Oleic Acid Inhibits Endothelial Nitric Oxide Synthase by a Protein Kinase C–Independent Mechanism
Abstract Many obese hypertensive individuals have a cluster of cardiovascular risk factors. This cluster includes plasma nonesterified fatty acid concentrations and turnover rates that are higher and more resistant to suppression by insulin than in lean and obese normotensive individuals. The higher fatty acids may contribute to cardiovascular risk in these patients by inhibiting endothelial cell nitric oxide synthase activity. To test this hypothesis, we quantified the effects of oleic (18:1[cis]) and other 18-carbon fatty acids on nitric oxide synthase activity in cultured bovine pulmonary artery endothelial cells by measuring the conversion of [3H]l-arginine to [3H]l-citrulline. Oleic acid (from 10 to 100 μmol/L) caused a concentration-dependent decrease in nitric oxide synthase activity at baseline and during ATP and ionomycin (Ca2+ ionophore) stimulation. At 100 μmol/L, linoleic (18:2[cis]) and oleic acids caused similar reductions of nitric oxide synthase activity, whereas elaidic (18:1[trans]) and stearic (18:0) acids had no effect. Oleic acid also inhibited the endothelium-dependent vasodilator response to acetylcholine in rabbit femoral artery rings preconstricted with phenylephrine (P<.05) but had no effect on the response to nitroprusside. The pattern of 18-carbon fatty acid effects on nitric oxide synthase activity in endothelial cells is consistent with activation of protein kinase C. Although oleic acid increased protein kinase C activity in endothelial cells, neither depletion of protein kinase C by 24-hour pretreatment with phorbol 12-myristate 13-acetate nor its inhibition with staurosporine eliminated the inhibitory effect of oleic acid on nitric oxide synthase. The vascular ring studies further indicate that oleic acid reduces the response to acetylcholine by inhibiting nitric oxide synthase activity rather than reducing the activation of guanylate cyclase or the effects of cGMP. Thus, elevated oleic acid values in obese hypertensive individuals may contribute to impaired endothelium-dependent vasodilation by a protein kinase C–independent mechanism.
- fatty acids, nonesterified
- nitric oxide
- endothelium, vascular
- endothelium-derived relaxing factor
- hypertension, obesity
Abdominal obesity is associated with a cluster of cardiovascular risk factors that includes hypertension, dyslipidemia, and impaired carbohydrate metabolism.1 2 3 Although several studies implicate hyperinsulinemia and resistance to insulin-mediated glucose disposal as central pathogenetic features of the cluster,4 5 other studies have been unable to confirm pathogenetic links between insulin and hypertension.6 7 8 9 10 11 An alternative explanation for the cluster is presented by the elevated concentrations and turnover of NEFAs, which are highly resistant to suppression by insulin in abdominally obese individuals, especially those who are also hypertensive.12 NEFAs may contribute to multiple components of the risk factor cluster because these lipids can increase vascular tone and blood pressure,13 14 15 impair carbohydrate metabolism,16 and stimulate synthesis of apolipoprotein B17 and triglyceride-rich very-low-density lipoprotein.18 Some adverse effects of NEFAs may be mediated by activation of PKC.19 In fact, cis-unsaturated NEFAs including oleic acids induce mitogenesis in human aortic smooth muscle cells by a PKC-dependent mechanism.20
Activation of PKC has been shown to impair endothelium-dependent vasodilation in vitro21 and inhibit NOS activity in cultured endothelial cells.22 Impairment of normal endothelial function can increase vascular resistance, neurovascular responses, blood pressure, and the risk of atherogenesis while impairing glucose disposal.23 24 25 26 Since NEFAs, especially cis-unsaturated fatty acids, activate PKC,19 20 the elevated NEFAs may contribute to risk factor clustering in obese hypertensive individuals by activating PKC and inhibiting NOS activity. As an initial step in testing this hypothesis, we undertook experiments to determine whether NEFAs inhibit NOS activity in BPAECs and endotheliumdependent vasodilation in vascular tissue and also whether the effects of NEFAs on NOS in cultured endothelial cells were mediated by activation of PKC.
