM Wako Pure Chemical Industries, Ltd. (Tokyo, Japan). Primary antibodies FAS (#3180), p-AMPK (#2535T), p-ACC (#11818T), and β-actin (#4967) were obtained from Cell Signaling Technology (Beverly, USA). Secondary antibodies, goat anti-rabbit IgG (H + L) antibody, and HRP conjugate (SA00001-2) were purchased from Cosmo Bio, Co. (Tokyo, Japan). The TaKaRa BCA Protein Assay Kit was obtained from Takara Bio, Inc. (Shiga, Japan). Silkworm rearing conditions and diet preparation Silkworms (hybrid race: Kin-Shu × Sho-Wa) were kindly provided by Atsumaru Holdings NSP Yamaga Plant (Atsumaru Inc., Kumamoto, Japan). Upon hatching, larvae were reared to the fifth instar on an artificial diet, SilkMate PM (Nosan Corporation), within plastic containers maintained at 25–27°C and 50–70% humidity. An artificial diet was prepared following the instructions provided by the feed company. SilkMate PM was blended with water at a 1:3 ratio and subsequently steamed in an autoclave at 105°C for 20 min. For the glucose diet, SilkMate PM, glucose, and water were mixed to achieve an equivalent weight of sugar, then subjected to autoclaving. Hemolymph and fat body collection methods Hemolymph (10 µL) was collected from the larva’s proleg using a 30 G needle (insulin syringe; Becton, Dickinson and Company) and immediately frozen on dry ice, then stored at − 20°C. The fat body was isolated from the larva’s dorsolateral region while in suspended animation by placing in a container filled with dry ice. After rinsing with PBS, drying, and scraping off with tweezers, the fat bodies were weighed and immediately frozen in liquid nitrogen, then stored at − 80°C. Determination of hemolymph trehalose and glucose levels The hemolymph trehalose concentration was determined with modifications to Tennessen’s method 47 . Hemolymph (2 µL) was diluted 100-fold in buffer (5 mM Tris, 137 mM NaCl, 2.7 mM KCl, and pH 6.7), then incubated at 80°C for 3 min before returning to room temperature. Hemolymph solution (100 µL) was mixed with 100 µL of porcine trehalase solution (10 mU/mL) diluted in buffer, incubated overnight at 37°C, and the resulting glucose production was measured using the Glucose CII Test Wako (FUJIFILM Wako Pure Chemical Industries, Tokyo, Japan), with trehalose concentration calculated from a calibration curve prepared using the same measurements. Glucose concentration was also measured using the Glucose CII Test Wako. For this, hemolymph (2 µL) was added to a frozen 96 well plate, followed by the addition of chromogenic solution (200 µL). After incubation at room temperature for 15 min, absorbance was measured at 505 nm. Determination of the neutral fat levels in hemolymph Neutral fat (NF) levels in the hemolymph were measured using LabAssay™ Triglycerides (FUJIFILM Wako Pure Chemical Industries, Tokyo, Japan). Hemolymph (2 µL) was added to a frozen 96-well plate, followed by the addition of chromogenic solution (300 µL). After incubating at room temperature for 5 min, absorbance was measured at 600 and 700 nm. Western blotting The fat body (frozen weight 50 mg) was crushed in 1 mL of lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 1% NP-40 Substitute, 0.5% Sodium Deoxycholate, 0.1% SDS, Protease inhibitor cocktail I, Phosphatase inhibitor cocktail II, pH 7.6) for 30 s using a polytron homogenizer. After incubating on ice for 30 min, the samples were centrifuged (14,000 g, 10 min, 4°C) and the protein concentration of the supernatant was determined using the BCA method. Each sample was diluted with sample buffer (125 mM Tris-HCl, 4% SDS, 20% glycerol, 0.004% bromophenol blue, 50 mM DTT, and pH 6.8) and water to adjust the protein concentration. The samples were then heat-treated at 90°C for 5 min and subjected to SDS-PAGE on 10% polyacrylamide gel. Proteins in the gel were transferred to a PVDF membrane, incubated with primary antibodies and secondary antibodies, and bands of the target proteins were detected using SuperSignal TM Chemiliuminescent HRP Substrates (Thermo Fisher Scientific Logo). Bands were quantified using Image J. Oil red O staining of the silkworm NF in fat bodies NF staining was performed with modifications to Matsumoto’s method 17 . Fat bodies were immediately immersed in 4% formaldehyde upon removal and fixed overnight at 4°C. The samples were rinsed in phosphate buffer (137 mM NaCl, 8.1 mM Na 2 HPO 4 , 2.68 mM KCl, 1.47 mM KH 2 PO 4 , and pH 7.4), treated with 60% 2-propanol for 1 min, and then stained with Oil red O stain solution (1.8 mg of Oil red O in 1 mL of 60% 2-propanol) for 20 min at room temperature. After being washed three times with 60% 2-propanol, the samples were immersed in 100% 2-propanol and sonicated for 30 min. Following centrifugation (10,000 g, 3 min), absorbance was measured at 490 nm, and triglyceride levels were calculated based on absorbance per gram of fat body. Measurement of Lipoprotein lipase activity Sample preparation for the Lipoprotein lipase (LPL) activity assay was performed using a modification of Ribeiro’s method 41 . Fat body and muscle (frozen weight 100 mg) were homogenized in 1 mL of ice-cold heparin solution (0.25 M Sucrose, 1 mM EDTA, 3 mM Tris-HCl, 2% BSA, 2 U/ml Heparin Sodium, and pH 7.5) using a polytron homogenizer. After incubation at 37°C for 1 h, the samples were centrifuged, and the supernatant was collected as the enzyme solution. Protein concentrations of the enzyme solutions were determined using the BCA method and then diluted in phosphate buffer (0.2 M Na 2 HPO 4 12H 2 O, 0.2 M NaH 2 O 4 2H 2 O, and pH 7.4) to adjust the protein concentration of each sample. LPL activity was assessed using a fluorescence method with 0.5 mM oleic acid 4-methylumbelliferyl solution dissolved in phosphate buffer as the substrate. Upon mixing of enzyme solution (100 µL) and substrate solution (100 µL) in a 96-well microplate, fluorescence intensity (ex/em = 355/460) was promptly measured for 30 min at 2 min intervals. The ratio of fluorescence intensity at 30 min to the fluorescence intensity at 0 min was used as the measure of LPL activity. Measurement of respiratory quotient The method was modified from the Hanatani method 48 . Measurements were performed using a metabolic measurement system designed for small animals (MK-5000RQ/MS model, Muromachi Machinery Corporation, Tokyo, Japan) at a room temperature of 26°C within a 12-h light–dark cycle. Silkworms fed with 10GD for 72 h were placed in groups of five in closed cages and provided with a ND, with or without the sample. Oxygen consumption (VO 2 ) and carbon dioxide production (VCO 2 ) were recorded every 5 min for 24 h. The respiratory quotient (RQ) was calculated as the ratio of VO 2 per VCO 2 . Statistical Analysis All data were presented as the mean ± SD of three replicates. Measurements of food intake and RQ were averaged across the five silkworms. The data were analyzed using a t-test, with as statistical significance set at p < 0.05 and p < 0.01. Declarations Conflict of interest: The authors declare no competing interests. Author Contribution Investigation, formal analysis, writing—original draft, YT; writing—review and editing, HPD; conceptualization, writing—review and editing, supervision, MY. Acknowledgement The authors would like to thank Atsumaru Holdings NSP Yamaga Plant, Atsumaru Yamaga Silk, for their kind gift of silkworms. We also thank Dr. Yasuhiko Matsumoto for his cooperation. This work was supported by JSPS KAKENHI Grant Number JP23804033. Data Availability The datasets used and/or analysed during the current study available from the corresponding author on reasonable request. References World Health Organization (WHO). Obesity and overweight. 2024. https://www.who.int/news-room/fact-sheets/detail/obesity-and-overweight Zhu, L. et al . Lean Yet Unhealthy: Asian American Adults Had Higher Risks for Metabolic Syndrome than Non-Hispanic White Adults with the Same Body Mass Index: Evidence from NHANES 2011–2016. Healthcare 9, 1518 (2021). https://doi.org/10.3390/healthcare9111518 Fan, J.G., Kim, S.U. & Wang, V.W.S. New trends on obesity and NAFLD in Asia. J Hepatol 67, 862–873 (2017) https://doi.org/10.1016/j.jhep.2017.06.003 Lee, M. et al . The cost-effectiveness of pharmacotherapy and lifestyle intervention in the treatment of obesity. Obes Sci Pract 6, 162–170 (2020). https://doi.org/10.1002/osp4.390 Ohishi, T. et al . The Beneficial Effects of Principal Polyphenols from Green Tea, Coffee, Wine, and Curry on Obesity. Molecules 26, 453 (2021). https://doi.org/10.3390/molecules26020453 Dellambra, E. et al . A. Non-Animal Models in Dermatological Research. ALTEX 36, 177–202 (2018). https://doi.org/10.14573/altex.1808022 Elbrense, H., Montaser, O., El-Aasr, M. & Meshrif, W.S. Potential anti-diabetic effect of certain plant extracts from the Egyptian flora on type II diabetes using Drosophila melanogaster as an animal model. IJCBR 5, 121–133 (2021). Lourido, F., Quenti, D., Salgado-Canales, D. & Tobar, N. Domeless receptor loss in fat body tissue reverts insulin resistance induced by a high–sugar diet in Drosophila melanogaster. Sci Rep 11, 3263 (2021). https://doi.org/10.1038/s41598-021-82944-4 Buescher, J.L. et al . Evidence for transgenerational metabolic programming in Drosophila. Dis Model Mech 6, 1123–1132 (2013). https://doi.org/10.1242/dmm.011924 Rovenko, B.M. et aI . High sucrose consumption promotes obesity whereas its low consumption induces oxidative stress in Drosophila melanogaster. J Insect Physiol 79, 42–54 (2015). https://doi.org/10.1016/j.jinsphys.2015.05.007 Reis, T. Effects of Synthetic Diets Enriched in Specific Nutrients on Drosophila Development, Body Fat, and Lifespan. PLos ONE 11, e0146758 (2016). https://doi.org/10.1371/journal.pone.0146758 Woodcock, K.J. et al . Macrophage-Derived upd3 Cytokine Causes Impaired Glucose Homeostasis and Reduced Lifespan in Drosophila Fed