Insulin resistance reduces arterial prostacyclin synthase and eNOS activities by increasing endothelial fatty acid oxidation (original) (raw)
Effect of FFAs on ROS production in arterial cells cultured in 5 mM glucose without insulin. As a cell culture model of IR, bovine aortic endothelial cells (BAECs) in 5 mM glucose without added insulin were incubated with different concentrations of oleic acid (Figure 2). Lower physiologic concentrations of oleic acid had no effect on ROS production (Figure 2A, bars 2 and 3). In contrast, incubation with higher physiologic concentrations of oleic acid similar to those found in insulin-resistant subjects increased ROS production in cells cultured in 5 mM glucose severalfold compared with cells cultured in 5 mM glucose alone, from 38 ± 1 nmol/ml (Figure 2A, bar 1) to 100 ± 8 nmol/ml for 800 μM oleic acid (Figure 2A, bar 4). Addition of insulin completely prevented increases in ROS production by 800 μM oleic acid (Figure 2A, bar 5), an effect that was reversed by addition of the PI3K inhibitor wortmannin (Figure 2A, bar 6). In these experiments, we used 1,000 μM of fatty acid–free albumin, a concentration somewhat higher than the normal range (530–833 μM), to ensure that the FFA fraction (the fraction unbound to albumin) would be similar to that found in insulin-resistant humans. In a separate experiment (data not shown), we found that reducing the concentration of fatty acid–free albumin while keeping the oleic acid concentration constant increased ROS production proportionally. This shows that the concentration of free, rather than total, fatty acids determines ROS production in aortic endothelial cells. Unlike hyperglycemia, which increases ROS production in both macrovascular and microvascular endothelial cells (10), FFA had no effect on ROS production by retinal microvascular endothelial cells (Figure 2B). At 30 mM glucose, exposure to 800 μM oleic acid caused a reduction of glucose flux through the tricarboxylic acid (TCA) cycle by 40%. However, this was still 3.7-fold higher than glucose flux at 5 mM (data not shown).
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 glucose alone.
Effect of CPT-I inhibition, uncoupling protein 1, and manganese superoxide dismutase on FFA-induced ROS production. To test our hypothesis that FFAs increase arterial endothelial cell ROS production by providing increased electron donors (NADH and FADH2) to the mitochondrial electron transport chain, the same mechanism previously shown to underlie hyperglycemia-induced ROS production (10), we first inhibited CPT-I with tetradecylglycidate (TDGA). Inhibition of this mitochondrial membrane enzyme, which catalyzes the rate-limiting step of fatty acid oxidation, completely inhibited FFA-induced ROS production by arterial endothelial cells cultured in 5 mM glucose (Figure 3). Eight hundred micromolar oleic acid increased ROS production 3-fold, from 39 ± 1 nmol/ml (Figure 3, bar 1) to 119 ± 2 nmol/ml (Figure 3, bar 2). However, when CPT-I was inhibited, the same concentration of oleic acid had no effect (Figure 3, bar 3). This shows that FFA-induced ROS production requires fatty acid oxidation. FFA-induced ROS production was similarly prevented in cells exposed to 800 μM oleic acid by overexpression of either uncoupling protein 1 (UCP-1) (Figure 3, bar 5), a specific protein uncoupler of oxidative phosphorylation capable of collapsing the proton electrochemical gradient that drives superoxide production (10), or manganese superoxide dismutase (MnSOD) (11), the mitochondrial isoform of this enzyme (Figure 3, bar 6). These data show that the mitochondrial electron transport chain is the source of FFA-induced ROS production, and that the initial ROS formed is superoxide.
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 alone.
Effect of FFA-induced ROS production on GAPDH activity, PKC activity, hexosamine pathway activity, and AGE formation. Since hyperglycemia-induced overproduction of superoxide by the mitochondrial electron transport chain has previously been shown to activate 3 major pathways of hyperglycemic damage in endothelial cells by inhibition of GAPDH activity (12), we next examined the effect of FFA-induced ROS production on these same parameters. Like hyperglycemia (12), 800 μM oleic acid decreased GAPDH activity by about 60% (Figure 4A, bar 2). FFA-induced inhibition of GAPDH was associated with activation of PKC (Figure 4B, bar 2), activation of the hexosamine pathway (Figure 4C, bar 2), and increased AGE formation. Inhibition of CPT-I prevented FFA-induced inhibition of GAPDH activity (Figure 4A, bar 3), and the downstream effect of GAPDH inhibition, activation of PKC (Figure 4B, bar 3), activation of the hexosamine pathway (Figure 4C, bar 3), and increased AGE formation. Overexpression of UCP-1 or MnSOD also completely prevented FFA-induced GAPDH inhibition (Figure 4A, bars 5 and 6), PKC activation (Figure 4B, bars 5 and 6), and hexosamine pathway activity (Figure 4C, bars 5 and 6). FFA-induced formation of AGEs was similarly inhibited by overexpression of UCP-1 or MnSOD (data not shown).
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 alone.
