Hrev_master Healthcare in Low-resource Settings 2024; volume 12(s1):13069 The effects of brown rice as functional food on lee index, adipose tissues and PRDM16 levels in obesity model Rattus norvegicus Jeany Pricelia,1 Putri Dwi Arini,1 Hazrina Putri Alifiyah,1 Riri Syabania,1 Inggita Kusumastuty,1,4 Etik Sulistyowati,2 Laksmi Sasiarini,3 Achmad Rudijanto,3 Dian Handayani1,4 1Department of Nutrition, Faculty of Health Sciences, Universitas Brawijaya, Malang, East Java; 2Department of Nutrition, Polytechnic of Health, Malang, East Java; 3Faculty of Medicine, Universitas Brawijaya, Malang, East Java; 4Metabolic Syndrome Research Group, Faculty of Health Sciences, Universitas Brawijaya, Malang, East Java, Indonesia Abstract Brown rice is a functional food known to improve Lee index, influencing PRDM16 levels in obesity conditions. Therefore, this study aims to determine the differences in Lee index, Brown Adipose Tissue (BAT) weight, White Adipose Tissue (WAT) weight, BeAT weight, total body fat, and PRDM16 levels of obese model rats with the intervention of brown and white rice, as well as g-oryzanol. A true experimental method was used with a post- test-only control group in vivo design. The obesity model was constructed with male Sprague Dawley rats (Rattus norvegicus), divided into five diet groups namely standard and HFHF diet con- trol, as well as HFHF + brown rice, HFHF + white rice, and HFHF + g-oryzanol combination diet. The experiment was carried out for 26 weeks, with details of 14 weeks to form an obese model and 12 weeks for the intervention. Before statistical correlation was test- ed, Lee index values, adipose tissues, and PRDM16 were ana- lyzed. The anthropometric data collection method was carried out by weighing before and after the intervention, while adipose tissue was collected by weighing after sacrifice. The immunofluores- cence method was used to collect the expression of PRDM16 and the mean of PRDM16 levels was analyzed in the ImageJ applica- tion. After the data collection process, analysis was performed using SPSS to determine possible differences in each group. Normally distributed data were analyzed using One-Way ANOVA, while those without normal distribution were assessed using the Kruskall-Walis method and the Mann Whitney-U advanced test, with a p-value of <0.05 considered significantly different. The result showed that there were differences among several groups regarding total body fat (p=0.012), WAT (p=0.026), and BAT (p=0.025). However, no differences were found between all groups regarding the Lee index (p=0.275), BeAT (p=0.079), and PRDM16 level (p=0.292). In conclusion, brown rice intervention did not significantly affect Lee index val- ues, the expression of PRDM16, and adipose tissue weights at the end of intervention. Introduction Obesity is a chronic metabolic disease caused by the pro- longed buildup of energy in the body, leading to fat tissue accumu- lation. The global cases are increasing each year, with the preva- lence reaching 650 million in 2016. In Indonesia, the prevalence among adults jumped from 6.4% to 21.8% between 2013 and 2018.1 Considering that obesity plays a significant role in devel- oping metabolic syndrome,2 fast and appropriate interventions are needed. Several factors can be a risk for obesity with diet being the most influential3 followed by genetics, lifestyle, physical activ- ity, environment, health, and psychology.3 Obesity interventions are focused on dietary habits, physical activity improvement, and lifestyle changes. In this context, highly aggressive methods capa- ble of causing side effects such as pharmacological and surgical are usually not recommended.4 However, obesity with complica- tions can be treated by using several methods consisting of diet, physical exercise, behavior modification, pharmacological thera- py, and bariatric surgery.4 Brown rice is one of the functional foods which has antiobesity and antihyperglycemic effects. Due to the intact outer skin layer and aleurone,5 brown rice has better potential nutritional content compared to white rice which con- tains dietary fiber, minerals, and g-oryzanol, a bioactive com- pound useful as an antiobesity agent.6,7 With this content, brown rice is capable of influencing parameters of obesity, including body anthropometry and PRDM16 levels. According to a previous study, obesity is not associated with total body