Fermented Rice Germ Extract Ameliorates Abnormal Glucose Metabolism via Antioxidant Activity in Type 2 Diabetes Mellitus Mice

: Rice germ is an abundant source of ferulic acid, which is known for its anti-oxidant activity. This study aimed to evaluate the regulatory effects of fermented rice germ extracts on hepatic glucose metabolism in C57BL/KsJ-db/db mice. Rice germ was fermented with Lactobacillus plantarum and extracted with 30% ethanol (RG_30E) or 50% ethanol (RG_50E). Mice were fed modiﬁed AIN-93 diets containing fermented rice germ extracts and ferulic acid for 8 weeks. RG_50E signiﬁcantly reduced food intake as well as liver weight and RG_30E and RG_50E improved glucose homeostasis, as indicated by fasting blood glucose levels and glucose tolerance. Hepatic triglyceride and total cholesterol levels were signiﬁcantly decreased in db/db mice fed RG_30E and RG_50E. The antioxidant capacity of RG_30E and RG_50E was conﬁrmed by a decrease in malondialdehyde levels and an increase in hepatic superoxide dismutase activity. The expression of genes related to glycolysis and gluconeogenesis was signiﬁcantly regulated by RG_30E and RG_50E. These results suggest that fermented rice germ extracts have the potential to regulate hypoglycemia and hepatic glucose metabolism in type 2 diabetes db/db mice.


Introduction
Diabetes mellitus is a metabolic disorder classified into insulin-dependent type 1 diabetes mellitus and non-insulin-dependent type 2 diabetes mellitus (T2DM) [1]. T2DM is a chronic condition characterized by continued hyperglycemia due to compromised glucose uptake into cells, a consequence of decreased insulin sensitivity. Previous studies have shown that persistent hyperglycemia can cause diseases in organs such as the liver, kidney, and heart. In particular, diabetes-induced liver damage accumulates over time, culminating in liver diseases such as fatty liver and non-alcoholic fatty hepatitis [2,3]. According to several studies, T2DM severely affects glucose metabolism and oxidative stress in the liver. First, under fasting conditions, the liver breaks down glycogen and produces glucose to maintain normal blood glucose levels. After a meal, liver cells synthesize glycogen to regulate blood glucose. When levels of blood glucose are high, it is taken up by liver cells via GLUT2, followed by its conversion into glucose-6-phosphate by glucokinase (GK), which is synthesized as glycogen. Conversely, under low blood glucose level, glucose-6-phosphatase (G6PC) and phosphoenolpyruvate carboxykinase (PEPCK) drive gluconeogenesis for glucose synthesis [4][5][6]. Second, recent studies have shown that the overproduction of reactive oxygen species (ROS), which results in oxidative stress, is involved in insulin resistance. Thus, compounds with antioxidant activity have been shown to improve blood glucose levels in T2DM patients [7,8]. Currently available

Preparation of Sample
Lactobacillus plantarum Korea Agricultural Culture Collection (KACC) 15357 was used as a fermentation strain, purchased from the Korean Collection for Type Cultures (KCTC, Korea). L. plantarum was cultured on MRS medium (Lactobacilli MRS broth, BD Difco, Germany) at 37 • C for 24 h. The culture medium was homogenized at 5000 rpm for 10 min and then sterilized at 95 • C for 15 min for fermentation. Thereafter, 3% (w/v) of the L. plantarum culture medium was inoculated into rice fermentation medium composed of 15% (w/v) rice germ and 15% (w/v) glucose. Fermented rice germ was produced via anaerobic fermentation for 24 h at 37 • C. The fermented rice germ was then extracted at 60 • C for 6 h using prethanol A (Duksan, Seoul, Korea). Prethanol A and fermented rice germ were mixed at a ratio of 1:1 and 1:2, followed by vacuum extraction and concentration using Cosmos 660 (Gyeongseo E&P, Incheon, Korea). The sample used in this study differed in the ratio of extracted prethanol A and fermented rice germ. The sample with a ratio of prethanol A and fermented rice germ of 1: 2 was denoted as RG_30E, and the sample with a ratio of 1:1 was denoted as RG_50E. For the dried powder, sample:maltodextrin was dried in a ratio of 1:4 and used in the experiment.

