Rice koji reduced body weight gain, fat accumulation, and blood glucose level in high-fat diet-induced obese mice

Animals and cell line

CE-2 as a normal diet and HFD composed of 60% of calories from lipids were purchased from CLEA Japan, Inc. (Tokyo, Japan) and Research Diets Inc. (New Brunswick, NJ, USA), respectively. Male C57BL/6 mice were purchased from CLEA Japan, Inc. at 7 weeks of age. After arrival, each mouse was housed in an individual cage and maintained on a 12-h/12-h light-dark cycle in a temperature-controlled room (22 ± 2 °C, 50% ± 10% humidity). The mice were allowed free access to water and a normal diet for acclimatization (1 week). After acclimatization, the mice were divided into 5 body weight-matched groups (n = 12 per group) as follows. Group-I (CE-rice) was fed CE-2; Group-II (HFD-rice), an HFD containing 10% (w/w) steamed rice powder; Group-III (HFD-Y), an HFD containing 10% (w/w) yellow koji powder; Group-IV (HFD-W), an HFD containing 10% (w/w) white koji powder; and Group-V (HFD-R), an HFD containing 10% (w/w) red koji powder. Body weight and food intake were measured every week. This study was approved by the ethical committee for animal experimentation at Kagoshima University Graduate School of Medical and Dental Sciences (Permit Number: MD10122). The L6 rat skeletal muscle cell line was obtained from American Type Culture Collection Inc. (ATCC, Rockville, MD, USA).

Preparation of rice koji

All rice koji were prepared in our laboratory. The seed cultures of Aspergillus oryzae and Aspergillus kawachii were purchased from Kawauchi Genichiro shoten (Kagoshima, Japan). The seed culture of Monascus anka was purchased from Akita Konno Co., Ltd. (Akita, Japan). Solid-state fermentation was performed to prepare koji as described in our previous paper (Yoshizaki et al., 2010a; Yoshizaki et al., 2010b). The koji preparation was freeze-dried, ground to powder, and stored at −80 °C until use.

Analysis of nutrient components

Moisture, protein, fat, ash, carbohydrate, and energy were analyzed by Japan Food Research Laboratories (Tokyo, Japan). Moisture and protein were measured by heating-drying method and Kjeldahl method, respectively. Fat and ash were measured gravimetrically after the extraction and the incineration, respectively. Carbohydrate was calculated by subtracting the amount of moisture, protein, fat, and ash from 100. The energy values were calculated by means of Atwater’s factors (protein and carbohydrate 4 kcal/g and fat 9 kcal/g, respectively). Glucose was measured according to the method described in our previous study (Okutsu et al., 2012). High-performance liquid chromatography (HPLC) consisted of a COSMOSIL Sugar-D column (i.d., 4.6 × 250 mm; Nacalai Tesque, Inc., Kyoto, Japan) and a refractive index detector (Shimadzu Co., Kyoto, Japan). The mobile phase consisted of CH₃CN:H₂O (75:25) at a flow rate of 1.0 ml/min. The column temperature was maintained at 40 °C. Citric acid was analyzed using HPLC consisting of an ion-exclusion column (Shim-pack SCR-102H; i.d., 8 × 300 mm × 2; Shimadzu Co.) and post-column pH-buffered electroconductivity detection (Shimadzu Co.). The mobile phase consisted of 4 mM of p-toluenesulfonic acid solution at a flow rate of 0.8 ml/min. A reaction mixture of 4 mM p-toluenesulfonic acid with 16 mM bis-tris and 80 µM ethylenediaminetetraacetic acid disodium salt (EDTA) was used as post-column reagent at a flow rate of 0.8 ml/min. The column temperature was maintained at 50 °C.

Blood sample analysis and isolation of tissue material

After 4 weeks of feeding on the respective diets, mice were fasted for 4 h. Then, blood samples were obtained from the orbital sinus under diethyl ether anesthesia at the end of the experiment. Mice were euthanized by cervical dislocation. Immediately after, liver, visceral white adipose tissue, epididymal white adipose tissue, and brown adipose tissue were removed and weighed. The entire sampling procedure was done in less than 2 min. Blood samples were collected and immediately centrifuged at 1,500 × g for 10 min at 4 °C. The serum was sampled and stored at −80 °C until analysis. Concentrations of total cholesterol (TC), high-density lipoprotein (HDL) cholesterol, triglyceride (TG), and glucose were measured using a clinical chemistry reagent kit (Cholesterol E test, HDL-Cholesterol test, and Triglyceride G test; Wako Pure Chemical Industries, Ltd., Osaka, Japan). Blood glucose was measured with the Nipro Freestyle Freedom (Nipro Co., Osaka, Japan). Concentrations of insulin and leptin were measured using an enzyme immunoassay (Mouse Insulin kit and Mouse leptin kit; Morinaga Institute of Biological Science, Inc., Yokohama, Japan). The adiponectin concentration was evaluated by a sandwich ELISA system (Mouse/Rat adiponectin ELISA kit; Otsuka Pharmaceutical Co. Ltd., Tokyo, Japan). Homeostasis model assessment of insulin resistance (HOMA-R) was calculated as (fasting glucose level (mg/dl) × fasting insulin level (ng/ml))/450.

