NAFLD is related to Post‐prandial Triglyceride‐enrichment of HDL Particles in Association with Endothelial and HDL Dysfunction

Abstract NAFLD is closely related with the metabolic syndrome (MetS) and increased risk of cardiovascular disease. Liver fat associates with post‐prandial hypertriglyceridemia, potentially contributing to triglyceride‐enrichment of high‐density lipoproteins (HDL‐TG), and subsequent HDL dysfunction. We assessed liver fat by MR spectroscopy, and its association with HDL physiochemical properties, and endothelial function, measured as flow‐mediated dilation (FMD), before and following three consecutive meals, in 36 men with type 2 diabetes mellitus (T2DM), with the MetS, and controls. Plasma triglycerides increased significantly following the meals (P < .001). Fasting HDL‐TG was highest in T2DM, relative to MetS and controls (P = .002), and increased post‐prandially in all groups (P < .001). HDL function was negatively associated with HDL‐TG following three meals (r = −.32, P<.05). Liver fat associated with HDL‐TG after three meals (r = .65, P < .001). HDL‐TG was independently associated with FMD following three consecutive meals (r = −.477, P = .003). We conclude liver fat is associated with post‐prandial HDL‐TG enrichment which was closely related with endothelial and HDL dysfunction.

(CETP), 4 is enhanced, yielding (the formation of) large, triglyceride-rich, cholesterol ester-core-depleted HDL particles. 5,6 These HDL particles are the preferred substrate for the enzyme hepatic lipase, which hydrolyzes HDL triglycerides and promotes hepatic HDL uptake. Liver fat content has been shown to be associated with alterations of the anti-atherogenic HDL subfractions, but not suggest a direct causal link per se. 5,7 There is evidence that functionally defective HDL is a cause of increased cardiovascular risk in NAFLD patients. 8,9 The association of post-prandial triglyceride elevations and impaired vascular endothelial function has also been established. 10 Previous studies showed that triglyceride-enrichment of HDL may alter anti-atherogenic capacities of this lipoprotein class, including anti-oxidative and anti-inflammatory properties, adversely affecting the ability of HDL to protect the endothelium and vascular reactivity. 11,12 However, the independent association of liver fat content and post-prandial HDL compositional and functional changes has hitherto not been addressed.
In the present study, we first investigated whether liver fat content is associated with post-prandial altered HDL composition, especially triglyceride content, and its anti-oxidative function. 13 Second, we assessed the interrelationship of liver fat, physicochemical properties of HDL particles and endothelial function in vivo, measured as flow mediated dilatation (FMD), following three consecutive meals during a 16 hours period in males with T2DM, males with the MetS and healthy controls.

| Subjects
Caucasian males, aged 40-65 years, with T2DM (n = 12) or with the MetS (n = 12), and 12 age-matched healthy males were recruited by advertisement and studied after obtaining written informed consent. Diet, sulphonylurea and/or metformin were the only glucose-lowering treatments allowed in the type 2 diabetic group. To disentangle the possible role of hyperglycaemia, males with the MetS had to meet three of five inclusion criteria based on NCEP/ATP III criteria, without having hyperglycaemia during a 75-g oral glucose tolerance test. Healthy control males were overweight (BMI >25 kg/m 2 ) without any other components of the MetS. Claustrophobia, excess alcohol intake (>20 units/wk), history of hepatitis and/or pancreatitis, abnormal liver and renal function tests (>2 times upper limits of normal), recent (<3 months) changes in weight (≥5%) and/or medication, history or current use of glucocorticosteroids, lipid-lowering drugs (including statins and fibrates), insulin and/or thiazolidinediones, were exclusion criteria.
Participants were instructed to omit their medication during the examination and to refrain from heavy physical activities during the previous 24h. The local ethics committee approved the study and the investigation conformed to the principles outlined in the Declaration of Helsinki.

