ABSTRACT Obesity is the most prevalent disease in the world which poses a serious risk for various chronic diseases. However, currently there are not any therapeutic agents that reduce body

weight without causing serious side effects. In order to prevent and/or treat obesity and related diseases through a nutraceutical approach, we created a resveratrol-enriched transgenic rice

accumulating 1.4 μg/g of resveratrol in its grain, DJ-526. Feeding of mice with the resveratrol-enriched rice DJ-526 showed excellent anti-obesity effect with reduction of body weights and

abdominal fat volumes compared to the control by 20.0% and 31.3%, respectively. Also, the consumption of the resveratrol-enriched rice DJ526 significantly improved the blood lipid profiles

HIGH-FAT DIET-FED MICE BY IMPROVING FAT ACCUMULATION AND METABOLIC PARAMETERS Article Open access 03 December 2020 GARCINIA MANGOSTANA EXTRACT AND CURCUMIN AMELIORATE OXIDATIVE STRESS,

DYSLIPIDEMIA, AND HYPERGLYCEMIA IN HIGH FAT DIET-INDUCED OBESE WISTAR ALBINO RATS Article Open access 31 March 2021 UNTARGETED METABOLOMICS-BASED NETWORK PHARMACOLOGY REVEALS FERMENTED BROWN

RICE TOWARDS ANTI-OBESITY EFFICACY Article Open access 30 March 2024 INTRODUCTION Obesity is a major health problem throughout the world and it is increasing both in prevalence and

severity1. According to the WHO, the current population of 1.6 billion overweight/obese people is expected to double by 2015. Pharmaceutical drugs such as orlistat, lorcaserin, sibutramine,

phentermine, diethylpropion, or fluoxetine have been developed for the treatment of obesity2,3. However, benefits of these pharmaceutical drugs do not outweigh the side effects of the drugs

in most cases4. Treatments of obesity with these drugs despite short-term benefits, is often associated with rebound weight gain after the cessation of drug use and serious side effects from

the medication. As such, it is necessary to develop a new type of anti-obesity treatment that treats obesity without long-term side effects. Because obesity is caused by a nutrient-uptake

imbalance where the amount of energy intake exceeds the amount of energy expended, one option to treat obesity effectively without side effect would be a nutraceutical approach5,6. Although

some have been described to have dietary effects, the impact on obesity and related diseases are not enough to be claim its efficacy7. For an effective treatment, it would be reasonable to

think that supplementation of nutrient ingredients having anti-obesity effect to an anti-obesity nutraceutical would generate a much more fortified anti-obesity nutraceutical. Resveratrol

(3,5,4′-trihydroxy-trans-stilbene) has been well recognized for its lipid-lowering function as well as calorie-restriction effect, proposing itself as one of the best anti-obese

ingredients8,9,10,11. To generate more effective and convenient way for dietary consumption of resveratrol other than red wine, one of the major research efforts in plant science was to

create transgenic cereal plants that accumulate resveratrol in their grains12,13. However, resveratrol was only detected at low levels in the leaves and stems except our previous work14. We

previously created the resveratrol-enriched rice by transferring the resveratrol biosynthesis gene, stilbene synthase (_STS_) to Dongjin rice through the expression validation approach15.

Among the 398 T1 small plantlets, one line of resveratrol-enriched rice plants, DJ-526, showed the excellent agricultural characteristic as well as accumulating a large quantity of

resveratrol in its grain. Parental plant of the resveratrol-enriched rice DJ-526, _Oryza sativa_ japonica variety Dongjin rice developed by the Rural Development Administration of Korea, has

characteristics of having grains rich in fiber and polyphenols with anti-obesity activity14. Thus, the resveratrol-enriched rice DJ-526 accumulating a large quantity of resveratrol in its

grains in addition to fiber and polyphenols could be an ideal nutraceutical to treat obesity and its related diseases. The purpose of this research was to investigate whether the

resveratrol-enriched rice DJ-526 is effective for obesity and its related diseases through a synergistic combination of the innate anti-obesity property of Dongjin rice and the

lipid-lowering property of transgenic resveratrol. Our animal experiments showed that the resveratrol-enriched rice DJ-526 has strong anti-obesity effects and significantly improved all

aspects of obesity-related diseases, suggesting its potential as an anti-obesity nutraceutical. RESULTS THE GRAINS OF THE RESVERATROL-ENRICHED RICE DJ-526 CONTAINED A LARGE AMOUNT OF

