Hrev_master Healthcare in Low-resource Settings 2023; volume 11(s1):11165 β-(1,3)-D-glucan from Pleurotus ostreatus correlates with lower plasma IL-6, IL-1β, HOMA-IR, and higher pancreatic beta cell count in High-Fat and High-Fructose Diet (HFFD) rats Alma Maghfirotun Innayah,1 Elvira Nur Sa’idah Hariani,2 Husnul Khotimah,1,3 Inggita Kusumastuty,2 Ema Pristi Yunita,4,5 Dian Handayani2,5 1Master Program in Biomedical Sciences, Faculty of Medicine, Universitas Brawijaya, Malang, Indonesia; 2Department of Nutrition Science, Faculty of Health Sciences, Universitas Brawijaya, Malang, Indonesia; 3Pharmacology Department, Faculty of Medicine, Universitas Brawijaya, Malang, Indonesia; 4Department of Pharmacy, Faculty of Medicine, Universitas Brawijaya, Malang, Indonesia; 5Research Center of Smart Molecule of Natural Genetic Resources (SMONAGENES), Universitas Brawijaya, Malang, Indonesia Abstract Introduction: The increasing consumption of high-fat and high-fructose foods contributes to the increasing prevalence of global obesity. Low-grade chronic inflammation in obesity is a significant risk factor for insulin resistance and type 2 diabetes. Therefore, this study aimed to determine the effect of β-(1,3)-D- glucan from oyster mushroom (Pleurotus ostreatus) extract on rats fed with a high-fat and high-fructose diet. Design and Methods: This experimental study was conducted on 35 male Sprague-Dawley rats aged eight weeks. The rats were divided into groups given a normal (N) diet, a high-fat and high- fructose diet (HFFD), D1 (HFFD+125 mg/kg BW β-glucan), D2 (HFFD+250 mg/kg BW β glucan), and D3 (HFFD+375 mg/kg BW β-glucan) with an intervention of 14 weeks. IL-6 and IL-1β levels were measured by the ELISA method, while HOMA-IR (Homeostatic Model Assessment for Insulin Resistance) was cal- culated by the fasting insulin (ng/mL) x fasting blood glucose (mg/dL)/405 formula. Pancreatic beta-cell counts were measured by hematoxylin and eosin (H&E) staining. Results: The results showed no differences in IL-6 and IL-1β between the treatment groups. However, there were significant differences in HOMA-IR and pancreatic beta-cell counts between groups. There were negative correlations between the dose of β- glucan and IL-6, IL-1β, and HOMA-IR levels. Also, there was a positive correlation between the dose of β-glucan and the number of pancreatic beta cells. Conclusions: Administration of β-(1,3)-D-glucan from oyster mushroom (Pleurotus ostreatus) extract prevented hyperglycemia and insulin resistance, also reduced inflammation in rats fed with HFFD regardless of weight gain. Introduction Obesity is a significant global health issue that triggers insulin resistance, the beginning of non-communicable diseases such as type 2 diabetes mellitus.1,2 In Indonesia, the prevalence of adult obesity increased from 10.5% in 2007 to 14.8% in 2013 and 21.8% in 2018. This condition was accompanied by an increase in diabetes mellitus based on the physician’s diagnosis, from 1.5% in 2013 to 2% in 2018. Furthermore, diabetes mellitus diagnosis based on blood glucose testing increased from 6.9% in 2013 to 8.5% in 2018.3 Changes in people’s consumption patterns to high- fat and high-fructose foods and drinks increase the prevalence of obesity.4 Fat tissue accumulation in overweight and obese condi- tions causes low-grade chronic inflammation by releasing cytokines such as TNF-α, IL-6, and IL-1β.5,6 Low-grade chronic inflammation is a significant risk factor for insulin resistance and type-2 diabetes mellitus. In this case, elevated IL-6 and IL-1β increase insulin resistance and type 2 diabetes risk.2,7,8 Prevention and treatment of metabolic syndrome is a strategy under development by developing functional food. An example is a well-known mushroom consumed for 3000 years and used in traditional Chinese and East Asian medicine.9,10 For their thera- peutic effects, some of the best well-known mushrooms are Ganoderma lucidum (Lingzhi mushroom) and Lentinula edodes (Shiitake Mushroom). A study showed that adding shiitake mush- rooms into a high-fat diet mixture could reduce plasma triglyc- eride (TG) levels, inhibit weight gain, and reduce fat deposition in rats.11 In Indonesia, one mushroom popularly cultivated is a white oyster mushroom (Pleurotus ostreatus), consumed as an alterna- tive for protein and a source of fiber.12 White oyster mushrooms have several bioactive components, such as β-glucan, a