Acides Gras Oméga-3 et Santé Yvon A. Carpentier Laboratoire de Chirurgie Expérimentale ULB Clinique des Lipides Hôpital Erasme

Structure et nomenclature des acides gras H3C COOH 18:0 Acide stéarique 9 H3C COOH 18:1n-9 Acide oléique H3C COOH 18:2n-6 Acide linoléique 6 3 H3C COOH 18:3n-3 Acide a-linolénique

Solide à température ambiante vs. Liquide à température ambiante

Metabolism of -linolenic acid From Cunnane SC, J Physiol Pharmacol, 1996

Trends in the intake of fatty acids in the UK % Energy PUFA to SFA ratio 1 0.8 0.6 0.4 0.2 50 40 30 20 10 Total fat SFA MUFA PUFA 1940 1950 1960 1970 1980 1990

Last 30 Years - Large Increase in the Intake of n-6 PUFAs in Industrialized Countries Between 1972 and 1998 the n-6 fatty acids rose from 4% to 6%. Upsurge of Asthma in the UK, Australia, New Zealand and Germany might be a related phenomenon. The unexplained increase in the incidence of edema, allergic rhinitis, and regional differences of inflammatory diseases within countries may relate to increased n-6 fatty acid intake. Roberts 1991, Dept. of Health UK 1994, Black and Sharp 1997, Lewis et al. 1996, Grimble 1998 Fü-Out-13b

Evolution des habitudes alimentaires Paléolithique « Western » AHA Lipides (% cal) 21 34 <30 Sat (% cal) <10 ~13 <10 P:S 1.4 ~0.5 >1.0 -3/ -6 1/1-1/2 1/10-1/20 1/1-1/5 Fibres (g/j) ~46 ~20 >25

DONNEES EPIDEMIOLOGIQUES Populations consommant bcp AG n-3 faible incidence de pathologies cardiovasculaires faible incidence de pathologies inflammatoires et allergiques faible incidence de cancers (colon, sein, prostate) faible incidence de lithiases rénales faible incidence de diabète type I allongement de durée de gestation incidence accrue d’insuffisance rénale chronique CORRELATION AVEC COMPOSITION EN ACIDES GRAS DES MEMBRANES CELLULAIRES ET FORMATION EICOSANOIDES

Rôles-clés en Santé Humaine Acides Gras oméga-3 Rôles-clés en Santé Humaine Développement cognitif Développement vision Réponses immunes/inflammatoires Grossesses & développement foetal Maladies neurodégénératives Aspects psychologiques Maladies cardio-vasculaires

Modes and sites of action of n-3 PUFA’s Formation of eicosanoids (tissue level, locally and at distance)

Membrane Pool (PL) Metabolic Pool (TG, FFA) - isoprostanes 2 Metabolic Pool (TG, FFA) 18:2 n-6 18:3 n-6 20:3 n-6 20:4 n-6 (LA) (GLA) (DGLA) (AA) Cyclo - oxygenase Lipoxygénases PGE 1 LT HETES PGI TXA 2 PGE 4 2 2

A major role of arachidonic acid is as a precursor for eicosanoids Arachidonic acid in cell membrane phospholipid Phospholipase A2 Free arachidonic acid COX 15-LOX 12-LOX 5-LOX PGG2 15-HPETE 12-HPETE 5-HPETE PGH2 15-HETE 12-HETE LTA4 5-HETE PGD2 LXA4 LTC4 LTB4 PGE2 PGF2a LTD4 LTE4 PGI2 TXA2

(ALA) (EPA) (DGLA) (DHA) Membrane Pool (PL) F3 - and F4 - isoprostanes Metabolic Pool (TG, FFA) 18:3 n-3 20:5 n-3 22:5 n-3 22:6 n-3 (ALA) (EPA) (DGLA) (DHA) Cyclo - oxygénases Lipoxygenases TXA3 LT5 PGI3 PGE3

Eicosanoids derived from n-3 (vs. n-6) PUFAs Less inflammatory Less thrombogenic Less chemo-attractive Weaker protection of gastro-intestinal mucosa Retard delivery (increase pregnancy duration)

Classic view of the anti-inflammatory action of long chain w-3 PUFA Arachidonic acid in membrane phospholipids DHA EPA Phospholipase A2 Free arachidonic acid 5-LOX COX-2 2-series PG and TX 4-series LT Inflammatory effects Inflammatory effects

