W9, A MEDICINAL PLANT OF THE EASTERN JAMES

W9, A MEDICINAL PLANT OF THE EASTERN JAMES
BAY CREE, MOBILIZES L6 MUSCLE GLUT4
TRANSPORTERS AND EXERTS ANTI-OBESITY AND
ANTIDIABETIC EFFECTS IN VIVO.
Hoda M. Eid 1,3,4, Antoine Brault 1,3,4, Meriem Ouchfoun 1,3,4, Farah Thong 5,
Diane Vallerand 1,3,4, Riya Ganguly5, John T. Arnason 2,4, Gary Sweeney 5, Pierre S.
Haddad 1,3,4
1
Natural Health Products and Metabolic Diseases Laboratory, Dept. of Pharmacology, Université de
Montréal, Montreal, QC, 2 Phytochemistry, Medicinal Plant and Ethnopharmacology Laboratory, Dept. of
Biology, University of Ottawa, Ottawa, ON, 3 The Institute of Nutraceuticals and Functional Foods (INAF),
4 Canadian Institutes of Health Research Team in Aboriginal Antidiabetic Medicines and Montreal
Diabetes Research Center,5 Dept. of Biology, York University, Toronto, ON
ABSTRACT
W9 has been identified among species used by the Cree of Eeyou Istchee of
northern Quebec to treat symptoms of diabetes. In a previous study, the ethanol
extract of W9 enhanced glucose uptake in C2C12 muscle cells via stimulation of
AMP-activated protein kinase (AMPK) pathway. In this study, we investigated the
effect of this product on the translocation of insulin-sensitive GLUT4 transporters
in skeletal muscle cells in culture. Treatment of L6 myotubes with W9 for 18 h
significantly increased glucose uptake and GLUT4 translocation to the cell
membrane. W9 increased phosphorylation of AMPK and P38 MAPK with no
indication of increased phosphorylation of Akt. To validate the effect of W9 in vivo,
the extract (1% in drinking water) was administered to KKAy mice for 10 days.
Glycemia and fluid intakes were significantly reduced by W9. Moreover, W9treatment increased levels of GLUT4 content in skeletal muscle, stimulated the
phosphorylation of ACC and increased the levels of PPAR-α in the liver of KKAy
mice. Administration of W9 to normal C57BL/6 had no effect on blood glucose
levels. The results of the present study confirm the potential of W9 for the
prevention and treatment of diabetes.
GLUT Family of Proteins
Nature Reviews Molecular Cell Biology 3, 267-277 (April 2002)
Name
Tissue distribution
Insulin
Function
sensitivity
GLUT1
Ubiquitous, erythrocytes and brain
No
Basal glucose transport, transport across blood-brain barrier
GLUT2
Liver, pancreatic β-cells, intestine, kidney
No
Intestinal absorption, renal re-absorption,
pancreatic and hepatic control of glucose homeostasis
GLUT3
Widely distributed in human tissues, restricted to brain
No
in other species. Immune cells.
Glucose transport into neurons in brain, basal transport in many
human cells.
GLUT4
Skeletal muscle, cardiac muscle, adipose tissue
Yes
Insulin-dependent glucose transport.
GLUT5
Intestine, testes, kidney.
No
Fructose transport.
GLUT6
Leucocytes, spleen, brain.
No
n. d.
GLUT7
Apical membrane of small and large intestine
No
Exhibits a low level of transport activity for fructose and
glucose.
GLUT8
Brain, heart
Yes
Role in neuronal proliferation and heart atrial activity.
GLUT9
Liver, kidney, intestine
No
Regulator of uric acid liver.
GLUT10 Liver, pancreas
No
n. d.
GLUT11 Different tissue types
No
Glucose, fructose transport, main substrate has not been
identified.
GLUT12 Heart, prostate, musle, small intestine, WAT
Yes
Glucose homeostasis.
GLUT13 Brain
No
Myoinositol transporter.
GLUT14 Testis
No
Most likely a glucose transporter.
Effect of Insulin on GLUT4
GLUT4 and Type 2 Diabetes
●
GLUT4 is responsible for facilitating the transport of glucose into
the cells in response to insulin.
●
Type 2 diabetes is associated with mutations and reduced
expression of GLUT4. As a result, glucose transport is
significantly impaired.
●
Drugs enhancing GLUT-4 translocation and/ or expression will
provide novel treatments for Type 2 diabetes.
Type 2 Diabetes
●
Type 2 Diabetes is a worldwide epidemic (220 million)
●
Canadian aboriginal populations :



Its prevalence is 3-4 times the canadian average
Its worsened by genetic predisposition, sedintary life and poor
compliance to western Rx
The need for culturally-adapted alternative : plants from their
Traditional Medicine
●
Ethno-botanical study previously identified 17 plants for the
treatment of Type 2 diabetes
●
In a previous study, the ethanol extract of W9 enhanced glucose
uptake in C2C12 muscle cells via stimulation of AMP-activated
protein kinase (AMPK) pathway
OBJECTIVES
●
Evaluate the downstream signalling of W9
●
Study the anti-diabetic activity of W9 in vivo
Figure 1: Insulin and non-insulin dependent pathways
regulating glucose transport
Inhibition
of
mitochondrial
respiration
ACC
Mitochondria
METHODS
In vitro studies
• We used L6 skeletal muscle cells wild type (WT) and transiently
transfected with GLUT4myc to measure :


