Emerging science on omega-3: A lipidomic view of omega-3 in health and disease. Health benefits of omega 3 fatty acids

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1 Emerging science on omega-3: A lipidomic view of omega-3 in health and disease Peter Meikle 04 May 2017 Health benefits of omega 3 fatty acids Can lower plasma triacylglycerol can reduce the risk of metabolic syndrome and type 2 diabetes? Have anti-inflammatory properties Can lower systemic inflammation Can reduce the risk of heart disease AHA Science Advisory Can reduce the risk of age related cognitive decline? Preclinical vs clinical trials Omega-3 fatty acids and incident type 2 diabetes Br J Nutr Jun;107 Suppl 2:S Omega-3 fatty acids and incident type 2 diabetes: a systematic review and meta-analysis. Wu JH 1, Micha R, Imamura F, Pan A, Biggs ML, Ajaz O, Djousse L, Hu FB, Mozaffarian D. The overall pooled findings do not support either major harms or benefits of fish/seafood or EPA + DHA on development of DM, and suggest that ALA may be associated with modestly lower risk. Reasons for potential heterogeneity of effects, which could include true biologic heterogeneity, publication bias, or chance, deserve further investigation. Omega-3 Polyunsaturated Fatty Acid (Fish Oil) Supplementation and the Prevention of Clinical Cardiovascular Disease Randomised control trials show evidence for omega-3 in cardiovascular disease May reduce CHD death in patients with a prior history of CHD May reduce CHD death in patients with a history of heart failure Evidence supports the reduction of recurrent atrial fibrillation No effect on recurrent non-fatal MI No benefit in CVD outcomes in patients with or at risk of diabetes mellitus No benefit in recurrent stoke in patients with prior stoke Effects thought to be through a reduction of ischemia induced sudden cardiac death Circulation Apr 11;135(15):e867-e884 Omega-3 Polyunsaturated Fatty Acid (Fish Oil) Supplementation and the Prevention of Clinical Cardiovascular Disease: A Science Advisory From the American Heart Association. Siscovick et al. American Heart Association Nutrition Committee of the Council on Lifestyle and Cardiometabolic Health; Council on Epidemiology and Prevention; Council on Cardiovascular Disease in the Young; Council on Cardiovascular and Stroke Nursing; and Council on Clinical Cardiology.

2 How do Omega-3 fatty acids exert health benefits Omega-3 fatty acid metabolites Protectins, resolvins and maresins have demonstrated anti-inflammatory effects o Can modulate the immune response and lower systemic inflammation Can influence other factors such as oxidation Can be oxidised and so protect other lipids/proteins (oxidative stress) Direct effect on cell receptors PPAR and other transcription factors Effects on cell behaviour mediated by composition of cell membranes Influence cell membrane fluidity (lipid rafts) o Via incorporation into phospholipid pools Plasma lipidomics at Baker Institute Clinical lipid measures Cholesterol, HDL-C, triglycerides, LDL-C Lipid composition of plasma Cholesteryl esters Di- and Triacylglycerols Phospholipids Ceramides Glycosphingolipids Sphingomyelin LCAT ApoA-I cholesterol cholesterol ester phospholipid triglyceride Modified lipids (oxidized, glycated) 1-palmitoyl-2-(5'-oxo-valeroyl)-sn-glycero- Phosphatidylcholine 3-phosphocholine Ceramide (POVPC) (d18:1/16:0) 16:0/18:0 Lysolipids Triacylglycerol 16:0/16:0/16:0 Free fatty acids 10,000 different lipids in humans (most will be in plasma at some level (>1000 abundant species) Metabolomics Laboratory (Analytical platforms) Metabolomics Laboratory (High throughput lipidomics) Lipid extraction (10µL plasma) 1 Addition of stable isotope/non-physiological standards Single phase BuOH/MeOH Centrifuge (supernatant) LC-MS/MS Sciex API 4000 Q/TRAP triple quadrupole mass spectrometer Agilent 1200 HPLC system Column = Agilent Zobax C-18 eclipse (1.8 um x 50 mm) Agilent 6490 triple quadrupole mass spectrometer Agilent 1290 HPLC system Column = Agilent Zobax C-18 eclipse plus (1.8 um x 50 mm) Lipid quantification (relative) 2 LC ESI-MS/MS Stable isotope dilution (non-physiological) Multiple reaction monitoring (scheduled) 350 lipid species (10 min) 1 Alshehry et al. An efficient single phase method for the extraction of plasma lipids. Metabolites. 2015;5:(2) Weir et al. Plasma lipid profiling in a large population-based cohort. Journal of lipid research. 2013;54:(10)