Resolvins & related compounds EPA DHA COX-2 (& presence of aspirin) E-series resolvins D-series resolvins, neuroprotectins etc. Anti-inflammatory; inflammation resolving

Possible sites of action of n-3 & n-6 PUFAs From J.A. Ross et al, Curr Opin Clin Nutr Metab Care, 1999

Modes and sites of action of n-3 PUFA’s Formation of eicosanoids (tissue level, locally and at distance) Components of membrane phospholipids (membrane physical properties & interaction with membrane proteins : cell level) Second messengers in signalling pathways (molecular level) Regulators of gene expression (transcription factors : molecular level)

Signalisation cellulaire: voie des MAP-Kinases

Chemotaxis Chemoattractants Adhesion Injury Eicosanoids Cytokines Inflammation Reactive species PAF

Nutrition et Pathologies Cardio-vasculaires Lipides plasmatiques Pression artérielle Tendance aux thromboses Résistance à insuline Oxydation Homocystéine Inflammation Fonction endothéliale Irritabilité ventriculaire NUTRITION MCV From WC Willett, 2004

Acides Gras Oméga 3 et le Coeur  triglycérides (4g/j)  arythmies  arrêts cardiaques  réactions inflammatoires  coagulation Synergie avec l’aspirine Pas d’effets secondaires Pas d’effet sur le cholestérol  morbidité et mortalité des maladies coronaires

The New England Journal of Medicine Volume 312 May 9, 1985 Number 19 THE INVERSE RELATION BETWEEN FISH CONSUMPTION AND 20-YEAR MORTALITY FROM CORONARY HEART DISEASE Daan KROMHOUT, PH.D., M.P.H., EDWARD B., BOSSCHIETER, M.D., AND COR DE LEZENNE COULANER, M.SC. 1960: town of Zutphen, the Netherlands 852 middle-aged men without CHD dietary history 20 year follow-up: 78 deaths from CHD (> 50%) mortality from CHD in subjects eating > 30 g fish/day

Effects of changes in fat, fish, and fibre intakes on death and myocardial reinfarction : diet and reinfarction trial (DART) M.L. Burr et al., The Lancet, 1989 2033 men having recovered from myocardial infarction (M.I.) Dietary intervention in secondary prevention of M.I. I. fat intake (to 30% energy intake) and P/S ratio (to 1.0) II. fatty fish intake (200-400g/week) III. cereal fibre intake (18g/day) 2 year follow-up : 29% REDUCTION OF MORTALITY (all causes) IN ‘‘FISH’’ GROUP II

Dietary intake and cell membrane levels of long-chain n-3 Polyunsaturated Fatty Acids and the risk of primary cardiac arrest D.C. Siscovick et al. « Compared with no dietary intake of eicosapentaenoic acid (C20:5n-3) and docosahexaenoic acid (C22:6n-3), an intake of 5.5 g of n-3 fatty acids per month (... equivalent of one fatty fish meal per week) was associated with a 50% reduction in the risk of primary cardiac arrest (odds ratio [OR], 0.5; 95% confidence interval [Cl], 0.4 to 0.8), after adjustment for potential confounding factors. » JAMA, 1995

Prevention and termination of arrhythmia by EPA J.X. Kang & A. Leaf, Proc Natl Acad Sci USA, 1994

Prevention of Sudden Cardiac Death by Dietary Pure w-3 Polyunsaturated Fatty Acids in Dogs Dog model of cardiac sudden death surgically, large myocardial infarct + inflatable cuff around left circumflex coronary art. (LCA) 1 month later: Treadmill running + LCA occlusion  Ventricular fibrillation and death in all controls & soybean oil (n = 7) Survival in 5/7 dogs infused for 1h with EPA 6/8 dogs infused for 1h with DHA 6/8 dogs infused for 1h with LNA G.E. Billman et al, Circulation, 1999

Résultats - Suppl. HP vs. 2 autres groupes: Association of n-3 polyunsaturated fatty acids with stability of atherosclerotic plaques : a randomised controlled trial 188 patients en attente endartériectomie carotide (7-189j) Suppl. HP (1.4g EPA/DHA/j) vs. Tournesol vs. Palme/Soja Résultats - Suppl. HP vs. 2 autres groupes:  EPA & DHA dans lipides de plaque (  incorporation avec temps) Plus de plaques avec couche fibreuse épaisse Moins de plaques avec couche fibreuse fine & inflammatoire Moins d’infiltration de macrophages dans plaques t/o rapide des acides gras dans lipides des plaques effet marqué de suppl. HP sur morphologie & fragilité des plaques F. Thies et al, The Lancet, 2003

