Ethics statement
All patients gave written informed consent, and study protocols were approved by the local ethics committees. The study of cohort I was approved by the Institutional Review Board of Dermatology and Hospital for Skin Diseases and the Ethics Committee of Suzhou Institute of Systems Medicine, Chinese Academy of Medical Sciences and Peking Union Medical College with the approval (2021) Linkuaishen (005) and (2023) Lunshen015, respectively. The study protocol for cohort II was approved by Marseille, Comité de Protection des Personnes, Sud Méditerranée II (identification 2014-A01302-45 and 2016-A00026-45; clinical trial identification: NCT02335996 and NCT02848703). All animal experimentation procedures applied to institutional rules and guidelines and were approved by the Gustave Roussy ethics committee (project numbers 2023_011_40501, 2023_053_44146 and 2024_040_50288).
Cell culture and reagents
Human neuroglioma H4 cells wild type (cat. no. HTB-148, ATCC) or stably expressing GFP–LC3 were cultured in Dulbecco’s modified Eagle medium (cat. no. 11995065, Thermo Fisher Scientific), supplemented with 10% (v/v) foetal bovine serum (cat. no. A5256701, Thermo Fisher Scientific), 100 U ml−1 penicillin and 100 μg ml−1 streptomycin (cat. no. 15140122, Thermo Fisher Scientific) at 37 °C in a humidified atmosphere with 5% CO2. Human hepatocellular carcinoma HepG2 cells wild type (cat. no. HB-8065, ATCC) or stably expressing GFP–LC3 were cultured in Eagle’s Minimum Essential Medium (cat. no. 30-2003, ATCC) supplemented as above. Plastic material was from Corning. Dialysed foetal bovine serum (cat. no. F0392) was from Thermo Fisher Scientific.
High-throughput screening
Two thousand H4 GFP–LC3 cells per well were seeded in 384-well µClear imaging plates (cat. no. 781091, Greiner Bio-One) 24 h before experimentation. Following compound treatment (Supplementary Table 1), cells were fixed with 4% paraformaldehyde containing 10 mM Hoechst 33342 (cat. no. H3570, Thermo Fisher Scientific) in phosphate-buffered saline (PBS) for 30 min at room temperature (RT), permeabilized with 0.1% Triton X-100 for 10 min and blocked with 5% bovine serum albumin (BSA) in PBS for 1–2 h, followed by overnight incubation with anti-ACBP antibody (cat. no. sc-376853, Santa Cruz Biotechnology) at 4 °C. After 2× washing with PBS, cells were incubated with AlexaFluor-conjugated secondary antibody (cat. no. A-11004; Thermo Fisher Scientific) for 2 h at RT. Cells were washed 3× before acquisition using an ImageXpressMicroXL bioimager (Molecular Devices) equipped with a 20× PlanApo objective (Nikon). Subsequently, images were analysed with R software (http://www.r-project.org/) using EBImage (https://bioconductor.org/) and MetaxpR (https://github.com/asauvat/MetaxpR) packages. Viability assessment, data processing and statistical evaluation were conducted as described in ref. 50.
Human cohort
Ninety-five dermatology patients with skin disease were included in cohort I (median age 46.8 years, range 11–93 years) (Supplementary Table 3). Subjects in the treatment group (n = 56) received glucocorticoid therapy, and patients (n = 3) who developed resistance to synthetic glucocorticoids were excluded. For cohort II (Supplementary Table 4), patients with ACTH-dependent Cushing’s syndrome (21 with ACTH-dependent pituitary Cushing’s syndrome, that is, Cushing’s disease, and 3 with ectopic ACTH secretion due to a bronchial carcinoid) (median age 56.5 years, range 22–73 years) were prospectively recruited from 2014 to 2017 at Marseille University Hospital, France. The ‘active’ group consisted of 11 newly diagnosed patients. The ‘remission’ group consisted of 13 patients in remission for at least 2 years, but no more than 6 years, regardless of the treatment modality.
