栎精对葡萄糖-6-磷酸酶基因表达和活性的影响
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摘要
目的:葡萄糖-6-磷酸酶(glucose-6-phosphatase,G-6-Pase)是糖代谢过程中一个重要的酶,由于它是糖异生和糖原分解的最后一步反应的限速酶,因此其基因表达水平及活性的变化直接影响到内生性糖的输出。事实上,2型糖尿病的高血糖同肝脏胰岛素抵抗所致肝糖过度输出有着紧密的关系。因此G-6-Pase是糖尿病治疗的一个潜在靶点。本实验旨在研究栎精,G-6-Pase的一种新型抑制剂,对该酶基因表达和活性的影响,为进一步探讨利用该酶的抑制剂作为糖尿病的治疗手段提供理论依据和实验基础。
方法:以胶原酶原位灌注消化的方法分离大鼠肝细胞,用含10%FBS的1640培养液进行原代培养,利用抗大鼠CK-18作为一抗,采用免疫组化方法对细胞进行鉴定。待细胞贴壁换用新鲜的含诱导剂和不同浓度抑制剂的培养液培养16小时后:1)提取细胞总RNA,采用半定量RT-PCR的方法测定G-6-pase两个亚基的mRNA的丰度;2)利用葡萄糖脱氢酶偶联反应测定酶的活性。
结果:1.采用以胶原酶原位灌注消化的方法分离的肝细胞产率及活率均比较高。细胞活率可达到85%以上。
2.免疫组化染色结果呈阳性着色(有阴性对照)。
3.大鼠肝细胞在高糖培养液(25mM Glu)中培养16小时后,G-6-Pase催化亚基和转运亚基mRNA的丰度和酶活性均明显增加。(P<0.05)
4.加入不同浓度的栎精与高糖联合培养16小时后,G-6-Pase的两个亚基的mRNA的丰度均下降(P<0.05),但没有呈现剂量依赖性的变化。
5.酶活性测定的结果与基因水平的结果相一致,加入栎精后,酶活性下降(P<0.05)。
Objective:Glucose-6-phosphatase (G-6-Pase) is an important enzyme in glucose metabolism. As a rate-limiting enzyme of the last reaction of glyconeogenesis and glycogenolysis, the change of G-6-Pase in gene expression and activity will affect the output of endogenic glucose directly. Infact, insulin resistance in liver contributes to the excessive hepatic output of glucose, which is highly correlated with hyperglycemia in type 2 diabetes. So, G-6-Pase is a potent therapeutic molecular target of diabetes. The purpose of this experiment is to study the effect of quercetin on G-6-Pase gene expression and activity and provide some theoretic and exprimental basement for developing the new type medicine for DM. Methods:The hepatocytes were isolated from adult Wistar rat by perfusion of the liver with collagenase . The isolated cells were cultured in 1640 with 10% FBS. We select anti-rat CK-18 as primary antibody to identify these cells by immunohistochemistry. After cell attachment ,the hepatocytes were incubated with 25mM glucose and different concentration of quercetin for 16 hours, then 1) total RNA was extracted and levels of mRNA expression of P36 and P46 were measured by RT-PCR;2) the enzymatic activity was detected by glucose dehydrogenase-coupled reaction. Result:1. The yield and the percentage viability of the isolated cell by perfusion of the liver with collagenase are good. The percentage viability of the isolated cell is about 85%.2. The result of immunohistochemistry is positive(with negative control).
3. After cultured with 25mM glucose for 16 huors, both mRNA of P36 and P46 and activity of G-6-Pase of rat hepatocyte increased significantly (p<0.05).4. Afrer incubated in medium cantaining different concentration of quercetinand glucose , the mRNA of two summits of G-6-Pase decreased (P<0.05), but it wasn't decreased depending on the concentration of quercetin.5. Quercetin can decrease the activity of G-6-Pase (p<0.05), which is concordant with the result of gene level.Conclusion:It has a good yield and percentage viability of the isolated cell by perfusion of the liver with collagenase. Both the mRNA level of the main component of G-6-Pase P36 and p46 increased in different extent after cultured with glucose, which accord with our design. Different concentration of quercetin can inhibit the mRNA of P36 and P46, but it isn't decreased depend on the concentration of quercetin. Quercetin can also inhibit the activity of G-6-pase (p<0.05),which is concordant with the result of gene level.
