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Am J Physiol Cell Physiol 294: C295-C305, 2008. First published October 31, 2007; doi:10.1152/ajpcell.00042.2007
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VASCULAR BIOLOGY

Diabetic HDL-associated myristic acid inhibits acetylcholine-induced nitric oxide generation by preventing the association of endothelial nitric oxide synthase with calmodulin

James White,1 Theresa Guerin,1 Hollie Swanson,2 Steven Post,2 Haining Zhu,3 Ming Gong,4 Jun Liu,1 William V. Everson,1 Xiang-An Li,1 Gregory A. Graf,5 Hubert O. Ballard,1 Stuart A. Ross,1 and Eric J. Smart1,4

Departments of 1Pediatrics and Kentucky Pediatric Research Institute, 2Pharmacology, 3Molecular and Cellular Biochemistry, 4Physiology, and 5Pharmaceutical Sciences, University of Kentucky, Lexington, Kentucky

Submitted 30 January 2007 ; accepted in final form 12 October 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
In the current study, we examined whether diabetes affected the ability of HDL to stimulate nitric oxide (NO) production. Using HDL isolated from both diabetic humans and diabetic mouse models, we found that female HDL no longer induced NO synthesis, despite containing equivalent amounts of estrogen as nondiabetic controls. Furthermore, HDL isolated from diabetic females and males prevented acetylcholine-induced stimulation of NO generation. Analyses of both the human and mouse diabetic HDL particles showed that the HDLs contained increased levels of myristic acid. To determine whether myristic acid associated with HDL particles was responsible for the decrease in NO generation, myristic acid was added to HDL isolated from nondiabetic humans and mice. Myristic acid-associated HDL inhibited the generation of NO in a dose-dependent manner. Importantly, diabetic HDL did not alter the levels of endothelial NO synthase or acetylcholine receptors associated with the cells. Surprisingly, diabetic HDL inhibited ionomycin-induced stimulation of NO production without affecting ionomycin-induced increases in intracellular calcium. Further analysis indicated that diabetic HDL prevented calmodulin from interacting with endothelial NO synthase (eNOS) but did not affect the activation of calmodulin kinase or calcium-independent mechanisms for stimulating eNOS. These studies are the first to show that a specific fatty acid associated with HDL inhibits the stimulation of NO generation. These findings have important implications regarding cardiovascular disease in diabetic patients.

fatty acid; lipoprotein; diabetes; signaling

CARDIOVASCULAR DISEASES are influenced by both genetic and environmental factors that can negatively or positively influence the development and progression of cardiovascular diseases, such as plasma levels of LDL and HDL (25, 26). High plasma levels of LDL promote the accumulation of cholesterol in vessel wall macrophages and the formation of atherosclerotic plaque (3, 11). In contrast, HDL is generally considered to protect from cardiovascular disease by virtue of its ability to mediate reverse cholesterol transport, that is, the transport of cholesterol from the periphery to the liver for uptake and clearance from the plasma (13). LDL, which is modified in the vessel wall, binds to several scavenger receptors, such as CD36 and SRA (28). The localization of these receptors in macrophages is thought to directly relate to the accumulation of cholesterol within macrophages and the development of foam cells (34). Similarly, a physiological HDL receptor, SR-BI, has been characterized and appears to be critically involved in the uptake of cholesteryl esters into hepatocytes and the subsequent clearance of sterol from the plasma (37). The role of scavenger receptors and their interactions with lipoproteins is much less studied in endothelial cells, despite the critical role that endothelial cell function plays in the initiation and progression of cardiovascular disease.

Cardiovascular diseases are often associated with other diseases, such as diabetes, and individuals with diabetes have an approximately fourfold higher risk of developing cardiovascular disease (12, 17, 29). The most prevalent type of diabetes in the United States is Type 2 diabetes. Lifestyle factors such as exercise and diet greatly influence the extent to which persons with Type 2 diabetes are at risk of developing cardiovascular disease (7). Diabetic persons often have an altered lipoprotein profile, with an increase in LDL and occasionally a decrease in HDL. However, the most dramatic change in plasma lipids is a large increase in triglycerides (35). The increase in triglycerides is attributed to altered fatty acid metabolism due to disruption of proper insulin signaling. A great deal of research has shown that trans fatty acids are prodiabetic and procardiovascular diseases (31). In contrast, it appears that omega-3 fatty acids may provide some protection against diabetes and cardiovascular disease (8, 19). The relative value or risk of saturated fats is more controversial. Furthermore, the effect of plasma fatty acid composition and concentration on HDL levels and function has not been extensively studied.

One of the striking pathophysiological features of diabetes is extensive vascular dysfunction, which often relates to the endothelium (20). For instance, diabetic persons often have poor peripheral circulation, which leads to ischemic injury, impaired wound healing, and impaired vision, all of which involve altered endothelial cell function that relates to endothelial nitric oxide (NO) synthase (eNOS) and the generation of NO (2, 21). Several studies have shown that HDL acting through its receptor, SR-BI, can stimulate eNOS to produce NO (4, 16, 24, 36). Interestingly, both calcium-dependent and calcium-independent mechanisms have been shown to account for HDL-mediated NO generation. Of particular relevance to the current study is the observation by Gong et al. (16), which established that estrogen, a relatively minor lipid component of HDL, stimulated NO generation and altered the function of the endothelium and the vessel. Importantly, this study showed that HDL could have an effect on the cardiovascular system independent of its role in reverse cholesterol transport and the regulation of plasma cholesterol levels.

