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How Oleic Acid in Olive Oil Reduces Blood Pressure.

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1 How Oleic Acid in Olive Oil Reduces Blood Pressure.
Pablo V. Escribá, University of the Balearic Islands, John E. Halver, University of Washington 1

2 PKA PKG AC PKA REGULATION OF BLOOD PRESSURE G proteins AMPc CROSS-TALK
VASCULAR SMOOTH MUSCLE AMPc PKA CROSS-TALK VASODILATION MUSCULAR RELAXATION G proteins GMPc PKG AC Endothelium-dependent AMPc PKA

3 GPCR-associated signaling GPCR-associated signaling
Blood Pressure is tightly controlled by several signaling systems. Many of them involve regulation of cell functions through G protein-coupled receptors (GPCRs). This slide shows a Gq protein-associated pathway involved in the regulation of cytosolic Ca2+. How Oleic Acid in olive oil reduces blood pressure. P.V. Escribá & J.E. Halver 3

4 G proteins: peripheral membrane proteins
G proteins propagate messages from membrane receptors to effectors that control cytosolic levels of second messengers (cAMP, cGMP, IP3, DAG, ions, etc.). Lipid-protein interactions regulate the localization and activity of G proteins and, therefore, cell signaling. How Oleic Acid in olive oil reduces blood pressure. P.V. Escribá & J.E. Halver

5 G PROTEIN-COUPLED RECEPTORS (GPCRs) INVOLVED IN THE REGULATION OF BP
CENTRAL NERVOUS SYSTEM : ADRENOCEPTORS α 2A α2B PERIPHERAL NERVOUS SYSTEM : CARDIAC MUSCLE: ADRENOCEPTORS α1 β1 β2 MUSCARINIC RECEPTORS M2 SMOOTH MUSCLE: ADRENOCEPTORS α1 β2 MUSCARINIC RECEPTORS M3

6 CARDIAC CELL SIGNALING
1 adrenoceptor 1 adrenoceptor Muscarinic M2 Gs protein Gq11 protein Gi/Go proteins (+) Adenylyl cyclase (+) Phospholipase C (-) Adenylyl cyclase (+) K+ Channels cAMP DAG IP3 (+) PKA (+) PKC (+) Ca2+ Cell hyperpolarization (+) Ca2+ channels contractility contractility contractility

7 VASCULAR CELL SIGNALING
2 adrenoceptor 1 adrenoceptor Muscarinic M3 Nitric Oxide Gs protein Gq11 protein (+) Adenylyl cyclase (+) Phospholipase C (+) Guanylyl cyclase cAMP DAG IP3 cGMP (+) PKA (+) PKC (+) Ca2+ (+) PKG Intracellular Ca2+ vasoconstriction Intracellular Ca2+ Vasorelaxation Vasorelaxation

8 Membrane lipid structure and G protein-membrane interactions
Binding of G proteins to model membranes (liposomes) determined by immunoblotting G protein heterotrimers prefer nonlamellar-prone membrane regions G protein a-monomers prefer lamellar-prone regions (higher PC content) How Oleic Acid in olive oil reduces blood pressure. P.V. Escribá & J.E. Halver

9 Membrane lipid structure and G protein-membrane interactions
G protein bg-dimers have a huge preference for nonlamellar-prone membrane regions (higher PE content). They take G protein a-monomers from nearby GPCRs. Binding of G proteins to model membranes (liposomes) determined by immunoblotting How Oleic Acid in olive oil reduces blood pressure. P.V. Escribá & J.E. Halver

10 Gg protein-membrane interactions
X-ray analysis DSC analysis FTIR analysis Membrane and aqueous phases Only in membrane Gg on membrane structure Membrane on Gg structure C-terminal region of Gg: Main player in G protein-lipid interactions. Membrane lipids affect G protein structure and vice versa. How Oleic Acid in olive oil reduces blood pressure. P.V. Escribá & J.E. Halver

11 A new model of G protein activity based on their interactions with lipids
Two views of the same process. From the membrane (above) or from the cytosol (right): receptors (R) are activated by agonists (a or AG) in hexagonal phase-prone regions (H), which are loaded with G proteins (G) thanks to Gbg. Upon activation, Ga goes to lamellar-prone regions (L) to regulate the activity of effectors (E, E1). How Oleic Acid in olive oil reduces blood pressure. P.V. Escribá & J.E. Halver

12 Gg protein-membrane interactions
GFP Gg2-GFP Nuclei Cells Merge WT MutC1 MutC3 MutC4 Altered Gg protein distribution after point mutations on the C-terminal region (Gg2-GFP fusion proteins) Cellular localization of Gg protein-GFP constructs by confocal microscopy C-terminal region of Gg: Main player in G protein-lipid interactions. How Oleic Acid in olive oil reduces blood pressure. P.V. Escribá & J.E. Halver