Animal experimentation
Six-week-old female and male C57BL/6J mice (Envigo) were group-housed and subjected to a 12 h light–dark cycle, under temperature-controlled specific pathogen-free (SPF) conditions with food and water ad libitum. Food intake per day per mouse was calculated by measuring chow weight semiweekly. Mice were kept for 1 week to acclimate upon arrival before commencing experiments. For ACBP/DBI autoimmunization, autoantibody production via active immunization was initiated by conjugating keyhole limpet haemocyanin (KLH, cat. no. 77649, Thermo Fisher Scientific) and mouse recombinant ACBP/DBI (KLH–ACBP) as described34. Wild-type 6-week-old female C57BL/6J mice were immunized via i.p. injections of 30, 30, 30 and 10 μg of KLH–ACBP, or KLH alone, emulsified (1:1) with Montanide ISA-51vg (cat. no. 36362/FL2R3, Seppic) on days 0, 7, 14 and 21, respectively. After 4 weeks, ACBP autoantibodies were assessed by subjecting plasma to immunoblotting against recombinant target protein. For further validation enzyme-linked immunosorbent assay (ELISA) was used to assess circulating ACBP levels. Starting from week 6, Cushing’s syndrome was induced by administration for 5 weeks of CORT (cat. no. 27840, Merck) dissolved in EtOH, at 100 μg ml−1 in drinking water. Final EtOH concentration was 0.66% (ref. 36). Water consumption was measured semiweekly, and CORT concentration was adjusted to maintain an average daily CORT exposure of approximately 500 μg per mouse. The control group received 0.66% vehicle in drinking water. C57BL/6 Gabrg2tm1Wul/J Gabra flox mice came from the Jackson Laboratory. All mice used for experimentation were female. C57BL/6J Acbpfl/fl with loxP sites flanking Acbp exon 2 was generated by Ozgene. To activate the CRE recombinase, tamoxifen was administered i.p. at a dosage of 75 mg kg−1 body weight per mouse per day for five consecutive days. Tamoxifen was diluted in 90% corn oil and 10% EtOH (v/v) at a concentration of 20 mg ml−1 and was agitated overnight at 37 °C. After tamoxifen administration, mice underwent a washout period of at least 1 week before the commencement of treatments. This knockout strategy was applied in combination with the expression of either a ubiquitous or hepatocyte-specific CRE recombinase (genotypes: UBC-cre-ERT2 or TTR-creTam, respectively). Following the procedure, mice were kept for at least a week before starting the treatment. For the neutralization of DBI by anti-ACBP mAb, experiments were conducted with 8-week-old female C57BL/6J mice. Passive immunization was performed by semiweekly i.p. injections of 5 mg kg−1 body weight anti-ACBP mAb (clone 7G4a, homemade) or isotype control (IgG2a, κ, cat. no. BE0085, Bio X Cell). For RES treatment, experiments were conducted with 8-week-old female C57BL/6J mice. RES (cat. no. HY-12216, MedChemExpress) was prepared in 10% dimethyl sulfoxide (DMSO), 40% PEG300, 5% Tween 80 and 45% drinking water (v/v), at a final concentration of 0.033 mg ml−1. For the T3 system, experiments were conducted with 8-week-old female C57BL/6J mice. The initial concentration of T3 (cat. no. T2877, Merck) was 3.3 μg ml−1and was adjusted to maintain an average daily exposure of approximately 10 μg per mouse. For citalopram treatment, experiments were conducted with 8-week-old female C57BL/6J mice. Citalopram (Seropram, Lundbeck) was administered at a concentration of 0.15 mg ml−1 (diluted in water). For all experiments described above, CORT was administered as described above. Water consumption, body weight and food intake were measured semiweekly. At the end of the fifth week, mice were killed, and tissues were collected and weighed.
