N U T RI TI O N RE S E ARCH 3 4 ( 2 0 14 ) 9 6 1–9 67
Available online at www.sciencedirect.com
ScienceDirect www.nrjournal.com
D-Sorbose
inhibits disaccharidase activity and demonstrates suppressive action on postprandial blood levels of glucose and insulin in the rat
Tsuneyuki Oku a, b, c,⁎, Yoko Murata-Takenoshita b , Yuko Yamazaki c , Fumio Shimura c , Sadako Nakamura a, b, c a b c
Graduate School of Human Health Science, University of Nagasaki Siebold, Nagayo, Nagasaki 851-2195, Japan Faculty of Nursing and Nutrition, Department of Nutrition, University of Nagasaki Siebold, Nagayo, Nagasaki 851-2195, Japan Institute of Food, Nutrition and Health, Jumonji University, Niiza, Saitama 352-8510, Japan
ARTI CLE I NFO
A BS TRACT
Article history:
In an attempt to develop D-sorbose as a new sweetener that could help in preventing lifestyle-
Received 19 April 2014
related diseases, we investigated the inhibitory effect of D-sorbose on disaccharidase activity,
Revised 15 September 2014
using the brush border membrane vesicles of rat small intestines. The inhibitory effect was
Accepted 24 September 2014
compared with that of
L-sorbose
and other rare sugars, and the small intestinal
disaccharidases in rats was compared with that of humans as well. In humans and the Keywords:
small intestines of rats, D-sorbose strongly inhibited sucrase activity and weakly inhibited
D-sorbose
maltase activity. Inhibition by D-sorbose of sucrase activity was similar to that of L-arabinose,
Human
and the Ki of D-sorbose was 7.5 mM. Inhibition by D-sorbose was very strong in comparison
Rat disaccharidase
with that of L-sorbose (Ki, 60.8 mM), whereas inhibition of D-tagatose was between that of D-
Postprandial blood glucose
sorbose and L-sorbose. The inhibitory mode of
Inhibitory effect
uncompetitive, and that of L-sorbose was competitive. To determine a suppressive effect on
D-sorbose
for sucrose and maltase was
Suppressive effect
postprandial blood levels of glucose and insulin via inhibition of sucrase activity, sucrose solution with or without D-sorbose was administered to rats. Increments in the blood levels of glucose and insulin were suppressed significantly after administration of sucrose solution with D-sorbose to rats, in comparison to administration of sucrose solution without D-sorbose. In contrast, the suppressive effect of L-sorbose on postprandial blood levels of glucose and insulin was very weak. These results suggest that D-sorbose may have an inhibitory effect on disaccharidase activity and could be used as a sweetener to suppress the postprandial elevation of blood levels of glucose and insulin. The use of D-sorbose as a sweetener may contribute to the prevention of lifestyle-related diseases, such as type 2 diabetes mellitus. © 2014 Elsevier Inc. All rights reserved.
1.
Introduction
D-sorbose is an isomer of D-fructose and termed a rare sugar because it is rarely found in nature [1]. Recently, however, mass
production of D-sorbose became possible when it was produced from glucose using specific enzymes as well as D-psicose and D-tagatose (also rare sugars) [2–4]. Matsuo and Izumori [5] reported that D-psicose inhibits intestinal α-glucosidase and
Abbreviations: ANOVA, analysis of variance; BBMV, brush border membrane vesicle; TGO, mixture of 10 U/mL glucose oxidase, 5 U/mL peroxidase, 0.05 mM, 4-aminoantipyrine, and 0.4 mM p-phenolsulfonic acid sodium salt in 0.5 M, Tris-HCl (pH 7.0). ⁎ Corresponding author at: Institute of Food, Nutrition and Health, Jumonji University, 2-1-28 Sugasawa, Niiza, Saitama, 352-8510 Japan. Tel.: +81 48 477 0555; fax: +81 48 478 9367. E-mail address: [email protected] (T. Oku). http://dx.doi.org/10.1016/j.nutres.2014.09.009 0271-5317/© 2014 Elsevier Inc. All rights reserved.