13 Oleic Acid Elaidic Acid Stearic Acid 18:1 n-9 cis 18:1 n-9 trans 18:0
Membrane fatty acids OA EA SA Oleic Acid Elaidic Acid Stearic Acid :1 n-9 cis :1 n-9 trans :0 How Oleic Acid in olive oil reduces blood pressure. P.V. Escribá & J.E. Halver

14 Effect of OA and EA on membrane structure by X-Ray diffraction
DEPE DEPE:OA (20:1, mole:mole) DEPE:EA (20:1, mole:mole) Oleic acid but not the structurally related fatty acids, elaidic and stearic acid, regulates membrane lipid structure. How Oleic Acid in olive oil reduces blood pressure. P.V. Escribá & J.E. Halver

15 Effect of OA and EA on G protein function
Oleic acid but not the structurally related fatty acids, elaidic and stearic acid, regulates G protein function. How Oleic Acid in olive oil reduces blood pressure. P.V. Escribá & J.E. Halver

16 Acute effects of VOO, TO, and OA on systolic BP
Normotensive rats Olive oil also induces short-term reductions of BP. Triolein and oleic acid have similar effects Acute effects of VOO, TO, and OA on systolic BP. Sprague–Dawley rats received one p.o. dose of vehicle (V), VOO (2 g·kg−1), TO (1 g·kg−1), or OA (1 g·kg−1). Systolic BP (expressed in mm Hg) was measured 2 h after administration (acute treatment). Each value represents mean ± SEM (n = 10). *, P < 0.05 and ***, P < vs. vehicle-treated rats (ANOVA). V, vehicle; VOO, virgin olive oil; TO, triolein; OA, oleic acid How Oleic Acid in olive oil reduces blood pressure. P.V. Escribá & J.E. Halver

17 Chronic effects of OA, elaidic acid and stearic acid on systolic BP
Normotensive rats Oleic acid, but not elaidic and stearic acids, induces BP reductions. Oleic acid is the fatty acid present in triolein and the main component of olive oil (70-80%). Chronic effects of OA, elaidic acid and stearic acid on systolic BP. Sprague–Dawley rats received vehicle (V), OA (1 g·kg−1), elaidic acid (EA; 1 g·kg−1), or stearic acid (SA; 1 g·kg−1) p.o. every 12 h for 14 days. Each value represents the mean ± SEM (n = 10). *, P < 0.05 vs. vehicle-treated rats (ANOVA). V, vehicle; OA, oleic acid; EA, elaidic acid; SA, stearic acid How Oleic Acid in olive oil reduces blood pressure. P.V. Escribá & J.E. Halver

18 Effects of VOO treatment on BP in SHRs
Olive oil and oleic acid (OA) reduce BP in hypertensive animals. Elaidic acid (EA) and stearic acid (SA) failed to reduce BP. Effects of VOO treatment on BP in SHRs. (A) Rats received vehicle or VOO (2 g·kg−1) p.o. every 12 h for 14 days. Time course of systolic BP (expressed in mm Hg) in vehicle-treated SHRs (Control) and VOO-treated SHRs (olive oil). (B) Chronic (14-day) effects of vehicle (Control), OA, stearic acid (SA), and elaidic acid (EA) on systolic BP of SHR. *, P < 0.05; **, P < 0.01; and ***, P < versus vehicle-treated SHRs (Student's t test in A and ANOVA in B). Hypertensive rats How Oleic Acid in olive oil reduces blood pressure. P.V. Escribá & J.E. Halver

19 Effects of VOO, TO, and OA treatments on total G protein αi2, αi3 and αq/11 subunits and PKCβ1 levels in aorta Olive oil, triolein and oleic acid regulate Gi and Gq protein levels and those of downstream effectors, such as phospholipase C (PLC). Effects of VOO, TO, and OA treatments on total G protein αi2, αi3 and αq/11 subunits and PKCβ1 levels in aorta. Sprague–Dawley rats received vehicle (V), VOO (2 g·kg−1), TO (1 g·kg−1), or OA (1 g·kg−1) p.o. every 12 h for 14 days. Insets show representative immunoblots of aortic G protein αi2 (A), G protein subunit αi3 (B), G protein subunit αq/11 subunit (C), and PLCβ1 (PLCβ1a + PLCβ1b) (D) after vehicle, VOO, TO, or OA administration. The amount of total protein loaded was 24 μg for Gαi2, αi3, and αq/11 subunits and 80 μg for PLCβ1. The columns represent the mean ± SEM (n = 5) of total protein levels quantified against standard curves and normalized to the protein content from vehicle-treated rats (taken as 100%). *, P < 0.05 and **, P < 0.01 vs. vehicle-treated rats (ANOVA). V, vehicle; VOO, virgin olive oil; TO, triolein; OA, oleic acid How Oleic Acid in olive oil reduces blood pressure. P.V. Escribá & J.E. Halver

