Patients with diet-induced obesity and type 2 diabetes are at greater risk for developing cardiovascular complications. An important complication is impaired blood vessel function. In a clinically relevant murine model of diet-induced obesity we showed that exposure to elevated free fatty acids (FFAs) reduces nitric oxide (NO) bioavailability leading to arterial dysfunction. My 2007 R15 sought to determine whether the signaling link between elevated FFAs and impaired NO bioavailability involves the lipid metabolite ceramide. Inhibition of ceramide synthesis with myriocin, or heterozygous deletion of dihydroceramide desaturase, an enzyme which catalyzes ceramide synthesis, prevented endothelial dysfunction and systemic hypertension, and preserved endothelial NO synthase (eNOS) phosphorylation in arteries from fat-fed mice. Molecular mechanisms whereby ceramide might lower NO bioavailability were examined using cultured endothelial cells. Palmitate induced repression of basal and agonist-stimulated eNOS phosphorylation, eNOS dimer formation, and NO production were restored following inhibition of ceramide synthesis. The ceramide-induced impairment of eNOS phosphorylation or dimer formation, respectively, was not the result of impaired kinase-mediated eNOS phosphorylation or superoxide anion-mediated peroxynitrite formation. [Instead, ceramide causes protein phosphatase 2A (PP2A) to associate directly with the eNOS/Akt complex, and this is concurrent with decreased basal and agonist-stimulated eNOS phosphorylation. New data obtained since the last submission suggest that PP2A attenuates eNOS phosphorylation by preventing phosphorylation of the pool of Akt that colocalizes with eNOS and / or by directly dephosphorylating eNOS.] In this renewal, Aim 1 will test the hypothesis that PP2A associates with and disrupts the Akt-Hsp90-eNOS complex as a consequence of de novo ceramide synthesis leading to impaired basal and agonist-stimulated eNOS phosphorylation.
Aim 2 will test the hypothesis that ceramide relieves the association of inhibitor 2 of PP2A (I2PP2A) with PP2A such that PP2A can translocate to the membrane and associate with eNOS.
Aim 3 will test the hypothesis that ceramide-induced, PP2A-mediated vascular dysfunction occurs in mice with diet-induced obesity. Results from these studies will provide mechanistic insight linking endogenous vascular ceramide biosynthesis to cardiovascular defects in a murine model of diet-induced obesity and type 2 diabetes, and present new targets for the treatment of vascular dysfunction in these prevalent conditions. Support for our research via the NIH R15 mechanism has provided >35 undergraduate researchers with valuable experience and each has continued to pursue an advanced degree in the biomedical sciences.

Public Health Relevance

Obesity can lead to type 2 diabetes and cardiovascular complications. Our research is focused on determining the mechanisms whereby obesity impairs blood vessel function. We will determine the mechanism whereby the fat metabolite ceramide impairs eNOS enzyme activity and precipitates arterial dysfunction.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Academic Research Enhancement Awards (AREA) (R15)
Project #
2R15HL091493-02A1
Application #
8366869
Study Section
Hypertension and Microcirculation Study Section (HM)
Program Officer
Maric-Bilkan, Christine
Project Start
2012-08-01
Project End
2015-07-31
Budget Start
2012-08-01
Budget End
2015-07-31
Support Year
2
Fiscal Year
2012
Total Cost
$436,594
Indirect Cost
$143,878
Name
University of Utah
Department
Miscellaneous
Type
Other Domestic Higher Education
DUNS #
009095365
City
Salt Lake City
State
UT
Country
United States
Zip Code
84112
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