3 Plasma lipid MRM experiment (20min LC gradient) %B Separation of isobaric and isomeric species XIC of +MRM (159 pairs): / amu Expected RT: 0.0 I e6 1.00e6 9.00e5 8.00e5 7.00e5 6.00e5 5.00e5 4.00e5 Phospholipids Separation of acyl / alkyl / alkenyl species of the same nominal mass PC(P-36:4) Max. 1.1e6 cps. Current capabilities >600 lipid species in 15 min e5 2.00e5 1.00e PC(35:5) PC(O-36:5) Time, min Separation of isobaric and isomeric species (phosphatidylcholine species) x10 6 +ESI x10 5 MRM Frag=380.0V CF=0.000 DF=0.000 CID@21.0 ( > ) 2016_02_13-TQC.d Smooth Cpd 146: PC 36:3: +ESI MRM Frag=380.0V CF=0.000 DF=0.000 CID@21.0 ( > ) FT03_0007 TQC 0745.d * * PC 36: PC 18:1/18:2 PC 16:0/20: Counts 6.78 vs. Acquisition 6.8 Time 6.82 (min) Counts vs. Acquisition Time (min) High throughput lipidomic profile Lipid class/subclass No. of species Parent ion Daughter ion dihydroceramide (dhcer) 6 [M+H] m/z ceramide (Cer) 41 [M+H] + Sphingoid specific m/z monohexosylceramide (MHC) 6 [M+H] m/z dihexosylceramide (DHC) 6 [M+H] /z trihexosylcermide phosphatidylcholine (THC) (PC) 686 [M+H] m/z sulphatide alkylphosphatidylcholine (Sul) (PC(O)) 22 6 [M+H] [M+H] m/z m/z G M1 ganglioside (GM1) 1 [M+2H] m/z alkenylphosphatidylcholine (PC(P)) 24 [M+H] G M3 ganglioside (GM3) 6 [M+H] m/z m/z sphingomyelin lysophosphatidylcholine (SM) (LPC) [M+H] m/z lysoalkylphosphatidylcholine (PC) (LPC(O)) [M+H] m/z phosphatidylethanolamine (PE) lysoalkenylphosphatidylcholine (LPC(P)) [M+H] [M+H] + NL, Da m/z phosphatidylinositol (PI) 32 [M+NH 4 ] + NL, Da lysophosphatidylinositol phosphatidylethanolamine (LPI) (PE) 36 8 [M+NH [M+H] + 4 ] + NL, NL, Da Da phosphatidylserine alkylphosphatidylethanolamine (PS) (PE(O)) 14 7 [M+H] + NL, Da phosphatidylglycerol alkenylphosphatidylethanolamine (PG) (PE(P)) 56 4 [M[M+H] + NH 4 ] + NL, Acyl specific Da bis(monoacylglycerol)phosphate (BMP) 1 [M + NH 4 ] m/z lysophosphatidylethanolamine (LPE) 14 [M+H] free cholesterol (COH) 1 [M + NH NL, Da 4 ] m/z cholesteryl lysoalkenylphosphatidylethanolamine ester (CE) (LPE(P)) 28 4 [M[M+H] + NH 4 ] + NL, m/z Da cholesteryl ester derivatives 18 [M + NH 4 ] /369.3 m/z acylcarnitine 14 [M+H] m/z diacylglycerol (DG) 20 [M + NH 4 ] + NL, fatty acid triaclyglycerol (TG) 44 [M + NH 4 ] + NL, fatty acid alkyl-diacylglycerol (TG(O)) 3 [M + NH 4 ] + NL, fatty acid TOTAL 602