Co-administration of DEX and mifepristone
Experiments were conducted with 8-week-old female C57BL/6J mice. DEX (cat. no. D4902, Merck) was diluted in 10% DMSO and 90% corn oil (v/v) and administered i.p. (5 mg kg−1 body weight). Mifepristone (cat. no. M8046, Merck) was diluted in drinking water containing 1% carboxymethyl cellulose (cat. no. 419281, Merck) with 0.20% Tween 80 (Cat. P4780, Merck) (v/v) and administered by oral gavage (120 mg kg−1 body weight). DEX was injected daily for 2 weeks. Mifepristone was administered from day 7 to day 14. For SAFit2 and fasting, experiments were conducted with 8-week-old female C57BL/6J mice. SAFit2 (cat. no. HY102080 MedChemExpress) was solubilized in vehicle (4% EtOH, 5% Tween80, and 5% PEG400 (v/v) in 0.9% saline (Veh-1)). CORT was dissolved in 100% EtOH, to a final EtOH concentration of 0.66% (Veh-2). Fasting was performed by removing food for 24 h. SAFit2 was injected i.p. at 40 mg kg−1per day, and CORT (500 µg per mouse) was given by oral gavage. Daily CORT exposure was adjusted to approximately 500 µg per mouse.
Pair feeding
C57BL/6J female mice were housed under standard conditions with a 12 h light–dark cycle and ad libitum access to water. Mice were randomly assigned to four treatment groups: vehicle, CORT, anti-ACBP/DBI antibody and CORT plus anti-ACBP/DBI antibody. To ensure controlled food intake, a pair-feeding protocol was implemented. Initially, baseline body weights and food consumption were measured over a 3-day period to establish average intake. The vehicle-treated group served as the control for food intake. The average daily food intake of the vehicle-treated group was calculated and used to determine the amount of food provided to the other groups. Mice in the CORT, anti-ACBP/DBI antibody and CORT plus anti-ACBP/DBI antibody groups were given the same amount of food consumed by the control group on the previous day. Food intake and body weights were recorded daily to ensure precise matching of food quantities across groups. Adjustments in food allocation were made on the basis of the control groups’ consumption. Daily CORT exposure was adjusted to approximately 500 µg per mouse.
Indirect calorimetry measurements
Indirect calorimetry was conducted using automated metabolic cages (Labmaster, TSE Systems GmbH), in which mice were individually housed for consecutive 7-day periods over 4 weeks. Each cage was equipped with bedding, and mice were provided unrestricted access to food and water. Food and water consumption was continuously monitored. Measurements included oxygen (O2) consumption, carbon dioxide (CO2) production, the respiratory exchange rate (RER = VCO2/VO2) and heat production. Locomotor activity, including ambulatory and fine movements as well as speed, was tracked using an infrared light beam-based system. O2 and CO2 volumes were assessed at the inlet ports of each cage and periodically calibrated against a reference empty cage. All measurements were conducted at 4-min intervals throughout the experiment, ensuring continuous recording during both light and dark phases.
Forced swim test
For the forced swim test (FST), mice were placed in a vertical glass cylinder filled with water and behaviour was observed for 5 min. Water temperature was maintained at 25 °C. Distinct phases of active swimming and immobility were documented. The time spent immobile during the test was considered an indicator of behavioural despair. Conversely, less time spent immobile suggested potential antidepressant effects.
Face angle assessment
Mice were anaesthetized and placed on a scaled matrix with a protractor. Bird’s-eye-view images were taken and then analysed to measure the angle between the edges of the two cheeks considering the tip of the nose as the vertex.