Animals and Diets
Five-week-old male C57BL/6KsJ-db/+ mice (db/+ mice) and C57BL/6KsJ-db/db mice (db/db mice) were purchased from JOONGAH BIO (Suwon, Korea). The experiment was approved according to the guidelines published by Kyungpook National University (KNU-2020-0045). The mice were housed in an environment of 23 ± 2 • C under a 12-h light/dark cycle. All mice were allowed free access to food and tap water during adaptation for 1 week and were then randomly divided into five groups (n = 8): db/+ control (db/+ mice and normal AIN-93 diet), db/db control (db/db mice and normal AIN-93 diet), RG_30E (db/db mice and modified diet with RG_30E), RG_50E (db/db mice and modified diet with RG_50E), and FA (db/db mice and modified diet with ferulic acid). Table 1 shows the composition of diets used in this experiment. The modified diets were based on AIN-93, prepared by mixing with ferulic acid (Sigma-Aldrich, St. Louis, MO, USA). The ferulic acid content was determined based on the amount of ferulic acid in fermented rice germ extracts (Supplementary Information). Body weight and food intake were measured every week for 8 weeks. After 8 weeks, the liver, epididymal adipose tissue, and kidney were excised and weighed. Blood samples were collected in EDTA tubes and centrifuged for Appl. Sci. 2021, 11, 3091 3 of 13 10 min at 1500× g and 4 • C to obtain plasma. All tissues and blood samples were stored at -80 • C until analysis.

Fasting Blood Glucose Level
Mice were fasted for 12 h prior to sample collection, and blood samples were obtained from the tail vein at 0, 3, 5, and 8 weeks. Blood glucose levels were measured using a New Accu-Chek Active GB glucometer (Roche Diagnostics, Mannheim, Germany).

Oral Glucose Tolerance Test (OGTT)
For the OGTT, glucose (2 g/kg body weight) was orally administered to mice that had been fasted for 12 h. Blood was collected from the tail vein at 0, 15, 30, 45, 60, 90, and 120 min. The blood glucose level was measured using a New Accu-Chek Active GB glucometer (Roche Diagnostics, Mannheim, Germany). The area under the blood glucose concentration-time curve (AUC) was calculated using the trapezoidal method [19] for 120 min.

Hepatic Lipid Level
Hepatic triglyceride (TG) and total cholesterol (TC) levels were measured using a commercial ELISA kit (Asan Pharmaceuticals, Seoul, Korea). Liver tissue lipids were extracted using a previously described method, with slight modifications [20]. Liver tissue was homogenized with Folch solution and centrifuged at 3000 rpm and 4 • C for 10 min. The supernatant was evaporated and dissolved in chloroform (Duksan, Seoul, Korea).

Lipid Peroxidation
Levels of erythrocyte and liver TBARS (Sigma-Aldrich, St. Louis, MO, USA) were determined according to a previously described method [21]. The livers (100 mg) of mice were homogenized in 1.15% KCl (Junsei Chemical Co., Ltd., Tokyo, Japan). A mixture of 0.9% thiobarbituric acid (Sigma-Aldrich, St. Louis, MO, USA), 8.1% sodium dodecyl sulfate (Sigma-Aldrich, St. Louis, MO, USA), and 20% acetic acid (Sigma-Aldrich, St. Louis, MO, USA), and distilled water was then added to the erythrocyte and tissue homogenate. The mixture was incubated at 95 • C for 1 h and cooled at room temperature. Distilled water and pyridine:n-butanol (1:15, v/v) were added and centrifuged at 4000× g and 4 • C for 10 min. The supernatant was removed, and absorbance was measured at 532 nm.