方法:以胶原酶原位灌注消化的方法分离大鼠肝细胞,用含10%FBS的1640培养液进行原代培养,利用抗大鼠CK-18作为一抗,采用免疫组化方法对细胞进行鉴定。待细胞贴壁换用新鲜的含诱导剂和不同浓度抑制剂的培养液培养16小时后:1)提取细胞总RNA,采用半定量RT-PCR的方法测定G-6-pase两个亚基的mRNA的丰度;2)利用葡萄糖脱氢酶偶联反应测定酶的活性。
结果:1.采用以胶原酶原位灌注消化的方法分离的肝细胞产率及活率均比较高。细胞活率可达到85%以上。
2.免疫组化染色结果呈阳性着色(有阴性对照)。
3.大鼠肝细胞在高糖培养液(25mM Glu)中培养16小时后,G-6-Pase催化亚基和转运亚基mRNA的丰度和酶活性均明显增加。(P<0.05)
4.加入不同浓度的栎精与高糖联合培养16小时后,G-6-Pase的两个亚基的mRNA的丰度均下降(P<0.05),但没有呈现剂量依赖性的变化。
5.酶活性测定的结果与基因水平的结果相一致,加入栎精后,酶活性下降(P<0.05)。
Objective:Glucose-6-phosphatase (G-6-Pase) is an important enzyme in glucose metabolism. As a rate-limiting enzyme of the last reaction of glyconeogenesis and glycogenolysis, the change of G-6-Pase in gene expression and activity will affect the output of endogenic glucose directly. Infact, insulin resistance in liver contributes to the excessive hepatic output of glucose, which is highly correlated with hyperglycemia in type 2 diabetes. So, G-6-Pase is a potent therapeutic molecular target of diabetes. The purpose of this experiment is to study the effect of quercetin on G-6-Pase gene expression and activity and provide some theoretic and exprimental basement for developing the new type medicine for DM. Methods:The hepatocytes were isolated from adult Wistar rat by perfusion of the liver with collagenase . The isolated cells were cultured in 1640 with 10% FBS. We select anti-rat CK-18 as primary antibody to identify these cells by immunohistochemistry. After cell attachment ,the hepatocytes were incubated with 25mM glucose and different concentration of quercetin for 16 hours, then 1) total RNA was extracted and levels of mRNA expression of P36 and P46 were measured by RT-PCR;2) the enzymatic activity was detected by glucose dehydrogenase-coupled reaction. Result:1. The yield and the percentage viability of the isolated cell by perfusion of the liver with collagenase are good. The percentage viability of the isolated cell is about 85%.2. The result of immunohistochemistry is positive(with negative control).
3. After cultured with 25mM glucose for 16 huors, both mRNA of P36 and P46 and activity of G-6-Pase of rat hepatocyte increased significantly (p<0.05).4. Afrer incubated in medium cantaining different concentration of quercetinand glucose , the mRNA of two summits of G-6-Pase decreased (P<0.05), but it wasn't decreased depending on the concentration of quercetin.5. Quercetin can decrease the activity of G-6-Pase (p<0.05), which is concordant with the result of gene level.Conclusion:It has a good yield and percentage viability of the isolated cell by perfusion of the liver with collagenase. Both the mRNA level of the main component of G-6-Pase P36 and p46 increased in different extent after cultured with glucose, which accord with our design. Different concentration of quercetin can inhibit the mRNA of P36 and P46, but it isn't decreased depend on the concentration of quercetin. Quercetin can also inhibit the activity of G-6-pase (p<0.05),which is concordant with the result of gene level.