4 Lipid (partial) Lipid (partial) Serine + palmitate fatty acid cholesterol Sphingolipid Serine + palmitate fatty acid cholesterol Sphingolipid Cardiolipin Cardiolipin CerS1-6 dihydroceramide DEGS SMGS ceramide UGCG monohexosylceramide B4GALT6 sphingomyelin G-3-P 1-acyl-G-3-P Cholesteryl ester Cho acyl-coa fatty alcohol DHAP P-Cho CDP-Cho Etn P-Etn CDP-Etn Plasmalogen Phosphatidylcholine Cholesteryl ester Glycerolipid Phosphatidylinositol CerS1-6 dihydroceramide DEGS SMGS ceramide UGCG monohexosylceramide B4GALT6 sphingomyelin G-3-P 1-acyl-G-3-P Cholesteryl ester Cho acyl-coa fatty alcohol DHAP P-Cho CDP-Cho Etn P-Etn CDP-Etn Plasmalogen Phosphatidylcholine Cholesteryl ester Glycerolipid Phosphatidylinositol dihexosylceramide A4GALT trihexosylceramide 1,2-diacyl-G-3-P 1-O-alkyl-2-acyl-glycerol EPT1 PEMT PC PE PE(O) Desaturase dihexosylceramide A4GALT trihexosylceramide 1,2-diacyl-G-3-P 1-O-alkyl-2-acyl-glycerol EPT1 PEMT PC PE PE(O) Desaturase PPAP2 CDS1 DG pgpa PG PGP DGAT cls CDP-DG pgsa Cardiolipin CDIPT TG PE(P) LPC LPE PLC PEMT PC(O) PC(P) 1-O-alkenyl-2-acyl-Gylcerol LPE(P) PPAP2 CDS1 DG pgpa PG PGP DGAT cls CDP-DG pgsa Cardiolipin CDIPT TG PE(P) LPC LPE PLC PEMT PC(O) PC(P) 1-O-alkenyl-2-acyl-Gylcerol LPE(P) PI LPI LPC(O) LPC(P) PI LPI LPC(O) LPC(P) Obesity, Impaired Glucose Tolerance and Diabetes Australia has an obesity epidemic Increase in type 2 diabetes 7.6% in 2000 will rise to 11.4% by 2025 More than 1/3 individuals will develop diabetes 1,000,000 new cases by 2025 Cost is in the $billions Obesity is an important risk factor for type 2 diabetes not all obese individuals will go on to develop diabetes. Changes in lipid play a key role in the development and progression of diabetes. What is the lipid profile characteristic of: Diabetes? Prediabetes? Obesity? What is the difference between: Diabetes and obesity? Diabetes and prediabetes? Can we classify/predict diabetes from the lipid profile? What is the influence of intervention (diet, drug) on the diabetiogenic lipid profile?