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N U TR I TION RE S E ARCH 3 4 ( 2 0 14 ) 9 6 1–9 67
suppresses glycemic responses after carbohydrate ingestion in rats. Furthermore, Iida et al [6] and Hayashi et al [7] demonstrated that D-psicose administration decreased the glycemic response to an oral maltodextrin test in normal adults. In addition, D-tagatose inhibits small intestinal disaccharidases as well as that documented for D-psicose [8,9]. Therefore, D-sorbose is expected to be a new sweetener that prevents metabolic syndrome and lifestyle-related diseases to the same extent as that seen for D-psicose and D-tagatose. However, the physiologic function of D-sorbose has not been clarified sufficiently because it has been difficult to obtain an adequate amount of D-sorbose for experiments in animals. Several rare sugars, such as D-tagatose and L-arabinose, are shown to have an inhibitory effect on small intestinal disaccharidases [9–11] as well as suppress the postprandial elevation of glucose levels caused by the ingestion of sucrose and starch hydrolysate in humans and rats [11]. The inhibitory effect by L-arabinose on sucrase activity has been applied to a new type of sweetener in which sucrose and L-arabinose were mixed in a ratio of 93:3. D-Sorbose appears to reveal an inhibitory effect for disaccharidases as well as that documented for D-tagatose and L-arabinose. L-Sorbose is an isomer of D-sorbose, but their physiologic functions appear to differ markedly. Furuse et al [12–14] reported that L-sorbose improved fasting blood glucose levels and reduced the number of hepatic deposits when diabetic mice were fed L-sorbose instead of sucrose. However, the physiologic function of D-sorbose, in terms of prevention of lifestyle-related diseases, remains unclear. This study used the brush border membrane vesicles (BBMVs) in the small intestines of rats to investigate the inhibitory effect of D-sorbose on disaccharidases and compared this effect with that of other rare sugars (L-sorbose, D-tagatose, and L-arabinose) whose inhibitory mode of action was previously reported [9,11]. In addition, inhibition upon small intestinal disaccharidases by D-sorbose was compared with that in humans and rats using small intestinal homogenates. Furthermore, to confirm the suppressive effect on postprandial blood levels of glucose and insulin via inhibition of sucrase activity, sucrose solution with or without D-sorbose was administered via the oral route to rats. After administration, the suppressive effect on incremental change in blood levels of glucose and insulin levels was measured. The aim of these experiments was to determine if D-sorbose, as a sweetener, affords so protection from lifestyle-related diseases, such as diabetes mellitus and obesity.
2.
Methods and materials
2.1.
Ethical approval of the study protocol
The protocol for the animal studies was approved by the Committee on Animal Experiments at the University of Nagasaki (Nagasaki, Japan). These experiments were conducted according to the Guidelines on the Care and Use of Laboratory Animals (National Institutes of Health, Bethesda, MD, USA) and the standards relating to the Care and Management of Experimental Animals (Notification number 88, from the Prime Minister's office). All experiments were carried out in the Public Health Nutrition Laboratory of the
Graduate School of Human Health Science, University of Nagasaki Siebold. The experiments using donated tissues from human intestines were conducted according to guidelines laid down in the Declaration of Helsinki. All procedures involving human subjects were approved by the Ethics Committee at the University of Nagasaki Siebold (received number 22, approval number 19) and Juzenkai Hospital in Nagasaki, Japan. All subjects provided written informed consent to participate in the study.
2.2.
Materials
D-Sorbose was purchased from Tokyo Chemical Industry Co, Ltd
(Tokyo, Japan), and L-sorbose was provided by Asahi Kasei Corp (Tokyo, Japan). L-Arabinose was purchased from Wako Pure Chemical Industries Ltd (Osaka, Japan), whereas D-tagatose was provided by Arla Foods Ingredients Japan (Tokyo, Japan). D-Erythritol, D-xylitol, and D-arabitol were obtained from Nikken Chemical Co, Ltd (Tokyo, Japan).
2.3. Determination of the inhibitory effect on sucrase and maltase using BBMVs of the small intestines of rats 2.3.1.
Rats
To prepare BBMVs from the small intestine, 15 rats (Wistar, male, 320 g; Clea Japan, Inc, Tokyo, Japan) were fed a standard solid diet (MF diets; Oriental Yeast Co, Osaka, Japan) and distilled water ad libitum for 5 days. The animals were kept in a temperature-controlled environment at 22°C to 24°C. The humidity was set at 50%, and the room was maintained on a 12-hour light-dark cycle (light, 08:00-20:00; dark, 20:00-08:00).
2.3.2. Preparation of BBMVs from the mucosae of the small intestines of rats Small intestinal BBMVs were prepared using a modified version of the methods of Kesseler et al [15]. The BBMVs obtained were suspended in an adequate volume of 50 mM phosphate buffer (pH 6.0) and stored at −80°C until the assay.