引文
1.司徒镇强,主编.细胞培养,世界图书出版社,1996,98-100
2.鄂征,主编.组织培养与分子细胞学技术,北京出版社,1995,83—97
3. Nam-on ku, Jiao Liao, and M. Bishr Omary. Phosphorylation of human keratin 18 serine 33 regulates binding to 14-3-3 proteins. EMBO, 1998, 17:1892
4.张蒙,苏映军,陈壁等.大鼠肝细胞原代长期培养的形态学观察.第四军医大学学报,1995,16(6):418
5. Martinez-Florez S, Gutierrez-Fernandez B. Quercetin attenuates nuclear factor-kappaB activation and nitric oxide production in interleukin-lbeta-activated rat hepatocytes. J Nutr, 2005,135:1359
6. JAMES D. FOSTER AND ROBERT C. NORDLIE. The Biochemistry and Molecular Biology of the Glucose-6-Phosphatase System. Experiment biology and medicine 2002;227:601-608
7. Van Sehaftingen E, Gerin I. glucose-6-phosphatase system. Biochem J, 2002, 362: 513. 532
8. Lei KJ, Pan CJ. Structure-function analysis of human glucose-6-phosphatase, the enzyme deficient in glycogen storage disease type la. J Biol Chem. 1995, 19: 270(20):11882-6.
9. Hemrika W,Wever R.A new model for the membrane topology of glucose-6-phosphatase: the enzyme involved in von Gierke disease. FEBS Left. 1997, 16;409(3):317-9
10. Ness GC, Sukalski KA. et al. Radiation inactivation analysis of rat liver microsomal glucose-6-phosphatase. J Biol Chem. 1989, 5;264(13):7111-4.
11. Lei KJ, Shelly LL. Mutations in the glucose-6-phosphatase gene that cause glycogen storage disease type la. Science. 1993, 22;262(5133):580-3.
12. Lange, A.J.,Argaud, D. Isolation of a cDNA for the catalytic subunit of rat liver glucose-6-phosphatase: regulation of gene expression in FAO hepatoma cells by insulin, dexamethasone and cAMP. Biochem Biophys Res Commun. 1994, 30;201(1):302-9.
13. Lei KJ, Pan CJ.Identification of mutations in the gene for glucose-6-phosphatase, the enzyme deficient in glycogen storage disease type la. J Clin Invest. 1994, 93(5):1994-9.
14. Khan A, Hong-Lie C, Landau BR. Glucose-6-phosphatase activity in islets from ob/ob and lean mice and the effect of dexamethasone. Endocrinology. 1995, 136(5):1934-8.
15. Chatelain F, Pegorier JP. Development and regulation of glucose-6-phosphatase gene expression in rat liver, intestine, and kidney: in vivo and in vitro studies in cultured fetal hepatocytes. Diabetes. 1998, 47(6):882-9.
16. Rajas F, Bruni N, Montano S, et al. The glucose-6 phosphatase gene is expressed in human and rat small intestine: regulation of expression in fasted and diabetic rats. Gastroenterology. 1999, 117(1):132-9.
17. Kishnani PS, Bao Y, Wu JY, et al. Isolation and nucleotide sequence of canine glucose-6-phosphatase mRNA: identification of mutation in puppies with glycogen storage disease type Ia. Biochem Mol Med. 1997, 61(2):168-77.
18. Marger MD, Saier MH Jr.A major superfamily of transmembrane facilitators that catalyse uniport, symport and antiport. Trends Biochem Sci. 1993, 18(1):13-20.
19. Gerin I, Veiga-da-Cunha M, Achouri Y, Collet JF, Van Schaftingen E. Sequence of a putative glucose 6-phosphate translocase, mutated in glycogen storage disease type Ib. FEBS Lett. 1997, 419(2-3):235-8.
20. Pan CJ, Lei KJ, Annabi B, Hemrika W, Chou JY. Transmembrane topology of glucose-6-phosphatase. J Biol Chem. 1998, 273(11):6144-8
21. Hiraiwa H, Pan CJ, Lin B, Moses SW, Chou JY. Inactivation of the glucose 6-phosphate transporter causes glycogen storage disease type 1b. J Biol Chem. 1999, 274(9):5532-6.
22. van de Werve G, Lange A, Newgard C, Mechin MC, Li Y, Berteloot A. New lessons in the regulation of glucose metabolism taught by the glucose 6-phosphatase system. Eur J Biochem. 2000, 267(6):1533-49.
23. Ihara K, Nomura A, Hikino S, Takada H, Hara T. Quantitative analysis of glucose-6-phosphate translocase gene expression in various human tissues and haematopoietic progenitor cells. J Inherit Metab Dis. 2000, 23(6):583-92.
