The aim was to investigate low-density lipoprotein (LDL) composition and Na+/K+ adenosine triphosphatase (ATPase) and Ca2+ ATPase activities and membrane fluidity measured by 1-(4-trimethylaminophenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) in platelets from obese patients and controls in order to identify, if any, platelet membrane's chemical–physical and/or functional modifications associated with compositional modification of circulating lipoproteins. Moreover, we studied the in vitro effect on both platelet transmembrane cationic transport and fluidity, by incubating LDL from 30 obese subjects with platelets from 30 control subjects. The analysis of the chemical composition of LDL from obese patients showed a significant increase in the percent content of total cholesterol (TC) and triglycerides (TGs) and in the mean levels of lipid hydroperoxides compared to controls' LDL. Platelet Na+/K+ ATPase and Ca2+ ATPase activities showed, respectively, a significant decrease and increase in patients compared to controls; minor significant, respectively, decreases and increases are shown also in control platelets incubated with LDL from obese patients. Anisotropy tested with TMA-DPH probe was significantly increased both in platelets from obese patients and in control platelets incubated with LDL from obese patients compared to control platelets. This study highlights that obesity induces remarkable modifications both in lipoproteins and platelets. Both platelet hyperfunction and quantitative/qualitative alterations in plasma lipoproteins, as well as an altered interaction between circulating lipoproteins and platelets, might play a relevant role in the increased prevalence of the early atherosclerotic lesions development in obese subjects. The present data point out that obesity might represent a major potentially modifiable risk factor for the onset of numerous complications, in particular cardiovascular ones.
Human obesity is a clinical condition characterized by an increased BMI resulting from a positive energy balance. Obesity has a multifactorial pathogenesis: both genetic and environmental factors, such as incorrect diet or sedentary life style, play a major role; even so, our knowledge on the role of physiological and molecular mechanisms involved in this pathology is still limited. Obesity-associated metabolic alterations could be at the basis of clinical complications: despite numerous studies the underlying molecular mechanisms are still conflicting. A wide-ranging understanding of its pathogenetic mechanisms is necessary for effective therapeutic interventions (1). Obesity is an important public health problem, because of the growing increase of its prevalence, and because of the associated increased morbidity and mortality. It is becoming a global epidemic in both children and adults; about 1.2 billion people in the world are overweight and at least 300 million of them are obese, according to the World Health Organization (2).
Obesity is associated with numerous comorbidities such as cardiovascular diseases, type 2 diabetes, hypertension, certain cancers, and sleep apnea/sleep-disordered breathing, and it also represents the most important risk factor for them (3).
Epidemiologic studies consistently have shown that obesity is a strong risk factor for coronary heart disease in both men and women (4).
The obese state has been recognized to accentuate the known risk factors for atherosclerotic disease as dyslipidemia, hypertension, glucose intolerance, and insulin resistance. Several studies have shown modifications of plasma lipids and lipoprotein metabolism in obese subjects. Hypercholesterolemia, high levels of triglycerides (TGs) and low-density lipoproteins (LDLs), and low levels of high-density lipoproteins (HDLs) are frequently observed in human obesity in both adult and pediatric patients (5,6). Moreover, alterations of lipoprotein heterogeneity (7,8,9) and lipoprotein lipid and apoprotein composition have been demonstrated in obese subjects (9). The modifications of lipoprotein levels and composition are associated with increased atherogenicity (10), and they are probably related to the greater risk of cardiovascular disease associated with obesity (11). Furthermore, several studies have demonstrated an increase in oxidative stress in obese subjects, with a higher susceptibility to lipid peroxidation of LDL isolated from obese subjects compared with healthy subjects (12,13,14,15,16).
Previous data suggest that the interaction between LDL and circulating cells, such as platelets, might play a central role in the development of atherosclerosis (17).
