Abstract
Context:
Postprandial hyperlipidemia has been suggested to contribute to atherogenesis by inducing proinflammatory changes in monocytes. Individuals with metabolic syndrome (MS), shown to have higher blood triglyceride concentration and delayed triglyceride clearance, may thus have increased risk for development of atherosclerosis.
Objective:
Our objective was to examine fasting levels and effects of a high-fat meal on phenotypes of monocyte subsets in individuals with obesity and MS and in healthy controls.
Design, Setting, Participants, Intervention:
Individuals with obesity and MS and gender- and age-matched healthy controls were recruited. Blood was collected from participants after an overnight fast (baseline) and at 3 and 5 hours after ingestion of a high-fat meal. At each time point, monocyte phenotypes were examined by multiparameter flow cytometry.
Main Outcome Measures:
Baseline levels of activation markers and postprandial inflammatory response in each of the three monocyte subsets were measured.
Results:
At baseline, individuals with obesity and MS had higher proportions of circulating lipid-laden foamy monocytes than controls, which were positively correlated with fasting triglyceride levels. Additionally, the MS group had increased counts of nonclassical monocytes, higher CD11c, CX3CR1, and human leukocyte antigen-DR levels on intermediate monocytes, and higher CCR5 and tumor necrosis factor-α levels on classical monocytes in the circulation. Postprandial triglyceride increases in both groups were paralleled by upregulation of lipid-laden foamy monocytes. MS, but not control, subjects had significant postprandial increases of CD11c and percentages of IL-1β+ and tumor necrosis factor-α+ cells in nonclassical monocytes.
Conclusions:
Compared to controls, individuals with obesity and MS had increased fasting and postprandial monocyte lipid accumulation and activation.
Obese subjects with MS had higher fasting levels of lipid-laden foamy monocytes and significant postprandial increases in activation markers on nonclassical monocytes after high-fat meal vs controls.
Postprandial plasma triglycerides are correlated with atherosclerosis and cardiovascular events (1, 2). Transient increase of blood triglycerides after a fat-containing meal, defined as postprandial hyperlipidemia, results from increased levels of triglyceride-rich lipoproteins (TGRLs), which include chylomicrons, chylomicron remnants, and very-low-density lipoproteins (3). Ingestion of a high-fat meal has been reported to increase proinflammatory cytokine levels, including IL-6, IL-18, and tumor necrosis factor-α, in the blood (4–6). Postprandial hyperlipidemia is also accompanied by increased leukocyte counts with concomitant production of proinflammatory cytokines, which may contribute to endothelial dysfunction (7–9). Postprandial leukocyte activation is a transient event, with leukocyte cell populations returning to baseline at about 8 hours after a fat load in lean volunteers (10). One mechanism by which leukocytes become activated after a high-fat meal is the direct interaction of leukocytes with TGRLs. TGRLs were also shown to contribute to the formation of lipid-laden macrophages, or “foam cells,” in atherosclerotic lesions in the arterial wall (11–13). In the traditional paradigm, atherogenic lipoproteins infiltrate into the arterial wall and are taken up by macrophages or foam cells. However, more recently, our group and others have shown that lipoproteins can interact directly with monocytes in the blood to form “foamy monocytes” (14, 15). Foamy monocytes elevated after a high-fat meal had increased expression of CD11c integrin, which facilitated monocyte adhesion on vascular cell adhesion molecule-1 (16). The increase in postprandial TGRLs was paralleled by upregulation of CD11b and reduction in CD62L, suggesting monocyte activation (10, 16, 17). Leukocytes activated by postprandial TGRLs could further interact with endothelium, thus contributing to atherosclerotic plaque formation (18).
Based on cell surface markers, monocytes are classified as classical (CD14++CD16–), intermediate (CD14++CD16+), and nonclassical (CD14+CD16+) (19). Intermediate and nonclassical monocytes, collectively known as CD16+ monocytes (19), constitute only 10% of total blood monocytes, but secrete high levels of proinflammatory cytokines and low levels of anti-inflammatory IL-10 (20–22). CD16+ monocyte counts were found to be significantly associated with body mass index (BMI) (23). Furthermore, CD16+ monocytes have been implicated in multiple inflammatory diseases, including atherosclerosis (21). However, little is known about postprandial changes in monocyte subsets and the effects of postprandial hyperlipidemia on activation markers in each monocyte subset.