Hepatic Catalase
Hepatic catalase activity was determined using a commercial ELISA kit (Thermo Fisher Scientific, Waltham, MA, USA). The liver was washed with ice-cold Dulbecco's Phosphate-Buffered Saline (DPBS) and homogenized with the assay buffer from the catalase assay kit. The homogenate was centrifuged at 10,000× g for 15 min, and the supernatant was assessed according to the manufacturer's protocol.

Statistical Analysis
Statistical analyses were performed using SAS 9.4 (SAS Institute, Cary, NC, USA). Data are presented as the mean ± standard error (SE). Data were analyzed via one-way analysis of variance (ANOVA), followed by Duncan's multiple range test. Differences were considered significant at p < 0.05. Table 2 shows the effects of ferulic acid and fermented rice germ extracts on body weight, food intake, and organ weight. The initial body weight was not significantly different between diabetic groups, except for the db/+ control. After 8 weeks, the final body weight decreased by 4.81% in the FA group compared to the db/db control. Neither RG_30E nor RG_50E affected body weight. However, food intake was significantly lower in the FA and RG_50E groups, with decreases of 14.22% and 12.24%, respectively, when compared to the db/db control group (p < 0.05). In addition, liver weight decreased by 10.73%, 6.92%, and 16.96% in the FA, RG_30E, and RG_50E groups, respectively, when compared to the db/db control (p < 0.05). The weight of epididymal adipose tissue tended to decrease in the FA, RG_30E, and RG_50E groups, but the decreases were not significant. Further, no significant decrease in kidney weight was observed.

Fasting Blood Glucose Levels
At week 0, there was no difference in blood glucose levels between all groups ( Figure 1). However, 8 weeks later, db/db mice had higher blood glucose levels than db/+ mice; additionally, there were differences between each group of db/db mice. At week 3, blood glucose levels in the RG_30E and RG_50E groups were significantly lower (29.68% and 25.71%, respectively) compared to the db/db control (p < 0.05). However, there was no significant difference between groups except the db/+ control group at 5 weeks. At 8 weeks, only RG_50E mice had a significantly lower blood glucose level compared to the db/db control, with a decrease of 12.37% (p < 0.05).
compared to the db/db control (p < 0.05). The weight of epididymal adipose tissue tended to decrease in the FA, RG_30E, and RG_50E groups, but the decreases were not significant. Further, no significant decrease in kidney weight was observed.

Fasting Blood Glucose Levels
At week 0, there was no difference in blood glucose levels between all groups ( Figure  1). However, 8 weeks later, db/db mice had higher blood glucose levels than db/+ mice; additionally, there were differences between each group of db/db mice. At week 3, blood glucose levels in the RG_30E and RG_50E groups were significantly lower (29.68% and 25.71%, respectively) compared to the db/db control (p < 0.05). However, there was no significant difference between groups except the db/+ control group at 5 weeks. At 8 weeks, only RG_50E mice had a significantly lower blood glucose level compared to the db/db control, with a decrease of 12.37% (p < 0.05). The effects of fermented rice germ extracts on blood glucose levels in db/+ mice and db/db mice over 8 weeks. db/+, C57BL/6KsJ-db/+ mice and normal diet; db/db, C57BL/6KsJ-db/+ Figure 1. The effects of fermented rice germ extracts on blood glucose levels in db/+ mice and db/db mice over 8 weeks. db/+, C57BL/6KsJ-db/+ mice and normal diet; db/db, C57BL/6KsJ-db/+ mice and normal diet; RG_30E, C57BL/6KsJ-db/+ mice and modified diet with RG_30E; RG_50E, C57BL/6KsJ-db/+ mice and modified diet with RG_50E; FA, C57BL/6KsJ-db/+ mice and modified diet with ferulic acid. Data with letters are significantly different (p < 0.05) according to Duncan's multiple range test. Figure 2 shows changes in blood glucose levels over time after a single administration of glucose (2 g/kg body weight) to mice at 8 weeks. A significant decrease in blood glucose levels was observed in FA (594.63 ± 19.21 mg/dL) and RG_50E (592.75 ± 13.27 mg/dL) mice at 60 min when compared to db/db controls (Figure 2a, p < 0.05). The RG_50E diet significantly decreased glucose levels by 20.68% compared to db/db control mice for 120 min (p < 0.05). In addition, the AUC value was significantly lower (5.14% decrease) in the RG_50E group compared to the db/db control (Figure 2b, p < 0.05). mice and normal diet; RG_30E, C57BL/6KsJ-db/+ mice and modified diet with RG_30E; RG_50E, C57BL/6KsJ-db/+ mice and modified diet with RG_50E; FA, C57BL/6KsJ-db/+ mice and modified diet with ferulic acid. Data with letters are significantly different (p < 0.05) according to Duncan's multiple range test. Figure 2 shows changes in blood glucose levels over time after a single administration of glucose (2 g/kg body weight) to mice at 8 weeks. A significant decrease in blood glucose levels was observed in FA (594.63 ± 19.21 mg/dL) and RG_50E (592.75 ± 13.27 mg/dL) mice at 60 min when compared to db/db controls (Figure 2a, p < 0.05). The RG_50E diet significantly decreased glucose levels by 20.68% compared to db/db control mice for 120 min (p < 0.05). In addition, the AUC value was significantly lower (5.14% decrease) in the RG_50E group compared to the db/db control (Figure 2b, p < 0.05).

