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ABSTRACT

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 Ganguly 5 , John T. Arnason 2,4 , Gary Sweeney 5 , Pierre S. Haddad 1,3,4

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ABSTRACT

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  1. Hoda M. Eid1,3,4, Antoine Brault1,3,4, MeriemOuchfoun1,3,4, Farah Thong 5,Diane Vallerand1,3,4, RiyaGanguly5, John T. Arnason2,4, Gary Sweeney 5, Pierre S. Haddad 1,3,4 1Natural Health Products and Metabolic Diseases Laboratory, Dept. of Pharmacology, Université de Montréal, Montreal, QC, 2Phytochemistry, Medicinal Plant and EthnopharmacologyLaboratory, Dept. of Biology, University of Ottawa, Ottawa, ON, 3 The Institute of Nutraceuticals and Functional Foods (INAF), 4Canadian Institutes of Health Research Team in Aboriginal Antidiabetic Medicines and Montreal Diabetes Research Center,5 Dept. of Biology, York University, Toronto, ON W9, a medicinal plant of the Eastern James Bay Cree, mobilizes L6 muscle Glut4 transporters and exerts anti-obesity and antidiabetic effects in vivo.

  2. ABSTRACT W9 has been identified among species used by the Cree of EeyouIstchee 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 W9for 18 h significantly increased glucose uptake and GLUT4 translocation to the cell membrane. W9increased 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,W9-treatment 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 W9for the prevention and treatment of diabetes.

  3. GLUT Family of Proteins Nature Reviews Molecular Cell Biology 3, 267-277 (April 2002)

  4. Effect of Insulin on GLUT4

  5. 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.

  6. Type 2 Diabetes • Type 2 Diabetesis a worldwideepidemic(220 million) • Canadian aboriginal populations : • Its prevalence is 3-4 times the canadianaverage • Its worsenedby geneticpredisposition, sedintary life and poorcompliance to western Rx • The need for culturally-adaptedalternative : plants fromtheirTraditionalMedicine • Ethno-botanicalstudypreviouslyidentified17 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

  7. OBJECTIVES • Evaluate the downstreamsignalling of W9 • Study the anti-diabeticactivity of W9 in vivo

  8. Figure 1: Insulin and non-insulin dependent pathwaysregulating glucose transport Inhibition of mitochondrial respiration ACC Mitochondria

  9. 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-phenylenediaminedihydrochloride (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)

  10. 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

  11. RESULTS

  12. W9 stimulates glucose uptake in L6 rat muscle cellsby inducing GLUT 4 translocation to the membrane Glucose Uptake(radioactive assay) GLUT 4 translocation(OPD assay) * * * * DMSO Insulin W9 DMSO Insulin W9 * : P<0.05 as compared to DMSO

  13. W9stimulates AMPK pathway in L6 muscle cells, but not the Insulinpathway AMPK pathway Insulin pathway phospho-AMPK phospho-Akt phospho-p38 MAPK b-Actin b-Actin DMSO Insulin AICAR W9 W9 DMSO AICAR

  14. 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) * Control W9 * : P<0.05 as compared to control

  15. W9 significantly decreases fluid intake glycemia in diabetic KKAy and normal C57BL/6J mice KKAy mice (Study #1) C57BL/6J mice(Study #2) * * Control W9 * : P<0.05 as compared to control

  16. W9 is not toxic, significantly decreases plasma triglycerides, but only tends to lower plasma insulin level in KKAy mice from study # 1

  17. Treatment of KKAy diabetic mice with W9 improved liver steatosis significantly $ Liver steatosis was evaluated histologically in control and W9 treated KKAy mice (Study # 1)

  18. W9 increased the levels of GLUT4 in skeletal muscles and those of PPAR-α in livers from diabetic KKAy mice Control W9 KKAy mice (Study #1) GLUT4 PPAR-a

  19. 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 KKAymice (p< 0.05). • Decreased cumulative fluid intake in diabetic KKAyand 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 KKAymice.

  20. ACKNOWLEDGEMENTS Study conducted in collaboration with the Cree Nation of Mistissini

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