5 AusDiab cohort: Cross sectional study of newly diagnosed patients at baseline Age Sex (%F) BMI Systolic BP Total Chol HDL Chol TRIGs NGT (n = 170) (+/- 13) (+/-3.1) (+/-21) (+/-1.0) (+/-0.4) (+/-0.6) IGT/IFG (n=64) (+/- 11) (+/-3.2) (+/-23) (+/-1.1) (+/-0.5) (+/-1.1) Diabetic (n = 117) (+/- 13) (+/-3.5) (+/-20) (+/-1.1) (+/-0.4) (+/-1.3) (Lipid analysis: API-4000 Q/TRAP ) Lipid extraction 10mL plasma Single phase CHCl 3 / MeOH / H 2 O BuOH / MeOH / H 2 O Multiple reaction monitoring (MRM) 2 x LC ESI-MS/MS experiments 351 participants 337 lipid species in total 7 days 118,000 measurements (Logistic regression analysis) Logistic regression (standardised to inter-quartile range) Diabetes against NGT o Adjust for age, sex, systolic BP, obesity Prediabetes against NGT o Adjust for age, sex, systolic BP, obesity Obese against non-obese (in NGT group only) o Based on median waist/hip ratio o Adjust for age, sex, systolic BP 282 known lipid species 142 associated with Diabetes (vs. NGT, p<0.05)* 116 associated with Prediabetes (vs. NGT, p<0.05)* 58 associated with Obesity (vs.non-obese, p<0.05)* o 108 associated with Obesity (vs. Non-obese, p<0.05) * Corrected for multiple comparisons (Benjamini-Hochberg) (Logistic regression analysis) dihydroceramide ceramide Chol monohexosylceramide dihexosylceramide HDL trihexosylceramide phosphatidylcholine LDL odd chain phosphatidylcholine alkylphosphatidylcholine TRIG alkenylphosphatidylcholine phosphatidylethanolamine phosphatidylserine phosphatidylinositol phosphatidylglycerol cholesterol ester diacylglycerol triacylglycerol glycosyltransferase Odds Ratio Odds Ratio serine + palmitate dihydroceramide multiple enzymes desaturase ceramide glycosyltransferase monohexosylceramide dihexosylceramide Diabetes vs. glycosyltransferase NGT* Prediabetes vs. NGT* trihexosylceramide Obese vs. non-obese (NGT only) (Adjusted for age, sex, SBP, obesity*)

6 Validation with San Antonio Family Heart Study (n=1200, 40 families) (Odds Ratio: Diabetes vs Obesity) dihydroceramide ceramide monohexosylceramide dihexosylceramide trihexosylceramide GM3 ganglioside sphingomyelin hydroxysphingomyelin phosphatidylcholine odd chain phosphatidylcholine alkylphosphatidylcholine alkenylphosphatidylcholine lysophosphatidylcholine lysoalkylphosphatidylcholine phosphatidylethanolamine phosphatidylglycerol phosphatidylinositol phosphatidylserine free cholesterol cholesterol ester diacylglycerol triacylglycerol Odds Ratio Cer 16:0 Cer 18:0 Cer 20:0 Cer 22:0 Cer 24:0 Cer 24:1 total ceramide Odds Ratio Diabetes vs. NGT (Adjusted for age, sex, SBP, obesity) AUSDIAB SAFHS dhcer 18:0 SM(OH) 20:1 LPC(O-18:0) oddpc 33:2 oddpc 35:2 oddpc 37:4 PC(O-34:2) PE 38:6 PE 40:7 PE 40:6 PS 40:6 PI 32:1 PI 34:0 PI 34:1 PI 38:6 PI 40:6 CE 16:2 CE 16:1 CE 24:2 DG 18:2/18: y = x R² = Kroger et al. Erythrocyte membrane phospholipid fatty acids, desaturase Djousse activity, 1.00L, Gaziano and dietary JM, fatty Buring acids JE, in Lee relation IM: to risk of type 2 diabetes in the Dietary European omega-3 Prospective fatty acids Investigation and fish consumption into Cancer and and risk Nutrition of type 2 (EPIC)- diabetes. Am J Clin NutrPotsdam 2011;93: Study. Am J Clin Nutr 2011;93: Diabetes vs. NGT* 0.25 Prediabetes vs. NGT* Obese vs. non-obese (NGT only) (Adjusted 4.00 for age, sex, SBP, obesity*) Odds Ratio (Diabetes) Odds Ratio (correlation to dietary n-3 PUFA) PE 38:6 PE 38:5 PE 40:7 PE 40:6 PS 40:6 PS 40:5 PI 38:6 PI 40:6 CE 22:6 CE 22:5 DG 16:0 22:5 DG 16:0 22:6 TG 18:1 18:1 22: Odds ratio Correlation P value E E E E E E E E E E E E E-11 Omega-3 and type 2 diabetes (Summary) Plasma lipid species are altered in obesity prediabetes and type 2 diabetes Ceramides are positively associated with T2D Alkenylphosphatidylcholines (plasmalogens) are negatively associated with T2D Omega-3 containing species correlate with dietary intake of omega-3 fatty acids Distribution into different lipid pools likely to also be a contributing factor Omega-3 containing species are positively associated with type 2 diabetes This may be a result rather than a cause of T2D The plasma lipidomic profile provides a detailed view of lipid and can be used to assess the effect of dietary intervention studies on metabolic health.