24. Lin B, Annabi B, Hiraiwa H, Pan CJ, Chou JY. Cloning and characterization of cDNAs encoding a candidate glycogen storage disease type 1b protein in rodents. J Biol Chem. 1998, 273(48):31656-60.
25. Gerin I, Veiga-da-Cunha M, Noel G, Van Schaftingen E. Structure of the gene mutated in glycogen storage disease type Ib. Gene. 1999, 227(2):189-95.
26. Fulceri R, Kardon T, Banhegyi G, Pralong WF, Gamberucci A, Marcolongo P, Benedetti A. Glucose-6-phosphatase in the insulin secreting cell line INS-I. Biochem Biophys Res Commun. 2000, 275(1):103-7.
27. Marcolongo P, Barone V, Priori G, Pirola B, Giglio S, Biasucci G, Zammarchi E, Parenti G, Burchell A, Benedetti A, Sorrentino V. Structure and mutation analysis of the glycogen storage disease type 1b gene. FEBS Lett. 1998, 436(2):247-50.
28. Yoshiuchi I, Shingu R, et al. Mutation/polymorphism scanning of glucose-6-phosphatase gene promoter in noninsulin-dependent diabetes mellitus patients. J Clin Endocrinol Metab. 1998, 83(3):1016-9.
29. Fronzo RA, Bonadonna RC, Ferrannini E. Pathogenesis of NIDDM. A balanced overview. Diabetes Care. 1992, 15(3):318-68.
30. Trinh KY, O'Doherty RM, Anderson P, Lange AJ, Newgard CB. Perturbation of fuel homeostasis caused by overexpression of the glucose-6-phosphatase catalytic subunit in liver of normal rats. J Biol Chem. 1998, 273(47):31615-20.
31. Li Y, Mechin MC, van de Werve G. Diabetes affects similarly the catalytic subunit and putative glucose-6-phosphate translocase of glucose-6-phosphatase. J Biol Chem. 1999, 274(48):33866-8.
32. Clore JN, Stillman J, Sugerman H. Glucose-6-phosphatase flux in vitro is increased in type 2 diabetes. Diabetes. 2000, 3un;49(6):969-74.
33. Perfetti R, Barnett PS, Mathur R, Egan JM. Novel therapeutic strategies for the treatment of type 2 diabetes. Diabetes Metab Rev. 1998, 14(3):207-25.
34. Herling A W , Burger H, Schubert G , et al. Alterations of carbohydrate an d lipid interm ediary metabolism during inhi- bition of glucose-6-phosphatnse in rats. Eur J Pharma-col, 1999, 386(1): 75-82
35. Lochhead P A, Coghlan M, Rice S Q, et al. Inhibition of GSK-3 selectively reduces glucose-6-phosphatase and phosphatase and phosphoenolypyruvate carboxykinase gene expression. Diabetes, 2001, 50: 937-946.
36. Westergaard N, Brand CL, Lewinsky RH, Andersen HS, Cart RD, Burchell A, Lundgren K. Peroxyvanadium compounds inhibit glucose-6-phosphatase activity and glucagon-stimulated hepatic glucose output in the rat in vivo. Arch Biochem Biophys. 1999, 366(1):55-60.
37. Westergaard N, Madsen P, et al. Identification of two novel and potent competitive inhibitors of the glucose-6-phosphatase catalytic protein. Diabetes Obes Metab, 2002,4(2): 96-105.
38. Castanas E , Kampa M, Hatzoglou A, Notas G, Oamianaki A, Bakogeorgou E, Gemetzi C, Kouroumalis E, Martin PM. Wine antioxidant polyphenols inhibit the proliferation of human prostate cancer cell lines. Nutr Cancer. 2000;37(2):223-33.
39. Lean ME, Noroozi M, Kelly I, Burns J, Talwar D, Sattar N, Crozier A. Dietary flavonols protect diabetic human lymphocytes against oxidative damage to DNA. Diabetes. 1999, 48(1):176-81.
40. Sanders RA, Rauscher FM, Watkins JB 3rd. Effects of quercetin on antioxidant defense in streptozotocin-induced diabetic rats. J Biochem Mol Toxicol. 2001, 15(3):143-9.