2.3.3. Inhibition experiments and determination of the inhibitory mode of sucrase and maltase The assay of hydrolyzing activity to observe inhibition was carried out according to the method of Oku et al [16], which partially modified Dahlqvist's method of using glucose oxidase [17]. Glucose was used as a standard. Substrates of sucrose and maltose were prepared at 112 mM in 0.1 M maleate-Na buffer (pH 6.0). Diluted BBMVs (0.1 mL) were pipetted into a small glass test tube. Then, 0.1 mL of 112 mM substrate was added to the test tube after preincubation for a few minutes at 37°C. After incubation for 10 to 30 minutes at 37°C, 2.4 mL of TGO reagent (containing 10 U/mL glucose oxidase, 5 U/mL peroxidase, 0.05 mM 4-aminoantipyrine, and 0.4 mM p-phenolsulfonic acid sodium salt in 0.5 M Tris-HCl [pH 7.0]) was added to the test tube to stop hydrolysis. The reactions by glucose oxidase and peroxidase were started simultaneously. The reaction mixture was incubated for an additional 10 minutes at 37°C. Then, 2 drops of 4 N NaOH were added to the test tube to stop the reactions. Optical absorbance was read at 500 nm, using a spectrophotometer (UV1240; Shimadzu, Kyoto, Japan).
N U T RI TI O N RE S E ARCH 3 4 ( 2 0 14 ) 9 6 1–9 67
To compare the degrees of inhibition for the disaccharidases in rat tissues, 20 μL of either 9.1 mM or 18.2 mM of one of the following was added to the reaction medium in a duplicate assay: D- or L-sorbose, L-arabinose, D-tagatose, D-erythritol, D-xylitol, or D-arabitol was added to the reaction medium in a duplicate assay. The same volume of distilled water was added as a control. Before the assay, preliminary studies were carried out to evaluate the appropriate concentration of inhibitors. To determine the mode of inhibition and the enzymeinhibition constant (Ki), 20 μL of 1.4 mM and 2.3 mM of D-sorbose, 18.2 mM of L-sorbose, and distilled water were added to the reaction medium. The concentration of substrate was prepared from 0 to 150 mM. The assay procedure was identical to that mentioned above. We determined the mode of inhibition and calculated the Ki values of D- and L-sorbose, using Lineweaver-Burk plots. The concentrations of D- and L-sorbose that we chose to use were based on our preliminary experiments.
2.4. Determination of inhibition by D- and L-sorbose of the activity of sucrase and maltase of human and rat small intestinal homogenates
1 mL of 112 mM maltose in 0.1 M maleate-Na buffer (pH 6.0) was added into the glass test tube, after preincubation for a few minutes at 37°C. The assay procedure was identical to that mentioned above.
2.5. Determination of protein concentration in BBMVs and homogenates Protein concentrations of BBMVs and homogenates were determined using the Bradford method [18].
2.6. Suppressive effect of D- and L-sorbose on elevation of plasma levels of glucose and insulin by sucrose administration in rats 2.6.1.
Preparation of human small intestinal homogenates
Five patients from Juzenkai Hospital donated fragments of human small intestines without malignant tissues. Patients provided written informed consent to donate these fragments of small intestine after resection of tumorous tissue in the small intestine. Immediately after resection, tissue fragments were soaked in ice-cold 0.9% NaCl and transferred to our laboratory. Fragments without lesions were cut and washed with ice-cold 0.9% NaCl. Three fragments were part of the lower ileum, 1 was the lower jejunum, and 1 was the upper jejunum. The mucosa was gently scraped-off with 2 slide glasses onto an ice-chilled glass plate, and then it was homogenized in a 19-fold volume of ice-cold 0.9% NaCl using a homogenizer (Polytron; Kinematica, Inc, Lucerne, Switzerland). It was stored at −80°C until use.
Calculations and statistical analyses
Preparation of rat small intestinal homogenates
For the preparation of the small intestinal homogenates, 5 male Wistar rats (200 g; Clea Japan, Inc) were fed a standard solid diet (MF diets; Oriental Yeast Co) and distilled water ad libitum for 7 days. The condition in the animal quarters was identical to that mentioned above. After an overnight fast, 5 rats were euthanized by decapitation. Small intestines from the duodenum to the ileum were immediately removed, slit open, and washed with ice-cold 0.9% NaCl. The mucosa was scraped off with slide glasses on ice-chilled glass plates, weighed, and homogenized in ice-cold 0.9% NaCl (10% wet wt/vol) using a homogenizer (Polytron; Kinematica, Inc). All homogenates were stored at −80°C until the assay. Before the assay, they were rehomogenized and diluted to appropriate concentrations.
2.4.3.
Oral administration of test substance to rats
After an overnight fast, rats were administered 1.5 mL of test solutions using a gastric tube, as based on our previous studies [9,19]. Test solutions were 0.3 g of sucrose per milliliter, 0.3 g of sucrose and 0.033 g of D-sorbose per milliliter, and 0.3 g of sucrose and 0.033 g of L-sorbose per milliliter. Blood (120 μL) was collected from the tail vein with 2 heparinized hematocrit tubes before and at 30-minute intervals for 3 hours after administration of test solutions. Plasma was obtained by centrifugation at 11 000 rpm for 5 minutes at room temperature, using a specific centrifuge for hematocrit tubes (3110; Kubota, Tokyo, Japan). Plasma levels of glucose and insulin were measured by Trinder's method using glucose oxidase [20] and enzyme-linked immunosorbent assay methods [21], respectively.