Postprandial response differs in lean and obese individuals. Both the maximum triglyceride increase and the magnitude of postprandial hyperlipidemia were found to be higher in individuals with atherosclerosis compared to controls (24). Moreover, obese individuals with type 2 diabetes have delayed TGRL clearance (25, 26).
The aim of the present study was to compare baseline levels of activation markers in each monocyte subset in lean controls and individuals with metabolic syndrome (MS). We also evaluated and compared the effect of a high-fat meal on inflammatory response of each monocyte subset between these two groups.
Materials and Methods
Subjects
Male and female volunteers were recruited at the Center for Cardiovascular Disease Prevention at Baylor College of Medicine, Houston, Texas, or by advertisement. Subjects with MS were identified by the Adult Treatment Panel III criteria (27): at least 3 of the following were present: 1) high-density lipoprotein cholesterol (HDL-C) below 40 mg/dl for men and below 50 mg/dl for women; 2) glucose of at least 100 mg/dl; 3) triglycerides of at least 150 mg/dl; 4) blood pressure (BP) of at least 130/85 mm Hg; 5) waist circumference more than 102 cm in men and more than 88 cm in women. Individuals with BMI 18.5–27.4 kg/m2 and fasting triglycerides lower than 150 mg/dl were used as normal controls. Individuals were excluded if they were younger than 18 years of age, pregnant, or breastfeeding, or had acute illnesses, chronic liver or renal disease, cancer, bone fractures, diabetes, endocrinologic causes of obesity, obvious inflammation, or a history of myocardial infarction within the past 6 months or any hospitalization within the previous 2 months. Informed consent was obtained from all subjects. The study was approved by the Institutional Review Board for Baylor College of Medicine and Affiliated Hospitals.
Study design
Subjects were asked to fast overnight (∼10 hours) prior to reporting to the Center for Cardiovascular Disease Prevention for the study. Weight, waist circumference, and BP were measured, and fasting venous blood was drawn (t = 0 hours). Subjects were then given a high-fat meal consisting of Sausage McMuffin with Egg and Bacon and Egg & Cheese Biscuit purchased from a McDonald's restaurant. The nutrient composition of the meal is shown in Supplemental Table 1. The meal was consumed within 20 minutes. Subjects returned for postprandial blood draw at 3 (t = 3 hours) and 5 hours (t = 5 hours) following ingestion of the meal. Participants were not allowed to consume other food or beverages other than water during the study.
Analytical methods
Blood samples were collected in 10.0-ml BD Vacutainer K2 EDTA tubes (Becton Dickinson). Whole blood (1.0 ml) was used to determine blood counts using the Horiba ABX-Pentra 60 C+ hematological analyzer. The rest of the blood was further centrifuged to isolate plasma. Lipid profile and glucose analysis were performed using the Beckman Coulter AU480 automated platform. Plasma total cholesterol, triglycerides, and HDL-C were measured by enzymatic methods. Samples exceeding the upper limit of linearity were diluted and measurements repeated. The new values were multiplied by the dilution factor to generate a final reportable value. For triglyceride levels of 400 mg/dl or lower, low-density lipoprotein cholesterol values were derived from the Friedewald equation. Glucose was measured using the hexokinase G-6-PDH assay with subsequent change in absorbance at 340/380 nm being proportional to the amount of glucose present in the sample. Samples exceeding the upper limit of linearity were diluted. Insulin concentrations were measured with a novel assay, Elecsys insulin assay (Roche Diagnostics), on an automated Cobas e411 analyzer.
