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Abstract
This study was conducted to evaluate the antihyperglycemic effects of three phytoestrogens, genistein, coumestrol, and enterolactone, in type 2 diabetic animals. Forty male C57BL/KsOlaHsd-db/db mice were used as a diabetic animal model. The animals were divided into four groups and fed a phytoestrogen-free AIN-76 diet (control), or one of three phytoestrogen-supplemented (3.75 mg/100 g diet) AIN-76 diets for six weeks. During the experimental period, fasting blood glucose levels were measured on week 0, 2, 5, and 6 of the experiment, and oral glucose tolerance tests were performed on the 5th week. After the experimental period, blood concentrations of HbA1c, insulin, and glucagon were measured, and hepatic glycogen content and glucose regulating enzyme activities were analyzed. Fasting blood glucose, HbA1c level, and the area under the blood glucose curve in the oral glucose tolerance test were significantly lower in all of the phytoestrogen-supplemented groups compared to the control group. Plasma glucagon levels were also significantly lower in all of the phytoestrogen-supplemented groups compared to the control group. Hepatic glycogen level was significantly higher in the coumestrol-supplemented group compared to the other groups. However, there were no significant differences in the activities of glucokinase and glucose-6-phosphatase between the groups. These results suggest that all of the three major phytoestrogens tested in the present study were effective in lowering blood glucose levels in type 2 diabetic animals. However, further studies need to be conducted to elucidate the exact mechanism for the hypoglycemic effects of phytoestrogens.
Figures and Tables
 | Fig. 1Blood glucose curves in oral glucose tolerance test in C57BL/KsOlaHsd-db/db mice. At the 5th week of the experimental period, the db/db mice were administered with glucose solution (0.1 g/100 g B.W) and the plasma glucose was determined at 0, 30, 60, 120 min after glucose load. DC: Diabetic control, GS: Genistein (3.75 mg/100 g diet), CS: Coumesterol (3.75 mg/100 g diet), EL: Enterolactone (3.75 mg/100 g diet). Values are mean ± S.E. Different letters on the top of the vertical bars indicate significant difference (p < 0.05) among 4 groups by Duncan's multiple comparison test.
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 | Fig. 2The effect of phytoestrogen supplementation on HbA1c level in C57BL/KsOlaHsd-db/db mice. The glycosylated hemoglobin values (HbA1c) are expressed as % of total hemoglobin. DC: Diabetic control, GS: Genistein (3.75 mg/100 g diet), CS: Cou-mesterol (3.75 mg/100 g diet), EL: Enterolactone (3.75 mg/100 g diet). Values are means with their standard errors indicated by vertical bars. Different letters on the top of the bars indicate significant difference (p < 0.05) among 4 groups by Duncan's multiple comparison test.
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 | Fig. 3The effect of phytoestrogen supplementation on hepatic glycogen level in C57BL/KsOlaHsd-db/db mice. DC: Diabetic control, GS: Genistein (3.75 mg/100 g diet), CS: Coumesterol (3.75 mg/100 g diet), EL: Enterolactone (3.75 mg/100 g diet).Values are means with their standard errors indicated by vertical bars. Different letters on the top of the bars indicate significant difference (p < 0.05) among 4 groups by Duncan's multiple comparison test.
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 | Fig. 4The effect of phytoestrogen supplementation on renal Nε-(carboxymethyl) lysine level in C57BL/KsOlaHsd-db/db mice. DC: Diabetic control, GS: Genistein (3.75 mg/100 g diet), CS: Coumesterol (3.75 mg/100 g diet), EL: Enterolactone (3.75 mg/100 g diet). CML: Nε-(carboxymethyl) lysine. Values are means with their standard errors indicated by vertical bars. 'ns' on the top of the bars indicates no significant difference (p < 0.05) among 4 groups by Duncan's multiple comparison test.
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Table 1
Composition of experimental diets
1) AIN-76 vitamin mix (g/kg mix): thiamin HCl 0.6, riboflavin 0.6, pyridoxine HCl 0.7, nicotinic acid 3, D-calcium pantothenate 1.6, folic acid 0.2, cyanocobalamin 0.001, retinyl palmitate 0.8 (500,000 IU/g), DL-α-tocopheryl acetate 20 (2,500 IU/g), cholecalciferol 0.0025, menaquinone 0.005, sucrose to make 1 kg 2) AIN-76 mineral mix (g/kg mix): calcium phosphate dibasic 500, sodium chloride 74, potassium citrate monohydrate 220, potassium sulfate 52, magnesium oxide 24, manganous carbohydrate 3.5, ferric citrate 6, zinc carbonate 1.6, cupric carbonate 0.3, potassium iodate 0.01, sodium selenite 0.01, chromium potassium sulfate 0.55, sucrose to make 1 kg. DC: Diabetic control, GS: Genistein supplemented group, CS: Coumesterol supplemented group, EL: Enterolactone supplemented group
Table 2
The effect of phytoestrogen supplementation on body weight, feed and water intakes in C57BL/KsOlaHsd-db/db mice during 6 weeks of experimental period
Table 3
The effect of phytoestrogen supplementation on fasting blood glucose concentration in C57BL/KsOlaHsd-db/db mice
DC: Diabetic control, GS: Genistein (3.75 mg/100 g diet), CS: Coumesterol (3.75 mg/100 g diet), EL: Enterolactone (3.75 mg/100 g diet), ns: not significantly different among 4 groups (p < 0.05)
Different superscripts in the same column indicate significant difference (p < 0.05) among 4 groups by Duncan's multiple comparison test
*Mean ± S.E.
Table 4
The effect of phytoestrogen supplementation on glucose tolerance in C57BL/KsOlaHsd-db/db mice
DC: Diabetic control, GS: Genistein (3.75 mg/100 g diet), CS: Coumesterol (3.75 mg/100 g diet), EL: Enterolactone (3.75 mg/100 g diet), AUC: Area under the curves in oral glucose tolerance test
Different superscripts in the same column indicate significant difference (p < 0.05) among 4 groups by Duncan's multiple comparison test
*Mean ± S.E.
Table 5
The effect of phytoestrogen supplementation on plasma insulin and glucagon levels in C57BL/KsOlaHsd-db/db mice
DC: Diabetic control, GS: Genistein (3.75 mg/100 g diet), CS: Coumesterol (3.75 mg/100 g diet), EL: Enterolactone (3.75 mg/100 g diet), ns: not significantly different among 4 groups (p < 0.05)
Different superscripts in the same column indicate significant difference (p < 0.05) among 4 groups by Duncan's multiple comparison test
*Mean ± S.E.
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