Immunoblot
For protein extraction, cells were washed twice with with PBS and collected in radioimmunoprecipitation assay buffer (cat. no. 89901, Thermo Fisher Scientific), subjected to ultrasonication for three pulses of 10 s on ice and then centrifuged for 10 min at 13,000g. Analogously 30 μg liver tissues and 60 μg adipose tissue were collected in Precellys lysing kits (cat. no. P000911-LYSK0-A, Bertin Technologies) with radioimmunoprecipitation assay buffer and protease/phosphatase inhibitors (cat. no. A32959, Thermo Fisher Scientific), followed by two cycles of homogenization for 20 s at 5,500 rpm using a Precellys homogenizer (Bertin Technologies). Then, samples were centrifuged at 13,000g for 30 min and supernatants were collected. The Bio-Rad BCA assay (DC Protein Assay Kit II, cat. no. 5000112, Bio-Rad) was used for protein concentration assessment. Loading buffer and reducing agent (cat. nos. NP0008 and NP0009, Thermo Fisher Scientific) were added before denaturation (100 °C for 15 min). After SDS–PAGE and electrotransfer to polyvinylidene fluoride membranes, unspecific binding sites were blocked for 1–2 h with 5% BSA at RT, followed by overnight incubation at 4 °C with primary anti-human ACBP/DBI antibody (1:500; cat. no. sc-376853, Santa Cruz Biotechnology), anti-mouse ACBP/DBI antibody (1:1,000; cat. no. ab231910, Abcam), anti-LC3B antibody (1:1,000; cat. no. ab192890, Abcam), anti-SQSTM1/p62 antibody (1:1,000; cat. no. ab109012, Abcam), anti-glucocorticoid receptor (D6H2L) XP antibody (1:1,000; cat. no. 12041, Cell Signaling Technology) or horseradish peroxidase (HRP)-coupled anti-β-actin antibody (1:2,000; cat. no. ab49900, Abcam). Membranes were washed and processed by incubation with HRP-coupled secondary antibody (1:2,000; cat. no. 4050-05 goat anti-rabbit IgG(H + L), mouse/human ads-HRP and cat. no. 1031-05 goat anti-mouse IgG(H + L), human ads-HRP, SouthernBiotech) for 1 h at RT. Imaging and quantification were conducted by using an ImageQuant LAS4000 and ImageJ software, respectively.
Mouse and human ACBP/DBI ELISA
Cells were treated and culture supernatants were collected, centrifuged at 500g for 5 min and stored at −80 °C until analysis. For in vivo experiments, mouse plasma was collected with lithium heparin separator (cat. no. 450535, Greiner Bio-One), then centrifuged at 1,500g for 10 min, and ACBP/DBI levels were measured by ELISA. Human anti-ACBP/DBI (cat. no. MBS768488, MyBioSource) and murine anti-ACBP/DBI capture antibodies (cat. no. ab231910, Abcam) diluted 1 µg ml−1 in PBS were used for coating high-binding 96-well plates (Corning) with 100 μl per well overnight at 4 °C. Subsequently, plates were washed twice with washing solution (0.05% Tween 20 (v/v) in PBS), and unspecific binding was blocked with 100 μl sterile blocking buffer (1% BSA and 0.05% Tween 20 (v/v) in PBS) for 2 h at RT. For sample assessment, 100 μl per well of either sample or standard (human serum at 1:50–1:75, murine plasma at 1:20 and cell culture supernatant at 1:4 dilution, with the flexibility to adjust as dictated by experimental requirements) was incubated for 2 h at RT and subsequently rinsed 3× with washing buffer. Then 100 μl per well human anti-ACBP/DBI (LS-C299614, Lifespan Biosciences) and murine anti-ACBP/DBI detection antibodies (cat. no. MBS2005521, MyBioSource) diluted 1 µg ml−1 in PBS were added for 1 h at RT followed by 3× rinsing with washing buffer. Subsequently, plates were incubated 30 min at RT with 100 μl of HRP-coupled avidin diluted in PBS (1/5,000 for human and 1/1,000 for murine samples). Subsequently plates were rinsed 4× with washing buffer. To visualize bound protein, 100 μl of 1-Step Ultra TMB-ELISA substrate solution (cat. no. 34029, Thermo Fisher Scientific) was added and incubated 10–30 min at RT in the dark. Then, 50 μl of stop solution (2 N H2SO4) was added and absorbance was measured at 450 nm using a FLUOstar OPTIMA microplate reader.