weight but specif- ically with the quantity of white adipose tissue (WAT)8 which accumulates the excessive energy into triglyceride form. There is also brown adipose tissue (BAT) playing an important role in metabolizing fatty acids to produce heat,9 although the amount is often lower than WAT in obese people.8 Specific interventions such as maintaining diet and physical activity can cause white adipocytes to form beige adipocytes in browning activity. This process indirectly affects the body weight of rats and improves the anthropometric profile in animal models of obesity. Positive regulatory domain zinc finger region protein 16 (PRDM16), a gene number 1p36.32 on the human chromosome, can inhibit the formation of WAT while increasing the formation of brown and beige adipocytes.10,11 It increases biogenesis in tissue Significance for public health Brown rice is considered a functional food that has been shown to impact the Lee index positively and may influence PRDM16 levels in cases of obesity. Research suggests that brown rice (BRG) may promote the enhancement of beige adipose tissue (BeAT), brown adipose tissue (BAT), and PRDM16 levels. However, further investigation is necessary to determine the optimal dietary ratio required to facilitate the browning process of white adipose tissue (WAT). [page 102] [Healthcare in Low-resource Settings 2024;12(s1):13069] Non -co mmerc ial us e o nly mitochondria and activates several transcription factors, such as PPAR-g, to enhance the expression of uncoupling protein 1 (UCP1), which potentially produces heat and stimulates browning in WAT. One effort to trigger browning is diet regulation through calorie restriction.12 Studies related to the relationship between brown rice and body fat have been carried out on both experimental animals and humans. The results showed that brown rice had a positive effect on glucose and body fat levels in 18 diabetes mellitus patients who were treated for 3 months.5 After the intervention was given, there was a decrease in poor blood glucose control by 27.7%, high fat by 22.2%, and high visceral fat index by 22.2%.5 In obese experimen- tal animals, brown rice effectively improved the Fermicutes/Bakteriodetes microbiota ratio.13,14 The supplementa- tion was proven to significantly reduce fat levels in the liver and prevent the development of NAFLD in obese model rats.15 g-oryzanol, one of the bioactive compounds in brown rice, plays a significant role in regulating body weight and preventing several diseases including hyperglycemia, hypertriglyceridemia, blood vessel disorders, and renal damage. It acts as an antioxidant and anti-inflammatory agent, suppressing inflammation in condi- tions of obesity.16 On average, brown rice contains 10-150 mg/100 g of γ-oryzanol.17 This presents an opportunity since the majority of Indonesians still consume white rice which potentially has a high glycemic index (79.6).18 In general, food with a high glycemic index contributes to obesity and type 2 diabetes mellitus. Studies regarding the effect of functional food on PRDM16 levels are limited. Therefore, this study aimed to examine differ- ences in Lee index, BAT weight, WAT weight, BeAT weight, total body fat, and PRDM16 levels in obesity model rats given brown and white rice as well as g-oryzanol interventions. The study nov- elty sets in examining the difference in PRDM16 levels and anthropometry of obesity model rats with functional food interven- tions of brown rice compared to white rice and g-oryzanol. The results are expected to offer insights into brown rice as a functional food against obesity and metabolic syndrome. In addition, this study can be used as a reference in developing functional foods at the human level. Materials and Methods Study design A true experimental method was used with a post-test-only control group in vivo design. The inclusion criteria were male Sprague Dawley (Rattus norvegicus) with age 10-12 weeks, body weight 200-250 g, in active condition (shiny fur, no alopecia, nor- mal extremities), and free of any treatment or chemical intake. This study was approved by the Health Research Ethics Committee, Faculty of Health Sciences, Brawijaya University No. 2020/UN10.F17.10.4/TU/2023. All procedures were carried out in collaboration with the Experimental Animal Care Laboratory, Faculty of Medicine, Universitas Brawijaya. Study subjects The samples were