polysac- charide group composed of D-glucose molecules bound to β-(1,3)- and (1,6)-D-glucan.13 β-glucan from oats have been explored earlier, which has been proven to improve insulin sensi- tivity and HOMA-IR and lowers blood glucose, HbA1C levels, and body weight in mice fed with a high-fat diet.14,15 Several species from the Pleurotus genus have been explored, including Pleurotus citrinopileatus, Pleurotus Sajor-caju, Pleurotus tuber-regium, and Pleurotus ostreatus, both in vitro and in vivo. They show positive effects on weight loss, prevent hyper- Article Significance for public health Changes in people's consumption patterns to high-fat and high-fructose food contribute to obesity and diabetes. White oyster mushroom has been widely con- sumed as alternative protein and dietary fiber. This in-vivo animal model study could be references for product development of β-glucan as nutraceuticals or white oyster mushrooms to prevent obesity and type 2 diabetes. [Healthcare in Low-resource Settings 2023; 11(s1):1165] [page 13] No n- co mm er cia l u se on ly glycemia and insulinemia, reduce gene expression of transcription factor IL-6 and IL-1β, and improve glucose tolerance.16–19 The increasing cultivation and consumption of oyster mushrooms in Indonesia have not been studied based on their benefits. This has limited the data on the benefits of β-glucan bioactive compounds from oyster mushrooms to prevent and treat metabolic syndrome. Therefore, this study aimed to investigate the effect of β-glucan from oyster mushroom (Pleurotus ostreatus) extract on IL-6, IL- 1β, and HOMA-IR levels in rats fed with High-Fat and High- Fructose Diet (HFFD). Design and Methods Animals and diet Our study was conducted using a Post Test Only Controlled Group Design. A sample of 35 male Sprague-Dawley (SD) rats aged five weeks were obtained from the animal resource center The National of Drug and Food Control, Jakarta-Indonesia. They were given three weeks of acclimatization to their new environ- ment, with ad libitum access to food and water. Furthermore, all rats were fed a normal diet of modified AIN-93M during the acclimatization period. They were divided into five groups based on the diet and the dose of β-glucan given, as shown in Table 1. The normal and high-fat diet pellet was based on the AIN-93M formula with modified ingredients amount.20 In this study, the nor- mal diet contained 4.1 kcal/grams, with 7% fat, while the high-fat diet contained 5.46 kcal/grams, with 36.95% fat. Fructose solution in 30% concentration was made by dissolving 300 grams fructose powder for each 1 L of water. The β-glucan from the oyster mush- room extract was produced based on the previous study.21 All experimental procedures have been approved by The Research Ethics Committee of the Faculty of Medicine, Universitas Brawijaya, Indonesia (136/EC/KEPK/07/2020). Food intake, body weight, and body composition Animals were weighed weekly in the fourteen-week interven- tion period. Food intake was measured by weighing the total food (g) provided to the rats and subtracting the remaining food (g) in the cage after 24 hours. After the intervention, the rats were sacri- ficed by ketamine + xylazine (0.1 mL/100 g BW) anesthetic agent after fasting for eight hours. Anthropometric measurement was conducted by weighing, measuring the body (naso-anal) length, and abdominal circumference. The Lee index was calculated by dividing the cubicle root of the weight (g) by the naso-anal length (mm) multiplied by 1000.22 Sample collection and parameters analysis The plasma samples were prepared for parameters analysis by collecting blood through the heart using syringes. The samples were directly inserted into a vacutainer containing ethylene diamine tetraacetic acid (EDTA) anticoagulant. The blood samples were centrifuged for 10 minutes at 10,000 rpm. Moreover, the plas- ma was aliquoted into several tubes using a micropipette. Plasma IL-6, IL-1β, and insulin levels were performed by Enzyme-linked immunosorbent assay (ELISA) following the manufacturer’s pro- tocol (Elabscience Rat IL-6 Cat No: E-EL-R1005; BT-Laboratory Rat IL-1β Cat No: E0119Ra; Elabscience Rat Insulin Cat No: E- EL-R3034). The pancreas was also obtained and preserved in 10% neutral buffered formaldehyde to examine pancreatic beta cell numbers. A histology examination was conducted by hematoxylin and eosin (H&E) staining and scanning with