how oleic acid in olive oil reduces blood pressure. pablo v. escribá, university of the balearic...

How Oleic Acid in Olive Oil Reduces Blood Pressure.

Pablo V. Escribá, University of the Balearic Islands, [email protected]

John E. Halver, University of [email protected]

VASCULAR SMOOTH MUSCLE

AM

P c

GM

P c

PKA

PKG

MUSCULARRELAXATION

VASODILATION CROSS-TALK

Endothelium-dependent

REGULATION OF BLOOD PRESSURE

AC

AM

P c

PKA

G proteins

How Oleic Acid in olive oil reduces blood pressure. P.V. Escribá & J.E. Halver

GPCR-associated signalingGPCR-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+.


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.

G PROTEIN-COUPLED RECEPTORS (GPCRs) G PROTEIN-COUPLED RECEPTORS (GPCRs) INVOLVED IN THE REGULATION OF BPINVOLVED 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

CARDIAC CELL SIGNALINGCARDIAC CELL SIGNALING

1 adrenoceptor

Gs protein

(+) Adenylyl cyclase

cAMP

(+) PKA

contractility

Gq11 protein

(+) Phospholipase C

DAG IP3

(+) PKC (+) Ca2+

contractility

Muscarinic M2

Gi/Go proteins

(+) Ca2+ channels

contractility

(-) Adenylyl cyclase

(+) K+ Channels

Cell hyperpolarization

1 adrenoceptor

VASCULAR CELL SIGNALINGVASCULAR CELL SIGNALING

1 adrenoceptor

Gs protein

(+) Adenylyl cyclase

cAMP

(+) PKA

Vasorelaxation

Gq11 protein

(+) Phospholipase C

DAG IP3

(+) PKC (+) Ca2+

vasoconstriction

Muscarinic M3

Nitric Oxide

Intracellular Ca2+

Vasorelaxation

(+) Guanylyl cyclase

2 adrenoceptor

cGMP

(+) PKG

Intracellular Ca2+

Membrane lipid structure and G protein-membrane interactions


G protein heterotrimers prefer nonlamellar-prone membrane regionsG protein -monomers prefer lamellar-prone regions (higher PC content)

Binding of G proteins to model membranes (liposomes) determined by immunoblotting

Membrane lipid structure and G protein-membrane interactions

G protein -dimers have a huge preference for nonlamellar-prone membrane regions (higher PE content).

They take G protein -monomers from nearby GPCRs.


Binding of G proteins to model membranes (liposomes) determined by immunoblotting

G protein-membrane interactions

Membrane on G structure

Membrane and aqueous phases

Only in membrane

G on membrane structure

FTIR analysisX-ray analysis DSC analysis


C-terminal region of G: Main player in G protein-lipid interactions.Membrane lipids affect G protein structure and vice versa.


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 G. Upon activation, G goes to lamellar-prone regions (L) to regulate the activity of effectors (E, E1).

G protein-membrane interactions


C-terminal region of G: Main player in G protein-lipid interactions.

GFP

G2-GFP

Nuclei

Cells

Merge

WT MutC1 MutC3 MutC4

Altered G protein distribution after point mutations on the C-terminal region (G2-GFP fusion proteins)

Cellular localization of G protein-GFP constructs by confocal microscopy

Membrane fatty acids

Oleic Acid Elaidic Acid Stearic Acid

18:1 n-9 cis 18:1 n-9 trans 18:0


OA EA SA

Effect of OA and EA on membrane structure by X-Ray diffraction

Oleic acid but not the structurally related fatty acids, elaidic and stearic acid, regulates membrane lipid structure.


DEPE DEPE:OA (20:1, mole:mole) DEPE:EA (20:1, mole:mole)

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.


Acute effects of VOO, TO, and OA Acute effects of VOO, TO, and OA on systolic BPon systolic BP


Normotensive rats

V, vehicle; VOO, virgin olive oil; TO, triolein; OA, oleic acid

Olive oil also induces short-term reductions of BP. Triolein and oleic acid have similar effects

Chronic effects of OA, elaidic acid Chronic effects of OA, elaidic acid and stearic acid on systolic BPand stearic acid on systolic BP


Normotensive rats

V, vehicle; OA, oleic acid; EA, elaidic acid; SA, stearic acid

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%).

Effects of VOO Effects of VOO treatment on treatment on BP in SHRsBP in SHRs


Hypertensive rats

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, TO, and OA treatments Effects of VOO, TO, and OA treatments on total G protein αion total G protein αi22, αi, αi33 and α and αq/11q/11

subunits and PKCβ1 levels in aortasubunits 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).

V, vehicle; VOO, virgin olive oil; TO, triolein; OA, oleic acid

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.

Consequences for human health

1. 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.

2. Because this effect has structural molecular bases, we can design molecules to regulate BP or reverse other pathological processes.



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!!!

Chronic effects of soybean oil, VOO, Chronic effects of soybean oil, VOO, or TO treatments on systolic BPor TO treatments on systolic BP


Normotensive rats

V, vehicle; SO, soy oil; VOO, virgin olive oil; TO, triolein

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.

OLEIC ACID ANALOGUES FOR TREATMENT OF BLOOD

PRESSURE

OLEIC ACID ANALOGUES FOR TREATMENT OF BLOOD

PRESSURE



Rational design of 2-hydroxyoleic acid

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).

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


2-Hydroxyoleic acid on BP in hypertensive animals


Effect of olive oilEffect of olive oil

Dose-dependent effects of 2-OHOA on systolic BP in SHRs




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.


2-Hydroxyoleic acid has no cytotoxicity

Lung

Kidney

Liver

Control 2OHOA Control 2OHOA

Aorta

Heart

S.I.


WKYveh

WKY2OHOA

SHRveh

SHR2OHOA

0

2

4

6

8

**

cGM

P le

vels

in a

ort

a

(pm

ol·m

g-1 p

rote

in)

cGMP is not involved in the effects of 2-hydroxyoleic acid

The levels of cGMP in normotensive (WKY) and hypertensive (SHR) rats were unrelated to 2-hydroxyoleic acid effects on BP

2-Hydoxyoleic acid on Gi proteins

Serum w/oPDGF

Serum with PDGF

Gi3

actin

Gi3

actin

48 h

BASAL

10%FBS OH50 OH100

PDGF


2-Hydroxyoleic acid induces reduced expression of G protein.This effect is PDGF-independent.

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

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.

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 ofhypertensive rats and do not change PKA activity in normotensive rats.


-Adrenoceptor signaling pathway

Epinephrine activates -adrenoceptors, which in turn activate a Gs protein. Gs proteins activate adenylyl cyclase, that produce cAMP and activate PKA.

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.

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.

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.

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.

How Oleic Acid in olive oil reduces blood pressure. P.V. Escribá & J.E. HalverFor references see:PNAS 105;13811(208)

how oleic acid in olive oil reduces blood pressure. pablo v. escribá, university of the balearic...

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