1. STEVIA SYMPOSIUM LEUVEN July 2009 EFFECT OF STEVIOSIDE ON ATHEROSCLEROSIS IN A MOUSE MODEL OF THE METABOLIC SYNDROME Paul Holvoet Atherosclerosis and Metabolism Unit Department of Cardiovascular Diseases University Leuven, Belgium

2. ATHEROSCLEROSIS GROUP

3. PART ONE: THE METABOLIC SYNDROME, OXIDIZED LDL AND CARDIOVASCULAR DISEASE PART TWO: STUDY OF ATHEROGENIC MECHANISMS IN A MOUSE MODEL OF THE METABOLIC SYNDROME PART THREE: EFFECT OF STEVIOSIDE ON ATHEROSCLEROSIS IN A MOUSE MODEL OF THE METABOLIC SYNDROME Structure of presentation

4. PART ONE: THE METABOLIC SYNDROME, OXIDIZED LDL AND CARDIOVASCULAR DISEASE

5. Ref. Groop L. et al,Journal of Internal Medicine, 2001, 250: 105-120. METABOLIC SYNDROME

6. METABOLIC SYNDROME AND CARDIOVASCULAR RISK Obesity High TG Low HDL-C Diabetes Hypertension METABOLIC SYNDROME CARDIOVASCULAR RISK

7. UNDERLYING MECHANISMS Oxidative Stress Lipoxygenases ↑ SOD ↓ Inflammation (IL-6↑, CRP↑) Oxidized LDL↑ Endothelial dysfunction ( ICAM- 1↑, VCAM-1↑, MCP- 1↑, PAI-1↑, NO↓) Infiltration of monocytes ↑ Adhesion Platelets↑ Thrombosis ↑ Foam cells Apoptosis Extracellular lipids TG Glucose Insulin Macrophages↑

8. METABOLIC SYNDROME AND OXIDIZED LDL We have measured oxidized LDL in 3,033 samples from the population-based Health ABC cohort. Participants (aged 70-79 Y) were recruited from a random sample of White and Black Medicare eligible adults living in the Memphis, TN and the Pittsburgh, PA vicinities from March 1997 – June 1998.

9. METABOLIC SYNDROME  Waist circumference ≥102 cm in men and ≥ 88 cm in women  Fasting triglycerides ≥ 150 mg/dL (1.70 mmol/L)  HDL-cholesterol <40 mg/dL (1.03 mmol/L) in men and < 50 mg/dL (1.29 mmol/L) in women  Blood pressure ≥ 130/85 mmHg or on antihypertensive medication  Fasting glucose ≥ 100 mg/dL (5.55 mmol/L) or on antidiabetic medication Persons with at least three of these components were defined as having metabolic syndrome Executive Summary of The Third Report of The National Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation, And Treatment of High Blood Cholesterol In Adults (Adult Treatment Panel III). JAMA 285:2486-2497, 2001

10. 0 1 23 4 56 Ox-LDL (mg/dl) * Ox-LDL (% LDL) ** Odds Ratio (95% CI) * Odds ratios for high ox-LDL for subjects with MetSyn compared with subjects without MS adjusted for age, sex, ethnicity, smoking and LDL-C. ** Adjusted for age, sex, ethnicity, smoking. Holvoet et al. Diabetes 2004; 53: 1068 Relation between MetSyn and oxidized LDL 2.0 (95% CI: 1.6-2.5) 2.6 (95% CI: 2.1-3.2)

11. Metabolic syndrome and oxidized LDL in Japanese population Yamagishi et al., Int J Cardiol. 2007;118:270-2

12. Relative Risk of Myocardial Infarction RR for MI compared with subjects without MS and adjusted for age, sex, ethnicity, smoking, and LDL-C. Holvoet et al. Diabetes 2004; 53: 1068 2.0 (95% CI: 1.4-2.9) 0123456 MetSyn Relative Risk (95% CI)

13. Relative Risk of Myocardial Infarction in Health ABC * RR for MI compared with subjects in the lowest quintile of ox-LDL and adjusted for age, sex, ethnicity, smoking, LDL-C and MetSyn Holvoet et al. Diabetes 2004; 53: 1068 2.0 (95% CI: 1.4-2.9) 1.9 (95% CI: 1.1-3.5) 0123456 MetSyn Ox-LDL * Relative Risk (95% CI)

14. CONCLUSIONS MetSyn, a risk factor of CHD, is associated with higher levels of circulating oxidized LDL that are associated with a greater disposition to myocardial infarction. This has been confirmed in the MONICA and the FRISC-II cohorts.