Olympus dot slide microscope at x400 magnification. The number of the pancreatic beta-cell is a count from Langerhans Island in 10 visual fields, assisted by OlyVia software.23 Fasting blood glucose (FBG) was measured using a glucometer (AUTOCHECK).24 Lastly, HOMA- IR index was calculated according to the formula fasting insulin (ng/mL) x fasting blood glucose (mg/dL)/405.25 Statistical analysis Food intake, anthropometric parameters, HOMA-IR, plasma IL-6, IL-1β, and insulin levels were presented as mean and stan- dard errors. Statistical analysis was performed using SPSS soft- ware (SPSS Inc version 25.0, Chicago, Ill, USA). Moreover, body weight gain, abdominal circumference, Lee Index, an intake of total energy, protein and carbohydrate, fasting blood glucose, plas- ma IL-6, and pancreatic beta-cell number were analyzed using a One-way ANOVA test. This was followed by a post hoc Tukey test for multiple comparisons. Food and fat intake, plasma insulin, plasma IL-1β, and HOMA-IR level were analyzed using the Kruskal Wallis test. It was followed by a post hoc Mann-Whitney U test to check different comparisons among the groups. The cor- relations between a dose of β-glucan given and fasting blood glu- cose, plasma insulin, plasma IL-1β, HOMA-IR, and pancreatic beta-cell number were analyzed using Spearman Correlation Test. The differences and correlations were considered significant when p-value <0.05. Results and Discussions After 14 weeks of intervention, the results showed a significant difference in weight gain between the normal and HFFD groups. The lowest Lee Index scores were found in groups D1 and D3, and no significant difference in the rats’ abdominal circumference. The measurement of food intake showed significant differences in the intake of fat and carbohydrates, as shown in Table 2. Furthermore, there were no significant differences in groups’ plasma IL-6 and IL-1β levels. Significant differences were found in fasting blood glucose levels (p = 0.000), insulin levels (p = 0.05), HOMA-IR (p = 0.005), and pancreatic beta-cell count (p = 0.000) between groups. The lowest blood glucose and HOMA-IR levels and the highest number of pancreatic beta cells were in group D2, as shown in Figures 1 and 2. The Spearman test results showed a negative correlation between β-glucan administration and levels of IL-6 (P = 0.032; r = -0.406), IL-1β (p = 0.018; r = -0.443), insulin (p = 0.025; r = - 0.423), and HOMA-IR (p = 0.039; r = -0.392). However, a positive correlation was found between β-glucan administration and pan- Article [page 14] [Healthcare in Low-resource Settings 2023; 11(s1):1165] Table 1. Animal grouping and treatment. Group Treatment Normal Normal diet HFFD (High-Fat and Fructose Diet) High-fat diet + 30% fructose solution (HFFD) D1 HFFD + 125 mg/kgBW β-glucan D2 HFFD + 250 mg/kgBW β-glucan D3 HFFD + 375 mg/kgBW β-glucan No n- co mm er cia l u se on ly Article Table 2. Anthropometric and food intake characteristics. Parameter N HFFD D1 D2 D3 P-value Anthropometric Body Weight Gain (g) 137.7 ± 52.5a 209.0 ± 16.8b 174.7 ± 23.9ab 188.4 ± 38.0ab 177.3 ± 53.2ab 0.034* Abdominal Circumference (cm) 18.6± 2.0 19.1 ± 0.9 19.3 ± 1.6 19.3 ± 1.1 19.0 ± 1.9 NS Lee Index 298.7± 7.6ab 291.3 ± 4.9a 288.8 ± 9.4a 308.2 ± 6.6b 288.1 ± 7.1a 0.000** Food Intake Feed intake (g) 18.93± 3.1a 11.43 ± 0.8b 10.96 ± 1.1b 12.49 ± 2.1b 11.01 ± 1.9b 0.001** Energy intake (kcal) 81.46 ± 9.4 94.77 ± 5.5 87.70 ± 7.7 92.35 ± 12.6 82.70 ± 12.6 NS Protein intake (g) 2.80 ± 0.3 3.00 ± 0.2 2.46 ± 0.3 2.80 ± 0.5 2.47 ± 0.4 NS Fat intake (g) 0.54 ± 0.1a 2.56 ± 0.2b 2.46 ± 0.3b 2.80 ± 0.5b 2.47 ± 0.4b 0.001** Carbohydrate intake (g) 10.89 ± 1.3ab 12.03 ± 1.2b 10.77 ± 1.3ab 11.90 ± 1.4b 9.49 ± 1.4a 0.007* *One Way Anova Test. *Kruskal Wallis Test. NS: non-significant. A significant difference if p<0.05. Post Hoc Test showed a significant difference between groups marked by different annotation (a,b,c). N: Normal diet. HFFD: High-fat diet + 30% fructose solution. D1: HFFD + 125 mg/kgBW β-glucan. D2: HFFD + 250 mg/kgBW β-glucan.. D3: HFFD + 375 mg/kgBW β-glucan. Figure 1. The Comparison of Levels of IL-6, IL-1β, FBG, Insulin, HOMA-IR, and Pancreatic Beta Cell Count between Groups. N: Normal diet, HFFD: High-fat diet + 30% fructose solution, D1: HFFD + 125 mg/kgBW β-glucan, D2: HFFD + 250 mg/kgBW β-glu- can, D3: HFFD + 375 mg/kgBW β-glucan Post Hoc Test showed a significant difference between