2.7. 2.4.2.
Animals
To observe the suppressive effect of D- and L-sorbose on increments in plasma levels of glucose and insulin by sucrose administration in vivo, 6 male Wistar rats (200-220 g body weight) were fed a standard solid diet (MF diets; Oriental Yeast Co) and distilled water ad libitum for 7 days. They were housed for 7 days under the conditions mentioned above.
2.6.2. 2.4.1.
963
Inhibition experiments on sucrase and maltase
The adequately diluted homogenate (0.1 mL) was pipetted into a small glass test tube. Then, 0.1 mL of 112 mM sucrose or
The activity of disaccharidases using rat intestinal BBMVs was calculated as specific activity (micromoles of substrate hydrolyzed per milligram protein per hour). We calculated the mean and SD values in duplicate assays and then compared them among inhibitors with the same concentration using analysis of variance (ANOVA) and Dunnett post hoc test vs control. The Ki values of D- and L-sorbose were calculated using Lineweaver-Burk plots. To compare the inhibition of sucrase and maltase between D- and L-sorbose in rat and human homogenates, we calculated the specific activity, mean value, SD value, and percentage of remaining activity by inhibition of D- and L-sorbose vs control. After this, inhibition was compared between D- and L-sorbose by ANOVA and Dunnett post hoc test vs control. In the in vivo experiments, differences in the plasma levels of glucose and insulin in 6 rats were compared at each time point using ANOVA and Tukey post hoc test. P < .05 was considered significant. Analyses were conducted using SPSS version 20.0 (IBM, New York, NY, USA).
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3.
Results
3.1. Inhibition by D- and L-sorbose of disaccharidase activity in the BBMVs of the small intestines of rats Using BBMVs in the small intestines of rats, Fig. 1 shows the inhibitory effect on the activity of sucrase and maltase by Dand L-sorbose, L-arabinose, D-tagatose, D-erythritol, D-xylitol, and D-arabitol. D-Sorbose, L-arabinose, and D-tagatose inhibited sucrase activity down to more than 50% at 9.1 mM, in comparison with that of control. Conversely, inhibition by L-sorbose was only 10%, and inhibition of sugar alcohols such as D-erythritol, D-xylitol, and D-arabitol was not that distinctive. In comparison with that of sucrose, D-sorbose at the same concentration showed weak inhibition of maltase activity, with inhibition at ≈36%. Inhibition by L-sorbose of maltase activity was less than 10%. L-Arabinose also showed weak inhibition, and the inhibition by D-tagatose and sugar alcohols of maltase activity was negligible.
3.2. Inhibitory mode of action by D- and L-sorbose of sucrase and maltase using BBMVs in the small intestine of rats
Specific Activity (µ moles substrate hydrolyzed/mg protein/hr)
Fig. 2 shows the inhibitory mode of action of D- and L-sorbose on sucrase in small intestinal BBMVs of rats using Lineweaver-Burk
150
Sucrase
plots. The inhibitory mode of action of D-sorbose on sucrase was uncompetitive because the inhibition line was parallel to the noninhibition line, whereas that of L-sorbose was competitive because the inhibition line crossed to the noninhibition line on the y-axis. In addition, the inhibitory mode of action by D-sorbose on maltase was uncompetitive (data not shown). The Ki values of D- and L-sorbose, L-arabinose, and D-tagatose, which inhibited sucrose activity, are summarized in the Table. The Ki was calculated from the Lineweaver-Burk equation. The Ki of D-sorbose was 7.5 mM and was much stronger than that of L-sorbose (68.0 mM). The Ki of L-arabinose was the smallest overall (2.6 mM), and the Ki of D-tagatose was 16.5 mM. The Ki of D-sorbose for maltase was 18.6 mM, and it was larger than that for sucrase (7.5 mM).