subjected to diet intervention with brown rice (BRG), white rice (WRG), and pure g-oryzanol (ORG) com- bined with high-fat high fructose (HFHF) diet. In addition, sam- ples had both positive (PG) and negative controls (NG). Positive control was a group of rats on HFHF diet, while the negative con- trol was on a standard diet. Instruments development and data collection Anthropometric data were collected by measuring the body weight and length of rats before and after intervention. Subsequently, BMI and Lee index were calculated using the data obtained. When Lee index value was > 0.3 then rats were catego- rized as obese. An analysis was further carried out to examine pos- sible differences in each group of rats before and after intervention. Data on the weight of adipose tissue were collected by weighing after rats were sacrificed. Adipose tissue weighed included WAT, BAT, BeAT, and Total Body Fat. Rats were sacrificed for the assessment of WAT, BAT, and BeAT. Fat tissues were converted to slides before heating (60� in 60 minutes) and soaking in xliol (2 x 10 minutes), absolute ethanol (2 x 10 minutes), ethanol 90% (1 x 5 minutes), ethanol 80% (1 x 5 minutes), and ethanol 70% (1 x 5 minutes). Subsequently, the slides were subjected to antigen retrieval process with citric buffer. Washing was carried out using PBS solution followed by incuba- tion with BSA 1% for 30 minutes at room temperature before incu- bation with primary antibody at 4°C overnight. PBS washing was initially performed before the use of secondary antibody and DAPI incubation. Fat slides were then subjected to a staining process using the immunofluorescence method. The staining results were examined using an immunofluorescence microscope and the ImageJ to select tissue sections and analyze the average PRDM16 levels. Data analysis Statistical analysis of data was conducted using IBM SPSS Statistics. The obtained data were tested for normality. For normal- ly distributed data, a homogeneity test was initially performed, and after meeting a p> 0.05, One-Way ANOVA analysis was conducted to determine the differences in three or more unpaired groups. For abnormal distribution, the data was analyzed using the Kruskal- Wallis test method to determine differences in three or more unpaired groups. A significant difference between the groups was indicated by a p<0.05. The analysis was continued with the Mann- Whitney U test carried out per two groups, and a p<0.05 indicated a significant difference. Results and Discussion Brown rice differs from other varieties due to the name which reflects the original color. This variety is subjected to only minimal processing, leaving the outer layer intact, including the rice bran.13 According to previous studies, brown rice has a high fiber content with seven times greater magnesium and manganese content than white rice.14 The fiber and mineral content has been proven to reduce gut microbiota dysbiosis, increase serum magnesium lev- els, as well as prevent rising serum magnesium levels in obese experimental animals.6,14 Based on the results, there was no significant difference between the control and intervention groups (Table 1). This was in contrast to a study conducted on the substitution of local brown rice varieties for anthropometry and improving blood glucose lev- els in patients with type 2 diabetes mellitus.19 The result showed that brown rice intervention significantly reduced body weight, BMI, body fat percentage, abdominal circumference, fasting blood glucose, 2-hour postprandial blood glucose, and HbA1C.19 The dif- ference between the results could be caused by a non-significant difference between the average energy food intake of rats in each group. However, the administration of white rice and g-oryzanol 4th International Nursing and Health Sciences Symposium [Healthcare in Low-resource Settings 2024;12(s1):13069] [page 103] Non -co mmerc ial us e o nly intervention showed a positive trend toward improving Lee index compared to positive control. Body weight results of experimental animals were also influ- enced by total body fat. Based on the results of statistical tests, sig- nificant total body fat differences were found between positive and negative control groups (p=0.001), negative control and white rice intervention (p=0.035), as well as negative control and g-oryzanol group (p=0.085). The highest average total body fat weight