Materials and Supplies
BPAECs were obtained from American Type Culture Collection at passage 16. Other materials included RPMI 1640 MEM (Fisher Scientific); ionomycin and staurosporine (Calbiochem); ATP, PMA, oleic and linoleic acids, stearic and elaidic acids, and the methyl β-cyclodextrin control for the oleic and linoleic acids dissolved in this vehicle (Sigma Chemical Co); [3H]l-arginine (Amersham); and AG50WX-8 Dowex 100-200 mesh (Bio-Rad). Sodium salts of fatty acids were prepared by dissolving the individual NEFA in 95% ethanol followed by the addition of 0.5N NaOH. The mixture was evaporated under nitrogen, and the NEFAs were reconstituted in water.
BPAECs (passages 17 through 21) were maintained in RPMI 1640 medium containing 20% fetal bovine serum at 37°C in a 5% CO2/95% air incubator. Cells were grown to confluence in 12-well plates and placed in MEM containing 725 μmol/L l-arginine, 0.5% fetal bovine serum, and 0.5% bovine serum albumin for 48 hours.
Determination of NOS Activity
NOS activity was measured by the conversion of [3H]l-arginine to [3H]l-citrulline after separation of these amino acids by ion-exchange chromatography as described.22 27 28 BPAECs were washed twice with phosphate buffer for removal of media and albumin. These cells were then exposed to various concentrations of four different 18-carbon NEFAs or vehicle for 15 minutes before addition of [3H]l-arginine (3 μCi/mL) because previous studies showed substantial uptake and/or incorporation of NEFAs within this time frame.29 BPAECs were incubated in [3H]l-arginine for 20 minutes. The effects of NEFAs on NOS activity under basal conditions as well as after stimulation with 100 μmol/L ATP and 2 μmol/L ionomycin for 20 minutes were determined. The reaction was terminated by washing the cells with ice-cold Ca2+-free buffer containing 5 mmol/L EDTA and adding 1 mL of 0.3 mol/L perchloric acid. NOS activity is expressed as picomoles [3H]l-citrulline produced per milligram of protein or as the percent conversion of [3H]l-arginine to [3H]l-citrulline.22 28 Cell protein was determined with the Lowry assay.30 The effects of various oleic acid concentrations on NOS activity were measured in triplicate on 7 separate days. All of the remaining NOS assays were performed in triplicate on 3 separate days (mean of nine separate values).
NOS Activity and Response to Oleic Acid After PKC Depletion
PKC was depleted by incubation of BPAECs in 200 nmol/L PMA for 24 hours. Depletion was confirmed by Western blot as described below. NOS activity was measured in these cells under basal conditions, during stimulation with ionomycin alone, and after incubation with various concentrations of oleic acid as described above.
NOS Activity During PKC Inhibition
BPAECs were exposed to 100 nmol/L staurosporine, a PKC inhibitor, or dimethyl sulfoxide vehicle in buffer for 30 minutes before the addition of 100 μmol/L oleic acid or 1 μmol/L PMA. Labeled arginine was then added and NOS activity determined.
Effects of Albumin on NEFA-Mediated Changes in NOS Activity
BPAECs were treated with 100 μmol/L oleic acid for 15 minutes in buffer containing either 100 or 300 μmol/L fatty acid–free albumin. Labeled l-arginine was added and NOS activity determined as described.