3.3. Comparison of the inhibitory effect between D- and L -sorbose on the activity of small intestinal disaccharidases in humans and rats Fig. 3 shows the inhibitory effect of D- and L-sorbose on the activity of sucrase and maltase in human and rat small intestinal homogenates. D-Sorbose inhibited sucrase activity in humans by ≈60% and that of rats by 70%. Conversely, L-sorbose only inhibited sucrase activity in humans and rats only by a few percent. In humans and rats, D-sorbose showed a weak inhibition of maltase activity (≈20%), in comparison with that of sucrase activity. Inhibition by L-sorbose of maltase activity was weaker (
Available online at www.sciencedirect.com
ScienceDirect www.nrjournal.com
D-Sorbose
inhibits disaccharidase activity and demonstrates suppressive action on postprandial blood levels of glucose and insulin in the rat
Tsuneyuki Oku a, b, c,⁎, Yoko Murata-Takenoshita b , Yuko Yamazaki c , Fumio Shimura c , Sadako Nakamura a, b, c a b c
Graduate School of Human Health Science, University of Nagasaki Siebold, Nagayo, Nagasaki 851-2195, Japan Faculty of Nursing and Nutrition, Department of Nutrition, University of Nagasaki Siebold, Nagayo, Nagasaki 851-2195, Japan Institute of Food, Nutrition and Health, Jumonji University, Niiza, Saitama 352-8510, Japan
ARTI CLE I NFO
A BS TRACT
Article history:
In an attempt to develop D-sorbose as a new sweetener that could help in preventing lifestyle-
Received 19 April 2014
related diseases, we investigated the inhibitory effect of D-sorbose on disaccharidase activity,
Revised 15 September 2014
using the brush border membrane vesicles of rat small intestines. The inhibitory effect was
Accepted 24 September 2014
compared with that of
L-sorbose
and other rare sugars, and the small intestinal
disaccharidases in rats was compared with that of humans as well. In humans and the Keywords:
small intestines of rats, D-sorbose strongly inhibited sucrase activity and weakly inhibited
D-sorbose
maltase activity. Inhibition by D-sorbose of sucrase activity was similar to that of L-arabinose,
Human
and the Ki of D-sorbose was 7.5 mM. Inhibition by D-sorbose was very strong in comparison
Rat disaccharidase
with that of L-sorbose (Ki, 60.8 mM), whereas inhibition of D-tagatose was between that of D-
Postprandial blood glucose
sorbose and L-sorbose. The inhibitory mode of
Inhibitory effect
uncompetitive, and that of L-sorbose was competitive. To determine a suppressive effect on
D-sorbose
for sucrose and maltase was
Suppressive effect
postprandial blood levels of glucose and insulin via inhibition of sucrase activity, sucrose solution with or without D-sorbose was administered to rats. Increments in the blood levels of glucose and insulin were suppressed significantly after administration of sucrose solution with D-sorbose to rats, in comparison to administration of sucrose solution without D-sorbose. In contrast, the suppressive effect of L-sorbose on postprandial blood levels of glucose and insulin was very weak. These results suggest that D-sorbose may have an inhibitory effect on disaccharidase activity and could be used as a sweetener to suppress the postprandial elevation of blood levels of glucose and insulin. The use of D-sorbose as a sweetener may contribute to the prevention of lifestyle-related diseases, such as type 2 diabetes mellitus. © 2014 Elsevier Inc. All rights reserved.
1.
Introduction
D-sorbose is an isomer of D-fructose and termed a rare sugar because it is rarely found in nature [1]. Recently, however, mass
production of D-sorbose became possible when it was produced from glucose using specific enzymes as well as D-psicose and D-tagatose (also rare sugars) [2–4]. Matsuo and Izumori [5] reported that D-psicose inhibits intestinal α-glucosidase and
Abbreviations: ANOVA, analysis of variance; BBMV, brush border membrane vesicle; TGO, mixture of 10 U/mL glucose oxidase, 5 U/mL peroxidase, 0.05 mM, 4-aminoantipyrine, and 0.4 mM p-phenolsulfonic acid sodium salt in 0.5 M, Tris-HCl (pH 7.0). ⁎ Corresponding author at: Institute of Food, Nutrition and Health, Jumonji University, 2-1-28 Sugasawa, Niiza, Saitama, 352-8510 Japan. Tel.: +81 48 477 0555; fax: +81 48 478 9367. E-mail address: [email protected] (T. Oku). http://dx.doi.org/10.1016/j.nutres.2014.09.009 0271-5317/© 2014 Elsevier Inc. All rights reserved.