Flow cytometry
Four staining protocols (tubes) were used at each time point of baseline, 3, and 5 hours after the high-fat meal for each individual. A total of 100 μl of whole blood was used for each protocol and stained with the following combinations of fluorescent antibodies: 1) CD14+CD16+CD11c+human leukocyte antigen (HLA)-DR+CX3CR1+CCR2+CD62L+IL-1β+viability dye; 2) CD14+CD16+CD11c+HLA-DR+CX3CR1+CCR2+CD62L+ TNF-α+viability dye; 3) CD14+CD16+CD11c+CCR2+CCR5+CD36+CD206+CD3+viability dye; 4) CD14+CD16+CD11c+Nile red+viability dye. The following antibodies were used for the analysis: Beckman Coulter HLA-DR-ECD, CD14-APC-Alexa Fluor 750, CD16-PC7; BioLegend CX3CR1-Alexa Fluor 647 (Clone 2A9-1); BD Pharmingen CD62L-FITC, CD11c-V450 (Clone B-ly6), CCR5-APC-Cy 7 (Clone 2D7/CCR5); and eBioscience CD206-PE (Clone 19.2). Samples were then incubated for 30 minutes in the dark at 4 C. Red blood cells were lysed and cells were fixed with the ImmunoPrep Reagent Kit (Beckman Coulter) on a TQ-Prep Workstation (Beckman Coulter). For intracellular staining, cells were incubated with Leukocyte Activation Cocktail with BD GolgiPlug (BD Pharmingen) in Roswell Park Memorial Institute medium for 5 hours at 37 C. Cell suspensions were then fixed and permeabilized with BD Cytofix/Cytoperm kit (BD Biosciences) and stained with IL-1β-PE (Clone CRM56) and TNF-α-PE (Clone MAb11) (eBioscience). Flow-Count Fluorospheres (Beckman Coulter) were added to cell suspension prior to analysis to allow a direct determination of absolute counts. Data were acquired on Beckman Coulter Gallios and analyzed with Kaluza 1.2 software (Beckman Coulter) to examine cell populations. Cells identified as viable were selected and all CD14+ monocytes were gated and further classified as classical (CD14++CD16–), intermediate (CD14++CD16+), or nonclassical (CD14+CD16+). Median fluorescence intensity (MFI) of CD62L, CD11c, CCR5, CCR2, CD206, CX3CR1, and HLA-DR and percentages of TNF-α+, IL-1β+, and Nile red+ cells were quantified in each monocyte subset. Tubes 1 and 2 were used to quantify the percentages of TNF-α and IL-1β, as well as MFI of HLA-DR, CD62L, and CX3CR1. Tube 3 was used to assess the percentages and concentration of the monocyte subsets as well as MFI of CCR5, CCR2, CD36, and CD206. Tube 4 was used to determine the percentages of Nile red+ cells.
Statistics
Data were analyzed using GraphPad Prism 5.0 software (GraphPad Software). Mean fasting differences were analyzed using independent two-sample t tests. Postprandial changes within groups were analyzed by repeated-measures ANOVA followed by Bonferroni post hoc test for pairwise comparisons between baseline and 3 hours, baseline and 5 hours, and 3 hours and 5 hours. Two experimental groups were compared using two-way ANOVA. P values ≤.05 were considered statistically significant.
Results
Subject characteristics and postprandial hyperlipidemia
The study included 11 subjects with MS and 11 age- and gender-matched lean controls. Subjects with MS had significantly higher BMI and greater waist circumference. Both systolic and diastolic BP were also higher in the MS group (Table 1). While HDL-C was significantly lower in the MS group, total plasma cholesterol and low-density lipoprotein cholesterol concentrations were not different between the two groups. Fasting plasma levels of triglycerides, glucose, and insulin were all significantly higher in the MS group than in control group (Table 1). With the ingestion of the high-fat meal in controls, plasma triglyceride concentration significantly increased from 89.1 ± 10.1 mg/dl at baseline to 162.3 ± 16.3 mg/dl at 3 hours after the meal and returned towards fasting level at 5 hours (Figure 1A). In contrast, in individuals with MS, postprandial triglycerides significantly increased from fasting levels of 217.0 ± 42.9 mg/dl to 302.0 ± 40.0 mg/dl at 3 hours, but only decreased to 295.5 ± 39.6 mg/dl at 5 hours (Figure 1A), indicating impaired triglyceride clearance. As expected, the ingestion of the high-fat meal increased insulin levels in both groups at 3 hours (Figure 1B). In addition to higher fasting insulin (Table 1), the MS group had significantly increased postprandial insulin response (P = .001, two-way ANOVA). Mean insulin concentration was 3 times higher in the MS group than the control group at 3 hours after the meal (Figure 1B). In contrast, the control group, but not the MS group, had significant reductions in glucose concentration at 3 and 5 hours after the meal (Figure 1C), consistent with insulin resistance in the MS group.
Table 1.