The goal of the current study was to determine whether HDL isolated from diabetic humans and mice could stimulate the generation of NO. We found that HDL isolated from diabetic persons (called diabetic HDL henceforth) not only failed to stimulate eNOS, but it also prevented stimulation by acetylcholine. Further analyses showed that a high level of myristic acid associated with diabetic HDL was responsible for the inhibitory effect. Additional studies demonstrated that HDL-associated myristic acid prevented calmodulin from associating with eNOS and thereby prevented the synthesis of NO. These findings have important implications regarding the relative risk of harboring high plasma levels of certain types of fatty acids. In addition, these data imply that HDL containing elevated levels of myristic acid may not be cardioprotective but instead may promote cardiovascular disease by interfering with the production of NO.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. Medium 199 (M199), basal medium Eagle (BME) vitamin mix, fetal bovine serum, glutamine, trypsin-EDTA, and penicillin/streptomycin were from Life Technologies (Grand Island, NY). The antibodies for eNOS and calmodulin were from BD Biosciences (San Diego, CA). [3H]arginine (specific activity, 52 Ci/mmol) was from DuPont. Bradford reagent was purchased from Bio-Rad (Hercules, CA). The mouse feed was obtained from Harlan-Teklad (Madison, WI). Dowex AG50WX-8 and Celite 545 were from Sigma (St. Louis, MO). The Ultra-Sensitive Estradiol radioimmunassay kit was from Diagnostic System Laboratories. C2-ceramide was from Calbiochem (San Diego, CA). Horseradish peroxidase-conjugated IgGs were supplied by Cappel (West Chester, PA). Super Signal chemiluminescent substrate was purchased from Pierce (Rockford, IL).

Cell culture. Human microvascular endothelial cells (CDC.EU/HMEC-1 from the National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, GA) were cultured in M199 medium supplemented with 100 U/ml penicillin/streptomycin, 0.5% (vol/vol) L-glutamine, BME vitamin mix (1 ml/100 ml M199), BME amino acid mix (1 ml/100 ml M199), and 10% (vol/vol) fetal bovine serum. On day 0, 50,000 cells were placed into 12-well plates and used on day 3 at ~60% confluency.

Insulin, triglycerides, and total cholesterol. Cholesterol and triglycerides were quantified by using commercially available kits from Wako Chemicals (Richmond, VA) per the manufacturer's instructions. Insulin was measured by ELISA with a kit from Linco Research (St. Charles, MO).

Isolation of lipoproteins. VLDL (density <1.006 g/ml), LDL (density = 1.019–1.05 g/ml), and HDL (density = 1.063–1.21 g/ml) were isolated from human or mouse plasma by sequential density gradient ultracentrifugation as previously described (30). SDS-PAGE, silver staining, and Western blotting of the appropriate lipoprotein was used to assay the purity of each lipoprotein fraction. In addition, lipoproteins were fractionated by size exclusion chromatography by using an Akta purifier UPC (GE Healthcare Bio-sciences, Uppsala, Sweden) (10, 14). Approximately 175 µl of the sample was injected into a 500-µl sample loop. The samples were eluted through two Superose 6 10/300 GL (GE) columns connected in series with buffer containing 200 mM sodium phosphate dibasic heptahydrate (pH 7.4), 50 mM sodium chloride, 0.03% (wt/vol) EDTA, and 0.02% sodium azide (wt/vol) at a flow rate of 0.35 ml/min. An elution profile of each sample was created by measuring the absorbance at 280 nm using an in-series flow cell. A total of 45.0 ml of eluted sample was collected in 0.275-ml fractions by using a Frac-920 (GE) fraction collector. The columns were flushed with 20.0 ml of elution buffer between each sample injection. The cholesterol concentration in each fraction was determined by using a Cholesterol E enzymatic colorimetric kit obtained from Wako Chemicals. All human blood was used according to Declaration of Helsinki principles. Donors provided written informed consent, and approval was obtained from the Institutional Review Board of University of Kentucky.

Animal models. All animals were housed in the University of Kentucky animal facilities. The animals were maintained in constant temperature conditions on a 14:10-h light-dark cycle (lights on at 0400) and were provided food and water ad libitum. The C57BL/6J wild-type mice, C57BL/ks wild-type mice, and C57BL/kslepr/db mice were obtained from Jackson Laboratory (Bar Harbor, ME) and used when 4 mo old. To induce hyperglycemia, 8-wk-old C57BL/6J wild-type mice were treated for 6 days with daily intraperitoneal injections of streptozotocin (60 mg/kg in a citrate buffer). Streptozotocin (Sigma) was prepared immediately before use in citrate buffer (pH 4.5). Streptozotocin readily dissolves in citrate buffer. The solution was filter sterilized by passing it through a 0.2-µm filter into a sterile 1.5-ml microcentrifuge tube. No less than 50 µl and no more than 100 µl was injected intraperitoneally into the mouse within 3 min of the preparation of the solution. The exact amount injected depended on the weight of the mouse. Control mice received an equal volume of citrate buffer. The animals were injected with the same dose for 6 consecutive days. The animals were tested for the extent of hyperglycemia on day 7 and once every 4 days thereafter. HDL was isolated 8 wk after the animals became diabetic. The commercial kit "OneTouch Ultra" from Johnson and Johnson was used to determine glucose levels. Food was removed for 3 h in the morning and then blood taken for the glucose measurement. Approximately 20 µl of tail vein blood was needed for the analysis. Animals with blood glucose levels <144 mg/dl were considered normoglycemic. Mice with blood glucose levels >350 mg/dl were classified as diabetic. All animal experiments were approved by the University of Kentucky Institutional Animal Care and Use Committee.