Effect of FFA-induced ROS production on PGI2 synthase activity in aortic endothelial cells. PGI2, produced mainly in endothelial cells, is, like eNOS-derived NO, an endogenous inhibitor of platelet aggregation and SMC proliferation. It is also a potent vasodilator (18). Since hyperglycemia inactivates PGI2 in aortic endothelial cells by reactive oxygen-mediated tyrosine nitration (17), we next examined the effects of FFA-induced ROS on both PGI2 synthase activity and PGI2 synthase tyrosine nitration. In cells cultured in 5 mM glucose, 800 μM oleic acid decreased PGI2 synthase activity by 95% (Figure 5A, bar 2). PGI2 protein levels did not change in the presence of oleic acid (data not shown). This inhibition was completely prevented by CPT-I inhibition (Figure 5A, bar 3), and by overexpression of either UCP-1 or MnSOD (Figure 5A, bars 5 and 6). Consistent with these data, the degree of PGI2 tyrosine nitration at tyrosine 430, a marker of the superoxide-derived ROS peroxynitrite (30), was increased 2-fold by 800 μM oleic acid (Figure 5B, bar 2). This increase was also completely prevented by CPT-I inhibition (Figure 5B, bar 3), and by overexpression of either UCP-1 or MnSOD (Figure 5B, bars 5 and 6).
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 alone.
Effect of FFA-induced ROS production on eNOS activity in aortic endothelial cells. NO production by eNOS is a critical antiatherogenic defense, mediating vasodilation, inhibition of platelet activation, monocyte and leukocyte adhesion, and SMC proliferation, and inhibition of atherosclerosis in the apoE knockout mouse (23). eNOS activity is suppressed in diabetes and IR (31, 32). Three mechanisms have been implicated, each of which is either a downstream consequence or a direct consequence of increased ROS production, including PKC activation (19), hexosamine pathway activation (20), and oxidative uncoupling of the eNOS dimer (21, 22). Since FFA-induced ROS production increases ROS production, PKC activity, and hexosamine pathway activity in aortic endothelial cells, we next examined the effect of FFA-induced ROS production on eNOS activity. In cells cultured in 5 mM glucose, 800 μM oleic acid decreased eNOS activity by about 60% (Figure 6, bar 2). eNOS protein levels did not change in the presence of oleic acid (data not shown). The inhibition was completely prevented by CPT-I inhibition (Figure 6, bar 3), and by overexpression of either UCP-1 or MnSOD (Figure 6, bars 5 and 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 alone.
Effect of FFA infusion on PGI2 synthase activity in rat aorta. Liposyn infusion caused FFA concentrations to rise rapidly to a steady-state level of 2.275 ± 0.176 mM, compared with a basal level of 0.379 ± 0.034 mM. There was no change in glucose or insulin concentrations during the Liposyn infusion. This in vivo exposure of aortic endothelial cells to elevated FFA concentrations had a dramatic effect on PGI2 synthase activity, however, reducing it in the aorta by 95% (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 controls.
Effect of FFA-induced ROS production on PGI2 synthase activity in fa/fa rat aortae. In order to evaluate our hypothesis in vivo, we chose the fa/fa rat as a model. These rats develop severe, early-onset obesity associated with IR, hyperinsulinemia, and hyperleptinemia (27, 33). However, they maintain normal glucose tolerance (34). In addition, it has been shown that these animals have a PI3K pathway–specific IR in their arteries (14). There was no difference in fasting blood glucose levels between fa/fa rats (120 ± 5 mg/dl) and lean controls (FA/fa [rats heterozygous for the mutant allele] or FA/FA [rats homozygous for the nonmutant allele]) (121 ± 4 mg/dl), nor in glycated hemoglobin levels (5.5% versus 5.4%). In aortae of fa/fa rats (Figure 8A, bar 2), PGI2 synthase activity was reduced by more than 95% compared with PGI2 synthase activity in aortae from lean controls (Figure 8A, bar 1); this was similar to what was observed previously in cultured aortic endothelial cells. Administration of the superoxide dismutase (SOD) mimetic manganese (III) tetrakis(4-benzoic acid) porphyrin (MnTBAP) for 1 week normalized PGI2 synthase activity in aortae of fa/fa rats (Figure 8A, bar 3), indicating that superoxide overproduction was responsible for the observed inhibition of aortic PGI2 synthase activity. To evaluate the role of increased fatty acid flux in the generation of excessive ROS, fa/fa rats were also treated with the antilipolytic agent nicotinic acid (NA), which decreases fatty acid release from adipose cells. NA treatment also completely normalized PGI2 synthase activity in aortae of fa/fa rats (Figure 8A, bar 4), suggesting that increased fatty acid flux due to IR was the source of the increased ROS that had inhibited the enzyme. However, since NA also causes changes in various lipoprotein fractions (35), another group of fa/fa rats were treated with etomoxir, an inhibitor of the rate-limiting enzyme for long-chain fatty acid oxidation, CPT-I (36). Inhibition of CPT-I restored PGI2 synthase activity in aortae of fa/fa rats to a level that was not significantly different from control (Figure 8A, bar 5), an effect identical to that observed with CPT-I inhibition in cultured aortic endothelial cells.