NOS Activity in BPAEC Lysates
BPAECs were washed twice with ice-cold phosphate buffer solution, scraped from the dishes with a rubber policeman, collected in tubes, and centrifuged at 2500g for 15 minutes in ice-cold phosphate buffer solution. The BPAECs were then homogenized for 1 minute with a Polytron cell homogenizer (Kinematica AG) in buffer containing 50 mmol/L Tris-HCl, 0.5 mmol/L EGTA, 1 mmol/L phenylmethylsulfonyl fluoride, 0.1% 2-mercaptoethanol, 1 mmol/L dithiothreitol, and 2 μmol/L leupeptin. After cell lysis the homogenate was treated with vehicle or 100 μmol/L oleic acid for 15 minutes at 22°C because initial studies showed that NOS activity declined rapidly between 5 and 20 minutes at 37°C. We found that NOS activity in the BPAEC lysates was linear between 5 and 20 minutes at the lower temperature. [3H]l-Arginine (3 μCi/mL) was then added in the presence of 1 mmol/L NADPH, 4 μmol/L flavin adenine dinucleotide, 10 μmol/L 5,6,7,8-tetrahydrobiopterin, 2 mmol/L CaCl2, and 50 nmol/L calmodulin. Thus, NOS activity was measured 5 and 20 minutes after addition of labeled arginine. The reaction was terminated by addition of 1 mL of 0.3 mol/L perchloric acid, and the conversion of [3H]l-arginine to [3H]l-citrulline was quantified.22 28
BPAECs were exposed to 200 nmol/L PMA for 24 hours, and measurements for PKC immunoreactivity (Western blot) were performed after exposure to PMA for 3, 6, 12, and 24 hours at 37°C in a 5% CO2 incubator. The cells were washed twice with phosphate-buffered saline and then scraped into cold 0.4% Triton X-100 lysis buffer containing (mmol/L) Tris-HCl 20 (pH 7.5), glycerophosphate 80, EDTA 5, EGTA 10, phenylmethylsulfonyl fluoride 1, dithiothreitol 2, and benzamidine 10 (lysis buffer). The suspended cells were homogenized by sonication for 10 seconds and centrifuged at 2000g for 10 minutes at 4°C. The supernatant was designated as whole-cell lysate. The protein contents were estimated by the Bradford protein assay.31 32 Equal amounts of protein were resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis on 7.5% Laemmli gels, electrophoretically transferred to polyvinylidene membranes (Millipore, Gelman Sciences), and then immunoblotted with rabbit polyclonal antisera against PKC (C4), a panPKC antibody, and against PKCζ (Santa Cruz Biotech Inc) at a 1:500 dilution. Anti-rabbit horseradish peroxidase–conjugated antibody was used as the secondary antibody. The blot was visualized with the ECL Western blotting system (Amersham International PLC).
PKC Enzyme Activity
Confluent BPAECs in 100-mm Petri dishes were incubated with 0.5% fetal bovine serum MEM for 48 hours to induce quiescence. The monolayer was exposed to oleic acid, PMA, and the PKC inhibitors staurosporine (10 nmol/L) and bisindolylmaleimide (4 μmol/L, in 0.1% fetal bovine serum MEM) for 10 minutes. Cells were scraped and prepared as described above. The supernatant was used as the enzyme preparation. The assay for PKC activity (32P transfer into lysine-rich histone) was modified from the procedure of Slivka et al.33 The reaction mixture contained 20 mmol/L HEPES, 150 μmol/L calcium chloride, 500 μmol/L type IIIS histone, 60 μg/mL phosphatidylserine, 6 μg/mL diolein, 50 μmol/L ATP (mixed with [γ-32P]ATP, 106 cpm), 10 mmol/L MgCl2 (pH 7.5), and 4 μg cytosolic protein in a final volume of 50 μL. Phosphorylation of histone was initiated by adding the enzyme preparation and terminated by transferring 40 μL of the reaction mixture to a 2×2-cm square of phosphocellulose paper (Whatman International Ltd). The filter paper was placed immediately in 150 mmol/L phosphoric acid and thereafter rinsed three more times for 30 minutes each. After the final wash, papers were transferred to a vial for liquid scintillation counting. The PKC-dependent reaction was calculated as the difference between activity in the presence and absence of calcium, phosphatidylserine, and diolein. Each enzyme preparation was analyzed in triplicate, and the results are expressed as picomoles ATP transferred per minute per milligram cytosolic protein.