NO synthesis assay. NOS activation was determined in intact cells as previously described (33). Briefly, the cells were plated into 12-well plates at 5,000 cells/well and grown to 60% confluency. The medium was replaced with serum-free medium for 16 h and then placed in phosphate-buffered saline at 37°C for 2 h. After the preincubation period, the PBS was removed from the wells and replaced with 400 µl PBS containing 0.75 µCi/ml L-[3H]arginine (basal buffer) and the indicated treatments. The cells were incubated at 37°C for 30 min. The NOS reaction was terminated by the addition of 500 µl of ice-cold 1 N TCA to each well. The cells were freeze fractured twice in liquid nitrogen for 2 min, followed by thawing at 37°C for 5 min, and scraped with a rubber spatula. The contents of each well were transferred to ice-cold glass test tubes. Ether extraction was performed three times with water-saturated ether to remove the TCA. The samples were neutralized with 1.5 ml of 25 mM HEPES, pH 8, applied to Dowex AG50WX-8 (Tris form) columns and eluted with 1 ml of 40 mM HEPES buffer, pH 5.5, containing 2 mM EDTA and 2 mM EGTA. L-[3H]citrulline was collected in scintillation vials and quantified by liquid scintillation counting. In individual experiments performed in 12-well plates, 3 wells were used for each treatment group. Findings were confirmed in at least six independent experiments. NOS activation in the intact cells was inhibited by 1 mM N-nitro-L-arginine (L-NNA). To ensure that the treatments did not affect the loading of the cells with L-[3H]arginine, the amount of L-[3H]arginine associated with the cells was determined. The cells contained 219,681 ± 7,995 dpm/well of L-[3H]arginine independent of the treatment and time (after 2 min).

Gas chromatography of fatty acids. HDL (200 µg) was extracted with 600 µl of chloroform containing 100 µg of butylated hydroxytoluene/ml. Next, 200 µl of the extract was mixed with 20 µl of 5 mg/ml heptadecanoic acid (17:0), an internal standard, and the sample was dried under a stream of nitrogen. The pellet was suspended in 100 µl of chloroform and 1 ml of BF3-methanol (10%, Supelco, Bellefonte, PA) and incubated at 60°C for 16 h. Fatty acid esters were extracted with 2 ml of chloroform, and the BF3 and methanol were removed from the chloroform by three 1-ml water washes. The sample (1 µl) was injected into the gas chromatography system (model 6890 GC G2579A system; Agilent, Palo Alto, CA) equipped with an OMEGAWAX250 capillary column 30 m (Supelco) and a flame ionization detector (FID). A mass-selective detector (model 5973, Agilent) was used to identify target peak. The GC program was as follows: injector, 1 µl at 10:1 split, 250°C; detector, FID, 260°C; oven: 160°C (5 min) to 220°C at 4°C/min, hold 30 min; carrier: helium, 1.2 ml/min.

HDL and fatty acid enrichment. HDL was loaded with fatty acid similar to the method used to label HDL with cholesteryl ester (18). In brief, fatty acid (1–50 mg) was dried onto 100 mg of Celite 545 and then incubated with purified HDL for 18 h at 37°C. At the end of the incubation, Celite was removed by centrifugation through 0.02-µm filters. The HDL was then reisolated by ultracentrifugation size exclusion chromatography to ensure that unincorporated fatty acid was removed. Control HDL was treated identically, with the exception that fatty acid was not added to the Celite.

Calcium imaging. In each experiment, cytosolic calcium was measured simultaneously in 12–14 fura-2-loaded cells by using a dual-excitation spectrofluorometric system (Zeiss AttoFluor Ratio Vision Workstation; Atto Instruments, Rockville, MD) (24). Before analysis, the cells were rinsed three times with PBS and loaded with 5 µM fura-2 AM for 10 min at 37°C. After loading, the cells were rinsed three times with PBS and were allowed to recover for at least 15 min. The coverslip was mounted in a closed perfusion chamber (Warner Instruments), placed on the stage of a Zeiss Axiovert inverted microscope fitted with a x40 fluorescence oil immersion objective, and constantly perfused by gravity-feed with ambient temperature PBS at 1–3 ml/min. In a typical experiment, basal (nonstimulated) fluorescence was monitored for 30–60 s before the addition of HDL (10 µg/ml), which was added by the rapid change-out of the bathing medium. After 15 min exposure to HDL, cells were again washed thoroughly with PBS and then exposed to ionomycin (1 µg/ml) by the rapid change-out of the bathing medium. Fluorescence was determined by using excitation wavelengths of 340 nm and 380 nm and an emission wavelength of 510 nm. Intracellular calcium levels were expressed as a 340:380 ratio.

Immunoprecipitation. Protein A-Sepharose beads were first blocked by incubation for 4 h at 4°C with cell lysate (200 µg/ml) plus 30 mg/ml of BSA in immunoprecipitation buffer (150 mM NaCl, 0.5% Triton X-100, and 50 mM Tris, pH 8.0). Blocked beads were then used to preclear the experimental fractions that had been adjusted to 0.5% (vol/vol) Triton X-100. Precleared fractions were incubated for 18 h at 4°C with the appropriate antibody before adding blocked, Protein A-Sepharose beads and incubating an additional 2 h at 4°C. The beads were collected by centrifugation, washed five times in high salt (500 mM NaCl) immunoprecipitation buffer, and then placed in Laemmli sample buffer. Immunoprecipitated proteins were detected by immunoblotting.

SDS-PAGE and Western blotting. Samples were concentrated by trichloroacetic acid precipitation and washed in acetone. Pellets were suspended in sample buffer that contained 1.2% (vol/vol) β-mercaptoethanol and heated at 95°C for 3 min before being loaded onto gels. Proteins were separated in a 12.5% SDS-polyacrylamide gel by using the method of Laemmli (22). The separated proteins were then transferred to polyvinylidene difluoride (PVDF). The PVDF was blocked in Tris-buffered saline (TBS) that contained 5% dry milk for 1 h at room temperature. Primary antibodies were diluted in TBS that contained 1% dry milk and incubated with the PVDF for 1 h at room temperature. The PVDF was washed 4 times, 10 min each in TBS + 1% dry milk. The secondary antibodies (all conjugated to horseradish peroxidase) were diluted 1/20,000 in TBS + 1% dry milk and incubated with the PVDF for 1 h at room temperature. The PVDF was then washed, and the bands were visualized by chemiluminescence. Sample buffer (5x) consisted of 0.31 M Tris, pH 6.8, 2.5% (wt/vol) SDS, 50% (vol/vol) glycerol, and 0.125% (wt/vol) bromophenol blue. TBS consisted of 20 mM Tris, pH 7.6, and 137 mM NaCl.