a Lipid-Rich Diet. Immunity 42, 133–144 (2015). http://dx.doi.org/10.1016/j.immuni.2014.12.023 Kayashima, Y. et al . Tea polyphenols ameliorate fat storage induced by high-fat diet in Drosophila melanogaster. Biochem Biophys Rep 4, 417–424 (2015). https://doi.org/10.1016/j.bbrep.2015.10.013 Azuma, M. et al . RNA-seq analysis of diet-driven obesity and anti-obesity effects of quercetin glucoside or epigallocatechin gallate in Drosophila adults. Eur Rev Med Pharmacol Sci 23, 857–876 (2019). https://doi.org/10.26355/eurrev_201901_16901 Wongchum, N. et al . Hydroethanolic Cyperus rotundus L. extract exhibits anti-obesity property and increases lifespan expectancy in Drosophila melanogaster fed a high-fat diet. J HerbMed Pharmacol 11, 296–304 (2022). https://doi.org/10.34172/jhp.2022.35 Asiimwe, O.H. et al . Anti-obesity effects of Erythrina abyssinica stem bark extract in flies exposed to a high fat diet. Heliyon 8, e09886 (2022). https://doi.org/10.1016/j.heliyon.2022.e09886 Matsumoto, Y. et al . Diabetic silkworms for evaluation of therapeutically effective drugs against type II diabetes. Sci Rep 5, 10722 (2015). https://doi.org/10.1038/srep10722 Lee, M.S., Kim, C.T. & Kim, Y. Green tea (–)-epigallocatechin-3-gallate reduces body weight with regulation of multiple genes expression in adipose tissue of diet-induced obese mice. Ann Nutr Metab 54, 151–157 (2009). https://doi.org/10.1021/jf402004x Serisier, S. et al . Effects of green tea on insulin sensitivity, lipid profile and expression of PPARalpha and PPARgamma and their target genes in obese dogs. Br J Nutr 99, 1208–1216 (2008). https://doi.org/10.1017/S0007114507862386 Bayliak, M.M. et al . Interplay between diet-induced obesity and oxidative stress: Comparison between Drosophila and mammals. Comp Biochem Physiol Part A Mol Integr Physiol 228, 18–28 (2019). https://doi.org/10.1016/j.cbpa.2018.09.027 Tchernof, A. & Després, J.P. Pathophysiology of Human Visceral Obesity: an update. Physiol Rev 93, 359–404 (2013). https://doi.org/10.1152/physrev.00033.2011 Ghareghani, P. et al . Aerobic endurance training improves nonalcoholic fatty liver disease (NAFLD) features via miR-33 dependent autophagy induction in high fat diet fed mice. Obes Res Clin Pract 12, 80–89 (2018). https://doi.org/10.1016/j.orcp.2017.01.004 Liu, C. et al . Flavonoid-Rich Extract of Paulownia fortunei Flowers Attenuates Diet-Induced Hyper liquidemia, Hepatic Steatosis and Insulin Resistance in Obesity Mice by AMPK Pathway. Nutrients 9, 959 (2017). https://doi.org/10.3390/nu9090959 Inamdar, S. et al . Vitexin alleviates non-alcoholic fatty liver disease by activating AMPK in high fat diet fed mice. Biochem Biophys Res Commun 519, 106–112 (2019). https://doi.org/10.1016/j.bbrc.2019.08.139 Prakash, S. et al . Amelioration of diet-induced metabolic syndrome and fatty liver with sitagliptin via regulation of adipose tissue inflammation and hepatic Adiponectin/AMPK levels in mice. Biochimie 168, 198–209 (2020). https://doi.org/10.1016/j.biochi.2019.11.005 Lee, H.A. et al . Ganoderma lucidum Extract Reduces Insulin Resistance by Enhancing AMPK Activation in High-Fat Diet-Induced Obese Mice. Nutrients 12, 3338 (2020). https://doi.org/10.3390/nu12113338 Li, T. et al . Milk Fat Globule Membrane Attenuates High-Fat Diet-Induced Obesity by Inhibiting Adipogenesis and Increasing Uncoupling Protein 1 Expression in White Adipose Tissue of Mice. Nutrients 10, 331 (2018). https://doi.org/10.3390/nu10030331 Grygiel-Górniak, B. Peroxisome proliferator-activated receptors and their ligands: nutritional and clinical implications - a review. Nutr J 13 (2014). https://doi.org/10.1186/1475-2891-13-17 Duval, C., Müller, M. & Kersten, S. PPARα and dysliquidemia. Biochim Biophys Acta Mol Cell Biol Lipids 1771, 961–971 (2007). https://doi.org/10.1016/j.bbalip.2007.05.003 Chen, W.L. et al . Fenofibrate lowers lipid accumulation in myotubes by modulating the PPARα/AMPK/FoxO1/ATGL pathway. Biochem Pharmacol 84, 522–531 (2012). https://doi.org/10.1016/j.bcp.2012.05.022 Yang, C.S. et al . Mechanisms of body weight reduction and metabolic syndrome alleviation by tea. Mol Nutr Food Res 60, 160–174 (2016). https://doi.org/10.1002/mnfr.201500428 Chen, Y.K. et al . Effects of Green Tea Polyphenol (–)-Epigallocatechin-3-gallate on Newly Developed High-Fat/Western-Style Diet-Induced Obesity and Metabolic Syndrome in Mice. J Agric Food Chem 59, 11862–11871 (2011). https://doi.org/10.1021/jf2029016 Grove, K.A., Sae-Tan, S., Kennett, M.J. & Lambert, J.D. (–)-Epigallocatechin-3-gallate Inhibits Pancreatic Lipase and Reduces Body Weight Gain in High Fat-Fed Obese mice. Obesiy 20, 2311–2313 (2012). https://doi.org/10.1038/oby.2011.139 Bose, M. et al . The Major Green Tea Polyphenol, (–)-Epigallocatechin-3-Gallate, Inhibits Obesity, Metabolic Syndrome, and Fatty Liver Disease in High-Fat–Fed Mice. J Nutr 138, 1677–1683 (2009). https://doi.org/10.1093/jn/138.9.1677 Murase, T., Misawa, K., Haramizu, S. & Hase, T. Catechin-induced activation of the LKB1/AMP-activated protein kinase pathway. Biochem Pharmacol 78, 78–84 (2009). https://doi.org/10.1016/j.bcp.2009.03.021 Takagaki, A. et al . Effects of Microbial Metabolites of (–)-Epigallocatechin Gallate on Glucose Uptake in L6 Skeletal Muscle Cell and Glucose Tolerance in ICR Mice. Biol Pharm Bull 42, 212–221 (2019). https://doi.org/10.1248/bpb.b18-00612 Li, F. et al . EGCG Reduces Obesity and White Adipose Tissue Gain Partly Through AMPK Activation in Mice. Front Pharmacol 9, 1366 (2018). https://doi.org/10.3389/fphar.2018.01366 Huang, J. et al . Green tea polyphenol EGCG alleviates metabolic abnormality and fatty liver by decreasing bile acid and lipid absorption in mice. Mol Nutr Food Res 62, 1700696 (2018). https://doi.org/10.1002/mnfr.201700696 Kapoor, M.P., Sugita, M., Fukuzawa, Y. & Okubo, T. Physiological effects of epigallocatechin-3-gallate (EGCG) on energy expenditure for prospective fat oxidation in humans: A systematic review and meta-analysis. J Nutr Biochem 43, 1–10 (2017). https://doi.org/10.1016/j.jnutbio.2016.10.013 Talal, S. et al . High carbohydrate diet ingestion increases post-meal lipid synthesis and drives respiratory exchange ratios above 1. J Exp Biol 224, jeb240010 (2021). https://doi.org/10.1242/jeb.240010 Ribeiro, L. & Fonseca, C.L.C. Occurrence of lipoprotein lipase in the fat body of Triatoma maculata. Comp Biochem Physiol B 52, 523–524 (1975). https://doi.org/10.1016/0305-0491(75)90229-1 Heusden, M.C.V. Characterization and Identification of a Lipoprotein Lipase from Manduca sexta Flight Muscle. Insect Biochem Mol Biol 23, 785–792 (1993). https://doi.org/10.1016/0965-1748(93)90066-2 Toprak, U., Hegedus, D., Doğan, C. & Güney, G. A journey into the world of insect lipid metabolism. Arch Insect Biochem Physiol 88 (2020). https://doi.org/10.1002/arch.21682 Forcheron, F., Basset, A., Carmine, P.D. & Beylot, M. Lipase maturation factor 1: Its expression in Zucker diabetic rats, and effects of metformin and fenofibrate. Diabetes Metab 35, 452–457 (2009). https://doi.org/10.1016/j.diabet.2009.05.004 Yoo, J. et al . Fenofibrate, a PPARα agonist, reduces hepatic fat accumulation through the upregulation of TFEB-mediated lipophagy. Metabolism 120, 154798 (2021). https://doi.org/10.1016/j.metabol.2021.154798 Tang, G. et al . Green Tea and Epigallocatechin Gallate (EGCG) for the Management of Nonalcoholic Fatty Liver Diseases (NAFLD): Insights into the Role of Oxidative Stress and Antioxidant Mechanism. Antioxidants 10, 1076 (2021). https://doi.org/10.3390/antiox10071076 Tennessen, J.M., Barry, W., Cox, J. & Thummel, C.S. Methods for studying metabolism in Drosophila. Methods 68, 105–115 (2014). https://doi.org/10.1016/j.ymeth.2014.02.034 Hanatani, S. et al . Acetate alters expression of genes involved in beige adipogenesis in 3T3-L1 cells and obese KK-Ay mice. J Clin Biochem Nutr 59, 207–214 (2016). https://doi.org/10.3164/jcbn.16-23 Additional Declarations No competing interests reported. Supplementary Files SupplementaryFigureobesityMurata.pdf Cite Share Download PDF Status: Published Journal Publication published 23 May, 2025 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 12 Aug, 2024 Reviews received at journal 08 Aug, 2024 Reviews received at journal 04 Aug, 2024 Reviewers agreed at journal 26 Jul, 2024 Reviewers agreed at journal 26 Jul, 2024 Reviewers invited by journal 24 Jul, 2024 Editor assigned by journal 24 Jul, 2024 Editor invited by journal 16 Jul, 2024 Submission checks completed at journal 13 Jul, 2024 First submitted to journal 11 Jul, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4724127","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":335715003,"identity":"dcf112c1-18e1-498a-b7d0-d90683cffc30","order_by":0,"name":"Mikiyo Wada","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA80lEQVRIiWNgGAWjYHACAyC2MUAT5CGoJQ2qJYF4LYfRteBTf/zwxs88FeeNDa4dfrrh4w8bBoMDzA8/MMjcwa3lTFqxNM+Z22YGt9PMbs5ISANqYTOWYOB5hlOL2YEcA2netts2BrcTzG7zJByu33CAwQzol8O4tZx/Y/ybt+0cUEv6N5AWoC3s3/BruZFjBrTlANBhOWZQLTz4bbG/8azMcs6ZZGPJ2zllN2ekpTFIHuYplkjA4xfJ/uTNN95U2Bn23U7fduODjQ0D3/H2jR8+9uAOMRBgQo03ZiBO7DmAVwvjD0yxH/i1jIJRMApGwYgCABT+WM2q9FhiAAAAAElFTkSuQmCC","orcid":"","institution":"Kumamoto University","correspondingAuthor":true,"prefix":"","firstName":"Mikiyo","middleName":"","lastName":"Wada","suffix":""},{"id":335715004,"identity":"56418f3e-60e2-4031-9ff6-9870c8198441","order_by":1,"name":"Yuki Murata","email":"","orcid":"","institution":"Kumamoto University","correspondingAuthor":false,"prefix":"","firstName":"Yuki","middleName":"","lastName":"Murata","suffix":""},{"id":335715005,"identity":"70269e90-a45a-4163-88d3-002f7d34d29d","order_by":2,"name":"Hari Prasad Devkota","email":"","orcid":"","institution":"Kumamoto University","correspondingAuthor":false,"prefix":"","firstName":"Hari","middleName":"Prasad","lastName":"Devkota","suffix":""}],"badges":[],"createdAt":"2024-07-11 12:29:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4724127/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4724127/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-96863-1","type":"published","date":"2025-05-23T15:57:40+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":61882251,"identity":"e0988989-75c3-4be1-bb30-f8298819f96b","added_by":"auto","created_at":"2024-08-06 15:38:58","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":531683,"visible":true,"origin":"","legend":"\u003cp\u003eDevelopment of hyperlipidemia and obesity in silkworms fed with 10% glucose diet.