Korean J Nutr.  2011 Aug;44(4):275-283. 10.4163/kjn.2011.44.4.275.

Effects of Phytoestrogens on Glucose Metabolism in C57BL/KsOlaHsd-db/db Mice

• Seo BH
• Kim KO
• Lee JH
• Lee HS

• KMID: 2268543
• DOI: http://doi.org/10.4163/kjn.2011.44.4.275

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.

Keyword

phytoestrogen; diabetes; hypoglycemic effect; db/db mice

MeSH Terms

4-Butyrolactone
Animals
Blood Glucose
Coumestrol
Diet
Fasting
Genistein
Glucagon
Glucokinase
Glucose
Glucose Tolerance Test
Glucose-6-Phosphatase
Humans
Hypoglycemic Agents
Insulin
Lignans
Liver Glycogen
Male
Mice
Models, Animal
Phytoestrogens
Plasma
4-Butyrolactone
Blood Glucose
Coumestrol
Genistein
Glucagon
Glucokinase
Glucose
Glucose-6-Phosphatase
Hypoglycemic Agents
Insulin
Lignans
Liver Glycogen
Phytoestrogens

Figure

• 

  Fig. 1 Blood 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.

• 

  Fig. 2 The 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.

• 

  Fig. 3 The 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.

• 

  Fig. 4 The 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.

Reference

1. Murkies AL, Wilcox G, Davis SR. Clinical review 92: Phytoestrogens. J Clin Endocrinol Metab. 1998. 83(2):297–303.

2. Lissin LW, Cooke JP. Phytoestrogens and cardiovascular health. J Am Coll Cardiol. 2000. 35(6):1403–1410.

3. Ohta N, Kuwata G, Akahori H, Watanabe T. Isolation of a new isoflavone acetyl glucoside, 6"-O-acetyl genistin, from soybeans. Agric Biol Chem. 1980. 44(2):469–470.

4. Setchell KD, Lawson AM, Conway E, Taylor NF, Kirk DN, Cooley G, Farrant RD, Wynn S, Axelson M. The definitive identification of the lignans trans-2,3-bis (3-hydroxybenzyl)-gamma-butyrolactone and 2,3-bis (3-hydroxybenzyl) butane-1,4-diol in human and animal urine. Biochem J. 1981. 197(2):447–458.

5. Shemesh M, Lindner HR, Ayalon N. Affinity of rabbit uterine oestradiol receptor for phyto-oestrogens and its use in a competitive protein-binding radioassay for plasma coumestrol. J Reprod Fertil. 1972. 29(1):1–9.

6. Prasad K. Hypocholesterolemic and antiatherosclerotic effect of flax lignan complex isolated from flaxseed. Atherosclerosis. 2005. 179(2):269–275.

7. de Kleijn MJ, van der Schouw YT, Wilson PW, Grobbee DE, Jacques PF. Dietary intake of phytoestrogens is associated with a favorable metabolic cardiovascular risk profile in postmenopausal U.S. women: the Framingham study. J Nutr. 2002. 132(2):276–282.

8. Kapiotis S, Hermann M, Held I, Seelos C, Ehringer H, Gmeiner BM. Genistein, the dietary-derived angiogenesis inhibitor, prevents LDL oxidation and protects endothelial cells from damage by atherogenic LDL. Arterioscler Thromb Vasc Biol. 1997. 17(11):2868–2874.

9. Chen Y, Wei X, Xie H, Deng H. Antioxidant 2-phenylbenzofurans and a coumestan from Lespedeza virgata. J Nat Prod. 2008. 71(6):929–932.

10. Coward L, Barnes NC, Setchell KDR, Barnes S. Genistein, daidzein, and their β-glycoside conjugates: antitumor isoflavones in soybean foods from American and Asian diets. J Agric Food Chem. 1993. 41(11):1961–1967.

11. Sarkar FH, Li Y. Soy isoflavones and cancer prevention. Cancer Invest. 2003. 21(5):744–757.

12. Lamartiniere CA, Murrill WB, Manzolillo PA, Zhang JX, Barnes S, Zhang X, Wei H, Brown NM. Genistein alters the ontogeny of mammary gland development and protects against chemically-induced mammary cancer in rats. Proc Soc Exp Biol Med. 1998. 217(3):358–364.

13. Ali AA, Velasquez MT, Hansen CT, Mohamed AI, Bhathena SJ. Modulation of carbohydrate metabolism and peptide hormones by soybean isoflavones and probiotics in obesity and diabetes. J Nutr Biochem. 2005. 16(11):693–699.

14. Ohno T, Kato N, Ishii C, Shimizu M, Ito Y, Tomono S, Kawazu S. Genistein augments cyclic adenosine 3'5'-monophosphate (cAMP) accumulation and insulin release in MIN6 cells. Endocr Res. 1993. 19(4):273–285.

15. Sorenson RL, Brelje TC, Roth C. Effect of tyrosine kinase inhibitors on islets of Langerhans: evidence for tyrosine kinases in the regulation of insulin secretion. Endocrinology. 1994. 134(4):1975–1978.

