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Background: Determination of lipoprotein lipase (LPL) activity is important for hyperchylomicronemia diagnosis, but remains both unreliable and cumbersome with current methods. Consequently by using human VLDL as substrate we developed a new LPL assay which does not require sonication, radioactive or fluorescent particles., Methods: Post-heparin plasma was added to the VLDL substrate prepared by ultracentrifugation of heat inactivated normolipidemic human serums, diluted in buffer, pH 8.15. Following incubation at 37°c, the NEFA (non esterified fatty acids) produced were assayed hourly for 4 hours. LPL activity was expressed as µmol/l/min after subtraction of hepatic lipase (HL) activity, obtained following LPL inhibition with NaCl 1.5 mmol/l. Molecular analysis of LPL, GPIHBP1, APOA5, APOC2, APOE genes was available for 62 patients., Results: Our method was reproducible (coefficient of variation (CV): intra-assay 5.6%, inter-assay 7.1%), and tightly correlated with the conventional radiolabelled triolein emulsion method (n = 26, r = 0.88). Normal values were established at 34.8 ± 12.8 µmol/l/min (mean ± SD) from 20 control subjects. LPL activities obtained from 71 patients with documented history of major hypertriglyceridemia showed a trimodal distribution. Among the 11 patients with a very low LPL activity (< 10 µmol/l/min), 5 were homozygous or compound heterozygous for LPL or GPIHBP1 deleterious mutations, 3 were compound heterozygous for APOA5 deleterious mutations and the p.S19W APOA5 susceptibility variant, and 2 were free of any mutations in the usual candidate genes. No homozygous gene alteration in LPL, GPIHBP1 and APOC2 genes was found in any of the patients with LPL activity > 10 µmol/l/min., Conclusion: This new reproducible method is a valuable tool for routine diagnosis and reliably identifies LPL activity defects.

Apolipoprotein CII (apoCII) is a specific activator of lipoprotein lipase and plays an important role in triglyceride metabolism. The aim of our work was to elucidate the regulatory mechanisms involved in apoCII gene modulation in macrophages. Using Chromosome Conformation Capture we demonstrated that multienhancer 2 (ME.2) physically interacts with the apoCII promoter and this interaction facilitates the transcriptional enhancement of the apoCII promoter by the transcription factors bound on ME.2. We revealed that the transcription factor STAT1, previously shown to bind to its specific site on ME.2, is functional for apoCII gene upregulation. We found that siRNA-mediated inhibition of STAT1 gene expression significantly decreased the apoCII levels, while STAT1 overexpression in RAW 264.7 macrophages increased apoCII gene expression. Using transient transfections, DNA pull down and chromatin immunoprecipitation assays, we revealed a novel STAT1 binding site in the -500/-493 region of the apoCII promoter, which mediates apoCII promoter upregulation by STAT1. Interestingly, STAT1 could not exert its upregulatory effect when the RXRα/T3Rβ binding site located on the apoCII promoter was mutated, suggesting physical and functional interactions between these factors. Using GST pull-down and co-immunoprecipitation assays, we demonstrated that STAT1 physically interacts with RXRα. Taken together, these data revealed that STAT1 bound on ME.2 cooperates with RXRα located on apoCII promoter and upregulates apoCII expression only in macrophages, due to the specificity of the long-range interactions between the proximal and distal regulatory elements. Moreover, we showed for the first time that STAT1 and RXRα physically interact to exert their regulatory function.

Aside from a decrease in the high-density lipoprotein (HDL) cholesterol levels, qualitative abnormalities of HDL can contribute to an increase in cardiovascular (CV) risk in end-stage renal disease (ESRD) patients undergoing chronic hemodialysis (HD). Dysfunctional HDL leads to an alteration of reverse cholesterol transport and the antioxidant and anti-inflammatory properties of HDL. In this study, a quantitative proteomics approach, based on iTRAQ labeling and nanoflow liquid chromatography mass spectrometry analysis, was used to generate detailed data on HDL-associated proteins. The HDL composition was compared between seven chronic HD patients and a pool of seven healthy controls. To confirm the proteomics results, specific biochemical assays were then performed in triplicate in the 14 samples as well as 46 sex-matched independent chronic HD patients and healthy volunteers. Of the 122 proteins identified in the HDL fraction, 40 were differentially expressed between the healthy volunteers and the HD patients. These proteins are involved in many HDL functions, including lipid metabolism, the acute inflammatory response, complement activation, the regulation of lipoprotein oxidation, and metal cation homeostasis. Among the identified proteins, apolipoprotein C-II and apolipoprotein C-III were significantly increased in the HDL fraction of HD patients whereas serotransferrin was decreased. In this study, we identified new markers of potential relevance to the pathways linked to HDL dysfunction in HD. Proteomic analysis of the HDL fraction provides an efficient method to identify new and uncharacterized candidate biomarkers of CV risk in HD patients.