Glucose uptake : for the specific uptake of 2-deoxy-D- glucose.
GLUT 4 translocation : using antibody-coupled colorimetric assay,
O-phenylenediamine dihydrochloride (OPD), and anti-myc antibody.
• We evaluated downstream signalling using western blot analysis:


Insulin-dependent pathway (phospho-Akt)
Insulin-independent pathway (phospho-AMPK, phospho-p38 MAPK
and phospho-ACC)
METHODS
In vivo studies
Animal models :
• Study # 1 : Diabetic KKAy mice


Group 1 mice received drinking tap water
Group 2 mice were administered with 1% W9 in drinking water
• Study# 2 : Normal C57BL/6J mice


Group 1 mice received drinking tap water
Group 2 mice were administered with 1% W9 in drinking water
Parameters measured :
• Blood parameters :



During study (every 2 days): glycemia and fluid intake were measured
Post sacrifice: toxicity, lipid profile were evaluated
Liver steatosis was evaluated histologically
• Western blot:
PPAR-a
GLUT4
RESULTS
W9 stimulates glucose uptake in L6 rat muscle cells
by inducing GLUT 4 translocation to the membrane
Glucose Uptake
GLUT 4 translocation
(OPD assay)
(radioactive assay)
Fold increaae in GLUT4 translocation
*
3
H-Deoxyglucose Uptake
(% of DMSO)
180
160
140
120
100
80
60
40
20
0
2
(% of DMSO)
*
200
*
*
Insulin
Insulin
W9
V.vitis
1
0
DMSO
DMSO
Insulin
Insulin
* : P<0.05 as compared to DMSO
W9
V.vitis
DMSO
DMSO
W9 stimulates AMPK pathway in L6 muscle cells,
but not the Insulin pathway
AMPK pathway
Insulin pathway
phospho-AMPK
phospho-Akt
phospho-p38 MAPK
b-Actin
b-Actin
W9 significantly decreases glycemia in diabetic KKAy
mice but had no effect in normal lean mice C57BL/6J
KKAy mice
(Study #1)
C57BL/6J mice
(Study #2)
35
Control
14
V.vitis
30
Control
V.vitis
12
20
*
15
10
5
Glycemia (mmol/L)
Glycemia (mmol/L)
25
10
8
6
4
2
0
0
-2
-1
0
1
2
3
4
5
6
7
8
9
10
-2
11
0
1
2
3
4
5
Days
Days
Control
* : P<0.05 as compared to control
-1
W9
6
7
8
9
10
11
W9 significantly decreases fluid intake glycemia in
diabetic KKAy and normal C57BL/6J mice
KKAy mice
(Study #1)
C57BL/6J mice
(Study #2)
300
300
Control
V.vitis
250
200
150
*
*
100
50
*
Cumulative fluid intake (mL)
250
Cumulative fluid intake (mL)
Control
V.vitis
200
150
100
*
50
0
0
-2
-1
0
1
2
3
4
5
6
7
8
9
10
-2
11
0
1
2
3
4
5
Days
Days
Control
* : P<0.05 as compared to control
-1
W9
6
7
8
9
10
11
W9 is not toxic, significantly decreases plasma triglycerides, but only
tends to lower plasma insulin level in KKAy mice from study # 1
Control
W9
AST (IU/L)
107.7 ± 14.5
92.0 ± 11.7
ALT (IU/L)
64.5 ± 8.7
56.0 ± 10.4
LDH (IU/L)
108.8 ± 9.4
71.5 ± 18.0
1280.7 ± 340.2
832.7 ± 219.9
112.4 ± 11.1
77.0 ± 7.9*
Triglycerides (mmol/L)
4.5 ± 0.8
2.9 ± 0.5*
Cholesterol (mmol/L)
2.3 ± 0.14
2.12 ± 0.19
HDL (mmol/L)
1.07 ± 0.09
1.09 ± 0.11
LDL (mmol/L)
0.5 ± 0.2
0.5 ± 0.0
Insulin (ng/mL)
35.32 ± 8.48
18.98 ± 4.22
Leptin (ng/mL)
27.12 ± 1.7
23.9 ± 0.99
Adiponectin (ug/mL)
18.17 ± 2.44
18.00 ± 2.2
Creatinine (IU/L)
Alkaline phosphatase (IU/L)
Treatment of KKAy diabetic mice with W9 improved
liver steatosis significantly
Steatosis$
Groups
0
1
2
3
Control
7
1
1
0
5
W9
7
3
1
0
3
Chi square
$
n
P < 0.05
Liver steatosis was evaluated histologically in control and W9 treated KKAy mice
(Study # 1)
W9 increased the levels of GLUT4 in skeletal muscles
and those of PPAR-α in livers from diabetic KKAy mice
Control
KKAy mice
(Study #1)
W9
GLUT4
PPAR-a
SUMMARY
W9 treatment
●
In vitro studies:




●
Increased glucose transport in L6 skeletal muscle cells to levels
similar to those of insulin.
Increased GLUT4 translocation in L6 muscle cells.
Did not stimulate phosphorylation of Akt (insulino-dependent ).
Increased phosphorylation of AMPK, p38 MAPK and ACC.
In vivo studies:





Decreased glycemia in diabetic KKAy mice (p< 0.05).
Decreased cumulative fluid intake in diabetic KKAy and normal
C57BL/6J (p< 0.05).
Decreased plasma triglyceride levels by 36% (p< 0.05).
Attenuated liver steatosis.
Increased GLUT4 content in muscle and PPAR-a content in liver
of diabetic KKAy mice.
ACKNOWLEDGEMENTS
Study conducted in collaboration
with the Cree Nation of Mistissini