Hepatic TG and TC Concentration
The effect of fermented rice germ extracts on hepatic lipid accumulation was assessed by measuring TG and TC levels ( Figure 3). Hepatic TG levels were significantly lower (decreased by 25.49%, 52.23%, and 53.33%, respectively) in FA, RG_30E, and RG_50E mice compared to the db/db controls (p < 0.05). Hepatic TC exhibited a tendency to decrease in the FA, RG_30E, and RG_50E groups compared to the db/db controls. A significant decrease was observed only in RG_50E mice, wherein TC levels were 37.05% lower (p < 0.05).

Hepatic TG and TC Concentration
The effect of fermented rice germ extracts on hepatic lipid accumulation was assessed by measuring TG and TC levels ( Figure 3). Hepatic TG levels were significantly lower (decreased by 25.49%, 52.23%, and 53.33%, respectively) in FA, RG_30E, and RG_50E mice compared to the db/db controls (p < 0.05). Hepatic TC exhibited a tendency to decrease in the FA, RG_30E, and RG_50E groups compared to the db/db controls. A significant decrease was observed only in RG_50E mice, wherein TC levels were 37.05% lower (p < 0.05).     Hepatic total cholesterol levels. db/+, C57BL/6KsJ-db/+ mice and normal diet; db/db, C57BL/6KsJ-db/+ mice and normal diet; RG_30E, C57BL/6KsJ-db/+ mice and modified diet with RG_30E; RG_50E, C57BL/6KsJ-db/+ mice and modified diet with RG_50E; FA, C57BL/6KsJ-db/+ mice and modified diet with ferulic acid. Data with letters are significantly different (p < 0.05) according to Duncan's multiple range test.

Discussion
This study was conducted to investigate the effects of fermented rice germ ex on hyperglycemia and hepatic glucose metabolism in T2DM mice. The db/db mice used as a T2DM model, as they develop obesity and dyslipidemia due to a mutated l receptor gene. Further, the db/db mouse is the most popular animal model used fo study of blood glucose, diabetes, and obesity. Currently, db/db mice are widely empl in T2DM research [4,8,9,22,23].
Previous studies have reported that ferulic acid and fermented rice germ extrac fect hyperglycemia and hepatic glucose metabolism in patients with diabetes. In pa lar, ferulic acid, a compound abundant in rice germ, was shown to regulate glucos tabolism in the liver [24,25]. Rice contains a variety of phenols, which are often me lized and not bioavailable. Fermentation using L. plantarum increases the amount of p Relative mRNA expression of phosphoenolpyruvate carboxykinase (Pepck). db/+, C57BL/6KsJ-db/+ mice and normal diet; db/db, C57BL/6KsJ-db/+ mice and normal diet; RG_30E, C57BL/6KsJ-db/+ mice and modified diet with RG_30E; RG_50E, C57BL/6KsJ-db/+ mice and modified diet with RG_50E; FA, C57BL/6KsJ-db/+ mice and modified diet with ferulic acid. Data with letters are significantly different (p < 0.05) according to Duncan's multiple range test.

