Insulin resistance reduces arterial prostacyclin synthase and eNOS activities by increasing endothelial fatty acid oxidation

Abstract

Insulin resistance markedly increases cardiovascular disease risk in people with normal glucose tolerance, even after adjustment for known risk factors such as LDL, triglycerides, HDL, and systolic blood pressure. In this report, we show that increased oxidation of FFAs in aortic endothelial cells without added insulin causes increased production of superoxide by the mitochondrial electron transport chain. FFA-induced overproduction of superoxide activated a variety of proinflammatory signals previously implicated in hyperglycemia-induced vascular damage and inactivated 2 important antiatherogenic enzymes, prostacyclin synthase and eNOS. In 2 nondiabetic rodent models--insulin-resistant, obese Zucker (fa/fa) rats and high-fat diet-induced insulin-resistant mice--inactivation of prostacyclin synthase and eNOS was prevented by inhibition of FFA release from adipose tissue; by inhibition of the rate-limiting enzyme for fatty acid oxidation in mitochondria, carnitine palmitoyltransferase I; and by reduction of superoxide levels. These studies identify what we believe to be a novel mechanism contributing to the accelerated atherogenesis and increased cardiovascular disease risk occurring in people with insulin resistance.

Figure 1. Schematic mechanism by which IR causes increased oxidation of FFA in arterial endothelial cells, activating proatherogenic signals and inhibiting antiatherogenic enzymes.

IR, insulin resistance; ACC, acetyl-CoA carboxylase; CPT-I, carnitine palmitoyltransferase I; AGE, advanced glycation end product; GlcNAc, N-acetylglucosamine; PGI₂, prostacyclin.

Figure 2. Effect of FFAs on ROS production by endothelial cells.

(A) Effect of FFAs on ROS production in arterial cells cultured in 5 mM glucose. Cells were incubated in 5 mM glucose without added insulin, plus the indicated concentrations of oleic acid (bars 1–4), and with oleic acid plus 3 nM insulin with and without pretreatment with 100 nM wortmannin (bars 5 and 6). Each bar represents the mean plus SEM of 4 separate experiments, each with n = 8. *P < 0.01 compared with cells incubated in 5 mM glucose alone. (B) Effect of FFAs on ROS production in retinal capillary endothelial cells cultured in 5 mM glucose. Cells were incubated in 5 mM glucose, 30 mM glucose, or 5 mM glucose plus oleic acid. Each bar represents the mean plus SEM of 4 separate experiments, each with n = 8. *P < 0.01 compared with cells incubated in 5 mM glucosalone.

Figure 3. Effect of CPT-I inhibition, UCP-1, and MnSOD on FFA-induced ROS production.

Cells were incubated in 5 mM glucose alone or in 5 mM glucose plus either oleic acid alone or oleic acid plus either tetradecylglycidate (TDGA), adenoviral vectors expressing uncoupling protein 1 (UCP-1), or adenoviral vectors expressing manganese superoxide dismutase (MnSOD). Each bar represents the mean plus SEM of 4 separate experiments, each with n = 8. *P < 0.01 compared with cells incubated in 5 mM glucose ale.

Figure 4. Effect of FFA-induced ROS production on GAPDH activity, PKC activity, and hexosamine pathway activity.

Cells were incubated in 5 mM glucose alone or in 5 mM glucose plus either oleic acid alone or oleic acid plus TDGA or adenoviral vectors expressing UCP-1 or MnSOD. (A)GAPDH activity. (B)PKC activity. (C)Immunoreactive protein-bound N-acetylglucosamine (GlcNAc). Each bar represents the mean plus SEM of 3 separate experiments, each with n = 3. *P < 0.01 compared with cells incubated in 5 mM glucose one.

Figure 5. Effect of CPT-1 and ROS inhibitors on PGI₂ inactivation by FFAs.

(A) Effect of FFA-induced ROS production on PGI₂ synthase activity. (B) Percent enzyme modified by 3-nitrotyrosine, a marker of the superoxide-derived ROS peroxynitrite. Cells were incubated in 5 mM glucose alone or in 5 mM glucose plus either oleic acid alone or oleic acid plus TDGA or adenoviral vectors expressing UCP-1 or MnSOD. Each bar represents the mean plus SEM of 3 separate experiments, each with n = 5. *P < 0.01 compared with cells incubated in 5 mM glucose ale.

Figure 6. Effect of FFA-induced ROS production on eNOS activity in aortic endothelial cells.

Cells were incubated in 5 mM glucose alone or in 5 mM glucose plus either oleic acid alone or oleic acid plus TDGA or adenoviral vectors expressing UCP-1 or MnSOD. Each bar represents the mean plus SEM of 3 separate experiments, each with n = 4. *P < 0.01 compared with cells incubated in 5 mM glucose ale.

Figure 7. Effect of FFA infusion on PGI₂ synthase activity in rat aorta.

Enzyme activity was determined in control and FFA-infused rats. Each bar represents the mean plus SEM of 4 rats per group. *P < 0.01 compared with control

Figure 8. Effect of inhibitors of lipolysis, CPT-1, and ROS on arterial PGI₂ inactivation in 2 animal models of insulin resistance.

(A) Effect of FFA-induced ROS production on PGI₂ synthase activity in insulin-resistant fa/fa rat aortae. Enzyme activity was determined in lean controls (FA/fa), fa/fa rats, and fa/fa rats treated with the SOD mimetic MnTBAP, the antilipolytic agent NA, or the CPT-I inhibitor etomoxir. Each bar represents the mean plus SEM of 6 rats per group. *P < 0.01 compared with lean controls. (B) Effect of FFA-induced ROS production on PGI₂ synthase activity in high-fat diet–induced insulin-resistant mouse aortae. Enzyme activity was determined in standard-diet controls, high-fat diet–induced insulin-resistant mice, and high-fat diet–induced insulin-resistant mice treated with the SOD mimetic MnTBAP, the antilipolytic agent NA, or the CPT-I inhibitor etomoxir. Each bar represents the mean plus SEM of 6 mice per group. *P < 0.01 compared with contrs.

Figure 9. Effect of inhibitors of lipolysis, CPT-1, and ROS on arterial eNOS inactivation in 2 animal models of insulin resistance.

(A) Effect of FFA-induced ROS production on eNOS activity in insulin-resistant fa/fa rat aortae. Enzyme activity was determined in lean controls (FA/fa), fa/fa rats, and fa/fa rats treated with the SOD mimetic MnTBAP, the antilipolytic agent NA, or the CPT-I inhibitor etomoxir. Each bar represents the mean plus SEM of 6 rats per group. *P < 0.01 compared with lean controls. (B) Effect of FFA-induced ROS production on eNOS activity in high-fat diet–induced insulin-resistant mouse aortae. Enzyme activity was determined in standard-diet controls, high-fat diet–induced insulin-resistant mice, and high-fat diet–induced insulin-resistant mice treated with the SOD mimetic MnTBAP, the antilipolytic agent NA, or the CPT-I inhibitor etomoxir. Each bar represents the mean plus SEM of 6 mice per group. *P < 0.01 compared with contrs.