20 Correlations between BP reductions (mmHg) and decrease of L-to-HII phase transition temperature (°C; A) or the dose of cis-MUFA (g/kg) administered to animals (B) Treatments: 1, VOO; 2, 2-hydroxyoleic acid; 3, TO; 4; OA; 5, stearic acid; 6; vehicle; 7, elaidic acid; 8, soybean oil. Decrease of BP correlates with the dose of cis-monounsaturated fatty acids (cis-MUFA). Fatty acids, rather than triacylglycerides, are involved in this effect. Correlations between BP reductions (mmHg) and decrease of L-to-HII phase transition temperature (°C; A) or the dose of cis-MUFA (g/kg) administered to animals (B). Each numbered point represents the treatment: treatment 1, VOO; treatment 2, 2-hydroxyoleic acid; treatment 3, TO; treatment 4; OA; treatment 5, stearic acid; treatment 6; vehicle; treatment 7, elaidic acid; treatment 8, soybean oil. Solid lines represent the regression of the correlations, whose correlation coefficients (r) and statistical significance (P) are shown in both cases. How Oleic Acid in olive oil reduces blood pressure. P.V. Escribá & J.E. Halver

21 Consequences for human health
Consumption of fats that contain cis-monounsaturated fatty acids (e.g., oleic acid) have positive effects on cardiovascular health, compared with saturated or trans-MUFA fats. Because this effect has structural molecular bases, we can design molecules to regulate BP or reverse other pathological processes. How Oleic Acid in olive oil reduces blood pressure. P.V. Escribá & J.E. Halver 21

22 Then, I can control cell functions by regulating membrane lipid structure and composition!!!
Sure. You may even design nutritional or pharmaceutical approaches to treat human pathologies!!! How Oleic Acid in olive oil reduces blood pressure. P.V. Escribá & J.E. Halver

23 Chronic effects of soybean oil, VOO, or TO treatments on systolic BP
Olive oil, but not soy oil, reduces blood pressure. Triolein, the main triacylglyceride in olive oil reproduces this effect. VOO is the oil obtained from olives by pressure at room temperature. Normotensive rats Chronic effects of soybean oil, VOO, or TO treatments on systolic BP. Sprague–Dawley rats received vehicle (V), soybean oil (SO; 2 g·kg−1), VOO (2 g·kg−1), and TO (1 g·kg−1), p.o. every 12 h for 14 days. Each value represents the mean ± SEM (n = 10). *, P < 0.05 and **, P < 0.01 vs. vehicle-treated rats (ANOVA). V, vehicle; SO, soy oil; VOO, virgin olive oil; TO, triolein How Oleic Acid in olive oil reduces blood pressure. P.V. Escribá & J.E. Halver

24 OLEIC ACID ANALOGUES FOR TREATMENT OF BLOOD PRESSURE
How Oleic Acid in olive oil reduces blood pressure. P.V. Escribá & J.E. Halver 24

25 Rational design of 2-hydroxyoleic acid
2-Hydroxyoleic acid (left) is an oleic acid (center) analog that keeps most structural properties of oleic acid and differs from elaidic acid (pink) in its interaction with membranes (right). How Oleic Acid in olive oil reduces blood pressure. P.V. Escribá & J.E. Halver

26 2-Hydroxyoleic acid on BP in hypertensive animals
Effect of olive oil Effects of 2-hydroxyoleic acid (2-OHOA) treatment (600 mg/kg every 12 h) on blood pressure (BP) and heart rate in spontaneously hypertensive rats (SHRs) and Wistar Kyoto (WKY) rats How Oleic Acid in olive oil reduces blood pressure. P.V. Escribá & J.E. Halver

27 2-Hydroxyoleic acid on BP in hypertensive animals
Dose-dependent effects of 2-OHOA on systolic BP in SHRs How Oleic Acid in olive oil reduces blood pressure. P.V. Escribá & J.E. Halver

28 2OHOA increases elasticity in aorta rings
Rat aorta rings were preincubated 1 hour in the presence (2OHOA) or absence (control) of 2-hydroxyoleic acid. Then, the contraction in response to noradrenaline was determined. How Oleic Acid in olive oil reduces blood pressure. P.V. Escribá & J.E. Halver

29 2-Hydroxyoleic acid has no cytotoxicity
Control 2OHOA Control 2OHOA Lung Aorta Heart Kidney Liver S.I.

