In vitro experiments
Table of Contents
biochemical binding assay
Biochemical binding of TLC-2716 and TLC-6665 to LXRα or LXRβ was determined by time-resolved fluorescence resonance energy transfer-based biochemical assays evaluating the concentration-dependent displacement of a N-terminally biotinylated coactivator NCOA3 to recombinant glutathione S-transferase (GST)-tagged LXRα or LXRβ ligand-binding domain (LBD). Briefly, different concentrations of TLC-2716 or TLC-6665 were incubated with an assay buffer (Tris/HCl buffer (pH 6.8) containing 240 mM KCl,1 µg µl−1 bovine serum albumin and 0.002% Triton X-100) containing 530 µg ml−1 recombinant GST-tagged LXRα/LXRβ LBD, 125 pg µl−1 GST-Tb cryptate (CisBio), 400 nM N-terminally biotinylated NCOA3 (coactivator) peptide, 1 μM 24(S)-25-epoxycholesterol (LXR agonist, only in the NCOA3 assay) and 2.5 ng µl−1 Streptavidin-XL665 (CisBio) for 1 h at 4 °C, following which fluorescence was measured in a VictorX4 multiplate reader (PerkinElmer Life Science) using 340 nm as excitation and 615 and 665 nm as emission wavelengths. Assays were performed in triplicate.
LXR GAL4 reporter transient transfection assays
LXRα and LXRβ activity status was determined via detection of interaction with coactivator and co-repressor proteins in mammalian two-hybrid experiments. HEK293 cells were transiently transfected with full length proteins of LXRα or LXRβ and their respective LBD domains under the pCMV-AD promoter(Stratagene). The cofactors, either the coactivator SRC1 or the co-repressor NCoR, were expressed as fusions to the DNA binding domain of the yeast transcription factor GAL4. Interaction was monitored via activation of a coexpressed Firefly luciferase reporter gene under control of a promoter containing repetitive GAL4 response elements (vector pFRLuc, Stratagene). Four hours after transfection, cells were incubated with a concentration range of TLC-2716 and TLC-6665 for 16 h, after which they were lysed, and luciferase activities were measured sequentially in the same cell extract using a BMG luminometer.
ABCA1 and SREBP1c luciferase reporter assays
HT-29 or HepG2 cells were stably transfected with pGL4 luciferase reporter plasmid containing the ABCA1 or SREBP1C, respectively, promoter region upstream of the Firefly luciferase reporter gene and incubated with a concentration range of TLC-2716 or TLC-6665 for 16 h as described above.
Human primary hepatocyte culture
Human primary Upcyte hepatocytes were cultivated in medium containing high concentrations of glucose (25 mM) and palmitate (100 nM) for 5 days in the presence or absence of TLC-2716, and intracellular lipids
Liver tissue was dipped into ice-cold PBS for 1 min and then fixed in 4% paraformaldehyde in PBS overnight for a maximum of 16 h. Afterward, samples were transferred to cold 70% ethanol/PBS to avoid prolonged fixation and embedded in paraffin before sectioning. Sections were stained with picrosirius red to visualize collagen.
Plasma samples were analyzed for levels of alanine aminotransferase, aspartate aminotransferase, cholesterol and triglycerides using respons 910 (Diasys Diagnostics Systems), as per the manufacturerS instructions.
Collected frozen liver samples were ground to a fine powder with a pestle and mortar under liquid nitrogen. An aliquot was subjected to lipid isolation using organic solvents (hexane and isopropanol), and, after extraction, triglyceride and cholesterol levels were quantified using commercially available kits (FUJIFILM Wako Chemicals Europe).An additional aliquot was used for collagen determination after acid hydrolysis (Total Collagen Assay, Quickzyme Biosciences).
The study was performed at physiogenex S.A.S. in accordance with ethical regulations, guide for the Care and Use of Laboratory Animals (revised 1996 and 2011, 2010/63/EU) and French laws.