41. Coldiron AD Jr, Sanders RA, Watkins JB 3rd. Effects of combined quercetin and coenzyme Q(IO) treatment on oxidative stress in normal and diabetic rats. J Biochem Mol Toxicol. 2002, 16(4):197-202.
42. Vessal M, Hemmati M, Vasei M. Antidiabetic effects of quercetin in streptozocin-induced diabetic rats. Comp Biochem Physiol C Toxicol Pharmacol. 2003,135C(3):357-64.
43. Anjaneyulu M, Chopra K. Quercetin, an anti-oxidant bioflavonoid, attenuates diabetic nephropathy in rats. Clin Exp Pharmacol Physiol. 2004, 31(4):244-8.
44. Anjaneyulu M, Chopra K. Quercetin, a bioflavonoid, attenuates thermal hyperalgesia in a mouse model of diabetic neuropathic pain. Prog Neuropsychopharmacol Biol Psychiatry. 2003,27(6):1001-5.
45. Gasparin FR, Salgueiro-Pagadigorria CL, Bracht L, Ishii-Iwamoto EL, Bracht A, Constantin J. Action of quercetin on glycogen catabolism in the rat liver. Xenobiotica. 2003, 33(6):587-602.
46. Gasparin FR, Spitzner FL, Ishii-Iwamoto EL, Bracht A, Constantin J. Actions of quercetin on gluconeogenesis and glycolysis in rat liver, ienobiotica. 2003, 33(9):903-11.
47. WP Halford. The essential prerequisites for quantitative RT-PCR. Nat Biotechnol, 1999, 17(9):835
48. Maria hlegre, Carlos J. Ciudad, et al. Determination of Glucose-6-phosphatase Activity using the Glucose Dehydrogenase
?Coupled Reaction. Analitical Biochemistry, 1988, 173:185-189
2.鄂征,主编.组织培养与分子细胞学技术,北京出版社,1995,83—97
3. Nam-on ku, Jiao Liao, and M. Bishr Omary. Phosphorylation of human keratin 18 serine 33 regulates binding to 14-3-3 proteins. EMBO, 1998, 17:1892
4.张蒙,苏映军,陈壁等.大鼠肝细胞原代长期培养的形态学观察.第四军医大学学报,1995,16(6):418
5. Martinez-Florez S, Gutierrez-Fernandez B. Quercetin attenuates nuclear factor-kappaB activation and nitric oxide production in interleukin-lbeta-activated rat hepatocytes. J Nutr, 2005,135:1359
6. JAMES D. FOSTER AND ROBERT C. NORDLIE. The Biochemistry and Molecular Biology of the Glucose-6-Phosphatase System. Experiment biology and medicine 2002;227:601-608
7. Van Sehaftingen E, Gerin I. glucose-6-phosphatase system. Biochem J, 2002, 362: 513. 532
8. Lei KJ, Pan CJ. Structure-function analysis of human glucose-6-phosphatase, the enzyme deficient in glycogen storage disease type la. J Biol Chem. 1995, 19: 270(20):11882-6.
9. Hemrika W,Wever R.A new model for the membrane topology of glucose-6-phosphatase: the enzyme involved in von Gierke disease. FEBS Left. 1997, 16;409(3):317-9
10. Ness GC, Sukalski KA. et al. Radiation inactivation analysis of rat liver microsomal glucose-6-phosphatase. J Biol Chem. 1989, 5;264(13):7111-4.
11. Lei KJ, Shelly LL. Mutations in the glucose-6-phosphatase gene that cause glycogen storage disease type la. Science. 1993, 22;262(5133):580-3.
12. Lange, A.J.,Argaud, D. Isolation of a cDNA for the catalytic subunit of rat liver glucose-6-phosphatase: regulation of gene expression in FAO hepatoma cells by insulin, dexamethasone and cAMP. Biochem Biophys Res Commun. 1994, 30;201(1):302-9.
13. Lei KJ, Pan CJ.Identification of mutations in the gene for glucose-6-phosphatase, the enzyme deficient in glycogen storage disease type la. J Clin Invest. 1994, 93(5):1994-9.
