NAHO 2011 Speaker Series, Ottawa, February 23, 2011 Pierre S. Haddad PhD Department of Pharmacology Université de Montréal This talk is dedicated to the memory of Elders Sam Awashish, René Coon Come, Smally Petawabano and Sally Matthews

1. Combining modern science and traditional medicine in the fight against Aboriginal type 2 diabetes: The experience of the CIHR Team in Aboriginal Anti-diabetic Medicines Pierre S. Haddad PhD Department of Pharmacology Université de Montréal http://www.taam-emaad.umontreal.ca NAHO 2011 Speaker Series, Ottawa, February 23, 2011 This talk is dedicated to the memory of Elders Sam Awashish, René Coon Come, Smally Petawabano and Sally Matthews

3. PREVALENCE OF DIABETES WHO

4. Acquired Diet, physical inactivity, Increased FFA, aging, glucotoxicity Decreased Insulin sensitivity Deficiency of  cells TYPE 2 DIABETES Type 2 diabetes (dysregulated high blood sugar) Genetic IR Mutations, anti-insulin antibodies, GLUT4 mutations Compensatory hyperinsulinemia (Insulin resistance) Postprandial hyperglycermia Type 2 diabetes (symptoms)

5. How is blood sugar controlled? TZD Metformin Skeletal Muscle Activation of AMPK Insulin Energy depletion Activation of Akt Glucose uptake GLUT4 expression & translocation Activation of Akt Fat Inhibition of AMPK Expression of C/EBP-  & PPAR  Activation of C/EBP-  Differentiation of adipocytes Accumulation of fat Hyperglycemia ACC activity Liver Activation of AMPK Activation of Akt Glucose production SREPB-1 expression Gene expression of lipogenic enzymes Fatty acid oxidation Fatty liver Hepatic insulin sensitivity Fatty acid synthesis

12. Problem 2: Diabetes in northern Quebec Cree community Years Modified from Cree Board of Health and Social Services of James Bay - CBHSSJB (2005) Crude prevalence of T2D in Eeyou Istchee area 21.6% 7.6% 25.5% 7.0% 17.7% 8.1% Kuzmina E, Lejeune P, Dannenbaum D, Torrie J. 2010. CREE Diabetes Information System CDIS): 2009 Annual Update. C hisasibi, Québec: Cree Board of Health and Social Services of James Bay 2009

14. Cree Nations Traditional Knowledge Medicinal plants CIHR Team in Aboriginal Antidiabetic Medicines (2003-2006) 2006-2011 Nutrition (Johns) Ethnobotany (Cuerrier) Phytochemistry (Arnason) Clinical/ Observational Trials (Chiasson, Yale) Education/ Integration (CBHSSJB) Pharmacology (Haddad, Prentki, Musallam, Bennett, Sirois) Toxicology (Foster)

15. Ethnobotanical study (4 communities) Collection of 17 species (potentially antidiabetic) Preparation of the extracts In vitro assays In vivo validation Isolation and identification of active principles Clinical studies Mechanisms of action Standardized plant extracts Quality controlled traditional preparations standardization Bioguided fractionation Toxicology All the species The most active species The active species PROJECT FLOW DIAGRAM

18. Ethnobotany results in Mistissini Leduc et al., J. Ethnopharmacol. 2006; 105(1-2):55-63

19. Larix laracenia Tamarack Watnagan (Inner bark) Picea mariana Black spruce Inaahtkw (Cones) Alnus incana Speckled alder Atushpi (Inner bark) Rhododendron groenlandicum Labrador Tea Kachichepukw (Leaves) Pinus banksiana Jack pine Ushchishk (Cones) Abies balsamea Balsam fir Inaasht (Inner bark) Sarracenia purpurea Pitcher plant Ayigadash (Whole plant) Sorbus decora Mountain ash Muskuannanatuk (Inner bark) Leduc et al., J. Ethnopharmacol. 2006; 105(1-2):55-63 Top 8 Mistissini plants screened

22. 1 Yield is expressed as (mass of recovered extract / mass dry plant material) x 100% 2 Total phenolics expressed as quercetin equivalents (μg) / mg extract Spoor et al., Can. J. Physiol. Pharmacol. 84:847-858, 2006 Phytochemistry (Arnason Lab: University of Ottawa) Total phenolics and identified marker compounds of each plant extract Plants % Yield 1 Total Phenolics (μg/mg) 2 Identified Phenolic Marker Compounds A. balsamea 15.3 % 97.6 p-coumaric acid, gallocatechin A. incana 26.1 % 305.9 catechins L. laricina 23.8 % 208.0 taxifolin, hydroxystilbenes P. mariana 21.0 % 163.7 p-coumaric acid, hydroxystilbenes P. banksiana 9.0 % 318.0 taxifolin, catechin, procyanidins R. groenlandicum 31.0 % 188.5 chlorogenic acid, catechins, procyanidins, quercetin glycosides S. purpurea 25.2 % 85.4 taxifolin, flavonol glycosides (quercetin, kaempferol, myricitin) S. decora 8.9 % 59.6 quercetin and quercetin glycosides