\u003c/p\u003e\n\u003cp\u003e(A) Experimental design. Silkworms were divided into groups fed either a normal diet (ND) or a 10% glucose diet (10GD) for 72 h (n = 3/group). (B) Food intake, (C) body weight, (D) fat body weight were measured every 24 h. (E) Photographs of the fat body at 72 h. (F) Neutral fat in the fat body and (G) in the hemolymph, (H) total sugar in the hemolymph, (I) bombyxin in the hemolymph were measured at 72 h (n = 5/group). Data represent mean ± SD. Significant differences between groups were evaluated using a t-test. ##p \u0026lt; 0.01, #p \u0026lt; 0.05 compared with the ND group, **p \u0026lt; 0.01, *p \u0026lt; 0.05, compared with the 10GD group.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4724127/v1/9e85df48bf3754a642edfd58.jpeg"},{"id":61882250,"identity":"7aaa40d5-f543-48b7-8765-2581f9a7e4ac","added_by":"auto","created_at":"2024-08-06 15:38:58","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":882091,"visible":true,"origin":"","legend":"\u003cp\u003eEvaluation of the lipid-lowering effect of various drugs in the hemolymph and fat body in the silkworm obesity and lipid metabolism disorder (SOLD) model.\u003c/p\u003e\n\u003cp\u003e(A) Experimental design. Silkworms were fed a normal diet (ND), a 10% glucose diet (10GD), or 10GD containing fenofibrate (FBT; 1, 2, and 20 mg/kg/days), epigallocatechin gallate (EGCG; 20, 200 and 400 mg/kg/days), AICAR (20, 200, and 400 mg/kg/days), metformin (MT; 10, 20, and 200 mg/kg/days), and pioglitazone (PG; 10, 20, and 200 mg/kg/days) for 72 h (n = 10/group). (B) fat body weight, (C) neutral fat in the fat body and (D) in the hemolymph were measured at 72 h (n = 4). (E) Western blotting analysis was performed to evaluate the expression levels of (F) fatty acid synthase (FAS), (G) phosphorylated acetyl-CoA carboxylase (p-ACC), (H) phosphorylated AMP-activated protein kinase (p-AMPK) in the fat body of the SOLD model fed 10GD containing FBT (20 mg/kg/days), EGCG (400 mg/kg/days), AICAR (400 mg/kg/days), MT (200 mg/kg/days), and PG (200 mg/kg/days) for 72 h. Relative expression levels of the proteins were calculated by normalization to β-actin expression. Data represent mean ± SD. Significant differences between groups were evaluated using a t-test (n = 3). ##p \u0026lt; 0.01, #p \u0026lt; 0.05 compared with the ND group, **p \u0026lt; 0.01, *p \u0026lt; 0.05, compared with the 10GD group.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4724127/v1/b38193be6754fadfa58471cf.jpeg"},{"id":61882252,"identity":"cf876e07-90dc-4bbd-b10d-66f19ca87c2a","added_by":"auto","created_at":"2024-08-06 15:38:58","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":575461,"visible":true,"origin":"","legend":"\u003cp\u003eLPL activity in the fat body and muscle, and respiratory quotient (RQ) of the silkworm obesity and lipid metabolism disorder model after drug administrated.\u003c/p\u003e\n\u003cp\u003e(A, B) Silkworms were fed a normal diet (ND), a 10% glucose diet (10GD), or 10GD containing FBT (20 mg/kg/days), EGCG (400 mg/kg/days), AICAR (400 mg/kg/days), MT (200 mg/kg/days), and PG (200 mg/kg/days), respectively for 72 h (n = 5/group). (A) LPL activity in fat bodies and (B) in muscles were measured at 72 h. (C) Experimental design: Silkworms were initially fed with a 10GD for 48 h (n = 5/group), followed by a switch to either a ND or ND supplemented with FBT (20 mg/kg/days), EGCG (400 mg/kg/days), AICAR (400 mg/kg/days), MT (200 mg/kg/days), and PG (200 mg/kg/days). (D) Respiratory exchange ratio was measured for 12 h after the diet switch. At the end of the measurement, fat bodies and hemolymph were collected, and (E) fat body weight, (F) neutral fat in the fat body and (G) in the hemolymph were measured. Data represent mean ± SD. Significant differences between groups were evaluated using a t-test. **p \u0026lt; 0.01, *p \u0026lt; 0.05 compared with the 10GD group.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4724127/v1/19edd4a3c48a1e868a0e128a.jpeg"},{"id":83460639,"identity":"012d4030-debb-48e6-8997-1bdbae871952","added_by":"auto","created_at":"2025-05-26 16:13:05","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2826146,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4724127/v1/c188e859-256c-4080-a1b1-5fd54118be15.pdf"},{"id":61882249,"identity":"83a452da-2b6b-41cd-9926-ddb6f370c6f1","added_by":"auto","created_at":"2024-08-06 15:38:58","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":263217,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigureobesityMurata.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4724127/v1/6a6b72334457f6b6b73ddfbb.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Development of a silkworms-based evaluation system for the extracts and compounds for their obesity and lipid metabolism improving activity","fulltext":[{"header":"Introduction","content":"\u003cp\u003eToday, more than 2.5\u0026nbsp;billion people worldwide are overweight or have obesity\u003csup\u003e1\u003c/sup\u003e, excessive fat accumulation, posing risk factors for various diseases like type II diabetes, dyslipidemia, cardiovascular disease, and non-alcoholic fatty liver disease (NAFLD). The World Health Organization (WHO) defines people with overweight as a body mass index (BMI) over 25 kg/m\u0026sup2; and people with obesity as over 30 kg/m\u0026sup2;. However, in Asia, individuals with a BMI\u0026thinsp;\u0026lt;\u0026thinsp;25 face increasing prevalence of hyperglycemia, impaired glucose tolerance, lipid metabolism disorder, and metabolic syndrome due to excessive visceral fat accumulation\u003csup\u003e2,3\u003c/sup\u003e. Most obesity cases are associated with lifestyle disorders, such as overeating and lack of exercise. Moreover, low-income countries witness a rapid rise in people with obesity due to high-carbohydrate diets centered on staple crops\u003csup\u003e1\u003c/sup\u003e. Weight loss can prevent or slow down the progression of obesity-related diseases, thus, improving dietary habits holds potential to reduce health issues and associated medical costs\u003csup\u003e4\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn recent years, various functional ingredients with anti-obesity and lipid metabolism improvement properties have been discovered in natural products; specifically, there are considerable reports on polyphenols found in plants. Tea polyphenols are known to inhibit lipid absorption, while tea catechins, chlorogenic acid in coffee, capsaicin in capsicum, and curcumin in turmeric inhibit fat accumulation and promote its degradation\u003csup\u003e5\u003c/sup\u003e. While these pharmacological activities have mostly been verified through experiments on mammals, there is a growing movement to abolish animal testing due to animal welfare concerns. Consequently, alternative methods to animal testing are being advocated\u003csup\u003e6\u003c/sup\u003e. The primary alternative method is cell culture systems, but accurately predicting biological reactions involving interactions between tissues and organs is challenging. Insects, however, have attracted attention as pathological models with no animal ethics issues.\u003c/p\u003e \u003cp\u003eInsects may appear vastly different from humans, but they possess tissues and organs crucial for glucose and lipid metabolism, with evolutionarily conserved cell signaling pathways. For instance, insects feature a midgut analogous to the mammalian digestive tract, and a fat body analogous to the mammalian liver and adipose tissue. \u003cem\u003eDrosophila melanogaster\u003c/em\u003e serves as a representative model organism that demonstrate diet-induced obesity similar to humans when fed high-sugar or high-fat diets, exhibiting characteristics, such as hyperglycemia, increased triglyceride (TG) levels, activation of inflammatory signals, and insulin resistance\u003csup\u003e7,8,9,10,11,12\u003c/sup\u003e. Consequently, \u003cem\u003eDrosophila\u003c/em\u003e are utilized as models for obesity and fatty liver disease to evaluate the effects of plant extracts and lactic acid bacteria\u003csup\u003e13,14,15,16\u003c/sup\u003e. However, their extremely small body size poses challenges in measuring food intake and collecting tissues. In mammals, the liver, muscle, and adipose tissue play crucial roles in lipid metabolism, underscoring the importance of assessing metabolic changes in each tissue in an insect model. Moreover, since food intake significantly influences fat accumulation and degradation, precise quantification becomes imperative. The silkworm, \u003cem\u003eBombyx mori\u003c/em\u003e, has attracted attention as an insect model due to its larger body size.