Rice (Oryza sativa japonica) Albumin Suppresses the Elevation of

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Rice (Oryza sativa japonica) Albumin Suppresses the Elevation of Blood Glucose and Plasma Insulin Levels after Oral Glucose Loading Shigenobu Ina, Kazumi Ninomiya, Takashi Mogi, Ayumu Hase, Toshiki Ando, Narumi Matsukaze, Jun Ogihara, Makoto Akao, Hitoshi Kumagai, and Hitomi Kumagai J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b00520 • Publication Date (Web): 26 May 2016 Downloaded from http://pubs.acs.org on June 5, 2016

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Journal of Agricultural and Food Chemistry

Suppressive effect of rice albumin on blood glucose and insulin elevation

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Rice (Oryza sativa japonica) Albumin Suppresses the Elevation of Blood

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Glucose and Plasma Insulin Levels after Oral Glucose Loading

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Shigenobu Ina1, Kazumi Ninomiya1, Takashi Mogi1, Ayumu Hase1, Toshiki Ando1,

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Narumi Matsukaze1, Jun Ogihara1, Makoto Akao1, Hitoshi Kumagai2, and Hitomi

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Kumagai1*

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Fujisawa-shi 252-0880, Japan

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Hitotsubashi, Chiyoda-ku, Tokyo 101-8347, Japan

Department of Chemistry and Life Science, Nihon University, 1866 Kameino,

Department of Food Science and Nutrition, Kyoritsu Women’s University, 2-2-1

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AUTHOR INFORMATION

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Corresponding Author

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*

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[email protected]

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Notes

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The authors declare no competing financial interest.

Phone:+81(0)466 84 3946. Fax: +81(0)466 84 3946. E-mail:

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ACS Paragon Plus Environment


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ABSTRACT: The suppressive effect of rice albumin (RA) of 16 kDa on elevation of

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blood glucose level after oral loading of starch or glucose and its possible mechanism

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were examined. RA suppressed the increase in blood glucose levels in both the oral

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starch tolerance test (OSTT) and the oral glucose tolerance test (OGTT). The blood

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glucose concentrations 15 min after the oral administration of starch were 144±6 mg/dL

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for control group and 127±4 mg/dL for RA 200 mg/kg BW group, while those after the

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oral administration of glucose were 157±7 mg/dL for control group and 137±4 mg/dL

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for RA 200 mg/kg BW group. However, in the intraperitoneal glucose tolerance test

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(IPGTT), no significant differences in blood glucose level were observed between RA

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and the control groups, indicating that RA suppresses the glucose absorption from the

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small intestine. However, RA did not inhibit the activity of mammalian α-amylase. RA

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was hydrolyzed to an indigestible high-molecular-weight peptide (HMP) of 14 kDa and

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low-molecular-weight peptides (LMP) by pepsin and pancreatin. Furthermore, RA

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suppressed the glucose diffusion rate through a semi-permeable membrane like dietary

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fibers in vitro. Therefore, the indigestible HMP may adsorb glucose and suppress its

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absorption from the small intestine.

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KEYWORDS: rice albumin, oral glucose and starch tolerance tests,

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intraperitoneal glucose tolerance test, digestibility, glucose adsorption

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INTRODUCTION

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Diabetes is a disease characterized by chronic hyperglycemia. It is associated with an

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increased risk of various complications, including cardiovascular disease, retinopathy,

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and nephropathy.1-3 Inhibition of amylase and glycosidase suppresses the elevation of

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blood glucose levels after the ingestion of starch-based food and therefore could be

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useful in the prevention and treatment of type II diabetes mellitus.4-6 Several substances

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that inhibit amylase and glycosidase have been used to develop functional foods for

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preventing diabetes. Tea polyphenols, such as epicatechins and theaflavins, inhibit the

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activities of α-amylase, maltase, and sucrase,7,8 while L-arabinose inhibits sucrase

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activity.9 However, the application of these substances is restricted because of their

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bitter or sweet taste. Alternatively, materials with no taste, such as proteins and

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polysaccharides, are useful for food applications because any flavor and taste can be

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added during processing.

