Fatty acids differentially modulate insulin-stimulated endothelial nitric oxide production by an Akt-independent pathway.
BACKGROUND
Insulin increases endothelial nitric oxide (NO) production by activating endothelial nitric oxide synthase (eNOS) through protein kinase B (Akt)-mediated phosphorylation of serine residue 1179 (p-eNOS serine 1179). Because fatty acids modulate insulin-stimulated Akt signaling cascades in smooth muscle cells, we hypothesized that fatty acids would differentially regulate endothelial Akt signaling, eNOS phosphorylation, and NO production.


METHODS
Porcine pulmonary artery endothelial cells (PAECs) were treated for 3 hours with 100 microM oleic (18:1) or eicosapentaenoic (20:5) acids or with an equivalent volume of ethanol vehicle (0.1%). PAECs were then treated with graded concentrations (10(9)-10(-5) M) of insulin or incubated overnight (24 hours) in culture medium without fatty acids before insulin treatment. Activation and phosphorylation of Akt and eNOS were determined by immunoblotting. NO production was measured with a chemiluminescence NO analyzer or with a NO-selective carbon fiber microelectrode.


RESULTS
Insulin-stimulated Akt phosphorylation, eNOS phosphorylation, and NO production. The phosphatidylinositol-3 kinase inhibitor wortmannin attenuated insulin-stimulated Akt activation and NO production. Treatment with the omega-3 fatty acid 20:5, but not 18:1, enhanced insulin-stimulated NO production but failed to alter insulin-stimulated Akt activation or eNOS serine 1179 phosphorylation.


CONCLUSIONS
Individual fatty acyl species have distinct effects on insulin-stimulated endothelial NO production. Although fatty acids alter Akt signaling in muscle cells, the current results indicate that fatty acids do not modulate endothelial NO production through alterations in insulin-stimulated, Akt-mediated eNOS phosphorylation.
Lynn
Matthew A
MA
Department ofMedicine, Veterans Affairs Medical Center, 151 -P 1670 Clairmont Rd, Decatur, GA 30033 , USA.
Rupnow
Heidi L
HL
Kleinhenz
Dean J
DJ
Kanner
William A
WA
Dudley
Samuel C
SC
Hart
C Michael
CM
D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D015153:Blotting, Western; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D004338:Drug Combinations; D015118:Eicosapentaenoic Acid; D004730:Endothelium, Vascular / Q000187:drug effects* / Q000378:metabolism; D005227:Fatty Acids / Q000494:pharmacology*; D015525:Fatty Acids, Omega-3 / Q000494:pharmacology; D005231:Fatty Acids, Unsaturated / Q000494:pharmacology; D007328:Insulin / Q000494:pharmacology*; D009569:Nitric Oxide / Q000378:metabolism*; D019301:Oleic Acid / Q000494:pharmacology; D000081082:Phosphoinositide-3 Kinase Inhibitors; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt; D011651:Pulmonary Artery; D013552:Swine; D000077191:Wortmannin
D000730:Androstadienes; D004338:Drug Combinations; D005227:Fatty Acids; D015525:Fatty Acids, Omega-3; D005231:Fatty Acids, Unsaturated; D007328:Insulin; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011518:Proto-Oncogene Proteins; D019301:Oleic Acid; D009569:Nitric Oxide; D015118:Eicosapentaenoic Acid; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D000077191:Wortmannin