\u003c/p\u003e \u003cp\u003eFifth instar silkworms are large, measuring about 4\u0026ndash;6 cm, facilitating the easy collection of hemolymph, fat bodies, and muscle. Moreover, these silkworms, daily, consume food equivalent to their own body weight, experiencing significant growth from 1 g after molting to 5 g within three days during which fat accumulates in the fat body. This makes silkworms ideal for verifying the effects of diet-induced inhibition of fat accumulation and degradation, a process that typically spans 10\u0026ndash;25 days in Drosophila, within a few days. Furthermore, when fed an artificial diet containing (10GD) for 18 h, silkworms display symptoms similar to human lipid metabolism disorders, including hyperglycemia, increased fat body weight, and increased TG levels in fat bodies and hemolymph\u003csup\u003e17\u003c/sup\u003e. Despite these similarities, currently, there are no established evaluation systems for lipid metabolism enhancers utilizing silkworms. Additionally, \u003cem\u003eDrosophila\u003c/em\u003e fed a high-fat diet supplemented with fenofibrate (FBT), a drug used to treat hyperlipidemia, exhibit lower TG levels in their bodies\u003csup\u003e16\u003c/sup\u003e, through the precise mechanism remains unclear.\u003c/p\u003e \u003cp\u003eIn this study, we established a silkworm with obesity and lipid metabolism disorder (SOLD) model by feeding the worms with a 10GD diet for 72 h. Then, we evaluated the usefulness of the model using the therapeutic effect of FBT and epigallocatechin gallate (EGCG), a functional anti-obesity ingredient.\u003c/p\u003e"},{"header":"Result","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eSilkworms develop obesity and dyslipidemia when fed a 10% glucose diet (10GD)\u003c/h2\u003e \u003cp\u003eSilkworms were divided into groups fed either a normal diet (ND) or a 10GD on the first day of the fifth instar (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). The 10GD group showed decreased food intake and body weight compared to the ND group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB and C). Fat body weight was higher in the 10GD group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD and E) showing a time-dependent increase, peaking at 72 h. Furthermore, levels of neutral fat in both the fat body (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF) and hemolymph (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG), as well as hemolymph sugar levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH) and bombyxin, a silkworm insulin-like peptide (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI), were significantly increased in silkworms fed with 10GD for 72 h compared to the ND group. These findings suggest that silkworms reared on a 10GD for 72 h develop a phenotype similar to obesity and lipid metabolism disorders observed in mammals.\u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eEvaluation of the effects of hyperlipidemia drugs and anti-obesity functional ingredients on lipid metabolism in the silkworm obesity and lipid metabolism disorder (SOLD) model\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo assess the efficacy of the SOLD model, silkworms were fed with 10GD supplemented with FBT, EGCG and AMP-activated protein kinase (AMPK) activator (AICAR), a target similar to EGCG, and metformin (MT) and pioglitazone (PG), both antidiabetic drugs that have no effect on improving lipid metabolism, for 72 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003eIn the SOLD silkworms, food intake and body weight decreased (Supplementary Fig.\u0026nbsp;1A and B), while fat body weight, NF levels in hemolymph and fat bodies, and hemolymph sugar levels significantly increased compared to the ND group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB\u0026ndash;D). In the FBT group, the hemolymph NF levels decreased in a concentration-dependent manner compared to the untreated group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). The EGCG group exhibited a concentration-dependent decrease in NF levels in the fat bodies and hemolymph compared to the untreated group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC and D). Moreover, hemolymph sugar levels tended to decrease for both compounds compared to the untreated group (Supplementary Fig.\u0026nbsp;1C). The AICAR group showed a significant decrease in NF levels in fat bodies (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Additionally, both the EGCG and AICAR groups showed a trend toward lower fat body weight (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). However, no significant improvement was observed in the MT and PG groups. These results indicate that the effects of FBT and EGCG on the SOLD model resemble their effects on mammals.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eMechanism of action underlying the effects of FBT and EGCG on the SOLD model\u003c/h2\u003e \u003cp\u003eTo clarify the mechanism of action of FBT and EGCG on the SOLD model, we evaluated the activation status of enzymes involved in lipid synthesis signaling in fat bodies using Western blotting (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). Compared to the ND group, the SOLD model showed increased activities of fatty acid synthase (FAS) activities (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF), along with decreased levels of the phosphorylated acetyl-CoA carboxylase (p-ACC) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG), and phosphorylated AMPK (p-AMPK) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH). The FBT group displayed a significant reduction in FAS and an upward trend in p-AMPK and p-ACC compared to the untreated group. Similarly, the EGCG group showed significantly lower FAS levels and higher p-AMPK and p-ACC levels compared to the untreated group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). These compounds act similarly to their effects in mammals. The AICAR group showed a decreasing trend in FAS and a significant increase in p-AMPK and p-ACC. While the MT group showed a significant decrease in FAS and an increase in p-AMPK, the PG group did not significantly differ from the untreated group.\u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eLPL activity\u003c/h2\u003e \u003cp\u003eIn mammals, FBT activates LPL promotes TG degradation in the blood, EGCG decreases LPL activation in adipose tissue18\u003csup\u003e21\u003c/sup\u003e, and green tea, rich in EGCG, increases LPL activation in muscle\u003csup\u003e19\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn the SOLD model, LPL activity showed no significant differences in either fat body (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA) or muscle (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB) compared to the ND group. However, in the FBT group, there was an increasing trend in muscle LPL activity. The EGCG group showed a significant decrease in fat body LPL activity (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and a significant increase in muscle LPL activity compared to the ND group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Furthermore, the AICAR group showed a significant decrease in fat body LPL and a tendency toward increased muscle LPL. Conversely, there were no significant changes observed in the MT and PG groups compared to the untreated group. These results suggest that FBT and EGCG regulate AMPK activation and LPL activity, indicating their action on LPL activity in the silkworm mirrors their effects on mammalian LPL.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eFatty acid oxidation activity\u003c/h2\u003e \u003cp\u003eFBT and EGCG are known to promote the β-oxidation of fatty acids in mammals. To determine whether this effect is present in the SOLD model, we measured the RQ as an indicator of fatty acid oxidation. Silkworms were initially fed with 10 GD for 48 h to induce obesity and dyslipidemia. Subsequently, from 48\u0026ndash;75 h, they were fed with ND mixed with FBT, EGCG, or AICAR. During this period, O\u003csub\u003e2\u003c/sub\u003e consumption and CO\u003csub\u003e2\u003c/sub\u003e emissions were monitored using a metabolic measurement system (MK-5000RQ/MS) designed for small animals (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). RQ was calculated by dividing CO\u003csub\u003e2\u003c/sub\u003e emissions by O\u003csub\u003e2\u003c/sub\u003e consumption (RQ\u0026thinsp;=\u0026thinsp;VCO\u003csub\u003e2\u003c/sub\u003e/VO\u003csub\u003e2\u003c/sub\u003e). Data collection was paused for the first 3 h after switching to the ND to allow the silkworms to acclimate to the measurement environment. Data were then collected continuously for 24 h from 51th to 75th h. RQ values represent the average of each 8-h period divided into 3 segments over the 24-h periods.\u003c/p\u003e \u003cp\u003eIn the SOLD model, RQ decreased over time from the start of measurements. In the non-treated group, it remained above 1 at 17\u0026ndash;24 h. Conversely, in the FBT, EGCG, and AICAR groups, the RQ at 17\u0026ndash;24 h was below 1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). The RQ at 17\u0026ndash;24 h was lowest in the EGCG and AICAR groups, which aligns with the observation that both groups exhibited the lowest NF levels in hemolymph and fat bodies (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE\u0026ndash;G).\u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eEstablishment of the SOLD model\u003c/h2\u003e \u003cp\u003eIn this study, we aimed to construct an evaluation system for screening compounds that could improve obesity and lipid metabolism disorders, using the silkworm model. Insects possess fat bodies that serve analogous functions of mammalian adipose tissue and liver. Sugars ingested are metabolized into trehalose within the fat body, stored in the hemolymph, or converted into TG and glycogen, which accumulate in the fat body\u003csup\u003e20\u003c/sup\u003e. Silkworms fed with 10GD showed a progressive increase in fat body weight over time (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD), alongside elevated levels of hemolymph bombyxin, suggesting enhanced trehalose uptake into fat bodies and increased fat synthesis. Moreover, the 10GD group exhibited higher levels of hemolymph sugar and NF in fat body and hemolymph (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF\u0026ndash;H). In humans, obesity is characterized by excessive body fat accumulation, typically with a BMI of \u0026ge;\u0026thinsp;25\u003csup\u003e1\u003c/sup\u003e, yet there is a growing population of individuals with high visceral fat levels despite a low BMI\u003csup\u003e21\u003c/sup\u003e. Silkworms fed with 10GD showed reduced overall body weight but increased fat body weight compared to those on a ND, suggesting that the SOLD model bears similarity to visceral fat obesity in human.