Endothelium-Dependent and -Independent Relaxation in Rabbit Aortic Rings
Six male New Zealand White rabbits (Rabbits, Ltd) weighing 3.5 to 4.0 kg were treated with 10 mg diazepam and isofluorine anesthesia according to a protocol approved by the Animal Research Committee at the Medical University of South Carolina. One femoral artery was excised and placed in chilled Krebs-Henseleit bicarbonate solution (mmol/L: NaCl 118, KCl 5.4, MgSO4 1.0, NaH2PO4 1.0, NaHCO3 25, and CaCl2 2.5). The fat and adventitia were dissected free, and the artery was sectioned into rings 4 mm in length. The rings were mounted and connected to force transducers (model FT03D), a polygraph (model 7D), amplifiers (model 7P122LC), and a recorder (all Grass Instrument Co) in a 10-mL water bath aerated with 95% O2/5% CO2 at 37°C.34 35 The rings were allowed to equilibrate for 30 minutes with a change of the Krebs-Henseleit solution every 15 minutes. All additions to the bath consisted of 10-μL volumes. Contractions were elicited to 80 mmol/L KCl and 10−6 mol/L phenylephrine. While the rings were contracted with phenylephrine, endothelium-dependent dilator responses to 10−8 to 10−4 mol/L acetylcholine were obtained. After washout of the phenylephrine and acetylcholine the rings were incubated in either 100 μmol/L water-soluble oleic acid or its methyl β-cyclodextrin vehicle for 30 minutes. The rings were contracted with 10−6 mol/L phenylephrine, and the cumulative relaxation responses to 10−8 to 10−4 mol/L acetylcholine were quantified. Pilot studies showed that the vasorelaxant responses to acetylcholine were abolished by 100 μmol/L L-NAME. Identical experiments were conducted with 10−9 to 10−5 mol/L sodium nitroprusside as the vasorelaxing agent.
Plasma NEFA Concentrations in Lean Normotensive and Obese Hypertensive Individuals
Seventeen subjects included 8 lean (body mass index <25.5 kg/m2) normotensive and 9 obese (body mass index >27 kg/m2) borderline to mild hypertensive male volunteers less than 45 years old who were described in previous reports.14 36 In brief, paid volunteers were recruited by advertisement and from the Hypertension Clinic at the Medical College of Wisconsin. All subjects received detailed verbal and written explanations of the study before participation and signed a written consent form approved by the Institutional Review Board and Clinical Research Center Advisory Committee. Volunteers discontinued all medications at least 3 weeks and followed a standardized diet for 1 week before study in the outpatient Clinical Research Center. NEFAs were measured by high-performance liquid chromatography37 of fasting plasma samples. Serum triglycerides and albumin were also measured in fasting samples. Ambulatory blood pressures were obtained with an Accutracker (Suntech Medical Instruments, Inc) as described,14 37 and mean blood pressure was calculated as the sum of diastolic pressure and one third pulse pressure.
Results are presented as mean±SEM. Single time point measurements were analyzed with Student’s t test for paired or unpaired data as appropriate. Time-series data were examined by the use of ANOVA with subsequent Scheffé’s F test (StatView ii, Abacus Concepts Inc). The concentration-force response relationships in rabbit aortic rings were analyzed by ANOVA. Values of P<.05 were accepted as statistically significant.
Effects of NEFAs on Endothelial NOS Activity
Oleic acid at 10, 50, and 100 μmol/L induced a concentration-dependent reduction of NOS activity in BPAECs under basal conditions as well as during stimulation with ATP and ionomycin (Fig 1⇓). Oleic acid concentrations greater than 100 μmol/L did not cause a significantly larger inhibition of NOS activity (data not shown). Oleic acid (100 μmol/L) did not alter cell protein or lactate dehydrogenase activity in BPAECs. [3H]l-Arginine uptake was not significantly affected by 10, 50, or 100 μmol/L oleic acid over 40 minutes, with values of 33.1±0.9 (control), 30.3±4.9, 28.7±4.0, and 31.5±4.0 pmol/mg protein, respectively.
In time-dependent studies ranging from 2 to 60 minutes, 50 μmol/L oleic acid inhibited basal and ATP-stimulated NOS activity by 34% and 45%, respectively, after 2 minutes of exposure. In contrast, inhibition of ionomycin-stimulated NOS activity was not observed until 15 minutes. Basal, ATP-stimulated, and ionomycin-stimulated NOS activities were all maximally inhibited 20 minutes after treatment with 50 μmol/L oleic acid at 46%, 61%, and 47%, respectively.
Effects of Different 18-Carbon NEFAs on NOS Activity
Incubation of BPAECs with 100 μmol/L of oleic (18:1[cis]) and linoleic (18:2[cis]) acids inhibited basal (data for oleic in Fig 1⇑) and ionomycin-stimulated NOS activities (Fig 2⇓), whereas elaidic (18:1[trans]) and stearic (18:0) acids did not. Extending the incubation with 100 μmol/L stearic acid to 24 hours also did not affect NOS activity (data not shown). Similarly to oleic acid, linoleic acid did not alter [3H]l-arginine uptake.
