2. Oxidative stress and caloric restriction Jan Škrha Professor of Medicine 3 rd Department of Internal Medicine, Laboratory for Endocrinology & Metabolism 1 st Faculty of Medicine, Charles University, Prague IFCC Congress, Berlin 2011

3. OBESITY AND CONSEQUENCES Overeating Weight increase Higher concentration of circulating FFA Insulin resistance Hyperinzulinemia ROS

4. FFA – GENERATOR OF INSULIN RESISTANCE AND OXIDATIVE STRESS Insulin FFA IRS-1 phosphoryl. serine/threonine FA-CoA IRS-1 phosphoryl. tyrosine DAG PKC ROS PI-3-kinase NFĸB activation GLUT 4 translocation ROS glucose transport inflammatory proteins Diabetes Care 27, 2004, 2253-9

5. I II Q-Q- IIIIV cyt c e-e- e-e- e-e- e-e- Succinate Fumarate NAD + NADH H 2 O+O 2 O 2 - O 2 Mn-SOD O2O2 H2OH2O ATP ADP + P UCP H + H + H + H + H + H + gradient generation Decrease of H + gradient SUPEROXIDE GENERATION IN MITOCHONDRIA Respiratory chain Substrate supply

6. Glucose GAPDH Polyols AGE PKC Hexosamine NFκB Mitochondria Nucleus Cytoplasm M.Brownlee, Diabetes 2005 UNIFYING MECHANISM FOR IMPAIRMENT CAUSED BY HYPERGLYCEMIA AND FFA FFA PARP ROS

7. ENDOTHELIAL CELL Hyperglycemia Intracellular glucose Reactive oxygen species in mitochondria polyols lipoxidation glycation PKCß hexosamines glycoxidation Endothelial dysfunction DIABETIC ANGIOPATHY oxidative stress GLUT 1

8. EXOGENOUS AGE´s ACCELERATE OXIDATIVE STRESS

9. EXOGENOUS AGEs

10. NITROSATIVE STRESS Endothelial dysfunction

11. MODULATION OF OXIDATIVE STRESS BY CALORIC INTAKE

12. I II Q-Q- IIIIV cyt c e-e- e-e- e-e- e-e- Succinate Fumarate NAD + NADH H 2 O+O 2 O 2 - O 2 Mn-SOD O2O2 H2OH2O ATP ADP + P UCP H + H + H + H + H + H + gradient generation Decrease of H + gradient REDUCED SUPEROXIDE GENERATION IN MITOCHONDRIA Respiratory chain Decreased substrate supply Gredilla et al., FASEB J 2001

13. Sirtuins – caloric restriction effectors (silent information regulators – SIRT1-7) family: NAD-dependent histone deacetylases - influencing aging, metabolic processes, tumorigenesis Reduced caloric intake: SIRT1 upregulation increases gluconeogenesis decreases adipocyte formation oposite to insulin sirtuins like „molecular sensor“ ActivatorsInhibitors increase life span and cell survival promote fat mobilization increase the mitochondria number stop tumor formation stop apoptosis stop neurodegenerative disease

14. Sirtuins – key regulators of oxidative stress increase of antioxidative defense: by upregulated MnSOD expression by reduced cellular hydrogen peroxide by enhanced mitochondrial glutathion capacity reduced mitochondrial ROS production Caloric restriction vs dietary composition reduced activity of complex I restriction of protein intake vs complex III in mitochondria (methionine content) decreased ROS production Crujeiras et al., Europ J Clin Invest 2008, Someya et al., Cell 2010, Sanz et al., J Bioenerg Biomembr 2004

15. Caloric restriction vs fasting Caloric restriction and short intermitent fasting Prolonged fasting beneficial effects: decreased ROS production mitochondrial protection harmful effects: superoxide anion release from hepatocytes decreased glutathione content Resistance to oxidative stress increaseddecreased Mattson et al., J Nutr Biochem 2005 Sorensen et al., Free Radic Res 2006

16. BIOMARKERS Biomarker Caloric restriction Lipid peroxidation Malondialdehyde (MDA) decreased F-2-isoprostanes decreased Nitrosative stress Nitrotyrosine decreased DNA damage 8-hydroxydeoxy-guanosine decreased Scavanger enzymes SOD decreased, no change, increased CAT decreased, no change, increased GPx inverse relationship to CAT GR Non-enzymatic scavengers GSH GSH/GSSG ratio increased Ascorbic acid no changes or increased α-tocopherol no changes (α-toc/chol+tg)

17. Increased oxidative stress Reduced oxidative stress Cycling of OX/RED forms

18. Comparison of the oxidative stress measures by obese and nonobese persons

19. OXIDATIVE STRESS IN OBESE SUBJECTS

22. DECREASED CALORIC INTAKE (for obese or overweight individuals) diabetic rats Wistar: 9 wks fed: group A – hypocaloric diet group B – free diet in group A: decreased TNF-α, IL-1, IL-4, IL-6 increased antiinflammatory mediators and IL-10 Prevention of diabetic complications by supression of inflammation due to decreased oxidative stress Ugochukwu et al., J Nutr Biochem 2007, 18, 120-126

23. COMBINATION OF DIET AND PHYSICAL ACTIVITY Roberts CK et al., Atherosclerosis 191, 2007, 98-106 Improved lifestyle in 19 children with overweight 2 wks: diet with reduced fat + increased fiber contents 2hrs daily physical training Results: decrease: cholesterol, TG, 8-iso-PGF-2, ICAM-1, selectin, CRP, myeloperoxidase, superoxide and H 2 O 2 increase: NO Improved parameters associated with atherosclerosis risk

24. CALORIC RESTRICTION & OXIDATIVE STRESS Patients: A/ obese Type 2 diabetic patients (n=9) B/ obese non-diabetic patients (n=9) Methods: VLCD diet (600 kcal), 1 week Laboratory methods: FFA, ß-HB, MDA, SOD, ascorbic acid, alpha-tocopherol, T-chol, HDL, LDL-chol., TG

25. CALORIC RESTRICTION & OXIDATIVE STRESS Diabetic pts: Age 55 (41-70) yrs. DM duration 12 ± 5 r. BMI 36.2 ± 1.6 kg/m 2 HbA 1c 10.2 ± 1.6 % Controls: Age 53 (35-69) r. BMI 37 ± 2.1 kg/m 2 HbA 1c 5.5 ± 0.4 %

26. 0,001 Low-calory diet Control persons

27. 0,050,001 Low-calory diet Control persons

28. 0,001 Low-calory diet Control persons

29. 0,0010,05 Low-calory diet Control persons

30. 0,01 Low-calory diet Control persons

31. 0,01 Low-calory diet Control persons

32. 0,01 Low-calory diet Control persons

33. 0,05 Low-calory diet

34. CALORIC RESTRICTION & OXIDATIVE STRESS Conclusions 1.Caloric restriction lowers LDL and HDL-cholesterol and triglycerides 2.Significant decrease of oxidative stress was found in obese non-diabetic pts (MDA decrease, SOD and ascorbic acid increase), these changes were only borderline in diabetic patients 3.Obese diabetic patients are more resistant to low calory diet in comparison with obese non-diabetic patients and they reduce oxidative stress less than non-diabetic persons

35. a) Oxidative stress is higher in obese than in nonobese persons b) Very low calory diet decreases the level of oxidative stress

37. Thank you