962
N U TR I TION RE S E ARCH 3 4 ( 2 0 14 ) 9 6 1–9 67
suppresses glycemic responses after carbohydrate ingestion in rats. Furthermore, Iida et al [6] and Hayashi et al [7] demonstrated that D-psicose administration decreased the glycemic response to an oral maltodextrin test in normal adults. In addition, D-tagatose inhibits small intestinal disaccharidases as well as that documented for D-psicose [8,9]. Therefore, D-sorbose is expected to be a new sweetener that prevents metabolic syndrome and lifestyle-related diseases to the same extent as that seen for D-psicose and D-tagatose. However, the physiologic function of D-sorbose has not been clarified sufficiently because it has been difficult to obtain an adequate amount of D-sorbose for experiments in animals. Several rare sugars, such as D-tagatose and L-arabinose, are shown to have an inhibitory effect on small intestinal disaccharidases [9–11] as well as suppress the postprandial elevation of glucose levels caused by the ingestion of sucrose and starch hydrolysate in humans and rats [11]. The inhibitory effect by L-arabinose on sucrase activity has been applied to a new type of sweetener in which sucrose and L-arabinose were mixed in a ratio of 93:3. D-Sorbose appears to reveal an inhibitory effect for disaccharidases as well as that documented for D-tagatose and L-arabinose. L-Sorbose is an isomer of D-sorbose, but their physiologic functions appear to differ markedly. Furuse et al [12–14] reported that L-sorbose improved fasting blood glucose levels and reduced the number of hepatic deposits when diabetic mice were fed L-sorbose instead of sucrose. However, the physiologic function of D-sorbose, in terms of prevention of lifestyle-related diseases, remains unclear. This study used the brush border membrane vesicles (BBMVs) in the small intestines of rats to investigate the inhibitory effect of D-sorbose on disaccharidases and compared this effect with that of other rare sugars (L-sorbose, D-tagatose, and L-arabinose) whose inhibitory mode of action was previously reported [9,11]. In addition, inhibition upon small intestinal disaccharidases by D-sorbose was compared with that in humans and rats using small intestinal homogenates. Furthermore, to confirm the suppressive effect on postprandial blood levels of glucose and insulin via inhibition of sucrase activity, sucrose solution with or without D-sorbose was administered via the oral route to rats. After administration, the suppressive effect on incremental change in blood levels of glucose and insulin levels was measured. The aim of these experiments was to determine if D-sorbose, as a sweetener, affords so protection from lifestyle-related diseases, such as diabetes mellitus and obesity.
2.
Methods and materials
2.1.
Ethical approval of the study protocol
The protocol for the animal studies was approved by the Committee on Animal Experiments at the University of Nagasaki (Nagasaki, Japan). These experiments were conducted according to the Guidelines on the Care and Use of Laboratory Animals (National Institutes of Health, Bethesda, MD, USA) and the standards relating to the Care and Management of Experimental Animals (Notification number 88, from the Prime Minister's office). All experiments were carried out in the Public Health Nutrition Laboratory of the
Graduate School of Human Health Science, University of Nagasaki Siebold. The experiments using donated tissues from human intestines were conducted according to guidelines laid down in the Declaration of Helsinki. All procedures involving human subjects were approved by the Ethics Committee at the University of Nagasaki Siebold (received number 22, approval number 19) and Juzenkai Hospital in Nagasaki, Japan. All subjects provided written informed consent to participate in the study.
2.2.
Materials
D-Sorbose was purchased from Tokyo Chemical Industry Co, Ltd
(Tokyo, Japan), and L-sorbose was provided by Asahi Kasei Corp (Tokyo, Japan). L-Arabinose was purchased from Wako Pure Chemical Industries Ltd (Osaka, Japan), whereas D-tagatose was provided by Arla Foods Ingredients Japan (Tokyo, Japan). D-Erythritol, D-xylitol, and D-arabitol were obtained from Nikken Chemical Co, Ltd (Tokyo, Japan).
2.3. Determination of the inhibitory effect on sucrase and maltase using BBMVs of the small intestines of rats 2.3.1.
Rats
To prepare BBMVs from the small intestine, 15 rats (Wistar, male, 320 g; Clea Japan, Inc, Tokyo, Japan) were fed a standard solid diet (MF diets; Oriental Yeast Co, Osaka, Japan) and distilled water ad libitum for 5 days. The animals were kept in a temperature-controlled environment at 22°C to 24°C. The humidity was set at 50%, and the room was maintained on a 12-hour light-dark cycle (light, 08:00-20:00; dark, 20:00-08:00).
2.3.2. Preparation of BBMVs from the mucosae of the small intestines of rats Small intestinal BBMVs were prepared using a modified version of the methods of Kesseler et al [15]. The BBMVs obtained were suspended in an adequate volume of 50 mM phosphate buffer (pH 6.0) and stored at −80°C until the assay.