Twenty-four-week-old DIO mice (Jackson Laboratories) were fed a HFD (Research Diets, D12492) for 18 weeks and dosed with vehicle (5% DMSO and 0.5% hydroxypropyl methylcellulose in phosphate-buffered saline), TLC-6665 (5 mg per kg (body weight)) or pioglitazone (30 mg per kg (body weight)) once daily by oral gavage for 4 weeks. Animals were subjected to a two-stage hyperinsulinemic-euglycemic clamp procedure after a 6-h fast and 2 h after the last dose. Briefly, animals received a bolus of D-[3-[3-[3-[3-3H]glucose (30 μCi per mouse) followed by D-[3-[3-[3-[3-3H]glucose (30 μci min−1 kg−1) infusion (2 μl min−1) for up to 210 min. Insulin was simultaneously infused at 8 mU kg−1 min−1 for the first 100 min and at 18 mU kg−1 min−1 for the last 110 min. Blood glucose was measured from the tip of the tail every 10 min by a glucometer. The glucose infusion rate was adjusted according to blood glucose levels until a first euglycemic steady state was reached (from ~70 to 100 min of infusion) and similarly adjusted until a second euglycemic steady-state was reached (from ~150 to 210 min of infusion). During both steady states, blood (5 μl) was collected frequently from the tail tip for 3H-radioactivity measurements and calculations of glucose flux.
A 26-week Good Laboratory Practice (GLP) toxicology study was conducted at Inotiv according to the protocol,
To initiate organoid formation, foregut cells were resuspended in Matrigel at a final concentration of 750,000 cells per ml. Fifty-microliter droplets of the cell-Matrigel mixture were plated and cultured in Advanced DMEM/F12 (Gibco) supplemented with B27, N2, 10 mM HEPES, 1% GlutaMAX, 1% penicillin-streptomycin, 5 ng ml−1 FGF2, 10 ng ml−1 VEGF, 20 ng ml−1 EGF, 3 μM CHIR99021, 0.5 μM A83-01 and 50 µg ml−1 ascorbic acid for 4 days. The medium was then replaced with the same basal formulation containing 2 μM retinoic acid and cultured for an additional 4 days.the organoids were maintained in hepatocyte culture medium (Lonza) for 6 days to promote hepatic maturation. To induce a steatohepatitis-like phenotype, HLOs were gently retrieved from the Matrigel and washed with PBS. HLOs were then exposed to 300 μM sodium oleate (Sigma) in hepatocyte culture medium for 3 days using ultra-low attachment six-well plates (Corning). For quantification of intracellular lipid accumulation, sHLOs were rinsed three times with prewarmed PBS and stained with 2 μM BODIPY 493/503 (Thermo Fisher Scientific) for neutral lipid detection, along with NucBlue Live readyprobes (Thermo Fisher Scientific) for nuclear counterstaining. Fluorescent images were acquired using a Keyence BZ-X710 automated fluorescence microscope. Lipid droplet volume was quantified using the Hybrid Cell Count request (Keyence) and normalized to nuclear signal intensity. For RNA sequencing, total RNA was extracted from iPS cell-derived HLOs using an RNeasy Plus Mini kit (Qiagen) following the manufacturer’s protocol.RNA quality were assessed using a fragment Analyzer (Advanced Analytical), and only samples with an RNA Quality Number of ≥8.0 were used for library preparation.
genotypes of GCKR variants were determined using an Illumina Infinium Global Diversity Array with the Enhanced PGx platform (Illumina) according to the manufacturer’s instructions. Genomic DNA was extracted from undifferentiated iPS cells using a QIAamp DNA Mini kit (Qiagen).Genotyping was performed at Cincinnati Children’s Hospital Medical Center Genomics Sequencing Facility using standard Infinium HTS assay protocols. Quality control steps included removal of samples with a call rate of <98%, excess heterozygosity or sex mismatch. Single-nucleotide polymorphisms (SNPs) with a call rate of <95%, Hardy-Weinberg equilibrium P of <1 × 10−6 or minor allele frequency of <1% were excluded from downstream analysis. Genomic positions were mapped to the GRCh38 (hg38) human reference genome.