groups marked by different annota- tion (a,b,c). [Healthcare in Low-resource Settings 2023; 11(s1):1165] [page 15] No n- co mm er cia l u se on ly creatic beta cell count (p = 0.002; r = 0.558), as shown in Figure 3. A positive correlation was also found between IL-6 levels and plasma IL-1β levels (p = 0.003; r = 0.533). The final anthropometric measurements showed no significant difference in the rats’ abdominal circumference between groups. The weight gain was inconsistent with the given dose, with group D2 having the highest weight gain. The results of the Lee Index in the group given the highest dose of β-glucan (375 mg/kg BW) showed the lowest value compared to other groups. These results are consistent with previous studies that showed no effect on weight changes in diabetic rats fed on a high-fat diet and STZ induction fed with oat β-glucan, oat starch, or whole oats.26 However, the results contradict another study, which stated that administering Pleurotus sajor-caju mushroom extract in rats fed with a high-fat diet could inhibit weight gain.19 In this study, the highest intake of carbohydrates, including fructose solution, was found in the HFFD group, and the lowest was in the D3 group. Previous studies showed that the fluid intake of rats given β-glucan was lower than the positive control group that experienced poly- dipsia.26 The group of rats fed with HFFD experienced symptoms of polydipsia through increased intake of fructose solution, increasing carbohydrate intake than the rats given β-glucan. A decrease in carbohydrate intake lowers the food efficiency ratio and energy balance. The lower total carbohydrate intake is related to inhibiting the adipogenesis process. This indicates the group given the highest dose of β-glucan had the lowest Lee Index.19 However, there was no significant difference in energy intake between groups. These results contradict previous studies, which showed that administering β-glucan from oats and Pleurotus sajor- caju extract could reduce energy intake.19,27 The energy density of normal feed and HFFD used in this study has a small difference. The difference in intake is more influenced by feed composition and consumption of fructose solution. Plasma IL-6 and IL-1β levels decreased in the group given β- glucan from the oyster mushroom extract. The correlation test results showed a negative correlation between β-glucan dose and plasma IL-6 and IL-1β levels. These results support previous stud- ies that concentrates from Pleurotus ostreatus could suppress IL-6 secretion in LPS-exposed mice, while Pleurotus tuber-regium extract could inhibit the release of IL-6 in LPS-induced cell lines.16,17 Another study on β-glucan from oat sources stated that giving a β-glucan intervention to rats with a high-fat diet reduced plasma IL-6 and IL-1β levels.26 Moreover, several in-vitro studies showed that β-glucan from sources such as Pleurotus sajor-caju, Ganoderma lucidum, and Poria cocos could suppress the produc- tion of IL-1β and TNF-α.28,29 This study found that β-glucan reduces pro-inflammatory cytokine levels, indicated by a signifi- cant negative correlation between the dose of β-glucan adminis- tered and plasma levels of IL-6 and IL-1β. The increase in pro- inflammatory cytokines in obesity and metabolic syndrome may occur by activating the transcription factor NF-kB. Further increas- es in circulating cytokine levels increase the risk of insulin resis- tance and type 2 diabetes. The mechanism of β-glucan in activating the anti-inflammatory pathway is not fully understood. However, β-glucan molecules can be recognized as pathogen-associated molecular patterns (PAMP) that bind to receptors such as dectin 1, complement receptor 3 (CR3), or toll-like receptors (TLRs).30 The interaction between β-glucan and TLR inhibits the activation of transcription factors NF-kB and AP-1, suppressing cytokine pro- duction and providing anti-inflammatory effects.16,31 This study did not determine the process that explains the anti-inflammatory mechanism of β-glucan from the oyster mushroom extract. This study showed significant differences between groups regarding fasting blood glucose, insulin, and HOMA-IR levels, and pancreatic beta-cell counts. Administering β-glucan from oys- ter mushroom extract (Pleurotus ostreatus) could reduce Fasting Blood Glucose (FBG), insulin, and HOMA-IR levels and increase pancreatic beta cells. These results