24. In vitro Pharmacology – in vitro (Haddad & Prentki Labs: University of Montreal) Glucose absorption Glucose production Glucose uptake Glitazone-like activity Insulin Secretion Primary anti-diabetic activity (glycemia-lowering activities) Glucose uptake Adipokines secretion Pancreas Liver Skeletal Muscle Fat Intestines FRACTIONATION

25. Mechanisms of action Skeletal Muscle Activation of AMPK Insulin Energy depletion Activation of Akt Glucose uptake GLUT4 expression & translocation Activation of Akt Fat Inhibition of AMPK Expression of C/EBP-  & PPAR  Activation of C/EBP-  Differentiation of adipocytes Accumulation of fat Hyperglycemia ACC activity Liver Activation of AMPK Activation of Akt Glucose production SREPB-1 expression Gene expression of lipogenic enzymes Fatty acid oxidation Fatty liver Hepatic insulin sensitivity Fatty acid synthesis

26. In vitro Pharmacology-Toxicology (Johns, Foster, Arnason & Bennett: McGill, University of Ottawa) CYP450 Pro/anti- Inflammation Neuroprotection against low & high glucose P-glycoprotein transport Toxicology (potential Herb-Drug interaction) Secondary anti-diabetic activity (protection against diabetic complications) Cardiomyocyte electrotoxicity Anti-oxidant activity Anti-glycation activity Heart Liver Brain Intestines Blood Cell-free assays

27. Pharmacology – in vivo (Haddad Lab: University of Montreal)

30. How is blood sugar controlled? Skeletal Muscle Activation of AMPK Insulin Energy depletion Activation of Akt Glucose uptake GLUT4 expression & translocation Activation of Akt Fat Inhibition of AMPK Expression of C/EBP-  & PPAR  Activation of C/EBP-  Differentiation of adipocytes Accumulation of fat Hyperglycemia ACC activity Liver Activation of AMPK Activation of Akt Glucose production SREPB-1 expression Gene expression of lipogenic enzymes Fatty acid oxidation Fatty liver Hepatic insulin sensitivity Fatty acid synthesis

31. 9 -10 of the 17 Cree anti-diabetic plants decrease hepatic glucose output by different mechanisms 9 plants significantly decrease G-6Pase Nachar, Haddad et al., unpublished % activation of GS 10 plants significantly activate GS % inhibition of G-6Pase Hepatic glucose production Expression of G-6Pase & PEPCK Activity of G-6Pase Hepatic glucose storage AMPK Akt Activity of GSK-3  Activity of GS Hepatic glucose output

33. MODE OF ACTION OF BOREAL PLANTS ? Altered Mitochondrial Respiration

34. Mechanism of action of Boreal plants: Insulin-dependent (p-Akt) and -independent (p-AMPK  and p-ACC) activities Martineau, Spoor, Haddad, et al., J. Ethnopharmacol. 2010

35. Mechanism of action of Boreal plants: Insulin-dependent (p-Akt) and -independent (p-AMPK  , p-ACC) activities Martineau, Spoor, Haddad, et al., J. Ethnopharmacol. 2010

36. Effect of Abies balsamifera on mitochondrial respiration Martineau, Spoor, Haddad, et al., J. Ethnopharmacol. 2010

37. Effect of plant extracts on mitochondrial respiration: Uncoupling Inhibiting Martineau, Spoor, Haddad, et al., J. Ethnopharmacol. 2010

38. L6-wt myotubes L6-myc myoblasts Glut-4 Translocation Assay Eid, Sweeney, Haddad et al., unpublished Insulin Plant Z Insulin Plant Z α-Glut4 α-myc (myc)

39. WORKING HYPOTHESIS FOR BOREAL PLANTS : Altered Mitochondrial Respiration ??

41. Screening for “ glitazone-like ” activity (Adipogenesis) Harbilas et al. Can. J. Physiol. Pharmacol. 2009 Boreal Plants from Cree Pharmacopeia W7 AD01