Baseline Characteristics of Study Participants
| Control (n = 11) | MS (n = 11) | P Value | |
|---|---|---|---|
| Age (y) | 42.5 ± 14.0 | 59.6 ± 7.7 | .151 |
| Gender (% male) | 82 (n = 9) | 82 (n = 9) | |
| Race (%Caucasian) | 64 (n = 7) | 73 (n = 8) | |
| BMI (kg/m2) | 24.6 ± 2.8 | 34.0 ± 7.1 | .001 |
| Waist circumference (inches) | 34.6 ± 4.0 | 43.4 ± 5.1 | .0002 |
| Systolic blood pressure (mm Hg) | 123 ± 18 | 142 ± 17 | .024 |
| Diastolic blood pressure (mm Hg) | 76 ± 11 | 88 ± 10 | .015 |
| Cholesterol (mg/dl) | 185 ± 45 | 190 ± 41 | .771 |
| HDL-C (mg/dl) | 51 ± 20 | 37 ± 9 | .05 |
| LDL-C (mg/dl) | 116 ± 33 | 120 ± 39 | .817 |
| Triglycerides (mg/dl) | 89 ± 34 | 217 ± 142 | .009 |
| Glucose (mg/dl) | 91.9 ± 7.1 | 111.8 ± 29.6 | .043 |
| Insulin (μU/ml) | 7.4 ± 3.5 | 20.0 ± 11.6 | .003 |
Abbreviation: LDL-C, low-density lipoprotein cholesterol.
Values are mean ± sd unless otherwise indicated.
Figure 1.
Postprandial triglyceride, insulin, and glucose response. Plasma concentration of (A) triglycerides, (B) insulin, and (C) glucose before and after the high-fat meal in controls and individuals with MS. Data shown as mean ± SEM. *P < .05, **P < .01 for comparison with baseline of the same group (one-way ANOVA). Differences between control and MS groups were assessed by two-way ANOVA. ns, not significant. #P < .05; ##P < .01.
Effect of high-fat meal on leukocyte counts and monocyte heterogeneity
Total leukocyte counts and differentials, including monocyte, lymphocyte, and granulocyte counts, were obtained in whole blood by automatic hematologic analyzer. In the fasting state, total leukocyte, lymphocyte, and monocyte counts tended to be higher in individuals with MS than in lean controls, whereas granulocyte counts were significantly higher in individuals with MS (Figure 2A).
Figure 2.
Changes in leukocyte counts after ingestion of the high-fat meal. Absolute counts of total leukocytes, lymphocytes, monocytes, and granulocytes were assessed by automatic method. A, Absolute blood leukocyte counts in the fasting state. Postprandial changes in (B) total leukocyte, (C) lymphocyte, (D) monocyte, and (E) granulocyte counts. Data shown as mean ± SEM. *P < .05, **P < .01 for comparison with baseline of the same group (one-way ANOVA). Differences between control and MS groups were determined by two-way ANOVA (P = .33 for leukocytes, P = .98 for lymphocytes, P = .41 for monocytes, and P = .09 for granulocytes).
In the control group, ingestion of a high-fat meal resulted in significant increases in total leukocyte, monocyte, and granulocyte, but not lymphocyte, counts at 3 hours (Figure 2, B–E), which corresponded to the peak time of lipemia. Total leukocyte and granulocyte counts decreased at 5 hours as compared to 3 hours, but were still significantly elevated compared to baseline levels (Figure 2, B and E). Leukocyte changes in the MS group showed a trend toward postprandial increases but did not reach statistical significance (Figure 2, B–E). Postprandial changes in leukocyte counts were not significantly different between the two groups, as assessed by two-way ANOVA (Figure 2, B–E).
We further assessed fasting absolute counts and postprandial changes in monocyte subsets by flow cytometry. Consistent with the results obtained from automated hematological analyzer, total monocyte counts tended to be higher but were not statistically different in the MS group compared to controls at baseline (data not shown). However, the concentration of nonclassical monocytes was significantly higher in fasting blood of individuals with MS than in lean controls (Figure 3A). Fasting classical and intermediate monocytes were not significantly different between controls and individuals with MS (Figure 3A). Ingestion of the high-fat meal increased or tended to increase each of the monocyte subsets in both the MS and control groups (Figure 3, B-D). While postprandial changes in classical and intermediate monocytes did not reach statistical significance in either group (Figure 3, B and C), the ingestion of the high-fat meal resulted in a significant increase in nonclassical monocytes at 3 hours in the control group (Figure 3D).
Figure 3.