mendelian randomization analysis
Critically important cis-eQTLs of LXRα (NR1H3) and LXRβ (NR1H2) in liver or blood were obtained from the GTEx (version 8, 208 individuals)36 or eQTLGen (31,684 individuals)65. Linkage disequilibrium clumping for SNPs was estimated using Plink (genetics.binaRies R package, window size 1 Mb, minor allele frequency ≥ 1%) on the 1000 Genomes reference panel66.Only one autonomous SNP was found within NR1H3 using the GTEx liver cis-eQTLs, whereas eight independent SNPs were identified using the eQTLGen blood cis-eQTLs, suggesting that the eQTLGen blood cis-eQTLs are more reliable for Mendelian randomization analysis. therefore, significant cis-eQTLs of LXR in blood extracted from eQTLGen were applied as exposures in the Mendelian randomization analysis. GWAS summary statistics for plasma lipid-related traits were derived from WES data in the UKBB and served as outcomes. The causal effects of NR1H3 and NR1H2 gene expression on lipid traits were estimated by Inverse Variance Weighted approach using the TwoSampleMR67,RNA-sequencing analysis
RNA was sequenced by BGI with the DNBSEQ platform. The quality of the reads was then verified using FastQC (version 0.11.9). Low-quality reads were removed,and no trimming was needed. Alignment was performed against the human genome (GRCh38, v113) following the STAR (version 2.73a) manual guidelines73. Normalized effective library sizes were calculated by trimmed meen of M values. The voom function of the Limma R package (version 3.60.0)74 was applied to transform gene counts for linear modeling with precision weights. The differential expression analysis for the transcriptome was performed using the R package Limma (version 3.60.0)74. Genes were ranked by log2 (fold change), and gene sets were extracted using the msigdbr R package (version 7.5.1)71. gene set enrichment analysis was performed using the clusterProfiler R package (version 4.12.6)Statistical Analysis Methods
The study employed non-parametric statistical tests to analyze data,specifically addressing potential issues with data distribution. Non-parametric tests are used when data does not meet the assumptions of parametric tests, such as normality. For analyzing changes from a baseline measurement, two statistical tests were used: the two-tailed Wilcoxon signed-rank test for comparisons within groups, and the two-tailed Mann-Whitney U-test for comparisons between groups. These tests do not assume a normal distribution of the data. To control for the risk of false positives due to multiple comparisons, the results were adjusted using the Benjamini-Hochberg (BH) procedure. The BH procedure is a method for controlling the false finding rate (FDR) in multiple hypothesis testing. National Center for Biotechnology Information provides detailed information on the BH procedure. The choice of non-parametric tests, like the Wilcoxon signed-rank and Mann-Whitney U tests, directly addresses the potential impact of skewed data or outliers. These tests are less sensitive to extreme values compared to parametric tests like the t-test or ANOVA.Skewed data violates the assumption of normality required for parametric tests, and outliers can disproportionately influence the results of those tests. such as,if a small number of participants experienced a very large betterment in a symptom,this could skew the mean and standard deviation,potentially leading to inaccurate conclusions if a parametric test were used. Non-parametric tests focus on ranks rather than absolute values, mitigating the influence of outliers. The study acknowledged a limitation related to sample size and the consideration of sex as a variable. Due to the small sample size within each group, the researchers did not include sex in the analysis. Small sample sizes reduce the statistical power of a study, making it harder to detect true effects and increasing the risk of Type II errors (false negatives). Self-reported sex was used in the study.The Centers for Disease Control and Prevention (CDC) defines sex as biological characteristics,while gender is a social construct. The study’s reliance on self-reported data acknowledges the complexities of these concepts. Additional details regarding the research design are available in the Nature Portfolio Reporting Summary, providing transparency and reproducibility of the study methods.Handling of Skewed Data and Outliers
Limitations: Sample Size and Sex as a Variable
Reporting Summary