was found in positive control (26.6 ± 9.2 g), followed by brown rice intervention group (19.27 ± 10.8 g) (Table 1). The result differed from a previous study where the consump- tion of brown rice affected the inhibition of fat accumulation, ulti- mately reducing the risk of central obesity in patients with type 2 diabetes mellitus.20 The variation between the results could be caused by a non-significant difference between the average dietary intake of rats in each group. Brown rice diet intervention group had a lower average intake compared to positive control, negative con- trol, and white rice intervention groups according to their average energy intake. Therefore, the bioactive content in brown rice did not provide significant benefits for reducing body weight and total body fat. As shown in Table 1, there were no significant differences between PRDM16 levels in the control and the intervention group (p=0.292). However, the highest levels were found in brown rice intervention group (9.5 ± 8 relative mRNA levels). Brown rice has antiobesity and antidiabetic properties, attributed to aminobutyric acid (GABA), inositols, tocotrienols, ferulic acid, oryzanol, and methanol.21,22 Compounds such as ferulic acid can block adipocyte production, while methanol potentially reduces the formation of transcription factors and adipogenic genes, including EHMT1 and CtBP1/2.21,22,23 These results were consistent with the previous study about PRDM16 stating that PRDM16 was found to play a crucial role in adipocyte differentiation.11 Antiobesity and antidia- betic intake of brown rice can cause adipocyte differentiation, including inhibition of production and transcription factors by increasing PRDM16 levels. Generally, white adipocytes are stored in the body in WAT, and in conditions of obesity, the composition tends to be more signifi- cant due to the accumulation of unused energy in the form of tria- cylglycerol.24 In this study, visceral and subcutaneous fat were taken as WAT samples, BAT was collected from the interscapular region, and BeAT was acquired from the testicular area. According to a previous study, visceral fat stores large circulating FFA and inflammatory markers, including IL-6, CRP, and TNF-α, which increase the risk of CVD.25 The smallest average weight of WAT was found in white rice intervention group (8.65 ± 6.54 g), while the largest was recorded in brown rice (11.3 ± 6.08 g). This result was not in line with PRDM16 levels which were significantly high in brown rice intervention group, suggesting WAT weight obtained might be influenced by several other tissues taken during the sac- rifice. A previous study about brown and beige adipose tissue explained that BeAT could appear in subcutaneous WAT storage, particularly in the anterior subcutaneous and inguinal areas.26 An ineffective diet was also implicated because the fiber, phe- nolic acid, and γ-oryzanol content of brown rice were not con- sumed to an extent capable of causing WAT browning.27 A study comparing different laboratory feeding methods in rats found that the group given AUTO, a daily feeding method set to reduce body weight by up to 90-95%, showed better results in weight loss com- pared to ad libitum feeding.28 Future studies should expand the investigation with a feeding method that has been regulated for weight loss. According to previous reports, PRDM16 has two mechanisms in WAT differentiation process.11 In the first mechanism, PRDM16 induces the formation of brown fat-related genes (PGC 1α, PGC- 4th International Nursing and Health Sciences Symposium [page 104] [Healthcare in Low-resource Settings 2024;12(s1):13069] Table 1. Data of diet, anthropometric, body fat, and PRDM16 Average. Non -co mmerc ial us e o nly 1β, PPARγ, uncoupling protein 1 (UCP1)) and forms a complex to cause thermogenesis effects on fat. This initiates the browning pro- cess, leading to the formation of BAT (Figure 1). High levels of PRDM16 in the brown rice intervention group resulted in compa- rable BAT weight. There was a significant difference in BAT between negative and positive control group, negative control and g-oryzanol intervention, as well as positive control and white rice intervention group (Table 1). The highest average weight of BAT was found in the brown rice intervention group (2.81 ± 1.6 g) fol- lowed by positive control (2.97 ± 1.27 g), and white rice interven- tion group (1.36 ± 0.42 g). The second mechanism is the formation of BeAT initiated by the conversion and secretion of the metabolite, β-hydroxybutyrate (BHB) from mature WAT.11,23 This activity potentially increases beige adipogenesis and UCP-1 stimulation of adiponectin.11,23 UCP-1 supports the formation of brown fat genes while reducing the expression of fiber precursors that cause fibrosis in fat tissue (Figure 1).11 Statistical analysis showed that there were no signifi- cant differences between groups regarding the beige adipose tissue (p-value=0.079) (Figure 2). However, the average BeAT weight of brown rice intervention group was greater than the other groups (5.17 ± 3.58 g). This is in line with the theory stating that BeAT tis- sue has higher expression of UCP1 and PRDM16 gene.29 Further studies are needed regarding the appropriate levels of brown rice to support WAT browning process in a specific time limit. Appropriate control of feeding and environmental conditions also needs to be considered to achieve optimal intervention results. Moreover, each rat has varying metabolic abilities, which greatly determines the results of using in vivo methods. Conclusions In conclusion, the intervention of brown and white rice, as well as pure g-oryzanol for 12 weeks did not show significant differ- ences in anthropometric changes and PRDM16 levels. This study should be further conducted with a feeding method that had been adjusted for weight loss based on body weight reference of rats to obtain more significant results. References 1. UNICEF. Overweight and Obesity Landscape Analysis in Indonesia. Jakarta: United Nations Children's Fund (UNICEF); 2019. 2. Han TS, Lean ME. A clinical perspective of obesity, metabolic syndrome, and cardiovascular disease. JRSM Cardiovasc Dis 2016;5:2048004016633371. 4th International Nursing and Health Sciences Symposium Figure 1. Mechanism of Functional Food Affecting the Components of Adipose Tissue. [Healthcare in Low-resource Settings 2024;12(s1):13069] [page 105] Correspondence: Dian Handayani, Department of Nutrition, Faculty of Health Sciences, Universitas Brawijaya, , Jl. Puncak Dieng, Kunci, Kalisongo, Kec. Dau, Malang, East Java, Indonesia, Postcode. Tel.:+62341569117 - Fax: +62341564755. E-mail: handayani_dian@ub.ac.id Key words: functional food; Lee index obese; PRDM16; total body fat. Contributions: all authors contributed equally to this study. Conflict of interest: the authors declare that there are no potential con- flicts of interest. Funding: this study was supported financially by Universitas Brawijaya. Ethics approval: this study was approved by the Health Research Ethics Committee, Faculty of Health Sciences, Brawijaya University No. 2020/UN10.F17.10.4/TU/2023. All procedures were carried out in col- laboration with the Experimental Animal Care Laboratory, Faculty of Medicine, Universitas Brawijaya. Conference presentation: part of this article was presented at the 4th International Nursing and Health Sciences Symposium, from 27th-28th of October 2023, Universitas Brawijaya, Malang, East Java, Indonesia. Acknowledgment: the authors are grateful to the Department of Nutrition, Faculty of Health Sciences, Universitas Brawijaya, Malang, and the Department of Nutrition, Polytechnic of Health, Malang, for pro- viding the best support. Received: 3 November 2023. Accepted: 28 June 2024. Early view: 10 September 2024. This work is licensed under a Creative Commons Attribution 4.0 License (by-nc 4.0). ©Copyright: the Author(s), 2024 Licensee PAGEPress, Italy Healthcare in Low-resource Settings 2024; 12(s1):13069 doi:10.4081/hls.2024.13069 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 product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher. Non -co mmerc ial us e o nly 3. Hendra C, Manampiring AE, Budiarso F. Risk Factors for Obesity in Adolescents in Bitung City. J e-Biomedik 2016;4:1- 5. 4. Douketis J. Screening, Prevention and Treatment of Overweight/Obesity in Adult Populations. Canada: McMaster University; 2013. 5. Kusumastuty I, Handayani D, Attamimi N, Affandy YIKD, Innayah AM, Puspitasari DA. Compliance of Brown Rice- Based Diet on Blood Glucose and Body Fat of Diabetes Mellitus Patients. Indonesian J Human Nutrition 2021;8:182- 194. 6. Sulistyowati E, Handayani D, Soeharto S, Rudijanto A. Serum mineral (Mg, Mn, and K) levels are associated with increasing the body mass index (BMI) and abdominal circumference. Obes Med 2019;15:100107. 