RESVERATROL One of the current challenges in creating transgenic plants is to make a desired gene or genes functional specifically in the targeted part of the transgenic plants. It has been

well known that piceid are accumulated as one of the major compounds in the plants having either natural or transgenic resveratrol biosynthetic pathways16. Because the health benefits of

piceid are insignificant and long-term effect on the physiology of human has not been completely understood17,18, we aimed to select a transgenic rice plant accumulating mostly resveratrol

rather than piceid in its grain. As we previously reported15, we first massively screened the resveratrol quantity of 398 T1 transgenic plants which contained the _AhSTS1_gene for

resveratrol production. After identifying the candidate T1 transgenic plants, the candidate T1 transgenic plants were transplanted into a rice paddy. By the thorough analyses of the

metabolic profile of the resveratrol and its related metabolites in the every portion of the transgenic plants, we selected a transgenic plant accumulating large quantity of resveratrol in

its grain, DJ-52615. Figure 1 showed the metabolic profiles of resveratrol and the its related compound, piceid, from the resveratrol-enriched rice DJ-526 using HPLC. As expected, neither

resveratrol nor piceid was detected in the HPLC analysis on the wild type Dongjin rice (Figure 1b). On the other hand, the grains of the resveratrol-enriched rice DJ-526 contained a

relatively high quantity of resveratrol compared with piceid (Figure 1c), whereas the high quantity of piceid compared with resveratrol was observed in the leaves. In fact, resveratrol

quantity in the grain of the resveratrol-enriched rice DJ-526, 1.4 μg/g, were close to the typical levels of resveratrol quantity in high-quality red wine, 0.8–5.8 μg/mL19. The preferential

distribution of the two related metabolites, high accumulation of resveratrol in the edible grains but low in the leaves, makes the resveratrol-enriched rice DJ-526 ideal as a

resveratrol-enriched cereal plant. THE RESVERATROL-ENRICHED RICE DJ-526 RICE SHOWED AN EXCELLENT ANTI-OBESITY EFFECT BY REDUCING BODY WEIGHTS AND ABDOMINAL FAT VOLUMES To investigate whether

the resveratrol-enriched rice DJ-526 has an anti-obesity effect through a synergistic effect of the innate characteristics of Dongjin and the transgenic resveratrol as we expected, the

efficacy of the resveratrol-enriched rice DJ-526 on obesity was examined using an _in vivo_ mouse model. The C57BL/6 inbred mice with diet-induced obesity were fed the high fat diet (HFD)

for 12 weeks in the control group or a modified HFD in the experimental group, in which the carbohydrate source was replaced with either Dongjin rice or the resveratrol-enriched rice DJ-526

(supplementary Table S1). Because body weight and abdominal fat volumes are the phenotypes of obesity, changes in the body weight and abdominal fat volumes in each mouse group were

periodically monitored under the continued HFD conditions. The food consumption rate was the same among different mouse groups during the experimental period. Figure 2 displayed the changes

in the body weight and abdominal fat volumes after dietary consumption of the resveratrol-enriched rice DJ-526. As shown in figure 2a, body weights were greatly reduced in mice fed with the

resveratrol-enriched rice DJ-526 by 20.0% compared to the control, slightly greater than Dongjin rice group. More significant data came from micro-CT image analyses on abdominal fat

deposition (Figure 2b). The total, visceral and subcutaneous fat volumes in the resveratrol-enriched rice DJ-526 group were 17.4%, 15.2% and 2.3%, respectively, which were significantly

lower than the fat volumes from the control (25.6%, 20.2% and 4.0%, respectively) and the Dongjin group (23.9%, 18.9% and 3.0%, respectively). The representative images of the micro-CT image

analyses on abdominal fat deposition clearly indicated that the total, visceral and subcutaneous fat accumulation was the lowest in the resveratrol-enriched rice DJ-526 group compared with

the other treatments (Figure 2c). Based on the experimental results of the efficacy of the resveratrol-enriched rice DJ-526 on body weight and abdominal fat volumes, it was concluded that

the resveratrol-enriched rice DJ-526 had an excellent anti-obesity effect. THE CONSUMPTION OF THE RESVERATROL-ENRICHED RICE DJ-526 SIGNIFICANTLY IMPROVED LIPID PROFILES AND BLOOD GLUCOSE

LEVELS WITH CLEAR ANTI-OBESITY EFFECT IN ANIMAL EXPERIMENTS UNDER HIGH-FAT DIET Obesity is not simply a consequence of overweight but is typically complicated by diabetes and hyperlipidemia.