Effects of Albumin on Oleic Acid–Mediated Changes in NOS Activity
At an equimolar ratio of fatty acid–free albumin to oleic acid (1:1), basal as well as ATP- and ionomycin-stimulated NOS activities were suppressed by 65.5±0.2%, 76.6±2.8%, and 36.8±7.6%, respectively (P<.05). These values for oleic acid–mediated inhibition of basal, ATP-, and ionomycin-stimulated NOS activities were comparable to the corresponding reductions observed in the absence of fatty acid–free albumin (56.3±6.8%, 70.5±4.0%, 46.2±1.1%; all P<.01). However, when the ratio of fatty acid–free albumin to oleic acid was increased to 3:1, oleic acid no longer inhibited NOS activity under either basal, ATP-, or ionomycin-stimulated conditions (+14.0±3.2%, +4.9±3.0%, −6.8±4.1%, respectively; P=NS).
Effects of Oleic Acid on NOS Activity in BPAEC Lysates
In contrast to the effects on intact cells, 100 μmol/L oleic acid compared with vehicle treatment did not inhibit NOS activity in cell lysates at either 5 minutes (0.78±0.20 versus 0.74±0.20 pmol/mg [3H]l-citrulline) or 20 minutes (3.26±0.58 versus 3.02±0.72 pmol/mg).
Effects of Oleic Acid on PKC Activity in BPAECs
Basal (control) PKC activity with the use of the assay method described was 968±192 pmol [32P]ATP transferred/min per milligram protein. The percent changes from control values in total PKC activity of BPAEC homogenates after exposure of intact cells to 100 μmol/L oleic acid or 100 nmol/L PMA (positive control) for 15 minutes are shown in Table 1⇓. The increased activity induced by these two PKC agonists was blocked by both staurosporine and bisindolylmaleimide.
Depletion of PKC: Effect on Basal NOS and Response to Oleic Acid
Incubation of BPAECs in 1 μmol/L PMA for 24 hours eliminated PKC immunoreactivity within 12 hours as defined by antibodies to both panPKC and against PKCζ (Fig 3⇓). Depletion of PKC with the phorbol ester PMA enhanced basal NOS activity (Fig 4⇓, top). Moreover, the inhibitory effects of oleic acid on NOS activity were unaffected by depletion of PKC (Fig 4⇓, top).
PKC Inhibition: Effect on NOS Activity and Response to Oleic Acid
Incubation of BPAECs with vehicle or 100 nmol/L staurosporine did not affect basal or agonist-stimulated NOS activity. Although oleic acid reduced basal and agonist-stimulated NOS activities (Fig 1⇑), staurosporine did not alter the inhibitory effects of oleic acid on either basal or ATP-stimulated NOS activity (Fig 4⇑, bottom). However, staurosporine partially reversed the inhibitory effect of oleic acid on ionomycin-stimulated NOS activity as shown (Fig 4⇑, bottom).
Effects of Oleic Acid on Endothelium-Dependent and -Independent Relaxation in Rabbit Femoral Artery Rings
As shown in Fig 5⇓, compared with vehicle (methyl β-cyclodextrin) 100 μmol/L oleic acid reduced the relaxation response to acetylcholine in rabbit femoral artery rings preconstricted with 1 μmol/L phenylephrine. Oleic acid did not significantly alter the overall concentration-force responses to sodium nitroprusside.
NEFA Concentrations in Obese Hypertensive and Lean Normotensive Volunteers
As shown in Table 2⇓ fasting plasma concentrations of oleic and stearic acids were significantly higher in obese borderline to mild hypertensive subjects than in lean normotensive control subjects, whereas linoleic acid values were not significantly different between the two groups. As expected, body mass index, blood pressure, and fasting triglycerides were greater in the obese than lean group. Serum albumin was not significantly different in obese hypertensive than lean normotensive subjects.