Statistical analysis. Least-squares ANOVA was used to evaluate the data with respect to sample, treatment, time, and their interactions by using the ANOVA procedure of Statistica. When appropriate, samples were compared by using Tukey's honestly significant difference test. Means were considered significantly different at P < 0.01.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
HDL isolated from diabetic women and diabetic female mice inhibit agonist stimulation of NO generation. Our previous studies showed that HDL containing estrogen binds to SR-BI and stimulates eNOS to produce NO (16). Because diabetic patients often have endothelial dysfunction that results in hypertension, hypercoagulation, and lack of angiogenesis in the peripheral vasculature (32), we tested the ability of HDL isolated from diabetic humans and diabetic mice to stimulate the production of NO. Both diabetic humans and mice (Table 1) had elevated fasting serum levels of glucose, insulin, triglycerides, and total cholesterol compared with nondiabetic controls. Serum obtained from nondiabetic and diabetic humans and mice was fractionated by ultracentrifugation to obtain lipoprotein-deficient serum (LPDS), HDL, LDL, and VLDL as we have done previously (16). We used an established human microvascular endothelial (HME) cell system (CDC.EU/HMEC-1 from the National Center for Infectious Diseases) to assay the ability of the isolated lipoproteins to stimulate eNOS in living cells (16). HME cells were incubated with 0.75 µCi/ml of [3H]arginine to label the intracellular arginine pool. Without removal of the radiolabel, the cells were then incubated with buffer only (basal), 50% serum, 50% LPDS, or 10 µg/ml of HDL, LDL, or VLDL isolated from nondiabetic females for 15 min at 37°C. Additional sets of cells were treated with either 1 µg/ml of ionomycin, a calcium ionophore, to determine maximal eNOS stimulation or with 2 µM acetylcholine, a physiological activator of eNOS. The cells were then processed to quantify the amount of [3H]arginine converted to [3H]citrulline as a measure of eNOS enzymatic activity (Fig. 1A). Ionomycin, acetylcholine, serum, and female HDL maximally stimulated NO production in this system (16). In contrast, LPDS, LDL, and VLDL did not increase the generation of NO. These data are consistent with our previous studies (16). As a control, we determined whether the serum fractions affected the ability of acetylcholine to stimulate NO production. Figure 1A also shows that the nonstimulatory fractions (LPDS, LDL, and VLDL) did not affect the ability of acetylcholine to stimulate NO production. Control experiments with 1 mM L-NNA, an eNOS inhibitor, showed that >99% of the generated citrulline was due to eNOS activity (data not shown). In agreement with previous studies, maximal stimulation of eNOS was achieved with 1 µg/ml of female HDL, whereas HDL isolated from males did not stimulate eNOS, even at concentrations up to 500 µg/ml (data not shown) (16).


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  		Table 1. Characterization of human and mouse sera

 

Figure 1
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  		Fig. 1. HDL isolated from diabetic humans inhibits agonist stimulation of nitric oxide (NO) production. Serum obtained from nondiabetic (A) and diabetic (B) women was fractionated to obtain lipoprotein-deficient serum (LPDS), HDL, LDL, and VLDL. Human microvascular endothelial (HME) cells were incubated with 0.75 µCi/ml of [3H]arginine for 15 min to label the intracellular arginine pool. Without removal of the radiolabel, the cells were then incubated with buffer only (basal), 50% serum, 50% LPDS, or 10 µg/ml of HDL, LDL, or VLDL isolated from nondiabetic or diabetic women for 15 min at 37°C. Additional cells were treated with 1 µg/ml of ionomycin or 2 µM acetylcholine. Finally, a set of cells was incubated with the indicated serum fraction and 2 µM acetylcholine. The cells were then processed to quantify the amount of [3H]arginine converted to [3H]citrulline as a measure of endothelial NO synthase (eNOS) enzymatic activity. The data are means ± SE of 5 independent experiments, with triplicate measurements in each experiment.

 
In contrast with the results obtained with HDL isolated from healthy volunteers, serum and HDL obtained from diabetic women did not stimulate NO production (Fig. 1B). We next tested whether HDL from diabetic women affected acetylcholine stimulation of NO production. Figure 1B shows that both serum (containing HDL) and the isolated HDL fraction completely inhibited acetylcholine-stimulated NO production. However, LPDS, LDL, and VLDL obtained from diabetic women did not alter acetylcholine stimulation. Similarly, HDL isolated from diabetic men and male mice also inhibited acetylcholine-induced NO generation (data not shown).

Similar observations were obtained by using HDL and serum isolated from both Type 1 mouse diabetic models (streptozotocin-induced) and Type 2 mouse diabetic models [leptin receptor-deficient mice (Leprdb)] (Table 2). In addition, lipoproteins isolated by size exclusion chromatography generated data similar to that generated by lipoproteins isolated by ultracentrifugation (data not shown). To ensure that these findings were not specific to acetylcholine receptors, we performed identical experiments using 2 µM histamine as the agonist to stimulate NO production and achieved similar results (data not shown).


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  		Table 2. Effect of diabetes on mouse HDL-induced nitric oxide production