Total RNA isolation and real-time PCR

Total RNA in the liver was prepared from frozen tissue using the NucleoSpin RNA II kit (Macherey-Nagel Ltd., Düren, Germany). The cDNA was synthesized from 1 µg of total RNA with the Prime Script RT reagent kit (Takara Bio Inc., Tokyo, Japan). Real-time PCR was performed using SYBR Premix ExTaq (Takara Bio Inc.) on a Thermal Cycler Dice real-time system (Takara Bio Inc.) for 40 cycles of 95 °C for 30 s and 60 °C for 30 s. Commercially available, prevalidated primer pairs were purchased from the Perfect Real-time primer Support System (Takara Bio Inc.). β-Actin (GenBank accession No. NM_007393.3) was used as endogenous control for all target genes. Fatty acid synthase (FAS; GenBank accession No. NM_007988.3), acetyl-coenzyme A carboxylase-α (ACACα; GenBank accession No. NM_NM_133360.2), acetyl-coenzyme A carboxylase-β (ACACβ; GenBank accession No. NM_133904.2), carnitine palmitoyltransferase 2 (Cpt2; GenBank accession No. NM_009949.2), acyl-Coenzyme A dehydrogenase, very long chain (Acadvl; GenBank accession No. NM_017366.3), acyl-Coenzyme A dehydrogenase, long-chain (Acadl; GenBank accession No. NM_007381.4), acyl-Coenzyme A dehydrogenase, medium chain (Acadm; GenBank accession No. NM_007382.5), and peroxisome proliferator-activated receptor-α (PPARα; GenBank accession No. NM_001113418.1) were the target genes. Gene expression results are expressed as the expression ratio relative to β-actin gene expression, which were determined according to the manufacturer’s instructions. Product specificity was verified by melting curve analysis.

Glucose uptake assay in L6 myotube cells

Steamed rice, white koji, or red koji powder (5 g) was added to 25 ml deionized water and homogenized at 11,000 rpm for 1 min. The homogenate was centrifuged at 21,500 × g for 10 min, and the supernatant was used as the extract. The extract was diluted in deionized water for use in the assay. The dilution ratios of these extracts are shown in the figures as the dilution factor (DF). Insulin (0.1 µM) was used in place of the extract as a positive control.

For the glucose uptake assay, monolayers of L6 myotube cells were grown in DMEM/high glucose medium containing 4.5 g/L glucose (HyClone; Thermo Fisher Scientific Inc., Logan, UT) in a humidified 5% CO₂ incubator at 37 °C. Cells (1 × 10⁵/ml) were seeded in 10-cm petri dishes and grown to 90% confluency. The cells were washed 3 times in phosphate-buffered saline (PBS). The culture medium was replaced for 3 h with DMEM without glucose medium (Invitrogen Co., Carlsbad, CA, USA), and the cells were trypsinized. A centrifugation step was performed to remove trypsin, and the cells were resuspended in DMEM without glucose medium at 2 × 10⁶ cells/ml. This culture medium (0.5 ml containing 1 × 10⁶ cells) was transferred to each test tube, and 0.5 ml DMEM without glucose medium and the extract or insulin solution (0.1 ml) were added to the tube. After preincubation at 37 °C for 5 min, 10 µl of 20 mM 2-(N-[7-nitrobenz-2-oxa-1,3-diazol-4-yl]amino)-2-deoxyglucose (2-NBDG; Invitrogen Co.) was added (Zou, Wang & Shen, 2005). Test tubes were incubated at 37 °C with 5% CO₂ for 30 min. The 2-NBDG uptake reaction was stopped by removing the incubation medium and washing the cells twice with pre-chilled PBS. Each test tube was subsequently resuspended in 500 µl pre-chilled PBS and measured on a fluorescence spectrometer (Hitachi F-2500; Hitachi, Ltd., Tokyo, Japan) with excitation and emission set at 480 and 520 nm, respectively. All data presented were obtained from at least 3 separate cell preparations.