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 controls.
Effect of FFA-induced ROS production on PGI2 synthase activity in aortae of high-fat diet–induced insulin-resistant mice. In order to exclude the effects of variables other than IR in the fa/fa rat model, these experiments were repeated in a different model of IR, the high-fat diet–induced mouse model. In addition to being a different species, this model does not have the potentially confounding variable of the fa/fa mutation in the leptin receptor. As shown in Figure 8B, the effects of FFA-induced ROS production on PGI2 synthase activity in aortae of these insulin-resistant mice were identical to those observed in the fa/fa rats. PGI2 synthase activity was reduced by more than 95% (Figure 8B, bar 2), compared with PGI2 synthase activity in aortae from standard-diet controls (Figure 8B, bar 1). Administration of MnTBAP for 1 week normalized PGI2 synthase activity in aortae of high-fat diet–induced insulin-resistant mice (Figure 8B, bar 3), indicating that superoxide overproduction was responsible for the observed inhibition of aortic PGI2 synthase activity. To evaluate the role of increased fatty acid flux in the generation of excessive ROS, high-fat diet–induced insulin-resistant mice were also treated with the antilipolytic agent NA, which decreases fatty acid release from adipose cells. NA treatment also completely normalized PGI2 synthase activity in aortae of these insulin-resistant mice (Figure 8B, bar 4), suggesting that increased fatty acid flux due to IR was the source of the increased ROS that had inhibited the enzyme. However, since NA also causes changes in various lipoprotein fractions (35), another group of high-fat diet–induced insulin-resistant mice were treated with etomoxir, an inhibitor of the rate-limiting enzyme for long-chain fatty acid oxidation, CPT-I (36). Inhibition of CPT-I restored PGI2 synthase activity in aortae of the insulin-resistant mice to a level that was not significantly different from control (Figure 8B, bar 5).
Effect of FFA-induced ROS production on eNOS activity in fa/fa rat aortae. Identical experiments to those described above were performed to determine the effect of FFA-induced ROS production on eNOS activity. eNOS activity in aortae of fa/fa rats (Figure 9A, bar 2) was reduced 78% compared with eNOS activity in aortae from lean controls (Figure 9A, bar 1). Administration of MnTBAP for 1 week normalized eNOS activity in aortae of fa/fa rats (Figure 9A, bar 3), indicating that superoxide overproduction was responsible for the observed inhibition of aortic eNOS. To evaluate the role of increased fatty acid flux in the generation of excess ROS, fa/fa rats were also treated with the antilipolytic agent NA, which decreases fatty acid release from adipose cells. NA treatment also completely normalized eNOS activity in aortae of fa/fa rats (Figure 9B, bar 4), suggesting that increased fatty acid flux due to IR was the source of the increased ROS that inhibited the enzyme. However, since NA also causes changes in various lipoprotein fractions (35), another group of fa/fa rats were treated with etomoxir, an inhibitor of the rate-limiting enzyme for long-chain fatty acid oxidation, CPT-I (36). Inhibition of CPT-I restored eNOS activity in aortae of fa/fa rats to a level that was not significantly different from control (Figure 9A, bar 5), an effect identical to that observed with CPT-I inhibition in cultured aortic endothelial cells. In some cell types, etomoxir activates PPARα (37), which can lead to increased eNOS transcription (38). Therefore, we determined protein levels of eNOS to confirm that effects measured in vivo were not an effect of etomoxir on eNOS expression. Our Western blot data (data not shown) showed no change in protein levels for eNOS protein and thus do not support a role for etomoxir-induced PPARα activation in our cells.
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 controls.
Effect of FFA-induced ROS production on eNOS activity in aortae of high-fat diet–induced insulin-resistant mice. As shown in Figure 9B, the effects of FFA-induced ROS production on eNOS activity in aortae of these insulin-resistant mice were identical to those observed in the fa/fa rats. eNOS activity was reduced by 47% (Figure 9B, bar 2), compared with eNOS activity in aortae from standard-diet controls (Figure 9B, bar 1). Administration of the SOD mimetic MnTBAP for 1 week normalized eNOS activity in aortae of high-fat diet–induced insulin-resistant mice (Figure 9B, bar 3), indicating that superoxide overproduction was responsible for the observed inhibition of aortic eNOS activity. To evaluate the role of increased fatty acid flux in the generation of excessive ROS, high-fat diet–induced insulin-resistant mice were also treated with the antilipolytic agent NA, which decreases fatty acid release from adipose cells. NA treatment also completely normalized eNOS synthase activity in aortae of these insulin-resistant mice (Figure 9B, bar 4), suggesting that increased fatty acid flux due to IR was the source of the increased ROS that had inhibited the enzyme. However, since NA also causes changes in various lipoprotein fractions (35), another group of high-fat diet–induced insulin-resistant mice were treated with etomoxir, an inhibitor of the rate-limiting enzyme for long-chain fatty acid oxidation, CPT-I (36). Inhibition of CPT-I restored eNOS activity in aortae of the insulin-resistant mice to a level that was not significantly different from control (Figure 9B, bar 5).