Thus, in atherosclerosis, an increase in the cholesterol/phospholipid ratio in plasma lipoproteins is associated with concomitant changes in blood platelets properties (18). Moreover, platelet functionality is directly influenced by lipoproteins (19), and platelets from patients with hypercholesterolemia display enhanced platelet reactivity, even though the exact mechanism of this effect is unclear. Platelet functionality modifications mainly afflict membrane fluidity and enzyme associated activity, such as Na+/K+ adenosine triphosphatase (ATPase) and Ca2+ ATPase; they are directly influenced by composition, properties, and molecular organization of membrane lipids (17,20,21).
Thus, the aim of this study was to investigate LDL composition and Na+/K+ ATPase and Ca2+ ATPase activities and membrane fluidity measured by fluorescent probe 1-(4-trimethylaminophenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) in platelets obtained from obese patients and controls in order to identify, if any, platelet membrane's chemical–physical and/or functional modifications associated with compositional modification of circulating lipoproteins. Moreover, the in vitro effects on both platelet transmembrane cationic transport and fluidity, by incubating LDL isolated from obese subjects with platelets obtained from control subjects were also studied.
These potential correlations could be useful to highlight further evidences of the key role played by the platelets–LDL interaction in the pathogenesis of obesity's clinical complications.
Methods and Procedures
The study was performed on 30 obese subjects (18 female and 12 male subjects) and 30 healthy control subjects (16 female and 14 male subjects), age matched, consecutively admitted between October 2006 and May 2007 to the Division of Endocrinology, Institute of Internal Medicine, Marche Polytechnic University, Ancona. Obese subjects were asked to participate at their first appointment in the Division of Endocrinology.
Clinical characteristics and plasmatic parameters of patients and controls are shown in Table 1.
Table 1. Clinical characteristics and plasmatic parameters of patients and controls
Inclusion criteria for obese subjects were BMI >30 kg/m2 and absence of both pathologies and risk factors rather than obesity. Control healthy subjects included in the study presented BMI <25 kg/m2 and absence of both other pathologies and risk factors.
Controls and patients were not taking lipid-lowering drugs, angiotensin-converting enzyme inhibitors, antioxidants, or other medication that could affect lipid metabolism. No individual had been treated with any medication known to affect platelet function during 2 weeks preceding the study. All controls were on a Mediterranean diet, while obese subject samples were obtained before a restricted diet. Furthermore, smokers, alcohol addicted, and subjects with a current or recent illness were excluded from the study.
All recruited subjects gave informed consent prior to the drawing of peripheral venous blood, the study was performed in accordance with the principles contained in the Declaration of Helsinki as revised in 2001, and it was approved by the Bioethical Committee of Marche Polytechnic University. We certify that all applicable institutional and governmental regulations concerning the ethical use of human volunteers were followed during this research.
Peripheral venous blood samples were drawn by venipuncture, after overnight fasting, using citrate dextrose or heparin as anticoagulants, respectively, in accordance with the two different methods for platelets or LDL isolation. Thus, each blood sample, obtained from both patients and controls, was divided into two aliquots: the one collected in anticoagulant citrate dextrose–containing Vacutainer tubes (Venoject; Terumo Europe NV, Leuven, Belgium) in order to isolate platelets, and the other collected in heparin-containing Vacutainer tubes to isolate LDL.
Isolation of human plasma LDL
Blood samples. were collected in heparin-containing Vacutainer tubes and plasma was prepared by centrifugation at 3,000 rpm for 15 min and thereafter used for the isolation of lipoproteins. LDL (density, 1.025–1.063 g/ml) were isolated by single vertical spin KBr density gradient ultracentrifugation for 90 min at 65,000 rpm as previously described (17); successively, LDLs were dialyzed at 4 °C for 24 h against 10 mmol/l phosphate-buffer saline, pH 7.4, containing a resin (Chelex 100; Sigma, St. Louis, MO) in order to remove any residual metallic ions. Lipoproteins were immediately used for the experiments.
The protein concentrations of LDL were determined by the method of Bradford (22), using serum albumin as standard.
The concentrations of TGs, phospholipids, and cholesterol were determined in LDL as previously described (23).