Western blotting analysis

The koji extract was the same sample as in the glucose uptake assay. Monacolin K, manscin, or citrate-Na buffer (pH 7.4) were also prepared to exposure for cells; monacolin K solutions (1–1000 µM), monascin solution (5–20 µM), and citrate-Na buffer (pH 7.4) (3–150 mM), respectively. Cells (1 × 10⁵ cells) were grown in 10-cm petri dishes with 5 ml DMEM/high glucose medium and 0.1 ml samples for 14 h. The L6 myotube cells were washed 3 times in PBS, trypsinized, and the trypsin was removed by centrifugation. Protein extracts were prepared by homogenizing the tissue in 50 mM Tris–HCl (pH 7.6) and 0.1% Triton X-100 supplemented with a protease inhibitor cocktail (F. Hoffmann-La Roche Ltd., Mannheim, Germany) and centrifuged at 21,500 × g for 30 min. The supernatant was used as the crude extract. The protein concentrations of extracts were determined using the Coomassie Protein Assay Reagent Kit (Thermo Fisher Scientific, Inc., CA, USA). Samples containing 60 µg of total protein were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using 10% gels and transferred to polyvinylidene fluoride (PVDF) membranes (Hybond-P; GE Healthcare UK Ltd., Buckinghamshire, UK). Membranes were probed with glucose transporter type 4 (GLUT4) and β-actin antibodies. The signal intensities were quantitated using the NIH Image J software (http://rsb.info.nih.gov/nih-image).

Statistical analyses

Data are expressed as the means ± SE. Statistical analyses were performed using SPSS software. Analysis of variance (ANOVA) was performed for the comparisons between groups. Significant differences (P < 0.05) between means were determined using Dunnett’s multiple comparison tests.

Nutritional components in steamed rice, yellow koji, white koji, and red koji

The nutritional components in the steamed rice, yellow koji, white koji, and red koji added to the HFD were investigated (Table 1). The carbohydrate content was lower in rice koji than in steamed rice, whereas the protein content was higher in rice koji. However, the total energy, fat, and ash were approximately the same among steamed rice, yellow koji, white koji, and red koji. All koji molds produced an abundance of glycolysis enzymes such as glucoamylase and α-amylase. Furthermore, white koji mold produced a large amount of citric acid during white koji manufacture. Thus, glucose and citric acid contents were measured. The glucose content in rice koji was 1,500–2,000-fold larger than that in steamed rice. The citric content of white koji was dramatically higher than in the other materials. Although yellow koji and red koji also contained much higher citric acid levels than did steamed rice, these values were one-tenth the amount of those found in white koji. Ingredient composition of the diets fed to mice is shown in Table 2.

Food intake and body and tissue weights of mice

Food intake of CE-rice diet and HFD was measured and expressed gram per day. CE-rice diet is the powder form and tended to be spilled out as shown in the variability of the amount of food measured (Table 3). The food intake rates for the HFD groups were 2.4–3.1 g per day and the energy intakes did not differ significantly between the HFD groups (Table 3).

The body weight increased gradually over the course of the experiment for all diet groups (Table 3). In particular, compared with the CE-rice diet, the HFD caused a significant increase in body weight. In groups receiving HFD, all rice koji significantly suppressed weight gain compared to HFD-rice diet. However, feed efficiency (weight gain/food intake × 100) was significantly reduced in the yellow koji group compared to the HFD-rice diet group. Liver weights were similar among all groups (Table 3). It was implied that HFD diet for 4 weeks did not induce the fatty liver. The HFD significantly increased the weights of total white adipose tissue compared with the CE-rice diet (Table 3). Furthermore, compared to HFD-rice diet, all rice koji significantly suppressed the weight of total white adipose tissue and epididymal white adipose tissue, in a manner similar to weight gain.

We performed blood analysis and gene expression profiling for these groups because the feed type-related differences on the mice appeared as the body weight gain after 4 weeks.

Lipid contents, adiponectin, and leptin levels in the serum of mice

Serum TC, HDL, and TG levels were measured to investigate the effects of rice koji on lipid metabolism (Table 4). After 4 weeks, the HFD had increased TC and HDL levels compared with those in the CE-rice diet group, while the TG level remained unchanged. Red koji was previously reported to reduce the levels of plasma natural lipid and TC (Trimarco et al., 2011). Significant differences were not found in TC, HDL, and TG levels between the HFD diet groups in this experiment. However, red koji showed a tendency to reduce the TG level in serum, consistent with previous studies.

Adiponectin decreases the plasma glucose level and enhances the utilization of fatty acids in muscle (Berg, Combs & Scherer, 2002; Nawrocki & Scherer, 2004). Leptin, which has an antifeeding effect, is secreted from adipocytes in response to the accumulation of lipids in cells (Campfield, Smith & Burn, 1996). Therefore, the influence of rice koji on the secretion of adiponectin and leptin was investigated (Table 4). Although red koji modestly decreased serum adiponectin, it did not significantly change in the experimental groups. The leptin level increased in the HFD groups; however, compared with the HFD-rice group, white koji significantly suppressed this increase.