Discussion
This study was conducted to investigate the effects of fermented rice germ extracts on hyperglycemia and hepatic glucose metabolism in T2DM mice. The db/db mice were used as a T2DM model, as they develop obesity and dyslipidemia due to a mutated leptin receptor gene. Further, the db/db mouse is the most popular animal model used for the study of blood glucose, diabetes, and obesity. Currently, db/db mice are widely employed in T2DM research [4,8,9,22,23].
Previous studies have reported that ferulic acid and fermented rice germ extracts affect hyperglycemia and hepatic glucose metabolism in patients with diabetes. In particular, 9 of 13 ferulic acid, a compound abundant in rice germ, was shown to regulate glucose metabolism in the liver [24,25]. Rice contains a variety of phenols, which are often metabolized and not bioavailable. Fermentation using L. plantarum increases the amount of physiologically active compounds such as ferulic acid, α-tocopherol, and γ-oryzanol in rice, improving its antioxidant activity [11,12,26]. In addition, ethanol can effectively extract phytochemicals or phenolic compounds from plants when used with water. This is related to polarity, as the use of an ethanol-water mixture lowers polarity, creating an environment where low-polarity phenol chemicals can be dissolved [27,28]. In this study, fermented rice germ was extracted with two different ethanol:water ratios to determine the more effective one with regard to glucose metabolism.
Diabetic mice fed diets with ferulic acid or other modified diets did not lose weight compared to the db/db controls. However, the significant decrease in food intake and liver weight observed in FA, RG_30E, and RG_50E mice indicated that ferulic acid and fermented rice germ extracts could regulate the diabetes-associated increase in food intake and liver weight. Epididymal adipose tissue weight was not significantly different between groups but showed a tendency to decrease in FA, RG_30E, and RG_50E mice ( Table 2). The intake of fermented rice germ, which is a rich source of minerals, vitamins, and phytochemicals, has been reported to reduce body weight, liver weight, and fat as a result of lower food intake [29,30].
Dyslipidemia, a condition characterized by elevated TG, very low-density lipoprotein (VLDL) cholesterol, and TC and decreased high-density lipoprotein (HDL) levels is a frequent symptom of T2DM patients [23]. RG_30E and RG_50E mice had lower hepatic TG and TC levels than those in the FA group. Further, RG_50E mice exhibited lower hepatic TC levels than their RG_30E counterparts ( Figure 3). Fermented rice was previously shown to reduce TC levels [31]. Lowering TG and TC levels can reduce the risk of developing cardiovascular disease in T2DM patients [32,33].
To confirm the effect of fermented rice germ extracts on hyperglycemia, blood glucose levels in the fasting state ( Figure 1) and OGTT ( Figure 2) were assessed. Lower blood glucose levels were observed in RG_30E and RG_50E mice than in those of the FA group. In particular, blood glucose levels were significantly lowered at 3, 5, and 8 weeks after RG_50E intake. Further, RG_50E significantly reduced the AUC value following oral glucose administration. Ferulic acid in rice germ was previously shown to suppress the activities of G6PC and PEPCK, both of which are involved in liver gluconeogenesis, highlighting the potential of this phenolic compound in T2DM [34,35].
To explore the involvement of ferulic acid in glycolysis and gluconeogenesis, we measured the expression of genes related to glucose metabolism in the liver. GK plays an important role in the regulation of glycolysis. Increased Gk expression decreases blood glucose levels by storing glucose in the form of glycogen or utilizing glucose. In contrast, G6pc and PEPCK are major enzyme regulators of gluconeogenesis. A decrease in G6pc and Pepck expression indicates reduced glucose synthesis in the liver, subsequently resulting in lower blood glucose levels [34,36,37]. We observed that Gk expression was significantly upregulated in FA, RG_30E, and RG_50E mice. In particular, Gk expression