Evaluation of lipid peroxidation of LDL
The extent of lipid peroxidation in LDL isolated from plasma of 30 controls and 30 obese subjects was evaluated by measuring the levels of lipid hydroperoxides by the ferrous oxidation xylenol orange assay, according to the method of Ferretti et al. (16). Briefly, aliquots of LDL (100 µg) resuspended in 10 mmol/l phosphate-buffer saline were incubated at 37 °C for 20 min with ferrous oxidation xylenol orange reagent (100 µmol/l xylenol orange, 250 µmol/l Fe2+, 25 mmol/l H2SO4, and 4 mmol/l butylated hydroxytoluene in 90% methanol). After 20 min of incubation at 37 °C, samples were centrifuged at 3,500 rpm for 15 min, and the absorbance of supernatant was evaluated at 560 nm. t-Butyl-hydroperoxide solution was used as the standard. The results are presented as micromoles of lipid hydroperoxides for milligram phospholipids (µmol/mg phL).
Blood samples were collected in Vacutainer tubes containing anticoagulant citrate dextrose (36 ml citric acid, 5 mmol/l KCl, 90 mmol/l NaCl, 5 mmol/l glucose, 10 mmol/l EDTA pH 6.8). In platelets isolation, appropriates steps were taken to ensure no white blood cell contamination. Platelets were isolated as previously described by Ferretti et al. (17). This method involved a preliminary centrifugation step (1,300 rpm per 10 min) to obtain platelet-rich plasma (PRP). Platelets were then washed three times in the antiaggregation buffer (Tris–HCl 10 mmol/l; NaCl 150 mmol/l; EDTA 1 mmol/l; glucose 5 mmol/l; pH 7.4) and centrifuged as above, to avoid any contamination with plasma proteins and to remove any residual erythrocytes. A final centrifugation at 4,000 rpm for 20 min was performed to isolate platelets. The platelet pellet was washed twice in phosphate buffered saline phosphate-buffer saline (containing NaCl 135 mmol/l, KCl 5 mmol/l, EDTA 10 mmol/l, Na2PO4 8 mmol/l, NaH2PO4·H2O 2mmol/l, pH 7.2) and platelets were immediately used for the experiments. The protein concentrations of platelet membranes were determined by the method of Bradford (22), using serum albumin as standard.
Platelet and LDL incubation
LDLs were sterilized on a 0.2-µm Millipore (Millipore, Billerica, MA) membrane before being incubated with platelets. Platelets from control subjects were incubated with native LDL from obese patients (100 µg LDL protein/ml) in Tyrode's buffer (137 mmol/l NaCl, 2.7 mmol/l KCl, 1 mmol/l MgCl2, 1 mmol/l CaCl2, 0.35 mmol/l NaH2PO4, 11.9 mmol/l NaHCO3, and 5.5 mmol/l glucose, pH 7.5) for 3 h at 37 °C. Before and after incubation the following parameters were evaluated: Na+/K+ ATPase and Ca2+ ATPase activities and membrane fluidity.
Ca2+ ATPase activity
Ca2+ ATPase activity was determined in platelet plasma membranes according to the method described by Mazzanti et al. (24), by measuring the inorganic phosphate (Pi) hydrolyzed from 1 mmol/l Na2ATP at 37 °C in the presence and absence of 0.15 mmol/l Ca2+. Platelet plasma membranes were obtained as previously described by Ferretti et al. (17). The ATPase activity determined in the absence of the Ca2+ was subtracted from total ATPase activity in order to calculate Ca2+ ATPase activity. Ca2+ ATPase activity results are expressed as µmol Pi/mg protein/90 min. Protein concentration of platelet membranes was determined with the Bradford BioRad protein assay using serum albumin as a standard (22).