15. OXIDIZED LDL and DEVELOPMENT of METABOLIC SYNDROME To establish the relation of oxidized LDL with metabolic syndrome in the general community

16. LIMITED ACCESS DATA SETS AVAILABLE TO SCIENTIFIC COMMUNITY Description of data on Public CARDIA website: http://www.cardia.dopm.uab.edu Procedures on how to request data: http://www.nhlbi.nih.gov/resources/deca/descriptions/cardia.htm For Information or Questions on how to request data: purkisek@nhlbi.nih.gov For Other Information or Questions, send an email to: CARDIAdataquestions@dopm.uab.edu

17. Copyright restrictions may apply. Holvoet, P. et al. JAMA 2008;299:2287-2293. DESIGN, SETTING, AND PARTICIPANTS CARDIA: a population- based, prospective, observational study 1,889 participants who were between the ages of 33- 45 years at year 15 (2000-2001) 5 years’ follow-up

18. Incidence of Y 20 metabolic syndrome by Y 15 oxidized LDL concentrations Quintiles of oxidized LDL Q1 <55.4 Q2 55.4-69.1 Q3 69.2-81.2 Q4 81.3-97.3 Q5 ≥97.4 P trend Cases/no.20/37739/37851/37357/38676/375 Ref. 2.1 (1.1-3.8) 2.4 (1.3-4.3) 2.8 (1.5-5.1) 3.5 (1.9-6.6) <0.001 Adjusted for age, gender, race, study center, cigarette smoking, physical activity, body mass index, and LDL-cholesterol Addition of C-reactive protein, adiponectin, and antihypertensive, antidiabetic, and cholesterol lowering medication did not materially reduce the ORs

19. Incidence of Y20 metabolic syndrome by Y 15 oxidized LDL and LDL-c concentrations 0 1 2 3 4 5 6 7 Q1Q2Q3Q4Q5 LDL-C Ox-LDL

20. Incidence of each component of METSYN at Y 20 by Y 15 oxidized LDL concentrations Quintiles of oxidized LDL Q1Q5P trend Abdominal obesity Ref.2.1 (1.2-3.6) 0.004 High fasting glucose Ref.2.4 (1.5-3.8) 0.002 High triglycerides Ref.2.1 (1.1-4.0) 0.008 High BPRef.0.9 (0.5-1.6) 0.36 Low HDL- cholesterol Ref.0.8 (0.4-1.8) 0.84 Adjusted for age, gender, race, study center, cigarette smoking, physical activity, body mass index, and LDL-cholesterol. Each row is a separate logistic regression model in which prevalent cases of the dependent variable were excluded.

21. CONCLUSIONS Our population-based data showed that higher concentrations of circulating oxidized LDL are associated with the incidence of metabolic syndrome and the accumulation of three of its risk factor constituents: abdominal obesity, hyperglycemia, and hypertriglyceridemia

22. CONCLUSIONS As yet, it is not possible to conclude whether oxidized LDL is a marker related to mechanistic underlying factors on the pathway to the development of metabolic syndrome, or whether it is by itself a functional intermediary in this pathway

23. PART TWO: STUDY OF ATHEROGENIC MECHANISMS IN A MOUSE MODEL FOR THE METABOLIC SYNDROME

24. Mouse model for the metabolic syndrome Mice with combined deficiency in LDL-receptor and leptin gene (DKO mouse) [ Mertens et al. Circ. 2003, 107: 1640] Metabolic syndrome Oxidative stress Inflammation Atherosclerosis Heart Function Insulin resistance Obesity Hypercholesterolemia HypertensionHypertrigyceridemia

25. How representative is this mouse model for human obesity and its associated risk for atherosclerosis? ??? MOUSE MODEL OF THE METABOLIC SYNDROME