 
HDLs isolated from diabetic humans and mice are enriched in myristic acid. Because estrogen associated with HDL induces the production of NO (16), we examined whether the amount of estrogen associated with HDL was decreased in diabetic women. The amounts of estrogen associated with HDL obtained from nondiabetic (0.023 ± 0.0003 pg estradiol/mg HDL) and diabetic (0.024 ± 0.0004 pg estradiol/mg HDL) women and nondiabetic (0.017 ± 0.0005 pg estradiol/mg HDL) and diabetic (0.019 ± 0.0005 pg estradiol/mg HDL) female mice were not different. Because there was no change in estrogen levels and because HDL isolated from both diabetic females and males inhibited acetylcholine and histamine stimulation of NO production, it seemed likely that another component of HDL was responsible for the phenomenon. Therefore, we analyzed the major protein and lipid components of nondiabetic and diabetic HDL. We extracted proteins from HDL isolated by ultracentrifugation, resolved them by SDS-PAGE, and detected the proteins by silver stain. Figure 2A shows that the qualitative protein composition of nondiabetic and diabetic HDL is similar. Apolipoprotein A-I (apoA-I) and apoA-II were identified by Western blot analysis (data not shown). The relative amounts of apoA-I and apoA-II in nondiabetic and diabetic HDL were within 5.2% of each other as determined by densitometry. Similar data were obtained for HDL isolated by size exclusion chromatography (data not shown). Figure 2B shows that HDLs fractionated by size exclusion chromatography have essentially identical profiles, which further indicates that nondiabetic and diabetic HDL has not undergone major structural modifications. Examination of the HDL particles by nondenaturing gradient gel electrophoresis did not detect any obvious heterogeneity between the different particles (Fig. 2C) (23). We also examined the major lipid components of the nondiabetic and diabetic HDLs. Table 3 shows that the cholesterol, phospholipid, triglyceride, and fatty acid contents of the HDLs were similar. The only significant difference between diabetic and nondiabetic HDL was a three- to fourfold increase in the amount of myristic acid associated with the diabetic HDL. Importantly, the oxidized phospholipid, oxidized-1-palmitoyl-2-arachidonyl-sn-glycero3-phosphorylcholine, which we previously showed can stimulate eNOS (15), was not detected in nondiabetic or diabetic HDL.


Figure 2
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  		Fig. 2. Analysis of diabetic and nondiabetic HDL. A: silver stain of the major protein components in HDL isolated from diabetic and nondiabetic humans and diabetic and nondiabetic mice. HDL isolated from diabetic and nondiabetic humans and mice [leptin receptor-deficient mice (Leprdb)] was delipidated, and the proteins were resolved by SDS-PAGE and visualized by silver stain. The identification of apolipoprotein (apo) A-I (apoA-I) and apoA-II (human and mouse) was done by Western blotting identical samples with the appropriate antibodies. The predominate apoprotein is apoA-I in both human and mouse HDL. In addition, mouse apoA-II is significantly smaller than human apoA-II (9). B: HDL3 isolated from diabetic and nondiabetic humans and diabetic and nondiabetic mice by ultracentrifugation was then resolved by size exclusion chromatography to determine whether subfractions of HDL were present. The profile of each sample was created by measuring the absorbance at 280 nm and plotted against the sample fraction. The data are representative of six unique samples for each group. mAU, milliabsorbance units. C: nondenaturing gradient gel electrophoresis of the indicated HDL particles. The data are representative of three unique samples from each group.

 

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  		Table 3. Characterization of normal and diabetic HDL particles

 
Myristic acid associated with HDL inhibits acetylcholine-induced NO generation. We previously showed that myristic acid-albumin complexes stimulate eNOS activity; therefore, it seemed unlikely that myristic acid associated with HDL would inhibit eNOS (38). To test whether HDL-associated myristic acid affected eNOS activation, HDL from nondiabetic women was isolated, enriched with myristic acid or palmitic acid, and reisolated by size exclusion chromatography. Figure 3A is a silver stain of electrophoresed HDL proteins, which shows that the major protein components of HDL are not significantly altered when HDL is enriched with myristic acid or palmitic acid. Figure 3B shows that the size exclusion chromatography profiles for nondiabetic HDL and HDL enriched with myristic acid or palmitic acid are essentially the same, which indicates that the fatty acid enrichment procedure did not cause major remolding of the HDL. Table 4 documents that, of the major lipids analyzed, myristic acid enrichment only increased myristic acid levels and palmitic acid enrichment only increased palmitic acid levels. Importantly, the method used to quantify fatty acids in Table 4 measures both esterified and free fatty acids. When just free fatty acids are measured, nondiabetic HDL contained 0.13 µg myristic acid/mg HDL, diabetic HDL contained 20.5 µg myristic acid, and myristic acid-enriched HDL contained 21.9 µg myristic acid. These data indicate that the increase in myristic acid is as a free fatty acid.


Figure 3
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  		Fig. 3. Analysis of nondiabetic HDL enriched with myristic acid or palmitic acid. A: silver stain of the major protein components in nondiabetic HDL and HDL enriched with myristic acid or palmitic acid. The HDL was delipidated, and the proteins were resolved by SDS-PAGE and visualized by silver stain. The identification of apoA-I and apoA-II (human and mouse) was done by Western blotting identical samples with the appropriate antibodies. The overall protein profile of the major protein components is the same in all three samples. B: nondiabetic HDL and HDL enriched with myristic acid or palmitic acid were resolved by size exclusion chromatography to determine whether subfractions of HDL were present. The profile of each sample was created by measuring the absorbance at 280 nm and plotted against the sample fraction. The data are representative of eight unique samples for each group.

 

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  		Table 4. Characterization of normal and HDL particles enriched with myristic acid or palmitic acid

 
HME cells were then incubated with 0.75 µCi/ml of [3H]arginine for 15 min to label the intracellular arginine pool. Without removal of the radiolabel, the cells were incubated with 1 µg/ml of ionomycin, 10 µg/ml of nondiabetic HDL, 10 µg/ml of nondiabetic HDL enriched with myristic acid or palmitic acid, 10 µM of myristic acid-albumin, or 10 µM of palmitic acid-albumin [a ratio of 0.5 fatty acid to albumin as we previously described (38)] for 15 min at 37°C. The same treatments were also tested in the presence of 2 µM acetylcholine to determine whether they affected acetylcholine-induced NO production. The amount of [3H]arginine converted to [3H]citrulline was quantified as a measure of eNOS enzymatic activity (Fig. 4). Consistent with results obtained with diabetic HDL, nondiabetic HDL enriched in myristic acid prevented acetylcholine stimulation of NO generation. As a critical control, HDL enriched with palmitic acid did not affect NO generation, which further indicates that the inhibition is due to the presence of myristic acid and not the fatty acid enrichment procedure. Importantly, myristic acid-albumin stimulated NO production as we previously reported (38). In contrast with myristic acid-HDL, myristic acid-albumin and palmitic acid-albumin complexes did not affect acetylcholine stimulation. Control experiments with 1 mM L-NNA, an eNOS inhibitor, showed that >99% of the generated citrulline was due to eNOS activity (data not shown).