Na+/K+ ATPase activity
The Na+/K+-activated Mg2+-dependent ATPase activity was determined in platelet membranes by the Mazzanti's method (24). Platelet plasma membranes were obtained as previously described by Ferretti et al. (17). The ATPase activity was assayed by incubating platelet membranes at 37 °C in the reaction medium containing MgCl2 (5 mmol/l), NaCl (140 mmol/l), KCl (14 mmol/l) in 40 mmol/l Tris–HCl, pH 7.7. The ATPase reaction was started by the addition of 3 mmol/l Na2ATP (200 µl) and stopped 20 min later, at 37 °C, by the addition of 1 ml of 15% trichloracetic acid. The tubes were centrifuged at 3,000 rpm for 10 min and the inorganic phosphate (Pi) hydrolyzed from reaction was measured in the supernatant by a colorimetric assay using KH2PO4 as standard according to Fiske and Subbarow (25). Enzyme activity was expressed as the difference in organic phosphate released in the presence and absence of 10 mmol/l ouabain (100 µl). The ATPase activity assayed in the presence of ouabain was subtracted from the total Mg2+-dependent ATPase activity to calculate the activity of the ouabain-sensitive Na+/K+ ATPase. Na+/K+ ATPase activity was expressed as µmol Pi/mg protein/60 min. Protein concentration of platelet membranes was determined with the Bradford BioRad protein assay using serum albumin as a standard (22).
Platelet plasma membrane fluidity was studied by determining the fluorescence anisotropy (reciprocal of fluidity) of the probe TMA-DPH (Sigma), which is incorporated at the lipid–water interface of the membrane bilayer (26). Membrane incubations with TMA-DPH were performed as described by Sheridan and Block (27). Briefly, 3 µl of TMA-DPH (10−3 mol/l) were incubated for 5 min at room temperature (23 °C) with 2 ml of platelet cells (2 × 106 cells/ml) in 50 mmol/l Tris–HCl buffer solution, pH 7.4. Fluorescence intensities (100 readings each) of the vertical and horizontal components of the emitted light were measured on a Perkin–Elmer MPF66 spectrofluorometer equipped with two glass prism polarizers (excitation wavelength 365 nm, emission wavelength 430 nm). Steady state fluorescence anisotropy (r) of TMA-DPH was calculated using the following equation: r = (IvG − Ih)/(Iv + 2Ih), where G is the instrumental factor that corrects the r value for an unequal detection of vertically (Iv) and horizontally (Ih) polarized light (28). Sample temperature was maintained at 37 °C using an external bath circulator (Haake F3). The fluorescence anisotropy values were expressed as arbitrary fluorescence numbers. Fluorescence anisotropy is a quantitative index of the freedom of rotation of the probe; a decrease in the r value indicates a higher mobility of TMA-DPH in the lipid microenvironment where it is located, i.e., increased membrane fluidity.
Statistical analysis was performed using the SAS statistical package (Statistical Analysis System Institute, Cary, NC). All experiments were carried out in duplicate and were repeated three times. Results are expressed as means ± s.d. Student's t-test was used to analyze the difference of results obtained in different experimental condition. Differences were considered significant with P < 0.05.
Table 1 shows the clinical characteristics and the plasmatic parameters of patients and controls. Fibrinogen values were not significantly increased in obese subjects compared to controls. Plasma levels of total cholesterol (TC) and HDL-cholesterol were not significantly increased in obese subjects compared to controls, whereas TC/HDL-cholesterol ratio was significantly higher in obese subjects in respect to controls (P < 0.05). Moreover, the mean plasma TG levels and LDL-cholesterol in obese patients were significantly higher than in the control values (P < 0.05).
As shown in Table 2, the analysis of the chemical composition of LDL from obese patients evidenced significant changes with respect to controls LDL. A significant increase in the percent content of TG (2.49 ± 1.01% vs. 4.63 ± 0.79%; P < 0.05) and TC (39.35 ± 7.45 % vs. 52.60 ± 7.85%; P < 0.05) was observed in LDL from obese patients compared with control ones. The levels of phospholipids and proteins were not significantly modified. Thus, the obese subjects appeared to exhibit an atherogenic change in lipoprotein metabolism with respect to healthy subjects included in the study.
Table 2. Analysis of the chemical composition of LDL from obese subjects and controls
The mean lipid hydroperoxides' levels associated with LDL isolated from control subjects and obese subjects were, respectively, 193.69 ± 48.91 and 238.73 ± 25.84 (µmol/mg phL), and were significantly increased in patients than in controls (P < 0.05) (Table 2).