support previous studies, which stated that administering oyster mushroom powder and extract could reduce FBG levels and improve pancreatic beta cells in dia- Article Figure 2. Histological Examination of Pancreatic Beta Cells between Groups. N: Normal diet, HFFD: High-fat diet + 30% fructose solu- tion, D1: HFFD + 125 mg/kgBW β-glucan, D2: HFFD + 250 mg/kgBW β-glucan, D3: HFFD + 375 mg/kgBW β-glucan. [page 16] [Healthcare in Low-resource Settings 2023; 11(s1):1165] No n- co mm er cia l u se on ly betic rats.32,33 Another in-vivo study in mice and rats fed with a high-fat diet showed that administering Pleurotus citrinopileatus and Pleurotus tuber-regium extracts could reduce fasting blood glucose levels.19,34 Administering β-glucan from sources such as oats and chitin reduces FBG and HOMA-IR levels insulin secre- tion and repairs pancreatic beta cells.15,26,35,36 Furthermore, this study found that β-glucan improves glycemic control, insulin resis- tance, and pancreatic beta cells. This is seen by the negative corre- lation between β-glucan dose and insulin and HOMA-IR levels and a positive correlation between β-glucan dose and pancreatic beta-cell counts. Several mechanisms concerning the effect of low- ering glucose and insulin occur through the ability of soluble fiber (β-glucan) to form a viscous layer on the gastrointestinal tract. This slows gastric emptying, digestion, absorption and reduces nutrient transport to enterocytes.35,36 Administering Pleurotus ostreatus extract also increased p-AMPK in muscle and adipose tissue. It leads to upregulation of the transcriptional regulator of the GLUT4 gene for increasing glucose uptake, providing an anti-hyper- glycemic effect.33 Moreover, β-glucan works as an antioxidant, protecting against pancreatic beta-cell apoptosis and increasing the production of hematopoietic stem cells (HSCs). This indicates HSCs could differentiate into special fibroblasts and liver, endothelial, and pancreatic cells. β-glucan becomes a potent molecule to improve glucose homeostasis in the body through hypoglycemic effects, improvement of insulin resistance, and decreased apoptosis of pancreatic cells.15,32,34 Decreased pro- Article Figure 3. Spearman Correlation Test Results between Dose of β-glucan administration and Levels of IL-6, IL-1β, insulin, HOMA-IR, and pancreatic beta-cell count. [Healthcare in Low-resource Settings 2023; 11(s1):1165] [page 17] No n- co mm er cia l u se on ly inflammatory cytokines also improved insulin resistance, where low IL-6 and IL-1β increased the expression and sensitivity of insulin receptor substrate, specifically IRS-1. This resulted in increased insulin-mediated glucose uptake into cells.37,38 β-glucan has various structural and molecular weight varia- tions, depending on its source and extraction procedure. The β-glu- can extracted from the genus Pleurotus has a high molecular weight. An example is Pleurotus tuber-regium, which contains β- glucan with a molecular weight of 5.76 x 104 - 77.4 x 104 g/mol, nearly similar with oat β-glucan, with a molecular weight of 15.6 x 104 - 68.7 x 104 g/mol.13,39 High molecular weight and viscosity β-glucans have hypocholesterolemic and hypoglycemic effects. In contrast, low molecular weight β-glucans have antioxidant and immunological effects.39 Studies on the functionality of molecular weight show pros and cons influenced by the source of β-glucan, the amount of daily food intake, and the dose of β-glucan given.13,39,40 This study did not determine the molecular weight of β-glucan. Therefore, further studies should analyze the molecular weight of Pleurotus ostreatus β-glucan and its functionality. The β- glucan structure is also associated with its functionality. It is com- posed of beta-D-glucose monomer units linked by glycosidic bonds at (1,3), (1,4), or (1,6), with or without branches.31 Brown algae, oats, and barley contain β-(1,3/1,4)-D-glucan that modulate microbiota, lowering cholesterol and blood glucose levels. Moreover, mushrooms and yeasts contain β-(1,3/1,6)-D-glucan that modulates the immune system and has antimicrobial and anti- cancer properties. Agrobacterium contains branchless β-(1,3)-D- glucan used as a thickening agent in food processing.31 The oyster mushroom extract used in this study had a β-(1,3)-D-glucan struc- ture.21 The result supports another study on oyster mushroom (Pleurotus ostreatus) using an alkaline extraction