1. STUDY ON SIGNAL TRANSDUCTION PATHWAY RESPONSIBLE FOR THE INSULIN MIMETIC ACTIVITY OF NEOHESPERIDOSE Taro Kimura, Eisuke Kato, Jun Kawabata Laboratory of Food Biochemistry, Division of Applied Bioscience, Graduate School of Agriculture, Hokkaido University, Kita-ku, Sapporo, Hokkaido 060-8589, Japan (5) Result Ⅲ : Inhibition of neohesperidose’s insulin mimetic activity with several inhibitors (2) (2) Research purpose (6) Summary Ⅰ Insulin mimetic activity of neohesperidose was observed in L6 and C2C12 cells. (4) Result Ⅱ : Detection of phosphorylated insulin receptor and IRS-1 Ⅱ Phosphorylation of insulin receptor or IRS-1 was not observed after stimulation by neohesperidose. (1) Introduction (1) Introduction Insulin mimetic activity is the ability to enhance glucose uptake from vessel to skeletal muscle cell which leads to lowering of blood suger level. This activity is useful for treating diabetes mellitus patients. We have previously focused on the insulin mimetic activity of kaempferol 3-O- neohesperidoside, which shows strong activity in nano molar range. 1 And the Structure Activity Relationship study revealed that the disaccharide neohesperidose is the key compound responsible for insulin mimetic activity. 2 (3) Result Ⅰ : Insulin mimetic activity of neohesperidose Yamamoto N et al. Anal Biochem, 351, 139–145 (2006). ■ Insulin mimetic activity assay of skeletal muscle cells C2C12 cell culture ↓ differentiation for 7 or 8days (in DMEM (+2% Horse Serum)) ↓ stimulation with insulin or samples for 1 hou ↓ incubating with 2-deoxy glucose for 30min ↓ rinse with KRPH buffer ↓ cell lyses ↓ freeze and thaw ↓ quantitation of 2-deoxy glucose Although called “insulin mimetic activity”, how neohesperidose induces its activity is not apparent. In this study, we tested the action of neohesperidose against L6 skeletal cell and C2C12 cell and then checked the phosphorylation state of IR and IRS-1 to see if insulin signaling pathway is concerned. Also, since there are altenative pathways related to glucose uptake, we tested several inhibitors to see their concernings. IRS-1 PI3K Insulin receptor Glucose GLUT4 Skeletal muscle cell PKB Insulin GLUT4 P 250 150 100 75 50 (kDa) ① ： Control (without stimulation) ② ： L6 cell was stimulated by 100 nM insulin(+) for 10min ③～⑥ ： L6 cell was stimulated by 0.1 nM neohesperidose(+) for 1, 2, 3, 4h Insulin receptorPhosphorylated insulin receptor antibody Anti insulin receptor β antibody (Cell Signaling Technology) anti phosphotyrosine antibody (MILLIPORE) Phosphorylation of insulin receptor or IRS-1 was not observed after stimulation by neohesperidose. ① ②③④ ⑤⑥ 250 150 100 75 50 (kDa) IRS-1 Phosphorylated IRS-1 antibody Anti IRS-1 antibody (Cell Signaling Technology) anti phosphotyrosine antibody (MILLIPORE) Neohesperidose showed insulin mimetic activity against L6 cell and C2C12 cell. Insulin is known to activate insulin receptor on cellular membrane by phosphorylation. And the signal is conducted through IRS-1, PI3K, PKB, and then GLUT4 (Glucose transporter 4) localized at endoplasmic reticulum migrates to cell surface and starts absorbing extracellular glucose into the cell. L6 cell culture ↓ differentiation for 8-12days (in DMEM (+2% FBS)) ↓ stimulation with insulin or samples for 4 hours ↓ incubating with 2-deoxy glucose for 30min ↓ rinse with KRPH buffer ↓ cell lyses ↓ freeze and thaw ↓ quantitation of 2-deoxy glucose ■ Method for detection of phosphorylation Cell lysate preparation ↓ stimulation with 100 nM insulin for 10 min or 0.1 nM neohesperidose for 1, 2, 3, 4h ↓ rinse with ice-cold PBS buffer ↓ incubation on ice with cell lysis buffer ※ ↓ scrape cells off the dish Immunoprecipitation SDS-PAGE Western blotting Insulin receptorIRS-1 antibodyAnti insulin receptor β antibody Anti IRS-1 antibody ※ cell lysis buffer 50 mM Tris / HCl pH 7.5 150 mM NaCl 1 mM EDTA 1 mM NaF 1 mM Na 3 VO 4 1 mM PMSF TritonX-100 (1.5%) Protease Inhibitor Cocktail ■ Insulin receptor detection ■ IRS-1 detection 1) Zanatta L, Rosso A, Folador P, Figueiredo MS, Pizzolatti MG, Leite LD, Silva FR, J. Nat. Prod., 71, 532–535 (2008). 2) Yamasaki K, Hishiki R, Kato E, Kawabata J, ACS Med. Chem. Lett., 2, 17-21 (2011). EX : 530nm EM : 590nm 250 150 100 75 50 (kDa) ① ②③④ ⑤⑥ ① ②③④ ⑤⑥ 250 150 100 75 50 (kDa) ① ②③④ ⑤⑥ L6 cellC2C12 cell 2 - deoxyglucose C2C12 cell LY294002 : PI3K inhibitor dorsomorphin : AMPK inhibitor GW9662 : PPARγ inhibitor Neohesperidose’s insulin mimetic activity was partially inhibited by three inhibitors related to different signaling pathway. Ⅲ Neohesperidose’s insulin mimetic activity was inhibited by three inhibitors related to different signaling pathway. Neohesperidose’s activity seems to be related to several pathways. 2 – deoxyglucose – 6P