Because the nature of obesity, we examined the efficacy of the resveratrol-enriched rice DJ-526 on the obesity-related diseases by monitoring changes in the levels of blood glucose,

triacylglycerol, total cholesterol, HDL-cholesterol, LDL-cholesterol using an _in vivo_ mouse model. The consumption of Dongjin rice resulted in some improvements in lipid profile and blood

glucose levels compared to the control, as expected from its endogenic nature. More importantly, the consumption of the resveratrol-enriched rice DJ-526 significantly improved the lipid

profiles as well as blood glucose. The consumption of the resveratrol-enriched rice DJ-526 lowered total cholesterol by 17.9% and LDL-cholesterol by 67.3%, while increasing HDL-cholesterol

by 34.2% compared to the control (Figure 3). Also, the consumption of the resveratrol-enriched rice DJ-526 lowered blood glucose by 13.9% and triacylglycerol by 49.3% compared to the control

(Figure 4). DISCUSSION Obesity is complicated with various diseases such as diabetes, hypercholesterolemia, hyperlipidemia, metabolic syndrome, _etc_1. Currently, obesity and its related

diseases became a major health problem throughout the world that is increasing both in prevalence and severity20. In spite of serious efforts to treat and prevent obesity and its related

diseases, an ideal solution for obesity has not been developed. Considering efficacy and their side effects, current pharmaceutical drugs to treat obesity and its related diseases do not

provide a solution for obesity and its related diseases so that only 6% of obese patients are treated pharmacologically21. We believe that the resveratrol-enriched rice DJ-526 is an ideal

nutraceutical solution to treat or prevent obesity. The anti-obesity effect resulting from the synergistic effect of Dongjin rice and resveratrol showed efficacy levels aiming to treat

obesity and its related diseases as much as the typical pharmaceutical drugs. Current pharmaceutical drugs only target individual aspects of obesity and its related diseases, such as blood

glucose, LDL/total cholesterol, or body weight. There is no pharmaceutical drug available to treat every aspect of obesity and its related diseases. We believe that the resveratrol-enriched

rice DJ-526 could be an ideal choice to target most, if not all, aspects of obesity and its related diseases. Due to the synergistic effect of the endogenous anti-obesity effect of the

Dongjin rice and the lipid-lowering effect of resveratrol, the resveratrol-enriched rice DJ-526 has more potent anti-obesity activity than Dongjin rice itself so that the

resveratrol-enriched rice DJ-526 can be used to treat and prevent obesity and its related diseases. Both the severity and prevalence of obesity and its related diseases, such as

cardiovascular diseases and diabetes, among many others, are more serious in developing countries than in developed countries. Limited medical care in developing countries make obesity and

its related diseases is a more serious issue in developing countries than in developed countries. We believe that the resveratrol-enriched rice DJ-526 could be an excellent alternative for

the management of obesity and its related diseases, not only in developed countries but also in developing countries. Finally our work provides an inspiration for a future development of

genetically modified crops. This work showed that a synergistic effect of the innate property of a host plant and a transgenic property significantly augmented the original anti-obesity

property of the Dongjin rice. We believe that a careful selection of host plant and transgene would lead to create a very interesting functional crop to manage many chronic diseases. METHODS

QUANTIFICATION OF RESVERATROL AND PICEID The eight-week-old leaves and mature grains of the wild-type cultivar Dongjin and the resveratrol-enriched rice DJ-526 rice plants were used to

determine the levels of resveratrol and the related resveratrol glucoside piceid as described previously15. After the fresh samples of the leaves and grains were freeze-dried, the samples

were homogenized to be extracted with 80% ethanol. 1 μL of the each sample was analyzed by a reverse phase HPLC equipped with a UV detector under the gradient condition (ACQUITY TUV, Waters,