Fasting concentration and postprandial changes in monocyte subsets in blood of controls and individuals with MS. Monocyte subsets were identified by flow cytometry based on the surface expression pattern of CD14 and CD16 into classical (CD14++CD16–), intermediate (CD14++CD16+), and nonclassical (CD14+CD16+) monocytes. Concentration of each subset was assessed using flow count beads. A, Concentration of monocyte subsets in fasting blood of control and MS groups. Baseline differences between control and MS groups were evaluated using independent two-sample t tests, **P < .01. Postprandial changes in (B) classical, (C) intermediate, and (D) nonclassical monocyte concentrations after the ingestion of a high-fat meal in controls and individuals with MS. *P < .05 for comparison with baseline of the same group (repeated measures ANOVA with Bonferroni posttests). ns, no significant difference between control and MS groups by two-way ANOVA.
Fasting and postprandial foamy monocyte formation
Hyperlipidemia may cause lipid accumulation in monocytes, leading to formation of lipid-laden foamy monocytes (14–16, 28, 29). We sought to determine if lipid-laden foamy monocytes correlated with blood lipid levels. At baseline, foamy monocytes, which stained positive for Nile red (15), were significantly increased in the blood of individuals with MS compared to controls (Figure 4A). Baseline Nile red+ monocytes further showed a positive correlation with fasting triglyceride levels (Figure 4B). Compared to baseline, Nile red+ foamy monocytes were significantly increased in the control group at 3 hours after the high-fat meal ingestion, corresponding to the peak of triglyceride concentration, and decreased at 5 hours to baseline level (Figure 4C). Nile red+ foamy monocytes tended to increase continuously in the MS group after the high-fat meal (Figure 4C); however, the increase at 3 hours and 5 hours did not reach statistical significance when compared to baseline.
Figure 4.
Fasting and postprandial changes in Nile red+ lipid-laden monocytes. A, Differences between % Nile red+ monocytes in control and MS groups in the fasting state assessed by independent two-sample t test, *P < .05. B, Nile red+ monocytes were positively correlated to fasting triglycerides (TG; Pearson r = 0.53, P = .02). C, Postprandial changes in Nile red+ monocytes, *P < .05 indicates significant increase between baseline and 3 hours in the control group by repeated measures ANOVA with Bonferroni posttests. ns, no significant difference between control and MS groups by two-way ANOVA.
Fasting and postprandial changes in monocyte phenotypes
We further quantified surface and intracellular inflammatory markers on the cells in each of the three monocyte subsets. At baseline, CD11c, a β2 integrin involved in monocyte adhesion to endothelial cells and atherogenesis (14, 16), was significantly higher on intermediate monocytes than on classical monocytes in the same individuals (Supplemental Figure 1A). CD11c on nonclassical monocytes was also significantly higher in controls and tended to be higher in the MS group when compared to classical monocytes in each group, which supports the proinflammatory nature of CD16+ intermediate and nonclassical monocytes. Compared to lean controls, individuals with MS had significantly increased CD11c expression on intermediate monocytes and tended to have increased CD11c on classical monocytes (P = .06) (Supplemental Table 2; Supplemental Figure 1A). Also consistent with the proinflammatory nature of CD16+ monocytes, CCR5 in controls and intracellular TNF-α and IL-1β expression in both groups were higher on/in intermediate and nonclassical monocytes than on/in classical monocytes (Supplemental Table 2; Supplemental Figure 1B–1D). Notably, although baseline levels of CCR5 and TNF-α were lower in classical monocytes than in intermediate and nonclassical monocytes in controls, individuals with MS had increases in TNF-α (P = .004) compared to controls (Supplemental Table 2). Classical monocyte levels of CCR5 and IL-1β tended to be higher in fasting blood of individuals with MS than that of controls, but the difference was not statistically significant (Supplemental Table 2). Compared to lean controls, individuals with MS also had elevated expression of HLA-DR (P = .05) on intermediate monocytes (Supplemental Table 2).