2.3.3. Inhibition experiments and determination of the inhibitory mode of sucrase and maltase The assay of hydrolyzing activity to observe inhibition was carried out according to the method of Oku et al [16], which partially modified Dahlqvist's method of using glucose oxidase [17]. Glucose was used as a standard. Substrates of sucrose and maltose were prepared at 112 mM in 0.1 M maleate-Na buffer (pH 6.0). Diluted BBMVs (0.1 mL) were pipetted into a small glass test tube. Then, 0.1 mL of 112 mM substrate was added to the test tube after preincubation for a few minutes at 37°C. After incubation for 10 to 30 minutes at 37°C, 2.4 mL of TGO reagent (containing 10 U/mL glucose oxidase, 5 U/mL peroxidase, 0.05 mM 4-aminoantipyrine, and 0.4 mM p-phenolsulfonic acid sodium salt in 0.5 M Tris-HCl [pH 7.0]) was added to the test tube to stop hydrolysis. The reactions by glucose oxidase and peroxidase were started simultaneously. The reaction mixture was incubated for an additional 10 minutes at 37°C. Then, 2 drops of 4 N NaOH were added to the test tube to stop the reactions. Optical absorbance was read at 500 nm, using a spectrophotometer (UV1240; Shimadzu, Kyoto, Japan).
N U T RI TI O N RE S E ARCH 3 4 ( 2 0 14 ) 9 6 1–9 67
To compare the degrees of inhibition for the disaccharidases in rat tissues, 20 μL of either 9.1 mM or 18.2 mM of one of the following was added to the reaction medium in a duplicate assay: D- or L-sorbose, L-arabinose, D-tagatose, D-erythritol, D-xylitol, or D-arabitol was added to the reaction medium in a duplicate assay. The same volume of distilled water was added as a control. Before the assay, preliminary studies were carried out to evaluate the appropriate concentration of inhibitors. To determine the mode of inhibition and the enzymeinhibition constant (Ki), 20 μL of 1.4 mM and 2.3 mM of D-sorbose, 18.2 mM of L-sorbose, and distilled water were added to the reaction medium. The concentration of substrate was prepared from 0 to 150 mM. The assay procedure was identical to that mentioned above. We determined the mode of inhibition and calculated the Ki values of D- and L-sorbose, using Lineweaver-Burk plots. The concentrations of D- and L-sorbose that we chose to use were based on our preliminary experiments.
2.4. Determination of inhibition by D- and L-sorbose of the activity of sucrase and maltase of human and rat small intestinal homogenates
1 mL of 112 mM maltose in 0.1 M maleate-Na buffer (pH 6.0) was added into the glass test tube, after preincubation for a few minutes at 37°C. The assay procedure was identical to that mentioned above.
2.5. Determination of protein concentration in BBMVs and homogenates Protein concentrations of BBMVs and homogenates were determined using the Bradford method [18].
2.6. Suppressive effect of D- and L-sorbose on elevation of plasma levels of glucose and insulin by sucrose administration in rats 2.6.1.
Preparation of human small intestinal homogenates
Five patients from Juzenkai Hospital donated fragments of human small intestines without malignant tissues. Patients provided written informed consent to donate these fragments of small intestine after resection of tumorous tissue in the small intestine. Immediately after resection, tissue fragments were soaked in ice-cold 0.9% NaCl and transferred to our laboratory. Fragments without lesions were cut and washed with ice-cold 0.9% NaCl. Three fragments were part of the lower ileum, 1 was the lower jejunum, and 1 was the upper jejunum. The mucosa was gently scraped-off with 2 slide glasses onto an ice-chilled glass plate, and then it was homogenized in a 19-fold volume of ice-cold 0.9% NaCl using a homogenizer (Polytron; Kinematica, Inc, Lucerne, Switzerland). It was stored at −80°C until use.
Calculations and statistical analyses
Preparation of rat small intestinal homogenates
For the preparation of the small intestinal homogenates, 5 male Wistar rats (200 g; Clea Japan, Inc) were fed a standard solid diet (MF diets; Oriental Yeast Co) and distilled water ad libitum for 7 days. The condition in the animal quarters was identical to that mentioned above. After an overnight fast, 5 rats were euthanized by decapitation. Small intestines from the duodenum to the ileum were immediately removed, slit open, and washed with ice-cold 0.9% NaCl. The mucosa was scraped off with slide glasses on ice-chilled glass plates, weighed, and homogenized in ice-cold 0.9% NaCl (10% wet wt/vol) using a homogenizer (Polytron; Kinematica, Inc). All homogenates were stored at −80°C until the assay. Before the assay, they were rehomogenized and diluted to appropriate concentrations.
2.4.3.
Oral administration of test substance to rats
After an overnight fast, rats were administered 1.5 mL of test solutions using a gastric tube, as based on our previous studies [9,19]. Test solutions were 0.3 g of sucrose per milliliter, 0.3 g of sucrose and 0.033 g of D-sorbose per milliliter, and 0.3 g of sucrose and 0.033 g of L-sorbose per milliliter. Blood (120 μL) was collected from the tail vein with 2 heparinized hematocrit tubes before and at 30-minute intervals for 3 hours after administration of test solutions. Plasma was obtained by centrifugation at 11 000 rpm for 5 minutes at room temperature, using a specific centrifuge for hematocrit tubes (3110; Kubota, Tokyo, Japan). Plasma levels of glucose and insulin were measured by Trinder's method using glucose oxidase [20] and enzyme-linked immunosorbent assay methods [21], respectively.