Milford, USA). The ACQUITY UPLC BEH-C18 1.7 μm column (2.1 mm × 100 mm, Waters) was used at a flow rate of 0.4 mL/min. The mobile phase was 10 to 90% acetonitrile (ACN). A gradient elution

was conducted as follows: 0 min, 10% ACN; 1.54 min, 10% ACN; 10 min, 15% ACN; 22 min, 25% ACN; 22.4 min, 90% ACN; and 25 min, 90% ACN; followed by re-equilibration of the column with 10% ACN

for 5 min prior to the next injection. Each metabolite peak of samples and standard chemical samples was calculated using the Empower software (Waters) to determine quantity of the

metabolites. The HPLC fractions of resveratrol and piceid were further verified using GC-MS analysis with the 6890/5973N GC/MS system (Agilent Technologies, Santa Clara, CA, USA) equipped

with an Rtx-5MS capillary column (30 mm × 0.25 mm I.D., 0.25 μm film thickness). ANIMAL EXPERIMENTS Six-week-old female C57BL/6 mice (Joongang Experimental Animal Co., Seoul, Korea) were

purchased and acclimatized for 2 weeks. Then, the animals were fed with a high-fat diet (HFD) containing 45% calorie as lard fat (D12451, Research Diets Inc., New Brunswick, NJ, USA) for 12

weeks. Mice with diet-induced obesity and related diseases were randomly divided and fed the HFD, modified HFD in which the corn and sucrose were replaced with Dongjin rice and modified HFD

in which the corn and starch were replaced with the resveratrol-enriched rice DJ-526. Food intake and body weight were measured regularly and blood samples were taken at indicated time

points. Fasting blood glucose was measured using Accu-check Glucometer (Roche, Indianapolis, IN, USA) and lipid profile analysis was performed using as enzymatic colorimetric method (Asan

Pharm., Yongjin, Korea). Fat volume was analyzed from anesthetized mice by high-resolution _in vivo_ micro-CT (Skyscan 1076; Skyscan, Konitech, Belgium). The total fat, visceral fat and

subcutaneous fat areas were analyzed with Micro-CT images at the level of the L1-L5 intervertebral disk using CTan Ver.1.10 software (Skyscan). All animal care and use were performed

strictly in accordance with the ethical guidelines by the Ethics Committee of Chonbuk National University Laboratory Animal Center and the animal study protocol was approved by the

institution. REFERENCES * Paul, C. et al. The epidemiology of overweight and obesity: public health crisis or moral panic? Int. J. Epidemiol. 35, 55–60 (2006). Article  Google Scholar  *

Ioannides-Demos, L. L., Piccenna, L. & McNeil, J. J. Pharmacotherapies for Obesity: Past, Current and Future Therapies. J. Obes. 2011, 179674 (2011). Article  Google Scholar  * Adan, R.

A. Mechanisms underlying current and future anti-obesity drugs. Trends. Neurosci. 36, 133–140 (2013). Article  CAS  Google Scholar  * Kang, J. G. & Park, C. Y. Anti-Obesity Drugs: A

Review about Their Effects and Safety. Diabetes Metab. J. 36, 13–25 (2012). Article  Google Scholar  * Luís, G. J. et al. Physical activity, energy balance and obesity. Public Health Nutr.

10, 1194–1199 (2007). Article  Google Scholar  * Shimizu, M., Kubota, M., Tanaka, T. & Moriwaki, H. Nutraceutical Approach for Preventing Obesity Related Colorectal and Liver

Carcinogenesis. Int. J. Mol. Sci. 13, 579–595 (2012). Article  CAS  Google Scholar  * Pittler, M. H. & Ernst, E. Dietary supplements for body-weight reduction: a systematic review. Am.

J. Clin. Nutr. 79, 529–536 (2004). Article  CAS  Google Scholar  * Lagouge, M. et al. Resveratrol improves mitochondrial function and protects against metabolic disease by activating SIRT1

and PGC-1 alpha. Cell 127, 1109–1122 (2006). Article  CAS  Google Scholar  * Baur, J. A. et al. Resveratrol improves health and survival of mice on a high-calorie diet. Nature 444, 337–342

(2006). Article  ADS  CAS  Google Scholar  * Rivera, L., Moron, R., Zarzuelo, A. & Galisteo, M. Long-term resveratrol administration reduces metabolic disturbances and lowers blood

pressure in obese Zucker rats. Biochem. Pharmacol. 77, 1053–1063 (2009). Article  CAS  Google Scholar  * Zhuang, H., Kim, Y. S., Koehler, R. C. & Dore, S. Potential mechanism by which

resveratrol, a red wine constituent, protects neurons. Ann. N. Y. Acad. Sci. 993, 276–286 (2003). Article  ADS  CAS  Google Scholar  * Leckband, G. & Lörz, H. Transformation and

expression of a stilbene synthase gene of Vitis vinifera L. in barley and wheat for increased fungal resistance. Theor. Appl. Genet. 96, 1004–1012 (1998). Article  CAS  Google Scholar  *

Stark-Lorenzen, P. et al. Transfer of a grapevine stilbene synthase gene to rice (_Oryza sativa_ L.). Plant Cell Rep. 16, 668–673 (1997). Article  CAS  Google Scholar  * Choi, H. et al.