The ingestion of a high-fat meal resulted in a significant reduction in CD62L on classical monocytes in the control group, which is suggestive of postprandial monocyte activation (Supplemental Figure 2A). Reduced levels of CD62L were also observed on intermediate and nonclassical monocytes, although the results were not statistically significant (Supplemental Figure 2A). Reduction in CD62L after the high-fat meal was also observed in the MS group; however, the results did not reach statistical significance (Supplemental Figure 2A). Although no postprandial changes in CD11c levels were detected on any monocyte subset in control subjects, elevations in postprandial triglycerides significantly increased levels of CD11c on nonclassical, but not classical and intermediate, monocytes of individuals with MS (Figure 5A, Supplemental Figure 2B). CD11c on nonclassical monocytes was upregulated by 30% within 3 hours after meal ingestion and remained elevated at 5 hours in individuals with MS (Figure 5A). Postprandial CD11c response was significantly higher in individuals with MS, who had significantly higher postprandial CD11c levels on nonclassical monocytes than controls (P = .008, two-way ANOVA) (Figure 5A). The effect of postprandial hyperlipidemia on blood monocyte inflammatory activation was further assessed by measuring intercellular IL-1β and TNF-α. Although no significant differences were observed at baseline between the MS and control groups, a significant increase was observed in the percentage of nonclassical monocytes expressing IL-1β or TNF-α at 3 hours after the high-fat meal in the MS group (Figure 5, B and C). However, this increase did not occur in the control group (Figure 5, B and C). In the MS group, IL-1β–expressing nonclassical monocytes increased from 23.3 ± 4.4% in the fasting state to 39.5 ± 6.8% at 3 hours after the meal (Figure 5B). Similar changes were observed in the percentage of nonclassical monocytes expressing TNF-α, which increased from 45.2 ± 7.5% at baseline to 62.7 ± 10.8% at 3 hours in the MS group (Figure 5C). Postprandial changes in TNF-α–expressing nonclassical monocytes were significantly greater in the MS group than the control group (P = .022, two-way ANOVA). In contrast, no significant postprandial effects were observed on the percentages of TNF-α- or IL-1β–producing classical and intermediate monocytes in either the MS or control group (Supplemental Figure 2, C and D).
Figure 5.
Inflammatory response following the ingestion of a high-fat meal in individuals with MS and controls. Cell surface expression of CD62L and CD11c and intracellular IL-1β and TNF-α expression on monocyte subsets were detected by flow cytometry. MFI of (A) CD11c on nonclassical monocytes. Percentage of nonclassical monocytes expressing intracellular (B) IL-1β and (C) TNF-α at baseline and after ingestion of the high-fat meal. Data are shown as mean ± SEM. *P < .05 for comparison with baseline of the same group (repeated measures ANOVA with Bonferroni posttests). ns, not significant. #P < .05, ##P < .01 by two-way ANOVA.
Discussion
Previous human studies have shown association of higher numbers of CD16+ intermediate or nonclassical monocytes with hyperlipidemia and cardiovascular disease (30–34). Studies have also revealed increased CD16+ monocytes, which also showed higher response to high-fat meal, in humans with obesity (23, 34).
Our data show that postprandial hyperlipidemia is associated with an alteration of circulating monocyte phenotypes consistent with monocyte activation that was more pronounced in nonclassical monocytes than other subsets. Fasting blood of individuals with MS was characterized by increased concentration of nonclassical monocytes with elevated expression of CD11c and intracellular TNF-α compared to control subjects. Fasting triglyceride levels were correlated to the percentage of lipid-laden “foamy” monocytes. Postprandial hypertriglyceridemia in individuals with MS resulted in upregulation of CD11c, IL-1β, and TNF-α on nonclassical monocytes. The ingestion of a high-fat meal also led to higher leukocyte counts, attributable to increases in granulocytes and classical and nonclassical monocytes.
Postprandial response was different in controls and individuals with MS. Consistent with previous studies showing delayed postprandial TGRL clearance in individuals with obesity and individuals with type 2 diabetes (35), we observed prolonged hypertriglyceridemia in individuals with MS; at 5 hours after meal ingestion, triglyceride concentration was comparable to that at 3 hours. In contrast, despite a higher percent increase in blood triglycerides from baseline to 3 hours in lean controls (80% in controls and 40% in MS group), triglycerides in controls were significantly reduced within 5 hours after meal consumption.
Individuals with MS had not only higher absolute levels of postprandial hyperlipidemia and delayed clearance, but also markedly different responses to glucose and insulin. Individuals with MS had impaired fasting glucose at baseline despite having almost a 3-fold increase in insulin levels. Both groups had an increase in insulin at 3 hours; the control group had a reduction in glucose at that time, whereas the group with MS had no significant changes and a slight numerical increase in glucose, despite a striking increase in insulin, consistent with marked insulin resistance.
