Lynn Matthew A, Rupnow Heidi L, Kleinhenz Dean J, Kanner William A, Dudley Samuel C, Hart C Michael
Department ofMedicine, Veterans Affairs Medical Center, 151 -P 1670 Clairmont Rd, Decatur, GA 30033 , USA.
J Investig Med. 2004 Mar;52(2):129-36. doi: 10.1136/jim-52-02-22.
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.
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.
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.
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.
胰岛素通过蛋白激酶B（Akt）介导的丝氨酸残基1179（p-eNOS丝氨酸1179）磷酸化激活内皮型一氧化氮合酶（eNOS），从而增加内皮一氧化氮（NO）的生成。由于脂肪酸可调节平滑肌细胞中胰岛素刺激的Akt信号级联反应，我们推测脂肪酸会对内皮Akt信号、eNOS磷酸化及NO生成产生不同的调节作用。
用100微摩尔油酸（18:1）或二十碳五烯酸（20:5）或等体积乙醇载体（0.1%）处理猪肺动脉内皮细胞（PAECs）3小时。然后用不同浓度（10⁹ - 10⁻⁵摩尔）的胰岛素处理PAECs，或在无脂肪酸的培养基中过夜培养（24小时）后再用胰岛素处理。通过免疫印迹法测定Akt和eNOS的激活及磷酸化情况。用化学发光NO分析仪或NO选择性碳纤维微电极测量NO生成量。
胰岛素刺激Akt磷酸化、eNOS磷酸化及NO生成。磷脂酰肌醇-3激酶抑制剂渥曼青霉素可减弱胰岛素刺激的Akt激活及NO生成。用ω-3脂肪酸20:5而非18:1处理可增强胰岛素刺激的NO生成，但未能改变胰岛素刺激的Akt激活或eNOS丝氨酸1179磷酸化。
不同的脂肪酸种类对胰岛素刺激的内皮NO生成有不同影响。尽管脂肪酸可改变肌肉细胞中的Akt信号，但目前结果表明脂肪酸并非通过改变胰岛素刺激的、Akt介导的eNOS磷酸化来调节内皮NO生成。