\u003c/p\u003e \u003cp\u003eThe pathways for fatty acid biosynthetic are shared between insects and mammals. In mice with high-sugar or high-fat diets induced obesity, there is an increase in the activity of key enzymes for fatty acid synthesis, such as FAS and ACC. Conversely, the activity of AMPK, a signaling protein that inhibits FAS and ACC, decreases\u003csup\u003e22,23,24,25,26,27\u003c/sup\u003e. In the SOLD model, compared to the ND group, there was an increase in FAS expression and a decrease in the inactive form of ACC (p-ACC), while the activated form of AMPK (p-AMPK), responsible for phosphorylating ACC, decreased (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF\u0026ndash;H). These results suggest that the SOLD model induces diet-induced obesity through mechanisms similar to those observed in mammals.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eUsefulness of the SOLD model as an animal model for obesity\u003c/h3\u003e\n\u003cp\u003eTo assess the utility of the SOLD model, we evaluated the effects of FBT, a drug for hyperlipidemia, and EGCG, known for its ability to inhibit fat accumulation in humans. FBT exhibits various lipid-improving effects, including lowering blood TG levels and increasing blood HDL cholesterol, attributed to the activation of peroxisome proliferator-activated receptor (PPAR)-α. PPARα is strongly expressed in tissues with active β-oxidation, such as liver, kidney, skeletal muscle, and heart, in humans\u003csup\u003e28\u003c/sup\u003e. Its activation increases the production and activity of LPL in muscle and adipose tissue by decreasing the production of ApoC III, thus, facilitating TG degradation in blood and free fatty acid uptake in tissues\u003csup\u003e29\u003c/sup\u003e. Furthermore, PPARα activation enhances β-oxidation and suppressing TG synthesis by activating AMPK in the liver and muscle\u003csup\u003e30\u003c/sup\u003e. In the SOLD model, administration of FBT led to a concentration-dependent decrease in hemolymph NF levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD) and a decrease in FAS alongside an increase in p-AMPK and p-ACC in fat bodies (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF\u0026ndash;H). Furthermore, the FBT-treated group showed an increasing trend in muscle LPL activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB) and a decreasing trend in RQ (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD) compared to the untreated group, suggesting an increase in β-oxidation. The effects and mechanisms of action of FBT observed in the SOLD model closely resembled those seen in mammals.\u003c/p\u003e \u003cp\u003eEGCG, a polyphenol abundant in green tea, is recognized for its ability to lower body weight and prevent obesity, diabetes, and cardiovascular diseases\u003csup\u003e31\u003c/sup\u003e. In mice with diet-induced obesity, EGCG decrease fat accumulation, blood TG, cholesterol, liver TG, blood glucose levels and the intestinal absorption of lipids\u003csup\u003e32,33,34\u003c/sup\u003e. EGCG primary mechanisms involve inhibiting fatty acid synthesis by activating AMPK in the liver, skeletal muscle and adipose tissue, as well as enhancing β-oxidation\u003csup\u003e35,36,37\u003c/sup\u003e; additional effects include decreased LPL activity in adipose tissue\u003csup\u003e18\u003c/sup\u003e, increased expression of PPARα in muscle\u003csup\u003e38\u003c/sup\u003e and a decrease in RQ\u003csup\u003e39\u003c/sup\u003e [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Additionally, green tea increases LPL activity in muscle tissue\u003csup\u003e19\u003c/sup\u003e. In the SOLD model, administration of EGCG led to a reduction in NF levels in the fat body and hemolymph (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC and D). Moreover, the EGCG-treated group showed increased p-AMPK and p-ACC in the fat body (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG and H), decreased fat body LPL activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA), increased muscle LPL activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB), and decreased RQ (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). The effects of EGCG on lipid metabolism in the SOLD model were consistent with those in the mammalian model with obesity.\u003c/p\u003e \u003cp\u003eIn this study, the effects of FBT and EGCG on the SOLD model demonstrated increased muscle LPL activity and decreased RQ. High RQ and low RQ indicate fat synthesis and β-oxidation, respectively\u003csup\u003e40\u003c/sup\u003e. Our results suggest that FBT and EGCG influenced myocytes LPL in silkworm, promoting the uptake of hemolymph NF and fat oxidation in muscle tissue. In the flying insect, LPL is found in the fat bodies, muscle and ovaries\u003csup\u003e41,42\u003c/sup\u003e. Diglycerides (DG), degraded by fat body lipase, bind to lipophorin, an insect lipoprotein in hemolymph and are uptake into the muscle cells via myocyte membrane LPL for ATP production\u003csup\u003e43,44\u003c/sup\u003e. Our report is the first to suggest that in the silkworm, LPL is expressed in muscle and involved in ATP production.\u003c/p\u003e \u003cp\u003eIn the SOLD model, the effective dose of FBT (10 mg/kg/day) for improving lipid metabolism was approximately one-tenth of that in mice (100 mg/kg/day\u003csup\u003e45\u003c/sup\u003e) and 3\u0026ndash;5 times that administered in humans (1.8\u0026ndash;2.7 mg/kg/day). The effective dose of EGCG in the SOLD model (100\u0026ndash;200 mg/kg/day) was about twice that used in mice (50\u0026ndash;100 mg/kg/day\u003csup\u003e36\u003c/sup\u003e) and approximately 10 times that used in humans (10 mg/kg/day). Despite these differences, the effective doses of both compounds in the SOLD model were comparable to those in mice or humans, suggesting their potential utility as alternative models. Furthermore, FBT and EGCG not only suppress fat accumulation by activating AMPK in the liver but also prevent the onset of NAFLD in mice\u003csup\u003e46, 47\u003c/sup\u003e. These effects were observed in the fat body of silkworms, suggesting that the SOLD model may offer a means to evaluate improved effects on NAFLD.\u003c/p\u003e \u003cp\u003e The SOLD model offers a means to evaluate lipid metabolism with a small sample, within a short timeframe, and at a low cost, and without encountering animal ethics issues. These results indicate the utility of the SOLD model for screening therapeutic drugs and functional food ingredients aimed at improving obesity and lipid metabolism disorders.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eChemicals\u003c/h2\u003e \u003cp\u003eTrehalase from porcine kidney and palmitoyl coenzyme A lithium salt was purchased from Sigma Aldrich Japan, Co, LLC. (Tokya, Japan). AICAR (S7863) was purchased from Selleck Chemicals (Houston TX, USA). (\u0026minus;)-Epigallocatechin gallate hydrate, pioglitazone hydrochloride, fenofibrate were obtained from Tokyo Chemical Industry, Co. (Tokyo, Japan). D-(+)-glucose were purchased from Nacalai Tesque, Inc. (Kyoto, Japan). Metformin hydrochloride, trehalose dihydrate, oil Red O, 4%-paraformaldehyde phosphate buffer solution, heparin sodium, 4-methylumbelliferyl oleate and 2-propanol were obtained from FUJIFILM Wako Pure Chemical Industries, Ltd. (Tokyo, Japan). Primary antibodies FAS (#3180), p-AMPK (#2535T), p-ACC (#11818T), and β-actin (#4967) were obtained from Cell Signaling Technology (Beverly, USA). Secondary antibodies, goat anti-rabbit IgG (H\u0026thinsp;+\u0026thinsp;L) antibody, and HRP conjugate (SA00001-2) were purchased from Cosmo Bio, Co. (Tokyo, Japan). The TaKaRa BCA Protein Assay Kit was obtained from Takara Bio, Inc. (Shiga, Japan).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eSilkworm rearing conditions and diet preparation\u003c/h2\u003e \u003cp\u003eSilkworms (hybrid race: Kin-Shu \u0026times; Sho-Wa) were kindly provided by Atsumaru Holdings NSP Yamaga Plant (Atsumaru Inc., Kumamoto, Japan). Upon hatching, larvae were reared to the fifth instar on an artificial diet, SilkMate PM (Nosan Corporation), within plastic containers maintained at 25\u0026ndash;27\u0026deg;C and 50\u0026ndash;70% humidity.\u003c/p\u003e \u003cp\u003eAn artificial diet was prepared following the instructions provided by the feed company. SilkMate PM was blended with water at a 1:3 ratio and subsequently steamed in an autoclave at 105\u0026deg;C for 20 min. For the glucose diet, SilkMate PM, glucose, and water were mixed to achieve an equivalent weight of sugar, then subjected to autoclaving.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eHemolymph and fat body collection methods\u003c/h2\u003e \u003cp\u003eHemolymph (10 \u0026micro;L) was collected from the larva\u0026rsquo;s proleg using a 30 G needle (insulin syringe; Becton, Dickinson and Company) and immediately frozen on dry ice, then stored at \u0026minus;\u0026thinsp;20\u0026deg;C. The fat body was isolated from the larva\u0026rsquo;s dorsolateral region while in suspended animation by placing in a container filled with dry ice. After rinsing with PBS, drying, and scraping off with tweezers, the fat bodies were weighed and immediately frozen in liquid nitrogen, then stored at \u0026minus;\u0026thinsp;80\u0026deg;C.