7 Plasmalogens as a potential therapeutic for cardiometabolic disease Plasmalogens and atherosclerosis vinyl ether polar head group Anti-atherogenic anti-oxidant anti-inflammatory cholesterol efflux Plasmalogens and cardiometabolic disease Baker IDI clinical cohort (n=220) Plasmalogens negatively associated with stable and unstable coronary artery disease (Meikle et al, ATVB. 2011;31(11): ) San Antonio Family Heart Study (n=1200) Plasmalogens negatively associated with type 2 diabetes Plasmalogens negatively associated with future CVE (particularly omega-3 species) (Meikle et al, PLoS One. 2013;8(9):e74341) LIPID / ADVANCE Study (n= 10,000) Plasmalogens negatively associated with future CVE (CVD death) o particularly omega-3 species Biosynthetic pathway of plasmalogens Dietary intake Long chain fatty alcohol Acyl-CoA Peroxisome Far-1*/2 1-alkyl-glycerol Acyl-CoA DHAP 1-acyl-DHAP 1-O-alkyl-DHAP DHAP-AT ADHAP-S 1-O-alkenyl-2-acyl-Gylcerol C-PT PLC PC-plasmalogen 1-O-alkyl-2-acyl-GPC C-PT AG kinase 1 desaturase 1-O-alkyl-2-acyl-Gylcerol 1-O-alkyl-2-acyl-G3P AADHAP-R PEMT AAG3P-AT 1-O-alkyl-G3P 1-O-alkyl-DHAP PE-plasmalogen 1-O-alkyl-2-acyl-GPE Cytosol E-PT PH AADHAP-R ER

8 Plasmalogen upregulation to prevent atherosclerosis in ApoE mice (Aliki Rasmiena) Analysis C57/BL6 (controls) ApoE -/- ApoE -/- GPx -/- batyl alcohol Lipid profiles of mouse plasma and heart Sudan IV staining for lesions/plaque in aorta Detection of inflammatory and oxidative markers in aorta and aortic sinus sections plasmalogen chow high fat diet +/- 2% batyl alcohol diet Weekly weighing and twice weekly scruffing 0 wk 6 wk 18 wk Batyl alcohol supplementation increased plasmalogen levels in plasma Batyl alcohol supplementation increased plasmalogen levels in heart Data are median (interquartiles) of N = 9 10 /group. indicates P<0.001 Data are median (interquartiles) of N = 9 10 /group. indicates P<0.001

9 Assessment of aortic lesions C57/BL6 ApoE -/- ApoE -/- GPx1 -/- Batyl alcohol supplementation attenuated atherosclerosis in ApoE deficient mice 0% BA -71% -69% 2% BA Data are mean SEM, expressed as % plaque area, n = 10/group. Data were analysed using student t-tests and compared to 0% BA treated group of the corresponding genotypes. indicates P< Diagram obtained from JACC Plasmalogen upregulation to prevent atherosclerosis in ApoE mice Batyl alcohol containing diet successfully elevated plasmalogen concentration in plasma and heart Plasmalogen up-regulation reduced atherosclerosis in ApoE -/- and ApoE -/- GPx -/- Differential effects on inflammation and oxidative stress were observed in ApoE -/- and ApoE -/- GPx -/- mouse model A lipidomic view of omega-3 in health and disease Plasma lipidomics provide a composite measure of environmental (dietary) and genetic influences on Plasma lipidomics can provide a detailed picture of metabolic health Plasma lipidomics can predict risk of disease Future studies will enable us to assess the effect of dietary interventions on plasma lipidomic profile and thereby on disease risk Such studies may help guide dietary recommendations in the future

10 Acknowledgments Baker Heart and Diabetes Institute Metabolomics Lab Baker Heart and Diabetes Institute Jonathan Shaw / Dianna Magliano Bronwyn Kingwell / Melissa Formosa Paul Nestel University of Indonesia Kevin Aristyo Gunawan Kevin Culham