14. Khan A, Hong-Lie C, Landau BR. Glucose-6-phosphatase activity in islets from ob/ob and lean mice and the effect of dexamethasone. Endocrinology. 1995, 136(5):1934-8.
15. Chatelain F, Pegorier JP. Development and regulation of glucose-6-phosphatase gene expression in rat liver, intestine, and kidney: in vivo and in vitro studies in cultured fetal hepatocytes. Diabetes. 1998, 47(6):882-9.
16. Rajas F, Bruni N, Montano S, et al. The glucose-6 phosphatase gene is expressed in human and rat small intestine: regulation of expression in fasted and diabetic rats. Gastroenterology. 1999, 117(1):132-9.
17. Kishnani PS, Bao Y, Wu JY, et al. Isolation and nucleotide sequence of canine glucose-6-phosphatase mRNA: identification of mutation in puppies with glycogen storage disease type Ia. Biochem Mol Med. 1997, 61(2):168-77.
18. Marger MD, Saier MH Jr.A major superfamily of transmembrane facilitators that catalyse uniport, symport and antiport. Trends Biochem Sci. 1993, 18(1):13-20.
19. Gerin I, Veiga-da-Cunha M, Achouri Y, Collet JF, Van Schaftingen E. Sequence of a putative glucose 6-phosphate translocase, mutated in glycogen storage disease type Ib. FEBS Lett. 1997, 419(2-3):235-8.
20. Pan CJ, Lei KJ, Annabi B, Hemrika W, Chou JY. Transmembrane topology of glucose-6-phosphatase. J Biol Chem. 1998, 273(11):6144-8
21. Hiraiwa H, Pan CJ, Lin B, Moses SW, Chou JY. Inactivation of the glucose 6-phosphate transporter causes glycogen storage disease type 1b. J Biol Chem. 1999, 274(9):5532-6.
22. van de Werve G, Lange A, Newgard C, Mechin MC, Li Y, Berteloot A. New lessons in the regulation of glucose metabolism taught by the glucose 6-phosphatase system. Eur J Biochem. 2000, 267(6):1533-49.
23. Ihara K, Nomura A, Hikino S, Takada H, Hara T. Quantitative analysis of glucose-6-phosphate translocase gene expression in various human tissues and haematopoietic progenitor cells. J Inherit Metab Dis. 2000, 23(6):583-92.
24. Lin B, Annabi B, Hiraiwa H, Pan CJ, Chou JY. Cloning and characterization of cDNAs encoding a candidate glycogen storage disease type 1b protein in rodents. J Biol Chem. 1998, 273(48):31656-60.
25. Gerin I, Veiga-da-Cunha M, Noel G, Van Schaftingen E. Structure of the gene mutated in glycogen storage disease type Ib. Gene. 1999, 227(2):189-95.
26. Fulceri R, Kardon T, Banhegyi G, Pralong WF, Gamberucci A, Marcolongo P, Benedetti A. Glucose-6-phosphatase in the insulin secreting cell line INS-I. Biochem Biophys Res Commun. 2000, 275(1):103-7.
27. Marcolongo P, Barone V, Priori G, Pirola B, Giglio S, Biasucci G, Zammarchi E, Parenti G, Burchell A, Benedetti A, Sorrentino V. Structure and mutation analysis of the glycogen storage disease type 1b gene. FEBS Lett. 1998, 436(2):247-50.
28. Yoshiuchi I, Shingu R, et al. Mutation/polymorphism scanning of glucose-6-phosphatase gene promoter in noninsulin-dependent diabetes mellitus patients. J Clin Endocrinol Metab. 1998, 83(3):1016-9.
29. Fronzo RA, Bonadonna RC, Ferrannini E. Pathogenesis of NIDDM. A balanced overview. Diabetes Care. 1992, 15(3):318-68.
30. Trinh KY, O'Doherty RM, Anderson P, Lange AJ, Newgard CB. Perturbation of fuel homeostasis caused by overexpression of the glucose-6-phosphatase catalytic subunit in liver of normal rats. J Biol Chem. 1998, 273(47):31615-20.