Blood glucose and serum insulin levels in the mice

C57BL/6J mice receiving HFD have exhibited severe obesity, hyperglycemia, and hyperinsulinemia (Harte et al., 1999). Herein, we presumed that glucose metabolism was altered by obesity in mice that fed on an HFD for 4 weeks and we therefore measured the blood glucose and serum insulin levels in all mice. A significant increase in blood glucose levels was observed in HFD-fed mice compared to those fed the CE-rice diet after 4 weeks. However, the blood glucose level was significantly lower in the white and red koji groups than in the HFD-rice group (Table 4). The HFD containing rice koji contained a small amount of glucose, approximately 1.5∼2.0% (w/w). Thus, our results showed that white and red koji efficiently suppressed the high blood glucose level induced by HFD. Red-mold-fermented-products have been reported to attenuate the development of diabetes and alleviate hyperglycemia (Shi & Pan, 2010), and our experiments confirmed this effect of red koji. Although the serum insulin level was not different among the experimental groups, the white and red koji groups tended to have lower insulin levels than those of the other HFD groups. HOMA-R, which was used to assess insulin resistance, was significantly lower in the white koji group than in the HFD-rice group. Although the red koji group showed no significant differences in HOMA-R, it tended to have lower HOMA-R than those of the other HFD groups, being reduced to almost the same level as that of the white koji group.

Expression of the fatty acid metabolism enzymes in mice

Rice koji presumably affect fat metabolism. To determine the molecules involved in fatty acid metabolism in HFD mice upon rice koji feed, we measured the hepatic gene expression of several genes involved in fatty acid metabolism. Fatty acid synthase (FAS), acetyl-CoA carboxylase α (ACACα), acetyl-CoA carboxylase β (ACACβ), carnitine palmitoyltransferase 2 (Cpt2), acyl-Coenzyme A dehydrogenase, very long chain (Acadvl), acyl-Coenzyme A dehydrogenase, long-chain (Acadl), acyl-Coenzyme A dehydrogenase, medium chain (Acadm) and peroxisome proliferator-activated receptor α (PPARα) was investigated herein. In the liver, the expression of FAS, ACACα, Cpt2, Acadvl, Acadl, Acadm and PPARα genes was not significantly different after 4 weeks of HFD feeding (Fig. 1). While the expression level ACACβ in the CE-rice group was significantly higher than the other HFD groups, it was not significantly different between HFD groups.

Effect of white and red koji on glucose uptake and GLUT4 protein expression in L6 myotube cells

It was reported that red mold-fermented products could delay the increase in the blood glucose levels in rats and that red koji protects against diabetes (Hsieh & Tai, 2003; Shi & Pan, 2010). This effect of red koji was also confirmed in this study and that of white koji was first recognized (Table 4). Skeletal muscle is the predominant tissue for insulin-stimulated glucose disposal (Koranyi et al., 1991). Glucose enters the muscle cell primarily by facilitated diffusion by utilizing glucose transporter carrier proteins. GLUT4 is the predominantly expressed glucose transporter isoform in muscle (Shepherd & Kahn, 1999), and whole body glucose disposal and GLUT4 expression are correlated in healthy subjects (Koranyi et al., 1991). Therefore, the glucose uptake activity and GLUT4 protein expression level were measured in muscle cells to investigate the mechanism by which white and red koji suppresses blood glucose. L6 myotube cells were exposed to various concentrations of white and red koji extracts or steamed rice extract. Treatment of L6 myotube cells with white and red koji resulted in a dose-dependent increase in 2-NBDG uptake, similar to that in insulin-stimulated cells (Fig. 2). We examined the expression of GLUT4 proteins in L6 myotube cells stimulated with white and red koji extracts. GLUT4 expression increased by approximately 1.5 and 1.6-fold in L6 myotube cells treated with the extract of white and red koji, respectively, compared with cells treated with steamed rice (Fig. 3).

To reveal the key compounds in red and white koji, it was investigated the effect of monacolin K, monascin and citric acid on the induction of GLUT4 protein in L6 myotube cells. Monacolin K is one of the most important compounds in red koji. A recent study has shown that monascin, which is a yellow pigment produced by Monascus sp., prevents fatty acid accumulation in high-fat diet-fed mice (Hsu et al., 2014). Citric acid is one of most abundant compounds in white koji. The exposure concentration was determined the previous results and citric acid concentration in white koji. Because concentrated citric acid solution is strongly acidic, cell damage was expected because of the low pH. Therefore, in this experiment, we used citrate-Na buffer (pH 7.4) to investigate the effect of citrate molecule. Our results showed monacolin K and monascin can induce GLUT4 expression (Fig. 4). Citric acid did not have this effect on L6 myotube cells.