26. Insulin resistance and diabetes Glucose Insulin Improved Deteriorated Lipid profile Inflammation Oxidative stress Hypertension High Low CVD risk Atherosclerosis Heart function Impaired Obese mice Weight loss Improved WEIGHT LOSS IS ASSOCIATED WITH DECREASE OF “CARDIOVASCULAR RISK” Verreth et al. Circ. 2004, 110: 3259

27. Plaque oxidized LDL ↓ Decreased oxidation of LDL –Increased expression of antioxidative genes: SOD1, SOD2, SOD3, catalase and glutathione reductase –Decreased infiltration of inflammatory cells  ROS production ↓ Increased removal of oxLDL LDL oxLDL oxidation oxysterols induce expression PPAR-γ induces expression activates transcription ABCA-1 cholesterol efflux to the RCT pathway HDL CD36 activates transcription mediates oxLDL uptake LXR 

28. Reduction in macrophages associated with increased insulin sensitivity Hypothesis 1: Insulin sensitivity in adipose tissues ↑  Oxidized LDL ↓  Accumulation of macrophages ↓ Hypothesis 2 Monocyte infiltration in adipose tissues ↓  Inflammatory state ↓ Oxidized LDL ↓  Insulin sensitivity ↑

29. PART THREE: EFFECT OF STEVIOSIDE ON ATHEROSCLEROSIS IN A MOUSE MODEL OF THE METABOLIC SYNDROME

30. Experimental Protocol 12 24 Age (weeks) Sacrifice and collection of: Blood Blood variables Aortic Arch Atherogenesis Tissues RNA qRT-PCR Start treatment Placebo (n=17) Stevioside 10 mg/kg/day (n=14) Stevioside

31. Weight and Blood Analysis All data are mean  SD per group. * P<0.05, ** P<0.01, vs. control DKO mice

32. Gene expression in adipose tissue C57BL6PlaceboStevioside 0.0 0.5 1.0 1.5 ** *** $$$ Insr (Ratio vs. C57BL6) Irs1 C57BL6PlaceboStevioside 0.0 0.5 1.0 1.5 *** $ (Ratio vs. C57BL6) C57BL6PlaceboStevioside 0.0 0.5 1.0 1.5 ** $$ Irs2 (Ratio vs. C57BL6) C57BL6PlaceboStevioside 0.0 0.5 1.0 1.5 *** $ Glut4 (Ratio vs. C57BL6) C57BL6PlaceboStevioside 0.0 0.5 1.0 1.5 *** $$ Fabp4 (Ratio vs. C57BL6) C57BL6PlaceboStevioside 0.0 0.5 1.0 1.5 *** $ Lxr  (Ratio vs. C57BL6) Adipocyte differentiation and Insulin signaling ↑ All data are mean  SD per group. *** P<0.001, ** P<0.01, * P<0.05 vs. C57BL6 mice (genetic background) $$$ P<0.001, $$ P<0.01, $ P<0.05 Stevioside-treated vs. control DKO mice

33. Increased adiponectin production is associated with improved insulin signaling Gene expression in adipose tissue All data are mean  SD per group. * P<0.05, ** P<0.01, vs. control DKO mice) Rs: Spearman correlation coefficients Adiponectin decreases the inflammatory and oxidative state in tissues

34. Antioxidative genes in visceral adipose tissue

35. Plaque size and composition Lipids Macrophages Oxidized LDL

36. Plaque size and composition All data are mean  SD per group. *** P<0.001, ** P<0.01, * P<0.05 vs. control DKO mice Decrease in macrophages and oxidized LDL is associated with a more stable plaque phenotype

37. Gene expression in aorta All data are mean  SD per group. *** P<0.001, ** P<0.01, * P<0.05 vs. C57BL6 mice (genetic background) $$$ P<0.001, $$ P<0.01, $ P<0.05 Stevioside- treated vs. control DKO mice

38. CONCLUSIONS Stevioside improves adipocyte differentiation associated with increased adiponectin production and improved insulin sensitivity This is associated with increased adiponectin production correlating with increased anti- oxidant SOD expression Improved anti-oxidative protection is associated with a decreased oxidized LDL and macrophage accumulation resulting in inhibition of atherosclerosis

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