31. Li Y, Mechin MC, van de Werve G. Diabetes affects similarly the catalytic subunit and putative glucose-6-phosphate translocase of glucose-6-phosphatase. J Biol Chem. 1999, 274(48):33866-8.
32. Clore JN, Stillman J, Sugerman H. Glucose-6-phosphatase flux in vitro is increased in type 2 diabetes. Diabetes. 2000, 3un;49(6):969-74.
33. Perfetti R, Barnett PS, Mathur R, Egan JM. Novel therapeutic strategies for the treatment of type 2 diabetes. Diabetes Metab Rev. 1998, 14(3):207-25.
34. Herling A W , Burger H, Schubert G , et al. Alterations of carbohydrate an d lipid interm ediary metabolism during inhi- bition of glucose-6-phosphatnse in rats. Eur J Pharma-col, 1999, 386(1): 75-82
35. Lochhead P A, Coghlan M, Rice S Q, et al. Inhibition of GSK-3 selectively reduces glucose-6-phosphatase and phosphatase and phosphoenolypyruvate carboxykinase gene expression. Diabetes, 2001, 50: 937-946.
36. Westergaard N, Brand CL, Lewinsky RH, Andersen HS, Cart RD, Burchell A, Lundgren K. Peroxyvanadium compounds inhibit glucose-6-phosphatase activity and glucagon-stimulated hepatic glucose output in the rat in vivo. Arch Biochem Biophys. 1999, 366(1):55-60.
37. Westergaard N, Madsen P, et al. Identification of two novel and potent competitive inhibitors of the glucose-6-phosphatase catalytic protein. Diabetes Obes Metab, 2002,4(2): 96-105.
38. Castanas E , Kampa M, Hatzoglou A, Notas G, Oamianaki A, Bakogeorgou E, Gemetzi C, Kouroumalis E, Martin PM. Wine antioxidant polyphenols inhibit the proliferation of human prostate cancer cell lines. Nutr Cancer. 2000;37(2):223-33.
39. Lean ME, Noroozi M, Kelly I, Burns J, Talwar D, Sattar N, Crozier A. Dietary flavonols protect diabetic human lymphocytes against oxidative damage to DNA. Diabetes. 1999, 48(1):176-81.
40. Sanders RA, Rauscher FM, Watkins JB 3rd. Effects of quercetin on antioxidant defense in streptozotocin-induced diabetic rats. J Biochem Mol Toxicol. 2001, 15(3):143-9.
41. Coldiron AD Jr, Sanders RA, Watkins JB 3rd. Effects of combined quercetin and coenzyme Q(IO) treatment on oxidative stress in normal and diabetic rats. J Biochem Mol Toxicol. 2002, 16(4):197-202.
42. Vessal M, Hemmati M, Vasei M. Antidiabetic effects of quercetin in streptozocin-induced diabetic rats. Comp Biochem Physiol C Toxicol Pharmacol. 2003,135C(3):357-64.
43. Anjaneyulu M, Chopra K. Quercetin, an anti-oxidant bioflavonoid, attenuates diabetic nephropathy in rats. Clin Exp Pharmacol Physiol. 2004, 31(4):244-8.
44. Anjaneyulu M, Chopra K. Quercetin, a bioflavonoid, attenuates thermal hyperalgesia in a mouse model of diabetic neuropathic pain. Prog Neuropsychopharmacol Biol Psychiatry. 2003,27(6):1001-5.
45. Gasparin FR, Salgueiro-Pagadigorria CL, Bracht L, Ishii-Iwamoto EL, Bracht A, Constantin J. Action of quercetin on glycogen catabolism in the rat liver. Xenobiotica. 2003, 33(6):587-602.
46. Gasparin FR, Spitzner FL, Ishii-Iwamoto EL, Bracht A, Constantin J. Actions of quercetin on gluconeogenesis and glycolysis in rat liver, ienobiotica. 2003, 33(9):903-11.
47. WP Halford. The essential prerequisites for quantitative RT-PCR. Nat Biotechnol, 1999, 17(9):835
48. Maria hlegre, Carlos J. Ciudad, et al. Determination of Glucose-6-phosphatase Activity using the Glucose Dehydrogenase
?Coupled Reaction. Analitical Biochemistry, 1988, 173:185-189