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Proteinaceous α-amylase inhibitors (α-AI) are widely distributed in cereals, such

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as wheat,10-12 barley,13 rye,14 maize,15 and rice.16 These are mostly resistant against

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insect α-amylase; however, some of them are known to also inhibit mammalian

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α-amylase. The intake of such α-AI suppresses the elevation of blood glucose level by

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inhibiting the digestion of starch in the small intestine.17 In particular, wheat albumin

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(WA), which is a water-soluble protein found in wheat seeds, has hypoglycemic effects

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attributable to its α-AI activity.18-20 Therefore, WA has already been used in foods for

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specified health uses (FoSHU) to prevent hyperglycemia, and is approved for use by the

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Japanese government. Rice (Oryza sativa japonica), which belongs to the same family

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(Poaceae family) as wheat, contains abundant rice albumin (RA); however the

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suppressive effect of RA on hyperglycemia remains so far uncertain. If RA also inhibits

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mammalian α-amylase similar to WA, it may be another promising substance for the 3



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suppression of blood glucose elevation. In this study, we investigated the suppressive effect of RA on elevation of blood

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glucose and plasma insulin levels by using the oral starch tolerance test (OSTT) and the

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oral glucose tolerance test (OGTT). In addition, we examined the mechanism of action

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of RA using the intraperitoneal glucose tolerance test (IPGTT) and measurement of

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α-AI activity, protein digestibility, and glucose diffusion rate through a semi-permeable

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membrane.

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MATERIALS AND METHODS Preparation of Rice and Wheat Albumin.

The RA and WA were

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prepared according to the methods described by Feng et al. with some

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modifications.16,21 Rice (Oryza sativa japonica cv. Nipponbare) or wheat (Triticum

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aestivum) flour (1 kg) was soaked in 5-fold (w/v) 100 mM citrate buffer (pH 6.0) and

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stirred at 4°C overnight. After centrifugation at 15,000 g for 15 min, the supernatant

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was heated at 80°C for 20 min. The heat-treated extract was cooled and centrifuged at

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15,000 g for 15 min. The heat-soluble proteins in the supernatant were precipitated by

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adding solid ammonium sulfate (40% saturation). The precipitate was collected by

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centrifugation at 15,000 g for 60 min, and then resuspended in distilled water. The

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suspension was dialyzed against distilled water, and centrifuged at 15,000 g for 15 min.

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The supernatant (crude albumin) was lyophilized and stored at -20°C until use.

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For purification of RA and WA, 1 g of crude albumin powder was dissolved in 40

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mL of distilled water. Subsequently, the solution was applied to a Sephadex G-50 gel

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filtration column (φ 2.5 cm × 100) and eluted with distilled water. For RA, the fractions

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of the second peak of optical density at 280 nm were collected. For WA, the inhibitory

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activity against α-amylase from porcine pancreas was measured for each fraction, and 4


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the fractions indicated more than 90% of α-amylase inhibitory activity were collected.

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After lyophilization, the RA and WA powder was stored at -20°C until use.

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The protein purity in the albumin powder was determined based on a standard

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curve of bovine serum albumin (BSA) using a BCA protein assay kit (Thermo Fisher

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Scientific, Kanagawa, Japan). The yield of RA and WA from flour (1 kg) was 0.8–1.2 g.

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The protein purity of the RA and WA powder was 85–95% and 80–90%, respectively.

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Animals.

Male 7-week-old Wistar rats were purchased from Japan SLC Inc.

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(Shizuoka, Japan). All rat experiments were performed in accordance with the

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Guidelines for Animal Experiments of the College of Bioresource Sciences of Nihon

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University (approval number: AP14B004). Rats were fed on a commercial diet (CE2,

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Clea Japan, Inc., Tokyo, Japan) for 1 week before the experiment. The room

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temperature and humidity were maintained at 23 ± 1°C and 50%, respectively, with a 12

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h light/dark cycle (8:00–20:00).

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Oral Starch and Glucose Tolerance Tests.