16. Liu D, Zhen W, Yang Z, Carter JD, Si H, Reynolds KA. Genistein acutely stimulates insulin secretion in pancreatic β-cells through a cAMP-dependent protein kinase pathway. Diabetes. 2006. 55(44):1043–1050.

17. Lee JS. Effects of soy protein and genistein on blood glucose, antioxidant enzyme activities, and lipid profile in streptozotocin-induced diabetic rats. Life Sci. 2006. 79(16):1578–1584.

18. Park SA, Kim MJ, Jang JY, Choi MS, Yeo J, Lee MK. Effect of genistein and daidzein on antioxidant defense system in C57BL/KsJ-db/db mice. J Korean Soc Food Sci Nutr. 2006. 35(9):1159–1165.

19. Béguin DP, Kincaid RL. 3-Hydroxy-3-methyl-glutaryl coenzyme A reductase activity in chicks fed coumestrol, a phytoestrogen. Poult Sci. 1984. 63(4):686–690.

20. Banskota AH, Nguyen NT, Tezuka Y, Nobukawa T, Kadota S. Hypoglycemic effects of the wood of Taxus yunnanensis on streptozotocin-induced diabetic rats and its active components. Phytomedicine. 2006. 13(1-2):109–114.

21. Velasquez MT, Bhathena SJ, Ranich T, Schwartz AM, Kardon DE, Ali AA, Haudenschild CC, Hansen CT. Dietary flaxseed meal reduces proteinuria and ameliorates nephropathy in an animal model of type II diabetes mellitus. Kidney Int. 2003. 64(6):2100–2107.

22. Prasad K. Secoisolariciresinol diglucoside from flaxseed delays the development of type 2 diabetes in Zucker rat. J Lab Clin Med. 2001. 138(1):32–39.

23. Shim JY, Kim KO, Seo BH, Lee HS. Soybean isoflavone extract improves glucose tolerance and raises the survival rate in streptozotocin-induced diabetic rats. Nutr Res Pract. 2007. 1(4):266–272.

24. Raba J, Mottola HA. Glucose oxidase as an analytical reagent. Crit Rev Anal Chem. 1995. 25(1):1–42.

25. Seifter S, Dayton S, Novic B, Muntwyler E. The estimation of glycogen with the anthrone reagent. Arch Biochem. 1950. 25(1):191–200.

26. Davidson AL, Arion WJ. Factors underlying significant underestimations of glucokinase activity in crude liver extracts: physiological implications of higher cellular activity. Arch Biochem Biophys. 1987. 253(1):156–167.

27. Swanson MA. Phosphatase of liver. 1. Glucose-6-phosphatase. J Biol Chem. 1950. 184(2):647–659.

28. Zarina S, Zhao HR, Abraham EC. Advanced glycation end products in human senile and diabetic cataractous lenses. Mol Cell Biochem. 2000. 210(1-2):29–34.

29. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976. 72(1-2):248–254.

30. Lee SM, Bustamante S, Flores C, Bezerra J, Goda T, Koldovský O. Chronic effects of an α-glucosidase inhibitor (Bay o 1248) on intestinal disaccharidase activity in normal and diabetic mice. J Pharmacol Exp Ther. 1987. 240(1):132–137.

31. Orland MJ, Permutt MA. Quantitative analysis of pancreatic proinsulin mRNA in genetically diabetic (db/db) mice. Diabetes. 1987. 36(3):341–347.

32. Stancoven A, McGuire DK. Preventing macrovascular complications in type 2 diabetes mellitus: glucose control and beyond. Am J Cardiol. 2007. 99(11A):5H–11H.

33. Gerich JE. Clinical significance, pathogenesis, and management of postprandial hyperglycemia. Arch Intern Med. 2003. 163(11):1306–1316.

34. Hanefeld M, Temelkova-Kurktschiev T. Control of post-prandial hyperglycemia-an essential part of good diabetes treatment and prevention of cardiovascular complications. Nutr Metab Cardiovasc Dis. 2002. 12(2):98–107.

35. Rahbar S. An abnormal hemoglobin in red cells of diabetics. Clin Chim Acta. 1968. 22(2):296–298.

36. Nathan DM, Singer DE, Godine JE, Harrington CH, Perlmuter LC. Retinopathy in older type II diabetics. Association with glucose control. Diabetes. 1986. 35(7):797–801.

37. Hers HG. Mechanisms of blood glucose homeostasis. J Inherit Metab Dis. 1990. 13(4):395–410.

38. Barzilai N, Rossetti L. Role of glucokinase and glucose-6-phosphatase in the acute and chronic regulation of hepatic glucose fluxes by insulin. J Biol Chem. 1993. 268(33):25019–25025.

39. Vedavanam K, Srijayanta S, O'Reilly J, Raman A, Wiseman H. Antioxidant action and potential antidiabetic properties of an isoflavonoid-containing soyabean phytochemical extract (SPE). Phytother Res. 1999. 13(7):601–608.