Moreover, platelet Na+/K+ ATPase (Figure 1) activity showed a significant decrease in obese patients compared to controls (P < 0.05), while minor significant decreases are shown also in control platelets incubated with LDL from obese patients compared to controls (P < 0.05); platelet Ca2+ ATPase (Figure 2) activity showed a significant increase in patients compared to controls (P < 0.05), while minor significant increases are shown also in control platelets incubated with LDL from obese patients compared to controls (P < 0.05).
Anisotropy tested with TMA-DPH probe (Figure 3) was significantly increased both in platelets from obese patients and in control platelets incubated with LDL from obese patients compared to control platelets (P < 0.05). Because anisotropy is inversely related to the fluidity of the probe's microenvironment, our results indicated lower fluidity of the external surface of the patients' platelet membrane.
The data obtained from Na+/K+ ATPase activity and TMA-DPH anisotropy showed a significant negative correlation (r = −0.92, P < 0.05).
In addition, a significant positive correlation between TMA-DPH anisotropy and LDL content of TG (r = 0.76, P < 0.05), TMA-DPH anisotropy and LDL content of TC (r = 0.89, P < 0.05) and TMA-DPH anisotropy and LDL content of lipid hydroperoxides (r = 0.84, P < 0.05) was observed.
A significant negative correlation between Na+/K+ ATPase activity and LDL content of TG (r = −0.79, P < 0.05), Na+/K+ ATPase activity and LDL content of TC (r = −0.88, P < 0.05) and Na+/K+ ATPase activity and LDL content of lipid hydroperoxides (r = −0.83, P < 0.05) was observed.
A significant positive correlation between Ca2+ ATPase activity and LDL content of TG (r = 0.75, P < 0.05), Ca2+ ATPase activity and LDL content of TC (r = 0.71, P < 0.05), and Ca2+ ATPase activity and LDL content of lipid hydroperoxides (r = 0.80, P < 0.05) was observed.
Data also showed a negative significant correlation between BMI and Na+/K+ ATPase activity (r = −0.68, P < 0.05); moreover, positive significant correlations were evidenced between BMI and Ca2+ ATPase activity (r = 0.73, P < 0.05), BMI and TMA-DPH anisotropy (r = 0.70, P < 0.05), BMI and LDL content of TG (r = 0.89, P < 0.05), BMI and LDL content of TC (r = 0.91, P < 0.05), BMI and LDL content of lipid hydroperoxides (r = 0.82, P < 0.05).
Despite numerous studies, pathogenetic mechanisms at the basis of obesity and molecular pathways related to both clinical complications and obesity-associated metabolic alterations are still to be clarified.
It is known that both platelets and lipoproteins are intimately involved in the pathogenesis of atherothrombotic diseases (29). Lipoproteins are important in the development of such diseases because they alter the properties of different circulating cells involved in atherosclerosis and thrombosis, such as platelets. Thus, the interactions of platelets with lipoproteins have been under intense investigation (30).
Therefore, conditions leading to altered platelet functions, such as severe disorders of plasma lipids, lead to augmented platelet reactivity and they are accompanied with an enhanced risk of atherosclerosis and thrombosis onset (31).
In our study, all the obese patients showed a significantly increased plasma content of TGs, LDL-cholesterol as well as a significant increase in TC/HDL-cholesterol ratio.
Moreover, as it concerns the LDL composition and their related chemical–physical modifications, we observed a significantly increased content of TGs and cholesterol as well as an increased content in lipid hydroperoxides in patients compared to controls. Thus, these data highlight that obesity induces important quantitative/qualitative modifications in lipoproteins. Because high plasma levels of LDL as well as modified LDL have been supposed to be a primary risk factor in atherosclerosis (32), the obese state might contribute to the development of atherosclerotic plaques.
A significant decrease in both Na+/K+ ATPase activity and membrane fluidity, as well as a significant increase in Ca2+ ATPase activity in platelets obtained from obese patients compared to controls has also been observed. A minor significant decrease in both Na+/K+ ATPase activity and membrane fluidity, as well as a minor significant increase in Ca2+ ATPase activity has been observed also in platelets from controls incubated with LDL from obese patients compared to controls.