Figure 4
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  		Fig. 4. Myristic acid associated with HDL inhibits NO generation. HDL isolated from nondiabetic humans was enriched with either myristic acid or palmitic acid and reisolated to remove unbound fatty acid as described in MATERIALS AND METHODS. HME cells were incubated with 0.75 µCi/ml of [3H]arginine for 15 min to label the intracellular arginine pool. Without removal of the radiolabel, the cells were then incubated with 1 µg/ml of ionomycin, 10 µg/ml of nondiabetic HDL, 10 µg/ml of nondiabetic HDL enriched with myristic acid or palmitic acid, 10 µM of myristic acid-albumin, or 10 µM of palmitic acid-albumin for 15 min at 37°C. The same set of treatments was also tested in the presence of 2 µM acetylcholine. The cells were processed to quantify the amount of [3H]arginine converted to [3H]citrulline. The data are means ± SE of 6 independent experiments, with triplicate measurements in each experiment.

 
To further determine whether the myristic acid associated with HDL was responsible for the inhibition of NO production by acetylcholine stimulation, we used nondiabetic HDL and generated particles containing increasing amounts of myristic acid as previously described (16). HME cells were incubated with 0.75 µCi/ml of [3H]arginine to label the intracellular arginine pool. Without removal of the radiolabel, the cells were incubated with 1 µg/ml of ionomycin and 10 µg/ml of nondiabetic HDL enriched with 10–30 µg of myristic acid ± 2 µM acetylcholine. The cells were processed to quantify the amount of [3H]arginine converted to [3H]citrulline as a measure of eNOS enzymatic activity. Figure 5 shows that myristic acid associated with HDL had a dose-dependent effect on preventing acetylcholine stimulation of NO generation.


Figure 5
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  		Fig. 5. Myristic acid associated with HDL inhibits acetylcholine-induced NO generation in a dose-dependent manner. HDL isolated from nondiabetic women was enriched with myristic acid and reisolated to remove unbound fatty acid, and the amount of myristic acid associated with the HDL was quantified. HME cells were then incubated with 0.75 µCi/ml of [3H]arginine for 15 min to label the intracellular arginine pool. Without removal of the radiolabel, the cells were then incubated with 1 µg/ml of ionomycin and HDL with increasing amounts of associated myristic acid ± 2 µM acetylcholine. The cells were processed to quantify the amount of [3H]arginine converted to [3H]citrulline. The data are means ± SE of 3 independent experiments, with triplicate measurements in each experiment.

 
Diabetic HDL does not alter the expression of eNOS, acetylcholine receptor, or acetylcholine-mediated calcium influx. The myristic acid associated with diabetic HDL was responsible for the inhibition of acetylcholine-mediated increases in NO production. One possible mechanism for the inhibition is that myristic acid associated with HDL induced a decrease in cellular eNOS and/or acetylcholine receptor levels. HME cells do not express the other isoforms of NOS (inducible NOS and neuronal NOS) (data not shown). To determine whether diabetic HDL directly altered the levels of eNOS or acetylcholine receptors, the cells were treated with diabetic HDL as described above and lysed, and 20 µg of protein was resolved by SDS-PAGE and analyzed by Western blot for eNOS and acetylcholine receptors. Figure 6A shows that diabetic HDL did not alter the cellular levels of eNOS or acetylcholine receptors or their localization to the plasma membrane (data not shown).


Figure 6
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  		Fig. 6. Diabetic HDL does not affect the levels of eNOS and acetylcholine receptors (AChR) or acetylcholine-mediated calcium influx. A: HME cells were treated with 1 µg/ml of ionomycin, 2 µM acetylcholine, 10 µg/ml of nondiabetic HDL, or 10 µg/ml of diabetic HDL in the indicated combinations. The cells were lysed, and 20 µg of protein was resolved by SDS-PAGE, analyzed by Western blot for eNOS and acetylcholine receptors, and visualized by chemiluminescence. The exposure times were 2 min for eNOS, 5 min for acetylcholine receptor, and 30 s for actin. The data are representative of 4 independent experiments. B: HME cells were loaded with 5 µM fura-2 AM for 10 min at 37°C. The cells were then incubated with 1 µg/ml ionomycin, 2 µM acetylcholine, nondiabetic HDL (10 µg/ml), diabetic HDL (10 µg/ml), or the indicated combinations of reagents for 15 min while being continuously monitored for calcium-induced fluorescence changes using excitation wavelengths of 340 nm and 380 nm and an emission wavelength of 510 nm. Intracellular calcium levels were expressed as a 340:380 ratio. The data are means ± SE of 3 independent experiments, with triplicate measurements in each experiment.

 
Another possible explanation for the lack of eNOS stimulation is that myristic acid associated with HDL prevents an increase in intracellular calcium. To test this, we loaded HME cells with fura-2 AM for 10 min at 37°C. The cells were then incubated with 1 µg/ml of ionomycin, 2 µM acetylcholine, nondiabetic HDL (10 µg/ml), diabetic HDL (10 µg/ml), or the indicated combinations of reagents for 15 min while being continuously monitored for calcium-induced fluorescence changes. In agreement with our previous data (24), Fig. 6B shows that the addition of nondiabetic HDL to the cells did not cause an increase in intracellular calcium. However, the addition of ionomycin or acetylcholine induced a calcium influx. Importantly, cotreatment with diabetic HDL and ionomycin or acetylcholine did not elicit a further increase in intracellular calcium. These data indicate that the mechanism of diabetic HDL inhibition is not related to the ability to increase intracellular calcium.