7. Witanto S. Identification of Gamma-Oryzanol Levels in n- Hexane Extract of Germinated Brown Rice (GBR) Sintanur Variety. Malang: Univ Brawijaya; 2019. 8. Park YM, Myers M, Potter, VJV. Adipose Tissue Inflammation and Metabolic Dysfunction: Role of Exercise. Missouri Medicine 2014;111:65-72. 9. Radhina A. Adipose Tissue Browning Process. Indonesian J Health Sci 2021;1:42-46. 10. Liu L, Chen Y, Chen J, et al. The relationship between PRDM16 promoter methylation in abdominal subcutaneous and omental adipose tissue and obesity. Clinical Nutrition 2021;40:2278-84. 11. Jiang N, Yang M, Han Y, et al. PRDM16 Regulating Adipocyte Transformation and Thermogenesis: A Promising Therapeutic Target for Obesity and Diabetes. Front Pharmacol 2022;13:1- 13. 12. Fabbiano S, Suárez-Zamorano N, Rigo D, et al. Caloric Restriction Leads to Browning of White Adipose Tissue through Type 2 Immune Signaling. Cell Metab 2016;24:434- 46. 13. Handayani D, Kusumastuty I, Innayah AM, et al. Substitution of local Indonesian varieties of brown rice on anthropometry and blood glucose level improvement in type 2 DM patients: a pilot project. J Public Health Res 2022;11:2283. 14. Sulistyowati E, Rudijanto A, Soeharto S, Handayani D. The identification of characteristic macro-and micronutrients and the bioactive components of Indonesian local brown rice as a functional feed in obesity nutrition therapy. Curr Nutr Food Sci 2020;16:494–500. 15. Matsumoto Y, Fujita S, Yamagishi A, et al. Brown rice inhibits development of nonalcoholic fatty liver disease in obese zuck- er (fa/fa) rats by increasing lipid oxidation via activation of retinoic acid synthesis. J Nutrition Dis 2021;151:2705-13. 16. Francisqueti FV, Ferron AJT, Hasimoto FK, et al. Gamma oryzanol treats obesity-induced kidney injuries by modulating the adiponectin receptor 2/PPAR-α axis. Oxid Med Cell Longev 2018;2018:1278392. 17. Francisqueti FV, Minatel IO, Ferron AJT, et al. Effect of gamma-oryzanol as therapeutic agent to prevent cardiorenal metabolic syndrome in animals submitted to high sugar-fat diet. Nutrients 2017;9:1299. 18. Shobana S, Lakshmipriya N, Bai MR, et al. Even minimal pol- ishing of an Indian parboiled brown rice variety leads to increased glycemic responses. Asia Pac J Clin Nutr 2017;26:829-36. 19. Handayani D, Kusumastuty I, Innayah AM, et al. Substitution of local Indonesian varieties of brown rice on anthropometry and blood glucose level improvement in type 2 DM patients: a pilot project. J Public Health Res 2021;11:2283. 20. Permatasari DI, Sutjiati E, Sulistyowati E. Effect of brown rice intervention on BMI and waist circumference in patients with type 2 diabetes mellitus. Indonesian J Human Nutr 2023;10:77-87. 21. Barathikannan K, Tyagi A, Shan L, et al. Antiobesity and antioxidative effect of fermented brown rice using in vitro with in vivo caenorhabditis elegans model. Life 2023;13:1-14. 22. Lim SM, Goh YM, Mohtarrudin N, Loh SP. Germinated brown rice ameliorates obesity in high-fat diet-induced obese rats. BMC Complement Altern Med 2016;16:140. 23. Chi J. Unraveling the interaction between beige adipocytes and the sympathetic nervous system. New York: The Rockefeller University; 2021. 24. Mulya A, Kirwan JP. Brown and beige adipose tissue. Endocrinol Metab Clin North Am 2017;45:605-21. 25. Fuster JJ, Ouchi N, Gokce N, Walsh K. Obesity-induced changes in adipose tissue microenvironment and their impact on cardiovascular disease. Circ Res 2016;118:1786-807. 26. Ziqubu K, Dludla PV, Mthembu SXH, et al. An insight into brown/beige adipose tissue whitening, a metabolic complica- tion of obesity with a multifactorial origin. Front Endocrinol (Lausanne) 2023;14:1114767. 27. Weng X, Sun M, Gao H, et al. Germinated brown rice, a whole grain with health benefits for common chronic diseases. Nutrition Food Sci J 2019;2:119. 28. Feige-Diller J, Krakenberg V, Bierbaun L, et al. The effects of different feeding routines on welfare in laboratory mice. Sec. Animal Behavior Welfare 2020;6:1-15. 29. Mishra BK, Madhu SV, Aslam M, et al. Adipose tissue expres- sion of UCP1 and PRDM16 genes and their association with postprandial triglyceride metabolism and glucose intolerance. Diabet Res Clin Pract 2021;182:1-10. 4th International Nursing and Health Sciences Symposium [page 106] [Healthcare in Low-resource Settings 2024;12(s1):13069] Non -co mmerc ial us e o nly