in the RG_50E group increased to a similar degree as in db/+ mice, which were non-diabetic controls. According to Son et al., mice fed ferulic acid were reported to regulate the hepatic glucose production by engaging in the activities of GK, G6PC, and PEPCK [34]. The result of this study also confirmed that ferulic acid significantly reduced the expression of the hepatic Gk gene. G6pc expression was significantly decreased in FA and RG_50E mice, while Pepck expression was lower in the FA, RG_30E, and RG_50E groups. These results indicate that the fermented rice germ extract regulates the expression of G6pc and Pepck, thereby inhibiting glucose synthesis.
Ferulic acid has both antihyperglycemic and antioxidant properties. It has been shown to alleviate hyperglycemia and oxidative stress by maintaining the redox balance and inhibiting mitochondrial apoptosis pathways [18,38,39]. Ferulic acid removes the free radicals present in aromatic rings, thus acting as an antioxidant and reducing MDA levels. The decrease in antioxidant enzyme activity in tissues promotes ROS-mediated lipid peroxidation and protein oxidation. As one such antioxidant enzyme, SOD inhibits ROS generation by converting superoxide anions into hydrogen peroxide [40,41]. We observed that ferulic acid and fermented rice germ extracts reduced MDA levels and improved catalase and SOD activity, confirming previous observations. While no effect on hepatic catalase activity was observed (Figure 4c), MDA levels in erythrocytes and the liver were significantly decreased in the FA and RG_50E groups, indicative of decreased lipid peroxidation (Figure 4a,b). Further, hepatic SOD activity was significantly increased in the FA, RG_30E, and RG_50E groups (Figure 4d). Thus, RG_50E significantly reduced MDA levels and enhanced SOD activity, exhibiting a greater antioxidant effect than RG_30E.
The db/db mice, which are animal models of type 2 diabetes, have mutation in the leptin receptor involved in dietary intake regulation and homeostasis [23]. In this study, compared to the db/db group, FA, RG_30E, and RG_50E did not confirm the tendency of a decrease in body weight. This is a limitation of this study. GABA is one of the compounds in rice germ [42]. Pu et al. reported that GABA was expressed in a subpopulation of neuropeptide Y (NPY) neurons involved in feed stimulation [43]. In particular, it was suggested that GABA and NPY interact to regulate of feeding. The relationship between GABA in rice germ and NPY must be a further study confirming the effect of weight loss by controlling food intake.
Yu et al. reported that rice germ contains large amounts of vitamin E and γ-oryzanol [44]. γ-oryzanol is a mixture of ferulic acid esters of phytosterols and triterpenoids [45]. Therefore, this study focused on ferulic acid which was known compound as hydrolyzed compound of γ-oryzanol, and it was conducted based on the study that antioxidants such as ferulic acid have antidiabetic effects [46,47]. This study has a limitation that fermented rice germ extracts still contained a small amount of ferulic acid. However, fermented rice germ extracts contain ferulic acid as well as various phenolic compounds and antioxidants, and it is expected that the effect of higher hepatic glucose metabolism was confirmed due to the interaction with other compounds. Since we have not verified the glucose metabolism between other compounds in fermented rice germ extracts, further research must be confirmed.
In conclusion, we demonstrated the efficacy of fermented rice germ extracts in improving hyperglycemia and hepatic glucose metabolism in T2DM mice. Fermented rice germ extracts lowered blood glucose levels as well as OGTT values and modulated hepatic glucose metabolism via regulating Gk, G6pc, and Pepck expression. In addition, fermented rice germ extracts lowered hepatic TG and TC as well as MDA levels, while enhancing SOD activity in liver tissue, highlighting their antioxidant effect. The current findings indicate the potential of fermented rice germ extracts for the prevention and alleviation of T2DM by regulating hepatic glucose metabolism.