42. Body Weight Food Intake *AD02 250 mg/kg and W7 125 and 250 mg/kg significantly prevent body weight gain compared to the control DIO group between day 20 and 60 of treatment Effects of AD02 & W7 on prevention of DIO AD02 125 mg/kg AD02 250 mg/kg W7 125 mg/kg W7 250 mg/kg AD02 125 mg/kg AD02 250 mg/kg W7 125 mg/kg W7 250 mg/kg Harbilas, Haddad et al., unpublished * Food intake significantly lower in animals treated with W7 250 mg/kg compared to DIO control group between day 30 and 60 of treatment

43. Body Weight Food Intake W7 W7 Harbilas, Haddad et al., unpublished Effects of W7 on DIO treatment W7 W7

45. Neuroprotection against low/ high glucose In vitro Anti-Inflammatory properties Inhibitor of ATP synthase 3 compounds Insulin secretion (at high glucose) AD01 Ouchfoun, Haddad et al., unpublished Harbilas et al., CJPP 2009 Martineau, Spoor et al., JEP 2010 Nistor et al., JEP 2010 Sugar absorption (10’) FRACTIONATION Sugar production Fat production Pancreas Liver Skeletal Muscle Fat Brain Intestines Blood Sugar uptake (1h) Sugar uptake (18h) Sugar uptake (1 & 18h)

46. Fractionation guided by stimulation of adipogenesis Fractionation : J-A Guerrero (Arnason) Bioassay : M Ouchfoun (Haddad) Leaves of AD01 Compound 1 Compound 2 Compound 3 Ouchfoun, Haddad et al., unpublished

47. Fractionation guided by stimulation of adipogenesis Compound 1 Compound 2 Compound 3

48. AD01 in DIO model

49. Weight (~6%) In vivo Glucose (~9 %) AD01 ( Ethanolic Extract) Fat hormons (~10%) Brains (Analysed by Bennett) Ouchfoun, Haddad et al., unpublished Whole Body Pancreas Blood Weight (~14%) Abdominal (~14%) Brown (~7%) Lipid content (~ 42 %) Insulin (~65%) Liver Fat Skeletal Muscle Brain

51. Pilot # 1 Original clinical Trial proposed Pilot # 2

52. « Putting Traditional Medicine First » Current all-inclusive observational studies 1. Inclusion criteria Pre-diabetic or Type 2 diabetes with duration  5 years since diagnosis Already treated with herbal remedies for diabetes or wishes to start de-novo treatment with herbal remedies Male or female 18 years or older HbA1C between <10% 2. Exclusion criteria Type 1 diabetes Type 2 diabetes patients requiring insulin therapy Type 2 diabetes >5 years duration? Renal disease with overt proteinuria (ACR > 30 mg/mmol) and/or eGFR < 60 ml/m Liver disease with ALT 3 times above upper limit of normal Unstable coronary artery disease (CAD) or uncontrolled hypertension Uncontrolled hypothyroidism Alcohol or drug abuse Pregnancy or lactation Any major illnesses Any medication that can impair glucose metabolism (i.e. glucocorticoids) Unable to sign voluntary consent 1. Efficacy HbA1C and Fructosamine Fasting plasma glucose 75 gram 2 hr post OGTT (pre-diabetic only) Fasting insulin level Capillary blood glucose profile Blood biochemistry/ Lipid/ Inflammatory profile Blood pressure Morphometric measurements 2. Safety Urea, creatinine, eGFR, and electrolytes Liver function tests (ALT, AST, GGT, bilirubin, and alkaline phosphatase, INR) Complete blood count ECG Urine analysis, ACR

60. ACKNOWLEDGEMENTS & TEAM CIHR-TAAM Annual retreat at Mistissini (2010) Haddad lab

61. MEGWETCH!!

63. AD01 strongly stimulated adipogenesis AD01

64. AD01 activates Insulin pathway in the skeletal muscle in vivo pAkt pAMPK pACC GLUT4 AMPK Akt GLUT4 expression & translocation Glucose uptake Ouchfoun, Haddad et al., unpublished

65. AD01 activates Insulin pathway and AMPK pathway in the liver in vivo pAkt pAMPK p-IKK  SREBP-1 Sugar production Akt Ouchfoun, Haddad et al., unpublished Hepatic insulin sensitivity Fatty acid oxidation AMPK Expression of SREBP-1 Fatty liver

66. AD01 tends to stimulate the adipogenic program in the abdominal fat in vivo p = 0.07 PPAR  C/EBP  C/EBP  Ouchfoun, Haddad et al., unpublished Adipogenesis Expression of C/EBP  Fat accumulation Expression of PPAR  Insulin sensitivity

67. Insulin actions and insulin resistance