We found elevated leukocyte counts, with significantly higher granulocyte absolute counts, in individuals with MS. Previous reports showed that increased leukocyte count is positively correlated with the incidence of atherosclerosis and hypertriglyceridemia and predicts the development of type 2 diabetes (36–38). Although total monocytes were not different between groups, individuals with MS had significantly higher fasting nonclassical CD16+ monocyte count. Increased CD16+ monocytes have been shown to have a strong correlation with BMI (23). Individuals with MS had a higher percentage of Nile red–stained monocytes, which is attributable to increased lipid content. Monocytes with lipid droplets, or “foam cells,” were significantly correlated with fasting triglyceride concentration. Lipid accumulation in circulating monocytes in mouse models has been shown to result in monocyte activation with higher expression of CD11c integrin (14). We demonstrated increased CD11c expression on intermediate monocytes in the MS group compared to controls. A recent report showed that CD11c expression on intermediate monocytes changed acutely in response to rise or fall of triglyceride levels (39). Higher CD11c expression on intermediate monocytes in individuals with MS may thus be explained by higher fasting triglycerides in this group compared to controls.
To evaluate the effect of a high-fat meal on monocyte activation, we fed the subjects a high-fat meal from McDonald's. This meal was chosen to be representative of a common meal in the Western diet consisting of a large percentage of calories from fat, especially saturated fat. Saturated fat produces inflammatory response in monocytes, stimulating the expression of proinflammatory cytokines and adhesion molecules (40, 41). We showed increases in monocytes and granulocytes at 3 hours after the meal in blood of control subjects. The same trend was observed in the MS group, but the results were not statistically significant. These results were consistent with previous studies showing increased leukocyte counts, primarily neutrophil counts, resulting from postprandial hyperlipidemia (8–10). Increases in leukocyte counts may suggest initiation of inflammatory response by postprandial TGRLs.
Previous reports demonstrated that postprandial leukocyte activation was increased in overweight individuals in the presence of insulin resistance (42). Furthermore, a high-fat meal induced a more prolonged postprandial inflammatory response in obese humans (5, 43). We found both higher magnitude and more prolonged inflammatory activation of monocytes in the MS group compared to controls. The changes in inflammatory markers may reflect postprandial triglyceride response. Higher triglyceride concentrations at 3 and 5 hours after the meal in the MS group paralleled CD11c expression levels. CD11c MFI on monocytes of individuals with MS was elevated at 3 and 5 hours after the meal compared to controls. Within the MS group, the most striking postprandial changes in monocyte activation markers were observed on nonclassical monocytes. A high-fat meal resulted in upregulation of CD11c as well as TNF-α and IL-1β expression on nonclassical monocytes. Indeed, CD16+ monocytes are known to be major producers of proinflammatory cytokines (20), and postprandial changes in TNF-α and IL-1β levels primarily reflects stimulation in intermediate and nonclassical monocyte subsets.
Our findings suggest higher baseline inflammation and exacerbated postprandial monocyte activation in hypertriglyceridemic individuals with MS. Activated monocytes with higher inflammatory and adhesion properties may lead to increased adhesion and transendothelial migration, contributing to progression of atherosclerosis and obesity-associated metabolic abnormalities.
Acknowledgments
The authors thank Lu Xu and Xiao-Yuan Dai Perrard (Baylor College of Medicine) for technical assistance, Kerrie Jara (Baylor College of Medicine) for editorial assistance, Margaret L. Jackson (Baylor College of Medicine) for help with sample collection, Jacob Couturier (Department of Internal Medicine, UT Health, Houston, Texas) for help with the flow cytometry, and Leif Peterson (Houston Methodist Research Institute) for help with statistical analysis. The authors also thank the human study participants.
This work was supported by National Institutes of Health grants T32 GM88129 and T32 HL007812 (to I.K.), R01 HL098839 (to H.W.), P30 AI36211 (to D.E.L.), and R01 DK078847 (to C.M.B.) and an American Heart Association award AHA16GRNT30410012 (to H.W.)
Disclosure Summary: The authors have nothing to disclose.
Footnotes
- BMI
- body mass index
- BP
- blood pressure
- HDL-C
- high-density lipoprotein cholesterol
- HLA
- human leukocyte antigen
- MFI
- median fluorescence assay
- MS
- metabolic syndrome
- TGRL
- triglyceride-rich lipoprotein
- TNF
- tumor-necrosis factor.
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