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eDetermination of hemolymph trehalose and glucose levels\u003c/h2\u003e \u003cp\u003eThe hemolymph trehalose concentration was determined with modifications to Tennessen\u0026rsquo;s method\u003csup\u003e47\u003c/sup\u003e. Hemolymph (2 \u0026micro;L) was diluted 100-fold in buffer (5 mM Tris, 137 mM NaCl, 2.7 mM KCl, and pH 6.7), then incubated at 80\u0026deg;C for 3 min before returning to room temperature. Hemolymph solution (100 \u0026micro;L) was mixed with 100 \u0026micro;L of porcine trehalase solution (10 mU/mL) diluted in buffer, incubated overnight at 37\u0026deg;C, and the resulting glucose production was measured using the Glucose CII Test Wako (FUJIFILM Wako Pure Chemical Industries, Tokyo, Japan), with trehalose concentration calculated from a calibration curve prepared using the same measurements. Glucose concentration was also measured using the Glucose CII Test Wako. For this, hemolymph (2 \u0026micro;L) was added to a frozen 96 well plate, followed by the addition of chromogenic solution (200 \u0026micro;L). After incubation at room temperature for 15 min, absorbance was measured at 505 nm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eDetermination of the neutral fat levels in hemolymph\u003c/h2\u003e \u003cp\u003eNeutral fat (NF) levels in the hemolymph were measured using LabAssay\u0026trade; Triglycerides (FUJIFILM Wako Pure Chemical Industries, Tokyo, Japan). Hemolymph (2 \u0026micro;L) was added to a frozen 96-well plate, followed by the addition of chromogenic solution (300 \u0026micro;L). After incubating at room temperature for 5 min, absorbance was measured at 600 and 700 nm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eWestern blotting\u003c/h2\u003e \u003cp\u003eThe fat body (frozen weight 50 mg) was crushed in 1 mL of lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 1% NP-40 Substitute, 0.5% Sodium Deoxycholate, 0.1% SDS, Protease inhibitor cocktail I, Phosphatase inhibitor cocktail II, pH 7.6) for 30 s using a polytron homogenizer. After incubating on ice for 30 min, the samples were centrifuged (14,000 g, 10 min, 4\u0026deg;C) and the protein concentration of the supernatant was determined using the BCA method. Each sample was diluted with sample buffer (125 mM Tris-HCl, 4% SDS, 20% glycerol, 0.004% bromophenol blue, 50 mM DTT, and pH 6.8) and water to adjust the protein concentration. The samples were then heat-treated at 90\u0026deg;C for 5 min and subjected to SDS-PAGE on 10% polyacrylamide gel. Proteins in the gel were transferred to a PVDF membrane, incubated with primary antibodies and secondary antibodies, and bands of the target proteins were detected using SuperSignal TM Chemiliuminescent HRP Substrates (Thermo Fisher Scientific Logo). Bands were quantified using Image J.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eOil red O staining of the silkworm NF in fat bodies\u003c/h2\u003e \u003cp\u003eNF staining was performed with modifications to Matsumoto\u0026rsquo;s method\u003csup\u003e17\u003c/sup\u003e. Fat bodies were immediately immersed in 4% formaldehyde upon removal and fixed overnight at 4\u0026deg;C. The samples were rinsed in phosphate buffer (137 mM NaCl, 8.1 mM Na\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e, 2.68 mM KCl, 1.47 mM KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, and pH 7.4), treated with 60% 2-propanol for 1 min, and then stained with Oil red O stain solution (1.8 mg of Oil red O in 1 mL of 60% 2-propanol) for 20 min at room temperature. After being washed three times with 60% 2-propanol, the samples were immersed in 100% 2-propanol and sonicated for 30 min. Following centrifugation (10,000 g, 3 min), absorbance was measured at 490 nm, and triglyceride levels were calculated based on absorbance per gram of fat body.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eMeasurement of Lipoprotein lipase activity\u003c/h2\u003e \u003cp\u003eSample preparation for the Lipoprotein lipase (LPL) activity assay was performed using a modification of Ribeiro\u0026rsquo;s method\u003csup\u003e41\u003c/sup\u003e. Fat body and muscle (frozen weight 100 mg) were homogenized in 1 mL of ice-cold heparin solution (0.25 M Sucrose, 1 mM EDTA, 3 mM Tris-HCl, 2% BSA, 2 U/ml Heparin Sodium, and pH 7.5) using a polytron homogenizer. After incubation at 37\u0026deg;C for 1 h, the samples were centrifuged, and the supernatant was collected as the enzyme solution. Protein concentrations of the enzyme solutions were determined using the BCA method and then diluted in phosphate buffer (0.2 M Na\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e 12H\u003csub\u003e2\u003c/sub\u003eO, 0.2 M NaH\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e 2H\u003csub\u003e2\u003c/sub\u003eO, and pH 7.4) to adjust the protein concentration of each sample.\u003c/p\u003e \u003cp\u003eLPL activity was assessed using a fluorescence method with 0.5 mM oleic acid 4-methylumbelliferyl solution dissolved in phosphate buffer as the substrate. Upon mixing of enzyme solution (100 \u0026micro;L) and substrate solution (100 \u0026micro;L) in a 96-well microplate, fluorescence intensity (ex/em\u0026thinsp;=\u0026thinsp;355/460) was promptly measured for 30 min at 2 min intervals. The ratio of fluorescence intensity at 30 min to the fluorescence intensity at 0 min was used as the measure of LPL activity.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eMeasurement of respiratory quotient\u003c/h2\u003e \u003cp\u003eThe method was modified from the Hanatani method\u003csup\u003e48\u003c/sup\u003e. Measurements were performed using a metabolic measurement system designed for small animals (MK-5000RQ/MS model, Muromachi Machinery Corporation, Tokyo, Japan) at a room temperature of 26\u0026deg;C within a 12-h light\u0026ndash;dark cycle. Silkworms fed with 10GD for 72 h were placed in groups of five in closed cages and provided with a ND, with or without the sample. Oxygen consumption (VO\u003csub\u003e2\u003c/sub\u003e) and carbon dioxide production (VCO\u003csub\u003e2\u003c/sub\u003e) were recorded every 5 min for 24 h. The respiratory quotient (RQ) was calculated as the ratio of VO\u003csub\u003e2\u003c/sub\u003e per VCO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eAll data were presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD of three replicates. Measurements of food intake and RQ were averaged across the five silkworms. The data were analyzed using a t-test, with as statistical significance set at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and p\u0026thinsp;\u0026lt;\u0026thinsp;0.01.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":" \u003ch2\u003eConflict of interest:\u003c/strong\u003e \u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e \u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eInvestigation, formal analysis, writing\u0026mdash;original draft, YT; writing\u0026mdash;review and editing, HPD; conceptualization, writing\u0026mdash;review and editing, supervision, MY.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThe authors would like to thank Atsumaru Holdings NSP Yamaga Plant, Atsumaru Yamaga Silk, for their kind gift of silkworms. We also thank Dr. Yasuhiko Matsumoto for his cooperation. This work was supported by JSPS KAKENHI Grant Number JP23804033.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe datasets used and/or analysed during the current study available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eWorld Health Organization (WHO). Obesity and overweight. 2024. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.who.int/news-room/fact-sheets/detail/obesity-and-overweight\u003c/span\u003e\u003cspan address=\"https://www.who.int/news-room/fact-sheets/detail/obesity-and-overweight\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhu, L. \u003cem\u003eet al\u003c/em\u003e. Lean Yet Unhealthy: Asian American Adults Had Higher Risks for Metabolic Syndrome than Non-Hispanic White Adults with the Same Body Mass Index: Evidence from NHANES 2011\u0026ndash;2016. Healthcare 9, 1518 (2021). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/healthcare9111518\u003c/span\u003e\u003cspan address=\"10.3390/healthcare9111518\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFan, J.G., Kim, S.U. \u0026amp; Wang, V.W.S. New trends on obesity and NAFLD in Asia. J Hepatol 67, 862\u0026ndash;873 (2017) \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.jhep.2017.06.003\u003c/span\u003e\u003cspan address=\"10.1016/j.jhep.2017.06.003\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLee, M. \u003cem\u003eet al\u003c/em\u003e. The cost-effectiveness of pharmacotherapy and lifestyle intervention in the treatment of obesity. Obes Sci Pract 6, 162\u0026ndash;170 (2020). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/osp4.390\u003c/span\u003e\u003cspan address=\"10.1002/osp4.390\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOhishi, T. \u003cem\u003eet al\u003c/em\u003e. The Beneficial Effects of Principal Polyphenols from Green Tea, Coffee, Wine, and Curry on Obesity. Molecules 26, 453 (2021). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/molecules26020453\u003c/span\u003e\u003cspan address=\"10.3390/molecules26020453\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDellambra, E. \u003cem\u003eet al\u003c/em\u003e. A. Non-Animal Models in Dermatological Research. ALTEX 36, 177\u0026ndash;202 (2018). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.14573/altex.1808022\u003c/span\u003e\u003cspan address=\"10.14573/altex.1808022\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eElbrense, H., Montaser, O., El-Aasr, M. \u0026amp; Meshrif, W.S. Potential anti-diabetic effect of certain plant extracts from the Egyptian flora on type II diabetes using Drosophila melanogaster as an animal model. IJCBR 5, 121\u0026ndash;133 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLourido, F., Quenti, D., Salgado-Canales, D. \u0026amp; Tobar, N. Domeless receptor loss in fat body tissue reverts insulin resistance induced by a high\u0026ndash;sugar diet in Drosophila melanogaster. Sci Rep 11, 3263 (2021). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41598-021-82944-4\u003c/span\u003e\u003cspan address=\"10.1038/s41598-021-82944-4\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBuescher, J.L. \u003cem\u003eet al\u003c/em\u003e. Evidence for transgenerational metabolic programming in Drosophila. Dis Model Mech 6, 1123\u0026ndash;1132 (2013). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1242/dmm.011924\u003c/span\u003e\u003cspan address=\"10.1242/dmm.011924\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRovenko, B.M. \u003cem\u003eet aI\u003c/em\u003e. High sucrose consumption promotes obesity whereas its low consumption induces oxidative stress in Drosophila melanogaster. J Insect Physiol 79, 42\u0026ndash;54 (2015). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.jinsphys.2015.05.007\u003c/span\u003e\u003cspan address=\"10.1016/j.jinsphys.2015.05.007\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eReis, T. Effects of Synthetic Diets Enriched in Specific Nutrients on Drosophila Development, Body Fat, and Lifespan. PLos ONE 11, e0146758 (2016). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1371/journal.pone.0146758\u003c/span\u003e\u003cspan address=\"10.1371/journal.pone.0146758\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWoodcock, K.J. \u003cem\u003eet al\u003c/em\u003e. Macrophage-Derived upd3 Cytokine Causes Impaired Glucose Homeostasis and Reduced Lifespan in Drosophila Fed a Lipid-Rich Diet. Immunity 42, 133\u0026ndash;144 (2015).\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e http://dx.doi.org/10.1016/j.immuni.2014.12.023\u003c/span\u003e\u003cspan address=\" 10.1016/j.immuni.2014.12.023\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKayashima, Y. \u003cem\u003eet al\u003c/em\u003e. Tea polyphenols ameliorate fat storage induced by high-fat diet in Drosophila melanogaster. Biochem Biophys Rep 4, 417\u0026ndash;424 (2015). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.bbrep.2015.10.013\u003c/span\u003e\u003cspan address=\"10.1016/j.bbrep.2015.10.013\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAzuma, M. \u003cem\u003eet al\u003c/em\u003e. RNA-seq analysis of diet-driven obesity and anti-obesity effects of quercetin glucoside or epigallocatechin gallate in Drosophila adults. Eur Rev Med Pharmacol Sci 23, 857\u0026ndash;876 (2019). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.26355/eurrev_201901_16901\u003c/span\u003e\u003cspan address=\"10.26355/eurrev_201901_16901\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWongchum, N. \u003cem\u003eet al\u003c/em\u003e. Hydroethanolic \u003cem\u003eCyperus rotundus\u003c/em\u003e L. extract exhibits anti-obesity property and increases lifespan expectancy in Drosophila melanogaster fed a high-fat diet. J HerbMed Pharmacol 11, 296\u0026ndash;304 (2022). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.34172/jhp.2022.35\u003c/span\u003e\u003cspan address=\"10.34172/jhp.2022.35\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAsiimwe, O.H. \u003cem\u003eet al\u003c/em\u003e. Anti-obesity effects of Erythrina abyssinica stem bark extract in flies exposed to a high fat diet. \u003cem\u003eHeliyon\u003c/em\u003e 8, e09886 (2022). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.heliyon.2022.e09886\u003c/span\u003e\u003cspan address=\"10.1016/j.heliyon.2022.e09886\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMatsumoto, Y. \u003cem\u003eet al\u003c/em\u003e. Diabetic silkworms for evaluation of therapeutically effective drugs against type II diabetes. Sci Rep 5, 10722 (2015). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/srep10722\u003c/span\u003e\u003cspan address=\"10.1038/srep10722\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLee, M.S., Kim, C.T. \u0026amp; Kim, Y. Green tea (\u0026ndash;)-epigallocatechin-3-gallate reduces body weight with regulation of multiple genes expression in adipose tissue of diet-induced obese mice. Ann Nutr Metab 54, 151\u0026ndash;157 (2009). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1021/jf402004x\u003c/span\u003e\u003cspan address=\"10.1021/jf402004x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSerisier, S. \u003cem\u003eet al\u003c/em\u003e. Effects of green tea on insulin sensitivity, lipid profile and expression of PPARalpha and PPARgamma and their target genes in obese dogs. Br J Nutr 99, 1208\u0026ndash;1216 (2008). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1017/S0007114507862386\u003c/span\u003e\u003cspan address=\"10.1017/S0007114507862386\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBayliak, M.M. \u003cem\u003eet al\u003c/em\u003e. Interplay between diet-induced obesity and oxidative stress: Comparison between Drosophila and mammals. Comp Biochem Physiol Part A Mol Integr Physiol 228, 18\u0026ndash;28 (2019). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.cbpa.2018.09.027\u003c/span\u003e\u003cspan address=\"10.1016/j.cbpa.2018.09.027\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTchernof, A. \u0026amp; Despr\u0026eacute;s, J.P. Pathophysiology of Human Visceral Obesity: an update. Physiol Rev 93, 359\u0026ndash;404 (2013). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1152/physrev.00033.2011\u003c/span\u003e\u003cspan address=\"10.1152/physrev.00033.2011\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGhareghani, P. \u003cem\u003eet al\u003c/em\u003e. Aerobic endurance training improves nonalcoholic fatty liver disease (NAFLD) features via miR-33 dependent autophagy induction in high fat diet fed mice. Obes Res Clin Pract 12, 80\u0026ndash;89 (2018). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.orcp.2017.01.004\u003c/span\u003e\u003cspan address=\"10.1016/j.orcp.2017.01.004\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu, C. \u003cem\u003eet al\u003c/em\u003e. Flavonoid-Rich Extract of Paulownia fortunei Flowers Attenuates Diet-Induced Hyper liquidemia, Hepatic Steatosis and Insulin Resistance in Obesity Mice by AMPK Pathway. Nutrients 9, 959 (2017). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/nu9090959\u003c/span\u003e\u003cspan address=\"10.3390/nu9090959\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eInamdar, S. \u003cem\u003eet al\u003c/em\u003e. Vitexin alleviates non-alcoholic fatty liver disease by activating AMPK in high fat diet fed mice. Biochem Biophys Res Commun 519, 106\u0026ndash;112 (2019). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.bbrc.2019.08.139\u003c/span\u003e\u003cspan address=\"10.1016/j.bbrc.2019.08.139\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePrakash, S. \u003cem\u003eet al\u003c/em\u003e. Amelioration of diet-induced metabolic syndrome and fatty liver with sitagliptin via regulation of adipose tissue inflammation and hepatic Adiponectin/AMPK levels in mice. Biochimie 168, 198\u0026ndash;209 (2020). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.biochi.2019.11.005\u003c/span\u003e\u003cspan address=\"10.1016/j.biochi.2019.11.005\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLee, H.A. \u003cem\u003eet al\u003c/em\u003e. Ganoderma lucidum Extract Reduces Insulin Resistance by Enhancing AMPK Activation in High-Fat Diet-Induced Obese Mice. \u003cem\u003eNutrients\u003c/em\u003e 12, 3338 (2020). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/nu12113338\u003c/span\u003e\u003cspan address=\"10.3390/nu12113338\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi, T. \u003cem\u003eet al\u003c/em\u003e. Milk Fat Globule Membrane Attenuates High-Fat Diet-Induced Obesity by Inhibiting Adipogenesis and Increasing Uncoupling Protein 1 Expression in White Adipose Tissue of Mice. Nutrients 10, 331 (2018). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/nu10030331\u003c/span\u003e\u003cspan address=\"10.3390/nu10030331\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGrygiel-G\u0026oacute;rniak, B. Peroxisome proliferator-activated receptors and their ligands: nutritional and clinical implications - a review. Nutr J 13 (2014). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1186/1475-2891-13-17\u003c/span\u003e\u003cspan address=\"10.1186/1475-2891-13-17\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDuval, C., M\u0026uuml;ller, M. \u0026amp; Kersten, S. PPARα and dysliquidemia. Biochim Biophys Acta Mol Cell Biol Lipids 1771, 961\u0026ndash;971 (2007). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.bbalip.2007.05.003\u003c/span\u003e\u003cspan address=\"10.1016/j.bbalip.2007.05.003\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen, W.L. \u003cem\u003eet al\u003c/em\u003e. Fenofibrate lowers lipid accumulation in myotubes by modulating the PPARα/AMPK/FoxO1/ATGL pathway. Biochem Pharmacol 84, 522\u0026ndash;531 (2012). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.bcp.2012.05.022\u003c/span\u003e\u003cspan address=\"10.1016/j.bcp.2012.05.022\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang, C.S. \u003cem\u003eet al\u003c/em\u003e. Mechanisms of body weight reduction and metabolic syndrome alleviation by tea. Mol Nutr Food Res 60, 160\u0026ndash;174 (2016). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/mnfr.201500428\u003c/span\u003e\u003cspan address=\"10.1002/mnfr.201500428\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen, Y.K. \u003cem\u003eet al\u003c/em\u003e. Effects of Green Tea Polyphenol (\u0026ndash;)-Epigallocatechin-3-gallate on Newly Developed High-Fat/Western-Style Diet-Induced Obesity and Metabolic Syndrome in Mice. J Agric Food Chem 59, 11862\u0026ndash;11871 (2011). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1021/jf2029016\u003c/span\u003e\u003cspan address=\"10.1021/jf2029016\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGrove, K.A., Sae-Tan, S., Kennett, M.J. \u0026amp; Lambert, J.D. (\u0026ndash;)-Epigallocatechin-3-gallate Inhibits Pancreatic Lipase and Reduces Body Weight Gain in High Fat-Fed Obese mice. Obesiy 20, 2311\u0026ndash;2313 (2012). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/oby.2011.139\u003c/span\u003e\u003cspan address=\"10.1038/oby.2011.139\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBose, M. \u003cem\u003eet al\u003c/em\u003e. The Major Green Tea Polyphenol, (\u0026ndash;)-Epigallocatechin-3-Gallate, Inhibits Obesity, Metabolic Syndrome, and Fatty Liver Disease in High-Fat\u0026ndash;Fed Mice. J Nutr 138, 1677\u0026ndash;1683 (2009). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/jn/138.9.1677\u003c/span\u003e\u003cspan address=\"10.1093/jn/138.9.1677\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMurase, T., Misawa, K., Haramizu, S. \u0026amp; Hase, T. Catechin-induced activation of the LKB1/AMP-activated protein kinase pathway. Biochem Pharmacol 78, 78\u0026ndash;84 (2009). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.bcp.2009.03.021\u003c/span\u003e\u003cspan address=\"10.1016/j.bcp.2009.03.021\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTakagaki, A. \u003cem\u003eet al\u003c/em\u003e. Effects of Microbial Metabolites of (\u0026ndash;)-Epigallocatechin Gallate on Glucose Uptake in L6 Skeletal Muscle Cell and Glucose Tolerance in ICR Mice. Biol Pharm Bull 42, 212\u0026ndash;221 (2019). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1248/bpb.b18-00612\u003c/span\u003e\u003cspan address=\"10.1248/bpb.b18-00612\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi, F. \u003cem\u003eet al\u003c/em\u003e. EGCG Reduces Obesity and White Adipose Tissue Gain Partly Through AMPK Activation in Mice. Front Pharmacol 9, 1366 (2018). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fphar.2018.01366\u003c/span\u003e\u003cspan address=\"10.3389/fphar.2018.01366\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHuang, J. \u003cem\u003eet al\u003c/em\u003e. Green tea polyphenol EGCG alleviates metabolic abnormality and fatty liver by decreasing bile acid and lipid absorption in mice. Mol Nutr Food Res 62, 1700696 (2018). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/mnfr.201700696\u003c/span\u003e\u003cspan address=\"10.1002/mnfr.201700696\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKapoor, M.P., Sugita, M., Fukuzawa, Y. \u0026amp; Okubo, T. Physiological effects of epigallocatechin-3-gallate (EGCG) on energy expenditure for prospective fat oxidation in humans: A systematic review and meta-analysis. J Nutr Biochem 43, 1\u0026ndash;10 (2017). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.jnutbio.2016.10.013\u003c/span\u003e\u003cspan address=\"10.1016/j.jnutbio.2016.10.013\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTalal, S. \u003cem\u003eet al\u003c/em\u003e. High carbohydrate diet ingestion increases post-meal lipid synthesis and drives respiratory exchange ratios above 1. J Exp Biol 224, jeb240010 (2021). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1242/jeb.240010\u003c/span\u003e\u003cspan address=\"10.1242/jeb.240010\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRibeiro, L. \u0026amp; Fonseca, C.L.C. Occurrence of lipoprotein lipase in the fat body of Triatoma maculata. Comp Biochem Physiol B 52, 523\u0026ndash;524 (1975). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/0305-0491(75)90229-1\u003c/span\u003e\u003cspan address=\"10.1016/0305-0491(75)90229-1\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHeusden, M.C.V. Characterization and Identification of a Lipoprotein Lipase from Manduca sexta Flight Muscle. Insect Biochem Mol Biol 23, 785\u0026ndash;792 (1993). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/0965-1748(93)90066-2\u003c/span\u003e\u003cspan address=\"10.1016/0965-1748(93)90066-2\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eToprak, U., Hegedus, D., Doğan, C. \u0026amp; G\u0026uuml;ney, G. A journey into the world of insect lipid metabolism. Arch Insect Biochem Physiol 88 (2020). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/arch.21682\u003c/span\u003e\u003cspan address=\"10.1002/arch.21682\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eForcheron, F., Basset, A., Carmine, P.D. \u0026amp; Beylot, M. Lipase maturation factor 1: Its expression in Zucker diabetic rats, and effects of metformin and fenofibrate. Diabetes Metab 35, 452\u0026ndash;457 (2009). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.diabet.2009.05.004\u003c/span\u003e\u003cspan address=\"10.1016/j.diabet.2009.05.004\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYoo, J. \u003cem\u003eet al\u003c/em\u003e. Fenofibrate, a PPARα agonist, reduces hepatic fat accumulation through the upregulation of TFEB-mediated lipophagy. Metabolism 120, 154798 (2021). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.metabol.2021.154798\u003c/span\u003e\u003cspan address=\"10.1016/j.metabol.2021.154798\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTang, G. \u003cem\u003eet al\u003c/em\u003e. Green Tea and Epigallocatechin Gallate (EGCG) for the Management of Nonalcoholic Fatty Liver Diseases (NAFLD): Insights into the Role of Oxidative Stress and Antioxidant Mechanism. Antioxidants 10, 1076 (2021). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/antiox10071076\u003c/span\u003e\u003cspan address=\"10.3390/antiox10071076\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTennessen, J.M., Barry, W., Cox, J. \u0026amp; Thummel, C.S. Methods for studying metabolism in Drosophila. Methods 68, 105\u0026ndash;115 (2014). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.ymeth.2014.02.034\u003c/span\u003e\u003cspan address=\"10.1016/j.ymeth.2014.02.034\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHanatani, S. \u003cem\u003eet al\u003c/em\u003e. Acetate alters expression of genes involved in beige adipogenesis in 3T3-L1 cells and obese KK-Ay mice. J Clin Biochem Nutr 59, 207\u0026ndash;214 (2016). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3164/jcbn.16-23\u003c/span\u003e\u003cspan address=\"10.3164/jcbn.16-23\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"hyperlipidemia, lipid metabolism, obesity, animal models, silkworm","lastPublishedDoi":"10.21203/rs.3.rs-4724127/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4724127/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAs lifestyle-related diseases like obesity, dyslipidemia, and non-alcoholic fatty liver disease are increasing globally, the demand for developing therapeutic agents and health foods remains high. However, there is a growing concern worldwide regarding the use of animals for biological testing. Herein, we developed a method using the silkworm, \u003cem\u003eBombyx mori\u003c/em\u003e, to evaluate the effects of compounds on improving obesity and lipid metabolism. A silkworm obesity and lipid metabolism disorder (SOLD) model, fed an 10% glucose diet for 72h showed increased fat body weight and accumulation of neutral fat in the hemolymph and fat body. Administration of fenofibrate reduced neutral fat levels in the hemolymph, and epigallocatechin gallate reduced neutral fat levels in the hemolymph and fat body. Silkworms with improved lipid metabolism, exhibited activation of lipoprotein lipase in muscle tissue, and decreased activities of fatty acid synthase and acetyl-CoA carboxylase, and activation of AMPK in the fat body. Furthermore, enhanced fatty acid beta-oxidation contributed to the promotion of lipolysis. These effects and mechanisms of action observed in silkworms are similar to those found in mammals. These results demonstrate the usefulness of the evaluation system in screening materials for their anti-obesity and lipid metabolism improvement effects using the SOLD model.\u003c/p\u003e","manuscriptTitle":"Development of a silkworms-based evaluation system for the extracts and compounds for their obesity and lipid metabolism improving activity","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-06 15:38:53","doi":"10.21203/rs.3.rs-4724127/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-08-12T04:21:04+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-08-08T08:12:28+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-08-04T09:11:58+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"177386488069855032646030477887985828936","date":"2024-07-27T03:35:03+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"171960284864288597744801067638094597735","date":"2024-07-27T01:51:47+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-07-24T17:55:46+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-07-24T12:24:43+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-07-16T05:27:43+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-07-13T07:02:33+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2024-07-11T12:27:52+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"c9746d05-9978-46cc-ba0a-8666aa445349","owner":[],"postedDate":"August 6th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":35553601,"name":"Biological sciences/Drug discovery"},{"id":35553602,"name":"Health sciences/Health care"}],"tags":[],"updatedAt":"2025-05-26T16:09:02+00:00","versionOfRecord":{"articleIdentity":"rs-4724127","link":"https://doi.org/10.1038/s41598-025-96863-1","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-05-23 15:57:40","publishedOnDateReadable":"May 23rd, 2025"},"versionCreatedAt":"2024-08-06 15:38:53","video":"","vorDoi":"10.1038/s41598-025-96863-1","vorDoiUrl":"https://doi.org/10.1038/s41598-025-96863-1","workflowStages":[]},"version":"v1","identity":"rs-4724127","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4724127","identity":"rs-4724127","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}