Comparative nutritional analysis for genetically modified rice, Iksan483 and Milyang204 and nontransgenic counterparts. J. Korean soc. Appl. Biol. Chem. 55, 19–26 (2012). Article  CAS 

Google Scholar  * Baek, S. H. et al. Creation of resveratrol-enriched rice for the treatment of metabolic syndrome and related diseases. PLOS One 8, e579302013 (2013). Google Scholar  *

Susanne, R. H. et al. Piceid (Resveratrol Glucoside) Synthesis in Stilbene Synthase Transgenic Apple Fruit. J. Agric. Food Chem. 54, 4633–4640 (2006). Article  Google Scholar  * Kimura, Y.,

Okuda, H. & Arichi, S. Effects of stilbenes on arachidonate metabolism in leukocytes. Biochim. Biophys. Acta. 834, 275–278 (1985). Article  CAS  Google Scholar  * Meng, X. et al. Urinary

and plasma levels of resveratrol and quercetin in humans, mice and rats after ingestion of pure compounds and grape juice. J. Agric. Food Chem. 52, 935–942 (2004). Article  CAS  Google

Scholar  * Souto, A. A. et al. Determination of _trans_-resveratrol concentrations in Brazilian red wines by HPLC. J. Food Comp. Anal. 14, 441–445 (2001). Article  CAS  Google Scholar  *

Flegal, K. M., Carroll, M. D., Ogden, C. L. & Curtin, L. R. Prevalence and trends in obesity among US adults, 1999–2008. JAMA. 303, 235–241 (2010). Article  CAS  Google Scholar  * Lee,

J. S., Sheer, J. L., Lopez, N. & Rosenbaum, S. Coverage of Obesity Treatment: A State-by-State Analysis of Medicaid and State Insurance. Public Health Reports 125, 596–604 (2010).

Article  Google Scholar  Download references ACKNOWLEDGEMENTS This work was supported by a grant from the Next-Generation BioGreen 21 program, Rural Development Administration (No.PJ009528)

and Technology Development Program for High Value-Added Food, Ministry of Agriculture, Food and Rural Affair (No.313040-03-1-CG000), Republic of Korea. AUTHOR INFORMATION Author notes * Baek

So-Hyeon, Chung Hea-Jong and Lee Heui-Kwan contributed equally to this work. AUTHORS AND AFFILIATIONS * National Institute of Crop Science, Rural Development Administration, Chonbuk, Iksan,

Korea So-Hyeon Baek * Department of Biomedical Sciences, Chonbuk National University Medical School, Chonbuk, Jeonju, Korea Hea-Jong Chung, Heui-Kwan Lee, Roshan D'Souza, Youngju Jeon 

& Seong-Tshool Hong * JINIS BDRD institute, JINIS Biopharmaceuticals Co., 948-9 Dunsan, Bongdong, Wanju, 565-902, Jeollabuk-do, South Korea Hyeon-Jin Kim * National Academy of

Agricultural Science, Rural Development Administration, Suwon, Gyeonggi, Korea Soon-Jong Kweon Authors * So-Hyeon Baek View author publications You can also search for this author inPubMed 

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CONTRIBUTIONS S.B., H.C., H.L., R.D. and Y.J. performed the experiments. H.K., S.K. and S.H. designed experiments, analyzed and interpreted the data and wrote the manuscript. ETHICS

DECLARATIONS COMPETING INTERESTS The authors declare no competing financial interests. ELECTRONIC SUPPLEMENTARY MATERIAL SUPPLEMENTARY INFORMATION Supplementary Table S1 RIGHTS AND

PERMISSIONS This work is licensed under a Creative Commons Attribution 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by/3.0/ Reprints and

permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Baek, SH., Chung, HJ., Lee, HK. _et al._ Treatment of obesity with the resveratrol-enriched rice DJ-526. _Sci Rep_ 4, 3879 (2014).

https://doi.org/10.1038/srep03879 Download citation * Received: 11 September 2013 * Accepted: 07 January 2014 * Published: 27 January 2014 * DOI: https://doi.org/10.1038/srep03879 SHARE THIS

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