RNA interference
Small interfering RNAs (siRNAs) were purchased from Horizon Discovery (ON-TARGETplus, GE Healthcare Dharmacon) and used following the manufacturer’s protocol and employing the following siRNA oligos:
siCtr, 5′-UAGCGACUAAACACAUCAA-3′;
siNR3C1-#1, 5′-GAACUUCCCUGGUCGAACA-3′;
siNR3C1-#2, 5′-GCAUGUACGACCAAUGUAA-3′;
siATG5, 5′-GGCAUUAUCCAAUUGGUUU-3′;
siATG7, 5′-CCAACACACUCGAGUCUUU-3′;
siTHRA, 5′-GUAUAUCCCUAGUUACCUG-3′;
siTHRB, 5′-GGACAAGCACCAAUAGUCA-3′.
Biochemical assays
ELISA kits were used for detecting biochemical indices, such as a mouse ALT ELISA kit (cat. no. ab282882, Abcam), mouse AST ELISA kit (cat. no. ab263882, Abcam), mouse insulin ELISA kit (cat. no. 10-1247-01, Mercodia), TG assay kit (cat. no. ab65336, Abcam), FFA assay kit (cat. no. ab65341, Abcam) and plasma CORT ELISA kit (cat. no. ab108821, Abcam). Plasma CORT samples were collected during the first hour of light at 8:00, and the collection process was performed under general anaesthesia using isoflurane inhalation. All procedures strictly followed the manufacturer’s protocol. For Luminex multiplex assays, plasma was collected in an EDTA anti-coagulant collecting tube with additional dipeptidyl peptidase IV inhibitor, protease inhibitor cocktail, aprotinin and serine protease inhibitor (Merck), then centrifuged for 10 min at 1,000g within 30 min of collection, aliquoted and stored at −80 °C. Mouse hormones were detected by using the mouse metabolic hormone magnetic bead panel (cat. no. MMHMAG-44K, Merck) and adiponectin single kit (cat. no. MADPNMAG-70K-01, Merck) by Luminex following the manufacturer’s protocol.
GTT and ITT
Mice were trained for tail pinch adaptation 1 week in advance. Animals were fasted for 6 h to perform a GTT. Blood for glycaemia measurement was collected from tail vein incisions 0, 15, 30, 60, 90 and 120 min after the injection of d-glucose (2 g kg−1 body weight, i.p.; cat. no. D3179, Merck). For ITT, animals were fasted 2–4 h before injection of insulin (NovoRapid, 0.5 U kg−1 body weight, i.p.). Blood was collected from tail cuts at 0, 15, 30, 60, 90 and 120 min, and glucose was measured using a precision glucometer (Accu-Chek Performa). Mice were monitored frequently, and hypoglycaemic shock was avoided by administration of 20% glucose solution. The HOMA-IR is calculated using the following formula: fasting plasma glucose (measured after 16 h of fasting, in millimolar) multiplied by fasting plasma insulin (measured after 16 h of fasting, in microunits per litre), divided by 22.5.
Histopathology
Fresh tissue was collected and fixed in 4% paraformaldehyde (or 10% formalin) for a maximum of 24 h at RT, then processed by serial paraffin embedment. Ten-micrometre-thick slices were obtained with a microtome. Standard haematoxylin–eosin staining was performed, and slides were scanned by a semi-automatic slide scanner (Nanozoomer 2.0 HT, Hamamatsu) equipped with 20× and 40× objectives. The images were analysed and quantified using Fiji software.
