Study design and participants
This single-center open-label study was carried out at the University Hospital of Dijon, France (Clinical trial reg. no. NCT02816099 ClinicalTrials.gov), and was approved by our regional ethics committee. The recruitments took place from June 2016 to October 2020, and written informed consent was obtained for all patients included in the study. We calculated that a sample size of 22 patients was necessary, considering a two-sided α error of 0.05, a β error of 0.2, an effect size of 0.8 and a standard deviation in CEC changes of 1.25%. We included 27 T1D patients with HbA1c > 75 mmol/mol (> 9%). Blood samples were withdrawn at inclusion and three months after a standard medical intervention to improve glycemic control (i.e. insulin introduction, pump therapy, therapeutic education). All subjects were older than 18 years and were not taking any treatment that could interfere with lipoprotein metabolism (lipid-lowering agents, anti-HIV agents, combined oral contraceptive pill, corticosteroids or retinoic acid). None were pregnant. Microvascular complications (retinopathy, nephropathy) were recorded. Three stages were used to define nephropathy: 0 for no nephropathy, 1 for microalbuminuria between 30 and 300 mg per day and 2 for proteinuria > 300 mg per day.
Blood was collected in the fasting state in BD Vacutainer tubes (Becton Dickinson, Franklin Lakes, USA) in dry tubes and in tubes with EDTA as anticoagulant and preservative. The serum was immediately used after centrifugation for routine lipid measurements. The plasma was immediately separated by centrifugation, and frozen at -80 °C until HDL isolation and subsequent analysis.
The HDL fraction (density = 1.063–1.210 g/mL) was isolated from 1.0 mL of plasma by sequential flotation ultracentrifugation at 4 °C using a 50.4 rotor in an Optima L80-XP ultracentrifuge (Beckman Coulter, Brea, CA, USA), as previously reported . Briefly, after ultracentrifugation at 33,000 rpm for 18 h (density adjusted at 1.063 g/mL with potassium bromide), the apolipoprotein (apo) B-containing fraction was withdrawn. Then, after a second ultracentrifugation at 40,600 rpm for 21 h (density = 1.210 g/mL), the HDL fraction was collected in 300 µL. The HDL fraction was extensively dialyzed three times against sterilized 0.01% (wt/vol.) EDTA endotoxin-free phosphate buffered saline (PBS) for 18 h at 4 °C in the dark, and then immediately used for analysis.
Routine analytical procedures
Total cholesterol, HDL-cholesterol, triglycerides, apolipoprotein AI (apoAI), total proteins, urea and creatinine were measured on a Dimension Vista analyzer using dedicated reagents (Siemens Healthcare Diagnostics, Deerfield, IL, USA). Free cholesterol and phospholipids were measured on the same analyzer but using reagents from Diasys (Condom, France). LDL cholesterolemia was estimated by the Friedewald equation, since triglyceridemia was below 3.88 mmol/L in each subject. Esterified cholesterol in each HDL fraction was calculated as the difference between total and free cholesterol. HbA1c was measured with a G8 HPLC Analyzer (Tosoh Bioscience, Tokyo, Japan). MPO was assessed by a commercial kit (Mercodia, Uppsala, Sweden) in plasma obtained from EDTA tubes. The estimated glomerular filtration rate (eGFR) was calculated with the Chronic Kidney Disease—Epidemiology Collaboration (CKD-EPI) equation.