In addition, our data evidenced significant negative correlations between the increased LDL content in TG, TC, lipid hydroperoxides, and Na+/K+ ATPase activity; the data also evidenced significant positive correlations between the increased LDL content in TG, TC, lipid hydroperoxides, and both increased TMA-DPH anisotropy and Ca2+ATPase activity.
Thus, it can be hypothesized that the increase in TGs, cholesterol, and lipid hydroperoxides content in LDL from obese patients enhances platelet aggregability, through both the release of arachidonic acid/augmented synthesis of thromboxane A2, and the increment in intracellular free Ca2+ concentration. This enhanced intracellular free Ca2+ concentration leads to an increase in Ca2+ ATPase activity of obese patients, as confirmed in this study, also by positive significant correlations between modifications in LDL composition and Ca2+ ATPase activity.
Moreover, the decrease in Na+/K+ ATPase activity observed in obese patients can be related to the diminished fluidity in the platelet membranes of the same patients, as demonstrated by a negative significant correlation between Na+/K+ ATPase activity and TMA-DPH anisotropy.
In particular, we investigated Na+/K+ ATPase activity that is a well-known factor playing a key role in cellular osmotic regulation through the maintenance of the transmembrane gradients of Na+ and K+ (33). Na+/K+ ATPase is also a marker of membrane function, as it is an integral membrane protein that greatly depends for its activity on the chemical–physical properties and composition of the microenvironment—membrane lipids—where it is embedded (34). Membrane fluidity, a submacroscopic property that is closely related to membrane molecular organization, is affected by various compositional factors such as the content in proteins, cholesterol and phospholipids, and their fatty acid composition (35). Thus, in association with membrane fluidity, Na+/K+ ATPase represents an index of membrane functionality being involved in the modulation of phospholipid and protein interactions (36). In addition, data regarding TMA-DPH anisotropy suggest that platelet membrane fluidity in obese patients was significantly lower than that in control subjects.
This study shows a decrease in the platelet Na+/K+ ATPase activity in patients as the enzyme is located in a less fluid cellular microenvironment due to alterations related to obesity (i.e., the increased TG, TC, and lipid hydroperoxides content in LDL), as confirmed both by negative significant correlations between modifications in LDL composition and Na+/K+ ATPase activity, and by positive significant correlations between the same LDL modifications and between the TMA-DPH anisotropy. Because the pump represents the key-enzyme of cellular osmotic regulation, its inhibition leads to altered internal and external membrane ionic concentrations (37). The presence of this ionic imbalance causes a derangement in cellular enzymatic systems, leading to membrane depolarization which in turn could compromise platelets survival (38). Because platelet membrane fluidity, strictly related to microviscosity, is a critical determinant of platelet aggregation and secretion, its decrease in obese patients leads us to hypothesize a crucial role in the pathogenesis of atherosclerosis.
In fact, in obesity we observed modified LDL which in turn, by interacting with platelets, could induce relevant structural and functional modifications for the development of atherosclerotic lesion, as confirmed by significant correlations between BMI and modifications in LDL composition, BMI and Na+/K+ ATPase activity, BMI and Ca2+ ATPase activity, BMI and TMA-DPH anisotropy. The effects of LDL from obese patients on enzymatic activities and structural organization of platelet membranes from controls seem to mimic the alterations observed in platelets from obese subjects.
Despite the fact that interactions between lipoproteins and platelets in different pathological conditions have been under intense investigation, data are not yet available on such interaction in obesity; thus the novelty of our research was to investigate platelet membrane's chemical–physical and/or functional modifications associated with compositional modification of circulating lipoproteins in obese patients.
In conclusion, this study highlights that obesity induces remarkable modifications on lipoproteins and platelets. Both platelet hyperfunction and quantitative/qualitative alterations in plasma lipoproteins, as well as an altered interaction between circulating lipoproteins and platelets, might play a relevant role in the increased prevalence of the early atherosclerotic lesions development in obese subjects. Thus, the present data point out that obesity might represent a major potentially modifiable risk factor for the onset of numerous complications, in particular cardiovascular ones. Further studies are in progress in obese patients during and after a hypocaloric diet.