2.7. 2.4.2.
Animals
To observe the suppressive effect of D- and L-sorbose on increments in plasma levels of glucose and insulin by sucrose administration in vivo, 6 male Wistar rats (200-220 g body weight) were fed a standard solid diet (MF diets; Oriental Yeast Co) and distilled water ad libitum for 7 days. They were housed for 7 days under the conditions mentioned above.
2.6.2. 2.4.1.
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Inhibition experiments on sucrase and maltase
The adequately diluted homogenate (0.1 mL) was pipetted into a small glass test tube. Then, 0.1 mL of 112 mM sucrose or
The activity of disaccharidases using rat intestinal BBMVs was calculated as specific activity (micromoles of substrate hydrolyzed per milligram protein per hour). We calculated the mean and SD values in duplicate assays and then compared them among inhibitors with the same concentration using analysis of variance (ANOVA) and Dunnett post hoc test vs control. The Ki values of D- and L-sorbose were calculated using Lineweaver-Burk plots. To compare the inhibition of sucrase and maltase between D- and L-sorbose in rat and human homogenates, we calculated the specific activity, mean value, SD value, and percentage of remaining activity by inhibition of D- and L-sorbose vs control. After this, inhibition was compared between D- and L-sorbose by ANOVA and Dunnett post hoc test vs control. In the in vivo experiments, differences in the plasma levels of glucose and insulin in 6 rats were compared at each time point using ANOVA and Tukey post hoc test. P < .05 was considered significant. Analyses were conducted using SPSS version 20.0 (IBM, New York, NY, USA).
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3.
Results
3.1. Inhibition by D- and L-sorbose of disaccharidase activity in the BBMVs of the small intestines of rats Using BBMVs in the small intestines of rats, Fig. 1 shows the inhibitory effect on the activity of sucrase and maltase by Dand L-sorbose, L-arabinose, D-tagatose, D-erythritol, D-xylitol, and D-arabitol. D-Sorbose, L-arabinose, and D-tagatose inhibited sucrase activity down to more than 50% at 9.1 mM, in comparison with that of control. Conversely, inhibition by L-sorbose was only 10%, and inhibition of sugar alcohols such as D-erythritol, D-xylitol, and D-arabitol was not that distinctive. In comparison with that of sucrose, D-sorbose at the same concentration showed weak inhibition of maltase activity, with inhibition at ≈36%. Inhibition by L-sorbose of maltase activity was less than 10%. L-Arabinose also showed weak inhibition, and the inhibition by D-tagatose and sugar alcohols of maltase activity was negligible.
3.2. Inhibitory mode of action by D- and L-sorbose of sucrase and maltase using BBMVs in the small intestine of rats
Specific Activity (µ moles substrate hydrolyzed/mg protein/hr)
Fig. 2 shows the inhibitory mode of action of D- and L-sorbose on sucrase in small intestinal BBMVs of rats using Lineweaver-Burk
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Sucrase
plots. The inhibitory mode of action of D-sorbose on sucrase was uncompetitive because the inhibition line was parallel to the noninhibition line, whereas that of L-sorbose was competitive because the inhibition line crossed to the noninhibition line on the y-axis. In addition, the inhibitory mode of action by D-sorbose on maltase was uncompetitive (data not shown). The Ki values of D- and L-sorbose, L-arabinose, and D-tagatose, which inhibited sucrose activity, are summarized in the Table. The Ki was calculated from the Lineweaver-Burk equation. The Ki of D-sorbose was 7.5 mM and was much stronger than that of L-sorbose (68.0 mM). The Ki of L-arabinose was the smallest overall (2.6 mM), and the Ki of D-tagatose was 16.5 mM. The Ki of D-sorbose for maltase was 18.6 mM, and it was larger than that for sucrase (7.5 mM).
3.3. Comparison of the inhibitory effect between D- and L -sorbose on the activity of small intestinal disaccharidases in humans and rats Fig. 3 shows the inhibitory effect of D- and L-sorbose on the activity of sucrase and maltase in human and rat small intestinal homogenates. D-Sorbose inhibited sucrase activity in humans by ≈60% and that of rats by 70%. Conversely, L-sorbose only inhibited sucrase activity in humans and rats only by a few percent. In humans and rats, D-sorbose showed a weak inhibition of maltase activity (≈20%), in comparison with that of sucrase activity. Inhibition by L-sorbose of maltase activity was weaker (