| Study design
After an overnight fast, participants were admitted in the research unit for a 16h period and received three consecutive, isocaloric

| HDL composition and function
HDL was isolated from EDTA plasma by ultracentrifugation for measurement of triglyceride, cholesterol and protein content. HDL triglyceride and HDL cholesterol content was expressed as nmol per gram protein (nmol/g). HDL-TG was calculated as the HDL triglyceride: total cholesterol ratio and expressed as nmol/nmol. and 530 nm (excitation and emission wavelength respectively). The intra-assay CV was 3.4%.

| Endothelial function
Before each blood collection, FMD was measured at the right brachial artery by a single observer using ultrasound (Wall-track System, PieMedical, Maastricht, The Netherlands), as previously described. 10 FMD refers to dilation of an artery following induced increased shear stress and release of nitric oxide by the endothelium, reflecting one of its functions.

| Liver fat content
Using a 1.5-T whole-body system MRI (Sonata; Siemens, Erlangen, Germany), liver fat content was measured after an overnight fast on a separate occasion within 2 weeks, at three positions in the liver and calculated by user-independent spectral quantification as previously described in detail. 14

| Statistical analysis
Results are presented as the mean ± SE or medians (interquartile range). Sixteen hour area under the curve (AUC 0-16h ) were calculated according to the trapezoid rule. Differences between groups were calculated using ANOVA and post-hoc analyses (Bonferroni).
Non-normally distributed data were log transformed. The associations of liver fat content, FMD and HDL cholesterol composition and function were assessed by univariate and multivariate linear regression analyses. A value of P < .05 was considered statistically significant.

| RE SULTS
The baseline characteristics of the T2DM, MetS and healthy groups are listed in Table 1. From the T2DM males, four were using diet only, six metformin only, one sulphonylurea only and one the combination as glucose-lowering therapy.  Values are means ± SE or median (interquartile range).

| D ISCUSS I ON
In the present study, we demonstrated that NAFLD is associated Our results suggest that NAFLD is related to exaggerated and prolonged post-prandial dysmetabolism, including HDL-TG enrichment that independently is associated with post-prandial endothelial dysfunction. We could only speculate whether this prolonged dysmetabolism is the result of an impaired clearance of TG-enriched HDL by the liver, or an increased TG enrichment of HDL by, for example, elevated CETP levels.
A limitation of the present study is that we isolated and examined the total class of HDL instead of the HDL 2 and HDL 3 subclasses.

| CON CLUS IONS
In men with T2DM and the MetS exposure to three consecutive meals produces exaggerated HDL-TG enrichment, which was closely associated with liver fat content, and HDL and endothelial dysfunction. Our findings may link liver fat accumulation and post-prandial dysmetabolism to the high CVD risk present in T2DM and the MetS.
Future studies should elucidate whether liver fat regression by therapeutic intervention may lead to an improvement of CVD risk.

CO N FLI C T O F I NTE R E S T S
All authors have no competing financial and non-financial interests.

AUTH O R CO NTR I B UTI O N S
MET, PGS and MD were involved in study concept and design, acquisition of data, analysis and interpretation of data, critical revision of the manuscript for important intellectual content and statistical analysis. BV and PJP were involved in drafting of the manuscript, critical revision of the manuscript for important intellectual content F I G U R E 1 The 16-h course of plasma glucose (A), triglyceride (B), apoB (C), HDL cholesterol (D), HDL-TG concentrations (E), and flowmediated dilatation (FMD)(F) after three high-fat mixed meals in T2DM (open circles), MetS (asterisk) and healthy males (solid circles). Bars (black, healthy males; grey, metabolic syndrome (MetS); white, type 2 diabetes mellitus (T2DM)) in the insets represent respective 16-h AUC values. The P value given for 16-h AUC difference (ANOVA). Data are mean ± SE. Scatter plots representing the relationship between liver fat content (G) and FMD (H), and HDL-TG enrichment following three high-fat mixed meals in the whole study population (groups are indicated by open circles (T2DM), asterisk (MetS) and solid circles (healthy males)). Pearson correlation coefficients are shown and statistical analysis. RPV was involved in drafting of the manuscript, and administrative and technical support.