method, which stated that the compound found was β-(1,3)-D-glucan.41 The study of β-glucan from chitin sources stated that it contains β-(1,3)-D- glucan, which improves blood glucose, triglyceride, and choles- terol levels and glucose tolerance in mice fed on a high-fat diet.36 Regardless of the structural variation, biological activity is the β- (1,3)-D-glucan core/backbone binding, which improves blood glu- cose control and dyslipidemic conditions. It also modulates immune responses and gut microbiota and improves obesity con- ditions.31 The findings concerning the correlation between differ- ences in branch structure and functionality of β-glucans are still limited. Therefore, further studies should examine the β-glucan structure with the desired therapeutic target. This study found that rats in the D2 group administered with 250 mg/kg BW β-glucan from oyster mushroom showed the best improvement effects in IL-6, IL-1β, HOMA-IR, and pancreatic beta-cell count. However, this mechanism was not elucidated in this study. Previous studies on the toxicity of β -glucans are still limited, such as giving the β-(1,3/1-6)-D-glucan with doses of 500, 1000, and 2000 mg/kg BW Sprague-Dawley rats for 90 days with- out side effects on anthropometric and hematological blood param- eters.42 Another study administered β-(1,3/1-4)-D-glucan with doses of 0.7, 3.5, and 7% through a mixture of feed on Wistar rats for 28 days. The results showed no side effects on growth, hema- tological abnormalities, and organ weight of rats.43 The levels of IL-6 in the D3 group were +9.9% higher, IL-1β -6.4% lower, HOMA-IR -3.3% higher. Additionally, pancreatic beta cells were - 0.7% lower than the normal group, with no significant difference. The β-glucan used in this study has a different β-(1,3)-D-glucan structure from the two previous studies, hence, it is necessary to explore the differences in structure and functionality. Conclusions This study showed that administering β-glucan with the structure of β-(1,3)-D-glucan from oyster mushroom (Pleurotus ostreatus) extract could reduce the levels of IL-6 IL-1β, FBG, HOMA-IR, and insulin. The administration could increase pancreatic beta-cell counts in HFFD-treat- ed rats, with the best effect reported in the dose of 250 mg/kg BW β-glu- can. Furthermore, the administration of β-glucans had a preventive effect on hyperglycemia and insulin resistance in inflammatory rats by induc- ing high-fat and fructose diets regardless of the weight gain. Further stud- ies should explore the influence of the structure and molecular weight of β-glucan as well as examine its mechanism as an anti-inflammatory, its prevention of hyperglycemia, and reduction of insulin resistance index. Article Correspondence: Dian Handayani, Department of Nutrition Science, Faculty of Health Sciences, Universitas Brawijaya, Malang, Indonesia, Jl. Veteran, Malang, East Java, Indonesia 65145, Tel.: +62341-569117, Fax +62341-564755. E-mail: handayani_dian@ub.ac.id Key words: β-(1,3)-D-glucan; HOMA-IR; IL-6; IL-1β; pancreatic beta cell. Acknowledgment: The authors thank the Faculty of Medicine and Institute of Research and Community Services Universitas Brawijaya for their support and motivation during this study. Contributions: Dian Handayani (DH), Inggita Kusumastuty (IK), and Ema Pristi Yunita (EY) designed and coordinated the study. Alma Maghfirotun Innayah (AI) and Elvira Nur Sa’idah Hariani (EH) con- ducted the experiments and biological assays. The first author and co- authors prepared and conducted the data analysis and wrote the manu- script. Dian Handayani (DH), Husnul Khotimah (HK), Inggita Kusumastuty (IK), and Ema Pristi Yunita (EY) assisted in data interpre- tation and contributed to the final manuscript. DH is a corresponding author. Conflict of Interests: The authors have declared no conflict of interest. Funding: This study was funded by the Institute of Research and Community Services Universitas Brawijaya (Decree: 437.4/UN10.C10/PN/2020). Clinical trials: All procedures were approved by the Research Ethics Committee of the Faculty of Medicine, Universitas Brawijaya, Indonesia (Ethics Approval Number: 136/EC/KEPK/07/2020). Availability of data and materials: All data generated or analyzed during this study are included in this published article. Informed consent: Not applicable. Conference presentation: Part of this paper was presented at the 2nd International