The principal finding of this study is that oleic acid inhibits endothelial cell NOS activity. These effects appear to be mediated predominantly by a PKC-independent mechanism. In these studies oleic acid inhibited both basal and agonist (ATP and ionomycin)-stimulated conversion of [3H]l-arginine to [3H]l-citrulline, which is stoichiometrically related to nitric oxide generation.22 Since basal and agonist-stimulated conversion of arginine to citrulline in BPAECs was negligible in the absence of Ca2+ or the presence of L-NAME,22 these observations suggest that oleic acid blunted NOS activity by inhibiting the constitutive Ca2+/calmodulin–dependent isoform (type III NOS).28
Despite reports which suggest that PKC activation could explain inhibition of NOS activity by oleic and linoleic acids,19 21 22 our results were not entirely consistent with this viewpoint. In support of a potential role for PKC were the findings that oleic (18:1[cis]) and linoleic (18:2[cis]) acids, which activate PKC,19 38 inhibited NOS, whereas stearic (18:0) and elaidic (18:1[trans]) acids, which are less effective at activating PKC,19 did not inhibit NOS (Fig 2⇑). Moreover, oleic acid increased PKC activity in BPAECs, and two PKC inhibitors, staurosporine and bisindolylmaleimide,39 prevented this activation (Table 1⇑). However, staurosporine did not blunt the inhibition of NOS activity by oleic acid in BPAECs under basal or ATP-stimulated conditions and only partially reversed the inhibitory effects of oleic acid on NOS activity in ionomycin-stimulated cells. Staurosporine blocked the inhibitory effect of PMA on NOS activity in our previous study.22
Further evidence against a major role for PKC is provided by studies on BPAECs in which PKC was depleted by 24-hour pretreatment with the phorbol ester PMA. Despite the absence of immunoreactive PKC (Fig 3⇑), the inhibitory effect of oleic acid on NOS was maintained (Fig 4⇑). Depletion of PKC with PMA prevented the inhibition of NOS activity by PMA in our earlier report.22 Also of note, 100 μmol/L oleic acid induced less than a 50% increase in PKC activity, whereas PMA caused a 200% increase in PKC activity (Table 1⇑). Thus, oleic acid was a relatively weak activator of PKC in BPAECs. Finally, PMA inhibited NOS activity in BPAEC lysates,22 whereas oleic acid did not. These observations suggest that the mechanism by which PMA and oleic acid inhibit the NOS activity of intact endothelial cells is different.
Although these data suggest that oleic acid inhibits NOS activity in BPAECs by a PKC-independent mechanism, the limitations of this conclusion require discussion. First, PKCζ, an isoform that is highly responsive to cis-unsaturated NEFAs,38 is relatively resistant to inhibition with staurosporine.40 Second, the PKCζ isoform does not have a phorbol ester binding site and is not downregulated by treatment with phorbol esters under some conditions.38 Despite the absence of a phorbol ester/diacylglycerol binding domain, some studies show that PKCζ immunoreactivity declines during prolonged exposure to phorbol ester,41 and our data (Fig 3⇑) are consistent with these reports. This phorbol ester–mediated downregulation of PKCζ has been attributed to phosphorylation of this atypical isoform by other PKC isoforms with a diacylglycerol/phorbol ester binding site.42 Consequently, the persistence of the inhibitory effect of oleic acid on NOS activity after PKC depletion strongly suggests that PKC, including the ζ isoform, is not significantly involved.
This study does not establish the mechanism by which oleic acid inhibited endothelial cell NOS. Fatty acids, including oleic acid, can modulate several membrane cation transport processes, including Ca2+.43 Since type III NOS in endothelial cells is a calcium/calmodulin–dependent enzyme, these data raise the possibility that oleic acid inhibited NOS by reducing the concentration or effect of intracellular Ca2+.
The potential physiological relevance of the observation that oleic acid inhibits NOS activity is supported by additional observations. First, oleic acid inhibited the relaxation of rabbit femoral artery rings to the endothelium-dependent vasodilator acetylcholine (Fig 5⇑).34 This result suggests that the inhibitory effect of oleic acid on NOS activity in cultured BPAECs extends to arterial tissue in vitro. Since some reports indicate that acetylcholine induces vasodilation in part by an endothelium-dependent but nitric oxide–independent mechanism,44 studies were performed in the presence of the NOS inhibitor L-NAME. L-NAME eliminated the vasorelaxation produced by acetylcholine, which suggests that the response depended on nitric oxide generation. Oleic acid did not affect the dilator response to nitroprusside, providing further indirect evidence that oleic acid decreased the vasorelaxant effect of acetylcholine by inhibiting NOS rather than reducing the activation of guanylate cyclase or the response to cGMP.45
Second, oleic acid inhibited endothelial NOS activity within the range of concentrations observed in vivo (Table 2⇑). Since fatty acids are highly bound to albumin,13 it is important to document the effects of fatty acids in a solution containing albumin on the capacity of oleic acid to inhibit NOS activity. In our experiments an equimolar ratio of fatty acid–free albumin to oleic acid, which approximates the ratio of albumin to total NEFAs in the fasting state (Table 2⇑),13 did not alter the effects of oleic acid on NOS activity.