After adaptation, the rats

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were randomly divided into 8 groups (n=7). The OSTT and OGTT were carried out

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under non-anesthesia conditions. After overnight fasting, a blood sample was collected

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(0 min) from the tail vein. A phosphate buffered saline (PBS) solution containing 1 g/kg

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body weight (BW) of starch or glucose with RA or WA (OSTT: 200 mg/kg BW of RA

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and WA; OGTT: 50–200 mg/kg BW of RA and 200 mg/kg BW of WA) was

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immediately administered by gavage (5 mL/kg BW). Four further blood samples were

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taken from the tail vein at 15, 30, 60, and 90 min after carbohydrate administration. All

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blood samples were collected in heparinized capillary tubes and were kept on ice until

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centrifugation (1,300 g, 30 min, 4°C) to separate the plasma. The plasma samples were

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stored at -80°C until the insulin assay. Blood glucose levels were analyzed with an

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Ascensia Autodisc Sensor-Dexter-Z II (Bayer Medical, Tokyo, Japan) using a small 5



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amount of blood obtained from the tail vein. The plasma insulin levels were measured

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with an ELISA kit (Levis rat insulin kit, Shibayagi, Gunma, Japan). The area under the

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curve (AUC) was calculated for blood glucose and plasma insulin according to the

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methods described by Wolever and Jenkins.22

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Intraperitoneal Glucose Tolerance Test.

The IPGTT was conducted

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under non-anesthesia conditions after overnight fasting. Immediately prior to IPGTT, a

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blood sample was drawn from the tail vein. A PBS solution containing 200 mg/kg BW

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of RA was administered orally by gavage (5 mL/kg BW), and 15 min later, 1 g/kg BW

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of glucose was administered via intraperitoneal (i.p.) injection (10 mL/kg BW). Further

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blood samples were collected at 0, 15, 30, 45, and 90 min after i.p. glucose injection.

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Preparation of plasma and measurements of blood glucose and plasma insulin levels

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were performed as described above.

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Measurement of α-Amylase Inhibitory Activity.

The inhibitory activity

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of RA and WA against α-amylase was measured according to the methods described by

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Foo and Bais with some modifications.23 Porcine pancreatic α-amylase and human

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salivary α-amylase were purchased from Sigma-Aldrich (Tokyo, Japan), while

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mealworm α-amylase was extracted according to the methods described by Buonocore

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et al.24 The following assay buffers were used for mammalian and mealworm α-amylase

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activity assays, respectively: 20 mM HEPES buffer at pH 6.9 containing 50 mM NaCl

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and 3 mM CaCl2 and 20 mM acetate-Na buffer at pH 5.4 containing 100 mM NaCl. We

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used 2-chloro-4-nitrophenyl-α-D-maltotrioside (G3-CNP) (Oriental yeast, Tokyo,

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Japan) as a substrate for α-amylase.

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For the measurement of the α-AI activity for each sample, 25 µL of 1.6 U/µL

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α-amylase solution and 25 µL of 1 µg/µL RA or WA solution were mixed in each well

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of 96-well microtiter plates. One amylase unit was defined as the amount of enzyme 6


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that liberates 1 mg of maltose from starch during a 3 min reaction at pH 6.9 and 20°C.

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After incubation for 30 min at 37°C, 50 µL of 2 mM substrate solution was added and

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further incubated for 10 min at 37°C. The reaction was stopped by adding 100 µL of

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10% Tris solution, and the absorbance of 2-chloro-4-nitrophenol at 405 nm produced

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from the cleavage of G3-CNP was measured. When using mealworm α-amylase

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solution, incubation was carried out at 25°C.

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Analysis of In Vitro Protein Digestibility.

The in vitro protein

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digestibility analysis was conducted according to the methods described by Kumagai et

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al. with some modifications.25 As a reference, casein (Sigma-Aldrich, Tokyo, Japan)

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was used. For RA, WA or casein, 100 mg was suspended in 10 mL of distilled water,

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adjusted to pH 2 with dilute HC1, and incubated at 37°C for 2 h together with 1 mg of

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porcine pepsin (Sigma-Aldrich, Tokyo, Japan). After incubation, the pepsin was

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inactivated by neutralization with 100 mg of NaHCO3. Subsequently, 10 mg of porcine

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pancreatin (Sigma-Aldrich, Tokyo, Japan) was added and incubated at 37°C for 2, 4,

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and 6 h. Then, 50 µL of each solution was collected and mixed with 950 µL of an

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SDS-sample buffer. These samples were used for SDS-PAGE analysis with 14%

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acrylamide gel, which was conducted according to the methods described by

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Laemmli.26 The gel was stained with Coomassie Brilliant Blue (CBB).

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Identification of Indigestible Rice Albumin by 2DE and LC-MS/MS

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Analysis.