Nursing and Health Sciences Symposium that took place at the Faculty of Medicine, Universitas Brawijaya, Malang, Indonesia. Received for publication: 5 December 2021. Accepted for publication: 10 May 2022. This work is licensed under a Creative Commons Attribution 4.0 License (by-nc 4.0). ©Copyright: the Author(s), 2023 Licensee PAGEPress, Italy Healthcare in Low-resource Settings 2023; 11(s1):11165 doi:10.4081/hls.2023.11165 Publisher's note: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organi- zations, or those of the publisher, the editors and the reviewers. Any prod- uct that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher. [page 18] [Healthcare in Low-resource Settings 2023; 11(s1):1165] No n- co mm er cia l u se on ly References 1. McArdle MA, Finucane OM, Connaughton RM, et al. Mechanisms of obesity-induced inflammation and insulin resistance: insights into the emerging role of nutritional strate- gies. Front Endocrinol (Lausanne) 2013;4:52. 2. Chen L, Chen R, Wang H, et al. Mechanisms linking inflam- mation to insulin resistance. Int J Endocrinol 2015;2015:1–9. 3. Kemenkes-RI. Hasil Utama Riskesdas 2018. Jakarta: Balitbangkes Kemenkes RI; 2018. 4. Pereira RM, Botezelli JD, da Cruz Rodrigues KC, et al. Fructose consumption in the development of obesity and the effects of different protocols of physical exercise on the hepat- ic metabolism. Nutrients 2017;9:405. 5. Rodríguez-Hernández H, Simental-Mendía LE, Rodríguez- Ramírez G, et al. Obesity and inflammation: epidemiology, risk factors, and markers of inflammation. Int J Endocrinol 2013;2013:1–11. 6. Kern L, Mittenbühler MJ, Vesting AJ, et al. Obesity-Induced TNFα and IL-6 Signaling: The Missing Link between Obesity and Inflammation—Driven Liver and Colorectal Cancers. Cancers (Basel) 2019;11:24. 7. Ballak DB, Stienstra R, Tack CJ, et al. IL-1 family members in the pathogenesis and treatment of metabolic disease: focus on adipose tissue inflammation and insulin resistance. Cytokine. 2015;75:280–90. 8. Bao P, Liu G, Wei Y. Association between IL-6 and related risk factors of metabolic syndrome and cardiovascular disease in young rats. Int J Clin Exp Med 2015;8:13491–9. 9. Guillamón E, García-Lafuente A, Lozano M, et al. Edible mushrooms: Role in the prevention of cardiovascular diseases. Fitoterapia 2010;81:715–23. 10. Wasser S. Medicinal mushroom science: Current perspectives, advances, evidences, and challenges. Biomed J 2014 Sep 2;37. 11. Handayani D, Chen J, Meyer BJ, et al. Dietary Shiitake mush- room (Lentinus edodes) prevents fat deposition and lowers triglyceride in rats fed a high-fat diet. J Obes 2011;2011:1–8. 12. Tjokrokusumo D. Jamur Tiram (Pleurotus ostreatus) untuk Meningkatkan Ketahanan Pangan dan Rehabilitasi Lingkungan. J Rekayasa Lingkung 2018;4:53–62. 13. Golak-Siwulska I, Kałużewicz A, Spiżewski T, et al. Bioactive compounds and medicinal properties of Oyster mushrooms (Pleurotus sp.). Folia Hortic 2018;30:191–201. 14. Zheng J, Shen N, Wang S, et al. Oat beta-glucan ameliorates insulin resistance in mice fed on high-fat and high-fructose diet. Food Nutr Res 2013;57:10.3402/fnr.v57i0.22754. 15. Cheng Y, Zhang J, Luo K, et al. Oat bran β-glucan improves glucose homeostasis in mice fed on a high-fat diet. RSC Adv 2017;7:54717–25. 16. Jedinak A, Dudhgaonkar S, Wu Q, et al. Anti-inflammatory activity of edible oyster mushroom is mediated through the inhibition of NF-κB and AP-1 signaling. Nutr J 2011;10:52. 17. Liu Y-W, Mei H-C, Su Y-W, et al. Inhibitory effects of Pleurotus tuber-regium mycelia and bioactive constituents on LPS-treated RAW 264.7 cells. J Funct Foods 2014;7:662–70. 18. Kanagasabapathy G, Chua KH, Malek SNA, et al. AMP-acti- vated protein kinase mediates insulin-like and lipo-mobilising effects of β-glucan-rich polysaccharides isolated from Pleurotus sajor-caju (Fr.), Singer mushroom, in 3T3-L1 cells. Food Chem 2014;145:198–204. 19. Sheng Y, Zhao C, Zheng S, et al. Anti-obesity and hypolipi- demic effect of water extract from Pleurotus citrinopileatus in C57BL/6J mice. Food Sci Nutr 2019;7:1295–301. 20. Reeves PG. Components of the AIN-93 diets as improvements in the AIN-76A diet. J Nutr 1997;127:838S-841S. 21. Yunita EP, Yuniar AM, Kusumastuty I, et al. The Effects of ß- glucan Extract from Oyster Mushroom (Pleurotus ostreatus) on Expression of Serum Malondialdehyde in Sprague dawley Rats Induced by HFHF Diet. J Phys Conf Ser 2020;1665:12035. 