Third, total NEFAs12 and oleic acid (Table 2⇑) values are higher in obese hypertensive than lean normotensive control individuals. Furthermore, NEFA concentrations and turnover during euglycemic hyperinsulinemia are significantly more resistant to suppression by insulin in obese hypertensive than either lean or obese normotensive individuals.12 Further studies will be required to determine whether the short-term effects of NEFAs on NOS in vitro are sustained in vivo. Near-maximal inhibitory effects of oleic acid in vitro were observed at a concentration of 100 μmol/L, which is similar to mean fasting values in lean normotensive subjects (Table 2⇑). Thus, additional studies are also required to determine whether the differences in oleic acid values between lean normotensive and obese hypertensive individuals are responsible for any group differences in endothelial function.
The findings of the present study suggest but do not prove that the NEFA abnormalities in high-risk abdominal obesity may inhibit endothelium-dependent vasorelaxation largely by a PKC-independent mechanism. If this can be established in future studies, then therapy to improve NEFA-mediated endothelial dysfunction may potentially ameliorate the metabolic25 and hemodynamic24 26 disturbances as well as atherosclerotic disease23 in these high-risk patients.
Selected Abbreviations and Acronyms
|BPAEC||=||bovine pulmonary artery endothelial cell|
|L-NAME||=||Nω-nitro-l-arginine methyl ester|
|MEM||=||minimum essential medium|
|NEFA||=||nonesterified fatty acid|
|NOS||=||nitric oxide synthase|
|PKC||=||protein kinase C|
|PMA||=||phorbol 12-myristate 13-acetate|
This research was supported by National Heart, Lung, and Blood Institute grant R01-43164 (B.M.E.), the Medical University of South Carolina Research Committee (R.K.D., M.E.U.), Medical University of South Carolina Clinical Research Center Fellowship Award (K.T.S.), and The Department of Veterans Affairs (T.L.G.). The authors are grateful for the technical assistance of Lyle Walsh and Ben Phillips with the aortic ring studies and Vickie Ribelin for secretarial assistance.
- Received June 13, 1995.
- Revision received July 18, 1995.
- Accepted August 2, 1995.
Stern M, Haffner S. Body fat distribution and hyperinsulinemia as risk factors for diabetes and cardiovascular disease. Arteriosclerosis. 1986;6:123-129.
Peiris A, Sothmann M, Hoffman R, Hennes M, Wilson C, Gustafson A, Kissebah A. Adiposity, fat distribution, and coronary heart disease. Ann Intern Med. 1989;110:867-872.
Reaven GM. Insulin resistance and compensatory hyperinsulinemia: role in hypertension, dyslipidemia, and coronary heart disease. Am Heart J. 1991;121:1284-1288.
Lind L, Lithell H, Pollare T. Is it hyperinsulinemia or insulin resistance that is related to hypertension and other metabolic cardiovascular risk factors? J Hypertens. 1993;11(suppl 4):S11-S16.
Hall JE, Brands MW, Kivlighn SD, Mizelle HL, Hildebrandt DA, Gaillard CA. Chronic hyperinsulinemia and blood pressure: interaction with catecholamines? Hypertension. 1990;15:519-527.
Anderson EA, Balon TW, Hoffman RP, Sinkey CA, Mark AL. Insulin increases sympathetic activity but not blood pressure in borderline hypertensive humans. J Clin Invest. 1992;87:2247-2252.
Neahring JM, Stepniakowski K, Greene AS, Egan BM. Insulin does not reduce forearm α-vasoreactivity in obese hypertensive or lean normotensive men. Hypertension. 1993;22:584-590.
Egan BM, Stepniakowski K, Nazzaro P. Insulin levels are similar in obese salt-sensitive and salt-resistant hypertensive subjects. Hypertension. 1994;23(suppl I):I-1-I-7.