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was dialyzed against distilled water to remove the low-molecular-weight peptides

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(LMP), and the high-molecular-weight peptide (HMP) was obtained after lyophilization.

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For 2-dimensional electrophoresis (2DE), 10 µg of HMP was dissolved in 155 µL of 60

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mM Tris-HCl buffer at pH 8.8 containing 0.5% (v/v) ZOOM Carrier Ampholytes pH

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3-10 (Thermo Fisher Scientific, Yokohama, Japan) and 0.02% (w/v) bromophenol blue.

RA was digested by pepsin for 2 h and pancreatin for 6 h. The hydrolysate

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Subsequently, isoelectric focusing (IEF) was performed using ZOOM IPG strip pH

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3-10NL (Thermo Fisher Scientific) on a ZOOM IPGRunner Mini-Cell (Thermo Fisher

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Scientific), and SDS-PAGE was performed in 4-12% 2D-NuPAGE Bis-Tris ZOOM gel

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(Thermo Fisher Scientific). The protein spots were visualized by CBB, cut into 1 mm3

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cubes, and destained with 30% (v/v) acetonitrile in 25 mM NH4HCO3 solution.

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Subsequent in-gel digestion was carried out according to the method of Mori et al..27

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LC-MS/MS analysis was carried out using a LCQ Deca XP ion trap mass

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spectrometer (Thermo Finnigan, San Jose, USA) equipped with a nano-LC electrospray

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ionization source (AMR, Tokyo, Japan) and interfaced on-line with a capillary HPLC

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system (Paradigm MS4, Michrom Bioresources, Auburn, USA). The peptides extracted

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from samples (10 µL) were injected into the analytical column (L-column Micro C18, φ

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0.2 mm x 50 mm, CERI, Tokyo, Japan), and eluted with buffer A (2% (v/v) acetonitrile

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and 0.1% (v/v) formic acid) and buffer B (90% (v/v) acetonitrile and 0.1% (v/v) formic

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acid) using a linear gradient from 5 to 80% buffer B over 30 min. The eluted peptides

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were directly infused into the electrospray ionization source and detected with the ion

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trap mass spectrometer.

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MS/MS data obtained were analyzed by SEQUEST (Bioworks v3.2, Thermo

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Fisher Scientific, San Jose, USA) that allows the correlation of experimental data with

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theoretical spectra obtained from known protein sequences. All spectra were searched

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against the database in FASTA-format from National Center for Biotechnology

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Information (NCBI).

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Homology Modelling and Structural Analysis of Rice Albumin.

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protein structure and amino-acid sequence homology of rice albumin were analyzed by

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automated protein structure homology-modelling server (SWISS-MODEL,

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http://swissmodel.expasy.org/, Swiss).28 For homology analysis, 0.19 wheat α-amylase 8


The

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inhibitor (Accession No.: BAA20139.1), major wheat α-AI, was chosen as a template

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protein.

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Analysis of Glucose Diffusion on a Dialysis System. The glucose

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diffusion analysis was performed according to the methods described by Ou et al.29 RA,

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carboxymethylcellulose (CMC; Sigma-Aldrich, Tokyo, Japan) and guar gum

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(Sigma-Aldrich, Tokyo, Japan) were used as samples, and glucose adsorbability was

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evaluated. In the upper chamber of the dialysis unit (Slide-A-Lyzer MINI Dialysis Unit

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with a cut-off molecular weight of 3,500; Thermo Fisher Scientific, Kanagawa, Japan),

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250 µL of a solution containing 100 mmol/L of glucose and 8 mg/mL of each sample

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was applied, and 2 mL of deionized water filled the lower chamber of the unit, which

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was kept at 37°C and constantly shaken at 100 cycle/min. After 30, 60, 90, 120 and 150

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min, 20 µL of the dialysate was collected in the lower chamber of the unit, and the

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glucose content was measured using the Glucose CII Test Wako (Wako Pure Chemical

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Industries, Osaka, Japan).

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Statistical Analysis.

The results are expressed as mean ± standard error.

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The data were analyzed by a one-way analysis of variance (ANOVA) using SPSS for

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Windows (SPSS Inc., Tokyo, Japan). Comparisons between each group were made by

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using the Tukey’s test when the ANOVA results were statistically significant (p

Rice (Oryza sativa japonica) Albumin Suppresses the Elevation of

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