Dietary Fish Oil Supplementation Reduces Myocardial Infarct Size in a Canine Model of Ischemia and Reperfusion 0.06 g/kg.day EPA for 6 weeks Occlusion of left circumflex coronary art. for 90 min followed by 6h of reperfusion Infarct size: 13.3% (w 3) vs 29.7% (control) (p<0.05); no difference in regional blood flow or oxygen consumption Protective mechanisms: inhibition of TXA2 inhibition of free radical production by leucocytes? H. J. Oskarsson et al., J Am Coll Cardiol, 1993

Acides Gras -3 et Endothélium Amélioration profil lipoprotéines Amélioration profil éicosanoïdes Effet sur membranes cellulaires (et Chol dans cavéoles) Modulation de facteurs de transcription nucléaire Amélioration défenses anti-oxidantes de cellule  vasorelaxation  coagulation,  act plaquettes & molécules adhésion  sensibilité aux cytokines & radicaux libres  activation CML

BENEFICIAL EFFECTS OF n-3 PUFA ( I ) essential for maturation of foetal CNS and retina (DHA) reduce inflammatory response anti-thrombotic effect prevent impaired cellular immunity when caused by PGE2 production decrease plasma triglyceride concentration (also post-prandial) decrease plasma free fatty acid concentration (also p-prandial)

BENEFICIAL EFFECTS OF n-3 PUFA ( II ) decrease cell reactivity to various stimuli (e.g. ventricular arrhythmia’s,...) prevent / reverse cancer & inflammatory cachexia increase tolerance to organ transplantation and improve function of the graft help maintaining adequate tissue microperfusion reduce cellular accumulation of fat (e.g., liver) potential interest for supplying n-3 PUFA to « acute » patients

Limitations to n-3 FA supply with Fish Oils Gastro-intestinal administration FO TG : poor substrate for pancreatic lipase slow & rather unefficient absorption Intravenous infusion FO TG : poor substrate for lipoprotein lipase slow plasma elimination ! a proportion of n-3 FA used for oxidative purposes !

Obésité, syndrome métabolique, et insulino-résistance Influence du patrimoine génétique 85% des diabétiques T2 sont obèses 30% des obèses sont diabétiques (T2)  prévalence Différences entre ethnies (NB: asiatiques) Influence facteurs comportementaux (nutrition, activité physique, …) Relation avec stress & inflammation

Le Syndrome Métabolique Combinaison de  3 facteurs: - obésité abdominale : tour taille > 94 cm (H) ou > 80 cm (F) - conc. triglycérides  1.7 mmol/L (150 mg/dL) ou traitt - conc. HDL-cholestérol < 1.03 mmol/L (40 mg/dL) H ou traitt < 1.28 mmol/L (50 mg/dL) F ou traitt - tension artérielle  130/85 mm Hg ou traitt - conc. glucose à jeun  5.56 mmol/L (100 mg/dL) ou traitt prévalence:  44% pour population USA > 50 ans (2003)  prévalence coronaropathies (>>  facteurs isolés)

FACTEURS ASSOCIES AU SYNDROME METABOLIQUE Insulino-résistance & risque diabète T2 Dépôts ectopiques de TAG (foie, muscles, … ) Altérations lipoprotéines (sd LDL athérogènes) Composante inflammatoire Dysfonction endothéliale  activité ortho-sympathique Etiologie multiple (nutrition, sédentarité, …)

Serum sialic acid concentration in 263 overweight women without (0) or with 1-3 other features of metabolic syndrome : insulin resistance; dyslipidemia; hypertension L. Browning, Proc Nut Soc, 2003

Gene variants, insulin resistance, and dyslipidaemia Hypotheses: primary effect of variants on insulin resistance or on dyslipidaemia M.Lakso, Curr Opin Lipidol, 2004

Interaction between dietary lipids and physical inactivity on insulin sensitivity and on intramyocellular lipids in healthy men 8 healthy male volunteers 60h complete bed rest + high fat or high CHO diet Hyperinsulinemic –euglycemic clamp (glucose disposal) 1H-magnetic resonance spectroscopy Bed rest + high fat :  glucose disposal (- 24%)  intramyocellular lipid content (+ 32%) R. Stettler et al, Diabetes Care, 2005