O’Shaughnessy IM, Myers TM, Stepniakowski K, Nazzaro P, Kelly TM, Hoffman RG, Egan BM, Kissebah AH. Glucose metabolism in obese hypertensives and normotensives. Hypertension. 1995;26:186-192.
Hennes MM, O’Shaughnessy IM, Kelly TM, Egan BM, Kissebah AH. Abnormal free fatty acid metabolism in upper-body obese hypertensive and normotensive subjects. Clin Res. 1994;42:422. Abstract.
Stepniakowski KT, Egan BM. Additive effects of hypertension and obesity to limit venous distensibility. Am J Physiol. 1995;268:R562-R568.
Stepniakowski KT, Goodfriend TL, Egan BM. Fatty acids enhance vascular α-adrenergic sensitivity. Hypertension. 1995;25(part 2): 774-778.
Kelley DE, Mokan M, Simoneau J-A, Manarino LJ. Interaction between glucose and free fatty acid metabolism in human skeletal muscle. J Clin Invest. 1993;92:91-98.
Cabezas MC, de Bruin TWA, de Valk HW, Shoulder CC, Jansen H, Erkelens DW. Impaired fatty acid metabolism in familial combined hyperlipidemia: a mechanism associating hepatic apolipoprotein B overproduction and insulin resistance. J Clin Invest. 1993;92:160-168.
Havel RJ, Kane JP, Balasse EO, Segel N, Basso LV. Splanchnic metabolism of free fatty acids and production of triglycerides of very low density lipoproteins in normotriglyceridemic and hypertriglyceridemic humans. J Clin Invest. 1970;49:2017-2035.
Murakami K, Chan SY, Routtenberg A. Protein kinase C activation by cis-fatty acid in the absence of Ca2+ and phospholipids. J Biol Chem. 1986;261:15424-15429.
Lu G, Morinelli TA, Meier KE, Rosenzweig SA, Egan BM. Oleic acid induced mitogenic signalling in human aortic smooth muscle cells: a role for protein kinase C. J Invest Med. 1995;43:375. Abstract.
Jin-Su CK, Brechtel G, Baron AD. Acute hypertension induced by L-NMMA causes insulin resistance in rats. Hypertension. 1993;22:420. Abstract.
Bredt DS, Snyder SH. Nitric oxide mediates glutamate-linked enhancement of cGMP levels in the cerebellum. Proc Natl Acad Sci U S A. 1989;86:9030-9033.
Khan WA, Blobe GC, Hannun YA. Activation of protein kinase C by oleic acid. J Biol Chem. 1992;267:3605-3612.
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951;193:265-275.
Slivka SR, Meier KE, Insel PA. Alpha 1-adrenergic receptors promote phophatidyl-choline hydrolysis in MDCK-D1 cells: a mechanism for rapid activation of protein kinase C. J Biol Chem. 1988;263:12242-12246.
Kenakin TP. Isolated tissue response systems. In: Pharmacologic Analysis of Drug-Receptor Interaction. New York, NY: Raven Press Publishers; 1987:52-84.
Nakanishi H, Exton JH. Purification and characterization of the ζ isoform of protein kinase C from bovine kidney. J Biol Chem. 1992;267:16347-16354.
Toullec D, Pianetti P, Coste H, Bellevergue P, Grant-Perret T, Ajakane M, Baudet V, Boissina P, Boursier E, Loriolle F, Duhamel L, Charon D, Kirilovsky J. The bisindolylmaleimide GF 109203X is a potent and selective inhibitor of protein kinase C. J Biol Chem. 1991;266:15771-15781.
Hug H, Sarre TF. Protein kinase C isoenzymes: divergence in signal transduction? Review article. Biochem J. 1993;291:320-343.
Bellan JA, McNamara DB, Kadowitz PJ. Differential effects of nitric oxide synthesis inhibitors on vascular resistance and responses to acetylcholine in cats. Am J Physiol. 1993;264:H45-H52.
Johnson RM, Lincoln TM. Effects of nitroprusside, glyceryl trinitrate, and 8-bromo-cyclic GMP on phosphorylase a formation and myosin light chain phosphorylation in rat aorta. Mol Pharmacol. 1985;27:333-342.