22. Malafaia AB, Nassif PAN, Ribas CAPM, et al. Obesity induc- tion with high fat sucrose in rats. ABCD Arq Bras Cir Dig (São Paulo) 2013;26:17–21. 23. Hariani ENS. The Effect of Beta-glucan from Oyster Mushroom Extract on Pancreatic Beta Cell Number in Male Sprague Dawley Rats given High-Fat High Fructose Diet (Unpublished Undergraduate Thesis). Universitas Brawijaya; 2021. 24. Firdaus MF. The Effect of Beta-glucan from Oyster Mushroom Extract on Fasting Blood Glucose Level in Male Sprague Dawley Rats given High-Fat High Fructose Diet (Unpublished Undergraduate Thesis). Universitas Brawijaya; 2021. 25. Roza NA V, Possignolo LF, Palanch AC, et al. Effect of long- term high-fat diet intake on peripheral insulin sensibility, blood pressure, and renal function in female rats. Food Nutr Res 2016;60:28536. 26. Zhu Y, Dong L, Huang L, et al. Effects of oat β-glucan, oat resistant starch, and the whole oat flour on insulin resistance, inflammation, and gut microbiota in high-fat-diet-induced type 2 diabetic rats. J Funct Foods 2020;69:103939. 27. Cheng HS, Ton S, Phang S, et al. Increased susceptibility of post-weaning rats on high-fat diet to metabolic syndrome. J Adv Res 2017;8:743–52. 28 Yoon H-M, Jang K-J, Han MS, et al. Ganoderma lucidum ethanol extract inhibits the inflammatory response by sup- pressing the NF-κB and toll-like receptor pathways in lipopolysaccharide-stimulated BV2 microglial cells. Exp Ther Med 2013;5:957–63. 29. Jeong J-W, Lee HH, Han MH, et al. Ethanol extract of Poria cocos reduces the production of inflammatory mediators by suppressing the NF-kappaB signaling pathway in lipopolysac- charide-stimulated RAW 264.7 macrophages. BMC Complement Altern Med 2014;14:101. 30. Jager J, Grémeaux T, Cormont M, et al. Interleukin-1beta- induced insulin resistance in adipocytes through down-regula- tion of insulin receptor substrate-1 expression. Endocrinology 2007;148:241–51. 31. Murphy EJ, Rezoagli E, Major I, et al. β-Glucan Metabolic and Immunomodulatory Properties and Potential for Clinical Application. J Fungi 2020;6:1–36. 32. Purbowati, Johan A, Rmd RAK. Pengaruh jamur tiram putih ( pleurotus ostreatus ) terhadap kadar glukosa darah , profil lipid dan kadar MDA pada tikus ( rattus norvegicus ) diabetes meli- tus. J Gizi Indones 2016;4:131–7. 33. Asrafuzzaman M, Rahman MM, Mandal M, et al. Oyster mushroom functions as an anti-hyperglycaemic through phos- phorylation of AMPK and increased expression of GLUT4 in type 2 diabetic model rats. J Taibah Univ Med Sci 2018;13:465–71. 34. Huang H-Y, Korivi M, Chaing Y-Y, et al. Pleurotus tuber- regium polysaccharides attenuate hyperglycemia and oxidative stress in experimental diabetic rats. Evidence-Based Complement Altern Med 2012;2012:1–8. 35. El Khoury D, Cuda C, Luhovyy BL, et al. Beta glucan: health benefits in obesity and metabolic syndrome. J Nutr Metab 2012;2012:1–28. 36. Delzenne NM, Neyrinck AM, Cani PD. Gut microbiota and metabolic disorders: how prebiotic can work? Br J Nutr Article [Healthcare in Low-resource Settings 2023; 11(s1):1165] [page 19] No n- co mm er cia l u se on ly 2013;109:S81–5. 37. Febbraio MA. Role of interleukins in obesity: implications for metabolic disease. Trends Endocrinol Metab [Internet]. 2014;25:312–9. Available from: https://doi.org/10.1016/ j.tem.2014.02.004 38. Shi J, Fan J, Su Q, et al. Cytokines and Abnormal Glucose and Lipid Metabolism. Front Endocrinol (Lausanne) 2019;10:703. 39. Du B, Meenu M, Liu H, et al. A concise review on the molec- ular structure and function relationship of β-glucan. Int J Mol Sci 2019;20:4032. 40. Wang Y, Harding S V, Eck P, et al. High-molecular-weight β- glucan decreases serum cholesterol differentially based on the CYP7A1 rs3808607 polymorphism in mildly hypercholes- terolemic adults. J Nutr 2015;146:720–7. 41. Baeva E, Bleha R, Lavrova E, et al. Polysaccharides from Basidiocarps of Cultivating Mushroom Pleurotus ostreatus: Isolation and Structural Characterization. Molecules 2019;24:2740. 42. Chen SN, Nan FH, Chen S, et al. Safety assessment of mush- room β-glucan: Subchronic toxicity in rodents and mutagenic- ity studies. Food Chem Toxicol 2011;49:2890–8. 43. Delaney B, Carlson T, Frazer S, et al. Evaluation of the toxicity of concentrated barley β-glucan in a 28-day feeding study in Wistar rats. Food Chem Toxicol 2003;41:477–87. Article [page 20] [Healthcare in Low-resource Settings 2023; 11(s1):1165] No n- co mm er cia l u se on ly