Mitochondrial dysfunction and type 2 diabetes Maintenance of normal glucose level : insulin responsiveness of skeletal muscle & liver defect  insulin resistance insulin secretion by pancreatic beta cells defect  hyperglycemia both defects may be caused by mitochondrial dysfunction Lowell BB & Shulman GI, Science, 2005

Insulin resistance associated to obesity and type 2 diabetes Molecular defects of insulin signaling in muscle  glucose disposal and transport (role of fatty acids & metabolites) Insulin resistance in liver  glucose output (overexpression of glucose-6-Pase) Visceral obesity & ectopic fat storage (e.g., muscle & liver) poor modulation of fat oxidation Non-alcoholic fatty liver disease (NAFLD) and steato-hepatitis (NASH)  lipolysis, oxidative stress, cytokine induction Altered activity of desaturases  9 &  5  6 desaturase DB Savage et al, Hypertension, 2005

N-3 long chain polyunsaturated fatty acids: a nutritional tool to prevent insulin resistance associated to type 2 diabetes and obesity ? RAT STUDIES N-3 PUFAs improve molecular defects of insulin signaling in muscle  IRS-1 phosphorylation  PI 3’-kinase act, GLUT-4 mRNA  muscle TAG content & LCFA CoA N-3 PUFAs improve insulin resistance in liver  expression & activity of G-6-Pase; normalize glucose output N-3 PUFAs improve visceral obesity & ectopic fat storage (muscle & liver)  fat oxid (PPAR)  lipogenesis, TG formation, & fat deposition

N-3 long chain polyunsaturated fatty acids: a nutritional tool to prevent insulin resistance associated to type 2 diabetes and obesity ? HUMAN STUDIES (Healthy volunteers) 3 week supplementation w FO (1.1 g EPA + 0.7 g DHA) Oral glucose load (after supplementation n-3 PUFA) :  insulin response (- 40%)  glycemic response  glucose oxidation  lipid oxidation  glycogen storage N.B.: no effect after short-term supplementation (need for n-3 FA incorporation in cell membranes ?) J. Delarue et al, Reprod Nutr Dev, 2004

Changes in AUC for insulin (final-baseline) after supplementation with n-3 PUFA vs. placebo in premenopausal non-diabetic subjects P < 0.05 Subjects : - age : 19-51 years - BMI : 24-44 kg/m2 Inflammatory status (IS) : - low IS : sialic acid < 2.00 mM - high IS : sialic acid > 2.20 mM L. Browning, Proc Nut Soc, 2003

Fish oil prevents the adrenal activation elicited by mental stress in healthy men 7 healthy volunteers 2 tests of mental stress, before & after suppl 7.2g FO/d (3wks) Before:  heart rate  blood pressure energy expenditure  plasma cortisol  plasma epinephrine plasma NEFAs After n-3 PUFA supplementation :  heart rate  blood pressure  energy expenditure  plasma cortisol  plasma epinephrine  plasma NEFAs blunting of sympatho-adrenal stimulation (at CNS level ?) potential role in prevention of insulin resistance J. Delarue et al, Diabetes Metab, 2003

Diabetogenic impact of long-chain omega-3 fatty acids on pancreatic beta-cell function and the regulation of endogenous glucose production Dietary saturated lipids in healthy subjects:  ins resist but  ins secretion  glucose tolerance maintained Substitution 7% dietary lipids with n-3 PUFA (4 weeks): reverses peripheral insulin resistance (normal glucose disposal) HOWEVER: no suppression of hepatic insulin resistance no suppression glucose output no compensatory insulin secretion (+ direct impairment ?)  glucose level MJ Holness et al, Endocrinology, 2003

Body weight modulates cholesterol metabolism in non-insulin dependent type 2 diabetics BMI, serum insulin and/or blood glucose levels:  cholesterol synthesis  bile acid synthesis  cholesterol excretion in bile  cholesterol t/o  cholesterol absorption  serum cholestanol & plant sterols PP Simonen et al, Obes Res, 2002

Lipoprotein metabolism in the metabolic syndrome Chylo LPL Chylo-R Visceral adipose tissue FA LPL CHO Apo B VLDL -TG VLDL TG FA FFA apoB CO2 IDL LDL Designed from PHR Barett & GF Watts, Curr Opin Lipidol, 2003

