Transcript スライド 1 - 北海道大学

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
(1) Introduction
(4) ResultⅡ: Detection of phosphorylated insulin receptor and IRS-1
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 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-Oneohesperidoside, 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
■Insulin receptor detection
(kDa) ① ② ③ ④
250
150
100
⑥ (kDa)
⑤
③
④
⑤
⑥
75
50
Insulin receptor
Anti insulin receptor β antibody
(Cell Signaling Technology)
antibody
②
250
150
100
75
50
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).
①
Phosphorylated insulin receptor
anti phosphotyrosine antibody
(MILLIPORE)
■IRS-1 detection
(2) Research purpose
(kDa)
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.
Insulin
Insulin receptor
P
IRS-1
PI3K
PKB
Glucose
⑤
⑥
(kDa) ①
250
150
100
75
50
50
IRS-1
Skeletal muscle cell
②
③
④
⑤
⑥
180
160
140
120
100
80
60
40
20
0
■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
Insulin receptor
antibody Anti insulin receptor
β antibody
IRS-1
Anti IRS-1
antibody
L6 cell
SDS-PAGE
Western blotting
※cell lysis buffer
50 mM Tris / HCl pH 7.5
150 mM NaCl
1 mM EDTA
1 mM NaF
1 mM Na3VO4
1 mM PMSF
TritonX-100 (1.5%)
Protease Inhibitor Cocktail
(5) Result Ⅲ: Inhibition of neohesperidose’s insulin mimetic activity with
several inhibitors
Neohesperidose showed insulin mimetic activity against L6 cell and C2C12 cell.
■Insulin mimetic activity assay of skeletal muscle cells
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
Anti IRS-1 antibody
(Cell Signaling Technology)
Phosphorylated IRS-1
anti phosphotyrosine antibody
(MILLIPORE)
Phosphorylation of insulin receptor or IRS-1 was not observed after stimulation
by neohesperidose.
2-DG uptake (% of control)
2-DG uptake (% of control)
2-DG uptake (% of control)
④
GLUT4
(3) ResultⅠ: Insulin mimetic activity of neohesperidose
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
③
GLUT4
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.
C2C12 cell
②
250
150
100
75
antibody
180
160
140
120
100
80
60
40
20
0
①
140
120
100
80
60
40
20
0
C2C12 cell
LY294002 : PI3K inhibitor
dorsomorphin : AMPK inhibitor
GW9662 : PPARγ inhibitor
2 - deoxyglucose
Neohesperidose’s insulin mimetic activity was partially inhibited by three
inhibitors related to different signaling pathway.
2 – deoxyglucose – 6P
(6) Summary
Ⅰ Insulin mimetic activity of neohesperidose was observed in L6 and
C2C12 cells.
Ⅱ Phosphorylation of insulin receptor or IRS-1 was not observed
after stimulation by neohesperidose.
EX : 530nm
EM : 590nm
Yamamoto N et al. Anal Biochem,
351, 139–145 (2006).
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.