Diabetic HDL prevents the interaction of eNOS and calmodulin. The enzymatic activity of eNOS, including its ability to interact with calmodulin (6), can be regulated at numerous steps. Although diabetic HDL did not prevent an increase in intracellular calcium, it was possible that diabetic HDL downregulated calmodulin or prevented calmodulin from interacting with eNOS. To test these possibilities, cells were incubated with 1 µg/ml of ionomycin, 2 µM acetylcholine, nondiabetic HDL (10 µg/ml), diabetic HDL (10 µg/ml), or the indicated combinations of reagents for 15 min. The cells were lysed, and 20 µg of protein was resolved by SDS-PAGE and analyzed by Western blot for eNOS and calmodulin. Figure 7A shows that diabetic HDL did not alter the cellular levels of eNOS or calmodulin. We next tested whether calmodulin interacts with eNOS by treating the cells as described above and then immunoprecipitating eNOS. The precipitated material was resolved by SDS-PAGE and analyzed by Western blot for eNOS and calmodulin. Figure 7B shows that ionomycin, acetylcholine, and normal HDL, but not diabetic HDL, induced the interaction of eNOS with calmodulin. In contrast, basal conditions and diabetic HDL did not induce an eNOS-calmodulin interaction. Normal HDL did not affect the ability of ionomycin and acetylcholine to promote eNOS-calmodulin interaction, but diabetic HDL completely inhibited eNOS-calmodulin interaction in the presence of ionomycin and acetylcholine. The effect of diabetic HDL on calmodulin appears to be specific for eNOS because diabetic HDL did not inhibit the activation of calmodulin kinase (data not shown).


Figure 7
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  		Fig. 7. Diabetic HDL prevents calmodulin interaction with eNOS. A: to determine whether diabetic HDL caused the downregulation of calmodulin, HME cells were incubated with 1 µg/ml ionomycin, 2 µM acetylcholine, nondiabetic HDL (10 µg/ml), diabetic HDL (10 µg/ml), or the indicated combinations of reagents for 15 min. The cells were lysed, and 20 µg of protein was resolved by SDS-PAGE and analyzed by Western blot for eNOS and calmodulin. The exposure times were 2 min for eNOS and 3 min for calmodulin. The data are representative of 3 independent experiments. B: HME cells were treated as described in A, the cells were lysed, and eNOS immunoprecipitated with eNOS IgG. The precipitated material was resolved by SDS-PAGE and analyzed by Western blot for eNOS and calmodulin. The exposure times were 1 min for eNOS and 2 min for calmodulin. The data represent 4 independent experiments.

 
Although the data clearly show that diabetic HDL prevents eNOS-calmodulin interaction, it is possible that diabetic HDL has other, noncalmodulin-related, effects on eNOS. To test this possibility, we stimulated eNOS with ceramide, which is independent of calcium-calmodulin but still requires other eNOS cofactors (24). HME cells were then incubated with 0.75 µCi/ml of [3H]arginine to label the intracellular arginine pool. Without removal of the radiolabel, the cells were incubated with 1 µg/ml of ionomycin, 2 µM acetylcholine, 2 µM C2-ceramide (24), nondiabetic HDL (10 µg/ml), diabetic HDL (10 µg/ml), or the indicated combinations of reagents for 15 min. The cells were then processed to quantify the amount of [3H]arginine converted to [3H]citrulline as a measure of enzymatic activity. Figure 8 shows that diabetic HDL inhibited calcium-calmodulin-dependent stimulation of eNOS (ionomycin and acetylcholine) but not calcium-independent stimulation of eNOS (C2-ceramide). These data provide further evidence that the action of diabetic HDL is related to the ability of eNOS to interact with calmodulin.


Figure 8
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  		Fig. 8. Diabetic HDL does not prevent ceramide-induced stimulation of eNOS. To determine whether diabetic HDL prevented noncalmodulin-dependent mechanisms of stimulating eNOS, HME cells were incubated with 0.75 µCi/ml of [3H]arginine to label the intracellular arginine pool. Without removal of the radiolabel, the cells were incubated with 1 µg/ml of ionomycin, 2 µM acetylcholine, 2 µM C2-ceramide, nondiabetic HDL (10 µg/ml), diabetic HDL (10 µg/ml), or the indicated combinations of reagents for 15 min. The cells were then processed to quantify the amount of [3H]arginine converted to [3H]citrulline as a measure of enzymatic activity. The data are means ± SE of 4 independent experiments, with triplicate measurements in each experiment.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The main finding from our studies is that, unlike nondiabetic HDL, HDL isolated from diabetic humans and mouse models of Type 1 and Type 2 diabetes does not activate eNOS and the production of NO. Furthermore, the presence of "diabetic HDL" inhibits the ability of acetylcholine, histamine, or ionomycin to stimulate eNOS activity in endothelial cells. Importantly, this was not due to any changes in acetylcholine receptor expression, eNOS expression, or intracellular calcium levels. Analysis of the HDL components showed increased amounts of myristic acid in the diabetic HDL versus the nondiabetic HDL. The augmented level of myristic acid in diabetic HDL appears to mediate this inhibition of eNOS because the addition of myristic acid to nondiabetic HDL produced similar inhibitory effects on eNOS. Importantly, the addition of palmitic acid to nondiabetic HDL did not affect eNOS activity, which indicates that the effect of myristic acid was specific and not due to the fatty acid enrichment procedure. Furthermore, the dose-dependent effect of myristic acid added to nondiabetic HDL further suggests that the inhibition of eNOS is dependent on the presence of the myristic acid. Our data show that the mechanism whereby diabetic HDL prevents eNOS activation involves the inability of calmodulin to associate with eNOS.