Visceral adipose tissue Lipoprotein metabolism in the metabolic syndrome : Effects of n-3 PUFA supplementation Chylo LPL Chylo-R Visceral adipose tissue FA LPL CHO Apo B VLDL -TG VLDL TG FA FFA apoB CO2 IDL LDL Designed from PHR Barett & GF Watts, Curr Opin Lipidol, 2003

N-3 Fatty Acids and Plasma Lipoproteins  plasma triglycerides  plasma VLDL-TG and VLDL-cholesterol  or  HDL-cholesterol, but  apo A-1  LDL-cholesterol (generally slight)  fraction of small dense LDL (phenotype B subjects)

Dietary effects on postprandial triglyceride levels SFA n-6 PUFA n-3 PUFA M. Weintraub et al, J Clin Invest, 1988

Differential effects of n-3 fatty acids Mildly hyperlipidemic subjects -  TAG conc. : 1.7g/d EPA+DHA > 9.5g/d ALA  LDL sensitivity to oxidation with 1.7g EPA+DHA YE Finnegan et al, AJCN, 2003 -  TAG conc. : comparable with 4g/d EPA and 4g/d DHA  LDL size : only with 4g/d DHA  forearm blood flow : only with 4g/d DHA TA Mori et al, AJCN & Circulation, 2000

48 obese insulin-resistant subjects Regulatory effects of HMG CoA reductase inhibitor and Fish Oils on apolipoprotein B-100 kinetics in insulin-resistant obese male subjects with dyslipidemia 48 obese insulin-resistant subjects 6 wks Atorva 40mg vs Omacor 4g vs Atorva+Omacor EPA/DHA :  plasma VLDL-apo B  VLDL-apo B secretion  VLDL to LDL conversion Atorva :  plasma apo B lipoproteins  FCR of VLDL-, IDL-, LDL-apo B Atorva + EPA/DHA : cumulative effects DC Chan, Diabetes, 2002

Healthy Swedish Subjects with mild hyperlipidaemia (after 4 weeks of Mediterranean vs. Swedish diet) P < 0.05 P < 0.05 Swedish Mediterranean P < 0.05 P < 0.05 A. Ambring et al, Clin Sci, 2004

Effect of weight loss and lifestyle changes on vascular inflammatory markers in obese women Randomized single-blind trial ;120 premenopausal obese women (BMI ≥ 30) with no diabetes, hypertension, or hyperlipidemia Low energy mediterranean diet +  physical activity vs. Healthy food + exercise K Esposito et al, JAMA, 2003

Le Syndrome Métabolique: stratégie Unique ? AG n-3 effet préventif, non thérapeutique, sur insulino-résistance  inflammation  défenses anti-oxydantes cellulaires  (modérée) pression artérielle  TAG  HDL-chol  sdLDL mais  LDL-chol Globale nutrition : association avec  AG saturés et  AGMI (alimentation type méditerranéen) amélioration contrôle pondéral activité physique si indiquée, association à hypolipémiants

Le syndrome métabolique : effets des fibres (FOS) chez le rat  satiété   prises alimentaires  absorption glucides & acides gras  glycémie & triglycéridémie post-prandiales  sécrétion insuline  phénomènes inflammatoires (NFkB)  possibilité effets complémentaires et/ou synergiques à ceux des AG w 3

Delivery of DHA and EPA via alpha linolenic acid is not efficient Delivery of DHA and EPA via increased fish oil intake is very efficient Current intake of fish oil EPA and DHA in the USA is ~1/3 – 1/6 of recommended levels

General Conclusions and recommendations Fish and Omega-3 fatty acid intake Clear benefits in secondary prevention Less strong evidence in primary prevention Include (fatty) fishes as part of healthy preventive diet Temporary limited intake in specific subgroups (pregnant women & children) Increased intake in other specific subgroups (high risk & hypertriglyceridaemia) Possibility to use (good quality) supplements

Fish and Omega-3 fatty acid intake : The unanswered questions Indications for primary prevention in high risk groups (renal failure, insulin-resistance, transplantation, …) ? Doses in different indications ? Effect of DHA vs. EPA ? Intake of ALA (and w3/w6 intake) ? Prevention of lipid peroxidation ? Optimising intestinal absorption & cell incorporation ?