Cholesterol efflux capacity (CEC)
The primary end point of this study was the change in CEC after the intervention to improve glycemic control. The capacity of HDLs to support cholesterol efflux was analyzed using the human monocyte cell line THP-1 (Sigma-Aldrich, Saint-Louis, MO, USA). THP-1 cells were maintained in RPMI 1640 complete medium (Gibco, New York, NY, USA) containing 4.5 g/L d-glucose, 2.383 g/L HEPES buffer, L-glutamine, 1.5 g/L sodium bicarbonate, 100 mg/L sodium pyruvate, and supplemented with 10% (vol./vol.) fetal bovine serum (Thermo-Scientific, Illkirch, France) and 1% (vol./vol.) penicillin–streptomycin (Thermo-Scientific). Cells were cultured in exponential phase at day 1. At day 2, cells were seeded for 48 h (37 °C, 5% CO2) into 96-well plates at density of 105 cells in 200 µL per well with 100 nmol/L phorbol 12-myristate-13-acetate in complete medium. At day 4, cells were charged for 1 h (37 °C, 5% CO2) with 10 nmol/L Bodipy-labelled cholesterol in ethyl acetate (TopFluor®, Sigma-Aldrich) in complete medium containing 2 µg/mL Sandoz 58–035 (Sigma-Aldrich) as acyl-CoA:cholesterol acyltransferase inhibitor, 20 mmol/L methyl-β-cyclodextrin (TCI, Paris, France) and 10 µmol/L TO901317 (Sigma-Aldrich) as liver X receptor (LXR) agonist to enhance the expression of ATP-binding cassette (ABC) transporters. After removal of medium, cells were incubated overnight in RPMI 1640 glutamax, 0.2% (vol./vol.) fatty acids free bovine serum albumin, 2 µg/mL Sandoz 58–035, 10 µmol/L TO901317. At day 5, HDL fractions (20 µg/mL apoAI) were incubated for 4 h (37 °C, 5% CO2) in 1X Opti-Klear™ live cell imaging buffer (Abcam, Cambridge, UK) containing 2 µg/mL Sandoz 58–035 and 10 µmol/L TO901317. After 4 h, 80 µL of supernatant were transferred into a black 96-well plate and fluorescence was read at 485/535 nm using a SPARK microplate reader platform (Tecan, Grödig, Austria). The remaining medium was withdrawn and cells were lysed by adding 100 µL/well of reporter lysis buffer (Promega, Madison, WI, USA). Eighty microliters of lysate were transferred into a black 96-well plate and fluorescence was read in the same conditions as the supernatant. Each fluorescence value in supernatant and in lysate was corrected by autofluorescence obtained in wells containing HDLs but without Bodipy cholesterol. Percent efflux was calculated by the following formula: [fluorescence in supernatant ÷ (fluorescence in supernatant + fluorescence in lysat)] × 100. All assays were performed in triplicate. All of the samples from one T1D patient (at baseline and post-intervention) were treated in the same runs to avoid bias due to inter-assay variations. To correct for inter-assay variations across plates, a plasma control from a healthy volunteer was included on each plate, and values for plasma samples from patients were normalized to this value in subsequent analyses.
Carbamylated HDL concentration
Carbamylated proteins were measured in isolated HDLs by a commercial sandwich ELISA (OxiSelect™, Cell Biolabs, San Diego, CA, USA) detecting carbamyl-lysine residues. Each sample was assayed in duplicate. The results were expressed as ng HDL carbamylated proteins / mg HDL total proteins.
In vitro carbamylation of HDLs
A pool of dialyzed HDLs isolated from healthy subjects (4 mg protein/mL) was incubated with 1 mmol/L and 10 mmol/L potassium cyanate (KCN) in PBS containing 100 μmol/L diethylenetriaminepentaacetic acid (Sigma-Aldrich) for 4 h at 37 °C, as previously described . Control HDLs were incubated under the same conditions with PBS instead of potassium cyanate. HDLs were then immediately and extensively dialyzed three times against sterilized 0.01% (wt/vol.) EDTA endotoxin-free PBS for 18 h at 4 °C in the dark, and then immediately used for analysis.
Data are reported as median [1st–3rd quartiles], otherwise indicated. Statistical calculations were performed using XLSTAT (version 2021.4.1). The primary end point was the change in CEC after the 3-month intervention. Skewness of each continuous variable was assessed using Pearson's first skewness coefficient, and values were log10 transformed before any statistical analysis to improve normality if necessary. The results from patients with T1D before and after the intervention were compared using the non-parametric Wilcoxon signed-rank test for paired samples. Univariate Spearman correlation coefficients were calculated to explore relationships between the changes in CEC after the intervention, and clinical and biological variables. A two-tailed probability level of 0.05 was considered as statistically significant.