One of the potential cardioprotective actions of HDL is thought to be its activation of eNOS leading to production of NO (16, 24, 27, 36). Similarly, the cardioprotective actions of estrogen include its ability to stimulate eNOS activity/NO production. HDL has been shown to be a carrier that can deliver estradiol to vascular cells (16). We previously examined the interplay between estradiol and HDL and found that HDL binding to its receptor, SR-BI, mediated the delivery of estradiol to caveolae and the subsequent activation of eNOS. Female HDL, but not male HDL, stimulated muscle strip relaxation, and this required SR-BI. In addition, HDL from premenopausal women, but not postmenopausal women, stimulated eNOS. In those studies, we showed that estradiol associated with HDL is a major contributor to the stimulation of eNOS activity by HDL (16). In the present study, we have shown that HDL isolated from both diabetic humans and diabetic mouse models no longer has the ability to stimulate eNOS and presumably eNOS-dependent cardioprotection. To our surprise, this was not due to a loss of estradiol, because the estradiol content of female diabetic HDL was similar to control female nondiabetic HDL. Furthermore, male diabetic HDL, which lacks appreciable estradiol, also inhibited eNOS activity.

The main change in diabetic HDL compared with nondiabetic HDL that we discovered was a three- to fourfold higher content of the saturated fatty acid myristic acid. To demonstrate that myristic acid was responsible for preventing HDL from activating eNOS, we showed that adding myristic acid to purified nondiabetic HDL prevented the HDL from stimulating eNOS activity. This result was unexpected in light of earlier studies in which we examined the effects of individual fatty acids, which were delivered using albumin as the carrier, on eNOS activity in endothelial cells (38). In contrast with the present study, myristic acid complexed with albumin specifically and potently stimulated the activity of eNOS (38), whereas other fatty acids had little or no effect. The exact reasons for these discrepancies are presently unknown but almost certainly lie with how myristic acid is presented to the cell. Our previous work showed that albumin-myristic acid binds to CD36 and elicits a novel signal transduction pathway that leads to the activation of AMP kinase, akt kinase, and ultimately eNOS. In contrast, we and others have previously shown that the scavenger receptor SR-BI mediates the binding of HDL to endothelial cells (1, 16), which does not stimulate AMP kinase or akt kinase activity. Thus, there are parallel and distinct pathways leading to eNOS activation.

Amazingly, the inability to stimulate eNOS was a consistent finding in all three sources of diabetic HDL tested (diabetic humans, streptozotocin-treated mice, and db/db mice). Interestingly, diabetic HDL also prevented acetylcholine-induced eNOS activity. The ability of a lipoprotein to inhibit acetylcholine-induced stimulation of eNOS activity is similar in nature to our prior work with oxidized LDL (oxLDL) (5, 33). We previously showed that oxLDL antagonizes acetylcholine-stimulated eNOS activation in a CD36-dependent fashion (5, 33). Mechanistically, this was mediated by depletion of caveolae cholesterol, which caused the mislocalization of eNOS and a disruption of caveolae signaling. By binding to SR-BI, HDL was able to restore both the cholesterol content of caveolae and acetylcholine stimulation of eNOS (33). The role of fatty acid interaction adds another level of complexity. The fatty acid stimulation of eNOS activity directly (not involving acetylcholine) by myristic acid delivered by albumin was shown to be mediated by CD36 (38). However, in the present study, myristic acid incorporated into HDL (or elevated in diabetic HDL) acts to both prevent HDL stimulation of eNOS and disrupt acetylcholine-induced eNOS activation. Both of these inhibitory effects are likely mediated by HDL binding to SR-BI but are beyond the scope of the present study and should be examined in future studies. However, unlike the disruption of eNOS signaling mediated by oxLDL, diabetic HDL does not act through mislocalization of eNOS and disruption of acetylcholine receptor signaling (data not shown), but instead the inhibition is mediated by preventing the interaction of eNOS with calmodulin.

The activation of eNOS is complicated and involves several distinct steps. eNOS can be activated by binding of acetylcholine or other agonists to their respective receptors. Binding of acetylcholine triggers a localized influx of calcium, causing calmodulin to displace caveolin from eNOS and allows calmodulin to bind to eNOS, thereby activating the enzyme. Alternatively, eNOS can be activated by other pathways that induce calcium influx or direct influx of calcium by ionophores such as ionomycin. Additionally, eNOS can be activated directly by phosphorylation by Akt kinase, AMP kinase (38), or in response to elevations in ceramide (24). Central to the activation of eNOS by several signals is the calcium-dependent binding of calmodulin. We have demonstrated a heretofore unknown mechanism in which diabetic HDL itself inhibits eNOS activation and also receptor-mediated eNOS activation. Diabetic HDL does not alter eNOS levels, interfere with acetylcholine receptors, or alter calcium influx. Instead, diabetic HDL acts by blocking the interaction between calmodulin and eNOS. Addition of the calcium ionophore ionomycin did not overcome the inhibition despite increases in intracellular calcium and calmodulin activation. In contrast, ceramide, a downstream mediator of non-calcium-dependent eNOS stimulation (24) activated eNOS to a similar extent in the presence or absence of diabetic HDL.

In the present study, we have discovered that a difference in the content of a single fatty acid, myristic acid, has a profound effect on the ability of HDL to stimulate eNOS activation. This clearly shows that the concentration of HDL may not be sufficient to indicate that lipoproteins are in a balance that is cardioprotective. The cardioprotective effect of HDL, which may be in part mediated by its effects on eNOS, can be affected by changing the composition of a single fatty acid. In addition, the estrogen-mediated activation of eNOS delivered by HDL was blocked by the presence of myristic acid in diabetic HDL, even though the content of estradiol was not changed. This result may explain some of the variability in estrogen studies, particularly in clinical studies in which HDL was obtained from postmenopausal women who had already developed cardiovascular disease, at a time when the composition of HDL is likely to be markedly different and its ability to activate eNOS impaired.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported, in part, by National Institutes of Health Grants HL-68509 (to E. J. Smart), DK-63025 (to E. J. Smart), and P20-RR-105592 (to T. Curry) and the Barnstable-Brown Endowment (to E. J. Smart).


   ACKNOWLEDGMENTS
 
We thank the Kentucky Pediatric Research Institute work group for providing invaluable advice and assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: E. J. Smart, Kentucky Pediatric Research Institute, Univ. of Kentucky, 423 Sanders-Brown, 800 South Limestone St., Lexington, KY 40536-0230 (e-mail: ejsmart{at}email.uky.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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