30 cGMP is not involved in the effects of 2-hydroxyoleic acid
8 6 levels in aorta protein) 4 -1 ·mg ** (pmol 2 cGMP WKY WKY SHR SHR veh 2OHOA veh 2OHOA The levels of cGMP in normotensive (WKY) and hypertensive (SHR) rats were unrelated to 2-hydroxyoleic acid effects on BP How Oleic Acid in olive oil reduces blood pressure. P.V. Escribá & J.E. Halver

31 2-Hydoxyoleic acid on Gi proteins
10%FBS OH50 OH100 Serum w/o PDGF Gi3 actin BASAL PDGF Serum with PDGF Gi3 actin 2-Hydroxyoleic acid induces reduced expression of G protein. This effect is PDGF-independent. How Oleic Acid in olive oil reduces blood pressure. P.V. Escribá & J.E. Halver

32 2-Hydroxyoleic acid regulates the expression of some PKA subunits
Effects of 2-hydroxyoleic acid treatment (600 mg/kg every 12 h) for 7 days on protein kinase A (PKA) subunit levels in aortas from SHRs and WKY rats 2-Hydroxyoleic acid regulates the expression of some PKA subunits How Oleic Acid in olive oil reduces blood pressure. P.V. Escribá & J.E. Halver

33 Effects of 2-hydroxyoleic acid treatment (600 mg/kg every 12 h) for 7 days on adenylyl cyclase (AC) activities in SHRs and WKY rat aortas 2-Hydroxyoleic acid treatments induce restoration of Adenyly cyclase activity in rat aorta. Adenyly cyclase produces cAMP, which in turn regulates PKA. How Oleic Acid in olive oil reduces blood pressure. P.V. Escribá & J.E. Halver

34 hypertensive rats and do not change PKA activity in normotensive rats.
Effects of 2-OHOA treatment (600 mg/kg every 12 h) for 7 days on PKA activity in aortas from SHRs and WKY rats 2-Hydroxyoleic acid treatments induce restoration of PKA activity in aorta of hypertensive rats and do not change PKA activity in normotensive rats. How Oleic Acid in olive oil reduces blood pressure. P.V. Escribá & J.E. Halver

35 b-Adrenoceptor signaling pathway
Epinephrine activates b-adrenoceptors, which in turn activate a Gs protein. Gs proteins activate adenylyl cyclase, that produce cAMP and activate PKA. How Oleic Acid in olive oil reduces blood pressure. P.V. Escribá & J.E. Halver

36 In addition, this effect was time- and concentration-dependent.
Effects of acute administration of the PKA inhibitor 8-bromo adenosine-3',5'-cyclic monophosphorothioate, Rp isomer (Rp-8-Br-cAMP), on systolic BP in vehicle-treated (control) and 2-OHOA-treated SHRs The use of a specific PKA inhibitor in rats treated with 2-hydroxyoleic acid reversed the hypotensive effect of this drug. This result demonstrates that the proposed mechanism of the hypotensive effects of 2-hydroxyoleic acid (via Gs protein, adenyly cyclase and PKA) operates in vivo. In normotensive animals (treated and untreated) and hypertensive rats not treated with 2-hydroxyoleic acid, no blood pressure reductions were observed (data not shown), indicating that the effect cannot be attributed to the PKA inhibitor itself. In addition, this effect was time- and concentration-dependent. How Oleic Acid in olive oil reduces blood pressure. P.V. Escribá & J.E. Halver

37 Effects of withdrawal of 2-OHOA on systolic BP and aortic PKA subunit levels in SHRs
After treatments with 2-hydroxyoleic acid, blood pressure started to rise to reach their initial high values about two weeks after the administration of the last dose. This slow recovery of blood pressure indicates that the modification induced by the fatty acid occurs on membrane lipids, whose turnover is slower. How Oleic Acid in olive oil reduces blood pressure. P.V. Escribá & J.E. Halver

38 Effects of 2-OHOA treatment (600 mg/kg every 12 h) for 7 days on Rho kinase II levels in aortas from SHRs and WKY rats Rho kinase also appeared to be markedly increased in hypertensive rats. Treatment with 2-OHOA reduced the expression of this protein to normal levels. This result indicates that besides PKA, vascular cell’s cytoskeleton is also regulated by this compound. How Oleic Acid in olive oil reduces blood pressure. P.V. Escribá & J.E. Halver

39 CONCLUSION The CIS structure of Oleic Acid fits neatly into the phospholipid structure of biomembranes, and alters receptor sites on the membrane surface, to regulate blood pressure. Q.E.D. For references see:PNAS 105;13811(208) How Oleic Acid in olive oil reduces blood pressure. P.V. Escribá & J.E. Halver


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