Gene expression analyses
For RNA extraction, the RNeasy Plus Mini kit (cat. no. 74134, QIAGEN) was used. About 25–30 mg of tissue was collected in lysis buffer (Buffer RLT Plus). The tissue was homogenized in two cycles using a Precellys homogenizer (Bertin Technologies) for 20 s at 5,500 rpm. The lysate was centrifuged and subjected to further purification procedures as described by the manufacturer. About 1 μg total RNA was reversed transcribed using the Maxima First Strand cDNA Synthesis Kit (cat. no. K1642, Thermo Fisher Scientific). Quantitative real-time PCR (qRT–PCR) was conducted by using PowerUp SYBR Green Master Mix (cat. no. A25776, Thermo Fisher Scientific) with a StepOnePlus Real-Time PCR System (Applied Biosystems, Thermo Fisher Scientific). The 2−ΔΔCT method was used for the analysis of real-time PCR data with the following primers (Eurofins Scientific):
ACBP/DBI forward primer: CAGAGGAGGTTAGGCACCTTA;
ACBP/DBI reverse primer: TATGTCGCCCACAGTTGCTTG;
GAPDH forward primer: TGTGGGCATCAATGGATTTGG;
GAPDH reverse primer: ACACCATGTATTCCGGGTCAAT;
Acbp/Dbi forward primer: GCTTTCGGCATCCGTATCAC;
Acbp/Dbi reverse primer: ACATCGCCCACAGTAGCTTG;
Gapdh forward primer: CGACTTCAACAGCAACTCCCACTCTTCC;
Gapdh reverse primer: TGGGTGGTCCAGGGTTTCTTACTCCTT.
Bulk RNA sequencing
RNA was isolated from murine liver tissue according to the manufacturer’s protocol (cat. no. 217004, miRNeasy mini kit, QIAGEN). Sequencing was conducted on a NovaSeq 6000 PE150 instrument, yielding paired-end reads of 2 × 150 base pairs, with a total of 40 million reads per sample (Novogene). Alignment and mapping to the GRCm39 (mm39) genome assembly were accomplished using HISAT2 (version 2.2.1). The resulting SAM file was processed by HTSeq-count (version 2.0.2) utilizing the union mode and including non-unique features for the generation of gene count tables. Software and websites used for analysis were RStudio (version 4.3.1), Cytoscape and https://string-db.org/.
Differential gene expression analysis
For gene expression comparison, volcano plots using the Enhanced Volcano R package, Venn diagrams using the Venn R package and heatmaps using the Complex Heatmap R package were generated. Differentially expressed genes (P ≤ 0.05 and |log2(fold change)| ≥1.0) were selected and further assessed by functional enrichment analysis, using various R packages, including clusterProfiler (v4.8.251), tidyverse, ggplot, forcats, biomaRt, stringr and org.Mm.eg.db. The gene background was defined using all sequenced genes.
Liver sample preparation and metabolite analysis
Approximately 30 mg of liver sample was homogenized as previously described52. For the extraction of endogenous metabolites, samples were mixed with 1 ml of ice-cold 90% methanol, 10% water (v/v) at −20 °C, along with a cocktail of internal standards and homogenized using a Precellys tissue homogenizer (Bertin Technologies), applying 3 cycles of 20 s at 6,500 rpm. After centrifugation (10 min at 15,000g, 4 °C), supernatants were divided into fractions and processed following established protocols. One fraction was used for short-chain fatty acid analysis (derivatization before injection). Another fraction was allocated to liquid chromatography–mass spectrometry (MS) analysis, while the third fraction was used for gas chromatography (GC)–MS analysis. Then, the analysis fractions were collected, dried (Techne DB3) at 40 °C and subsequently kept at −80 °C. Widely targeted analysis was conducted using a 7890A GC system (Agilent Technologies) coupled to a QQQ 7000C triple quadrupole mass spectrometer (Agilent Technologies) for GC–MS/MS. For the analysis of polyamines, short-chain fatty acids and bile acids, liquid chromatography-MS/MS was used utilizing a 1290 UHPLC system (Agilent Technologies) coupled to a QQQ 6470 triple quadrupole mass spectrometer (Agilent Technologies). Furthermore, a pseudo-targeted analysis was performed using an ultra-high performance liquid chromatography-high-resolution mass spectrometry system (UHPLC-HRMS), using a Dionex U3000 system coupled with an Orbitrap q-Exactive mass spectrometer (Thermo Fisher Scientific). All data were processed with the GRMeta in R (version 4.0) package (https://github.com/Kroemerlab/GRMeta). Data analysis and visualization were performed using AreaQCorrLog2Cen in R (version 4.2.1).
Data analysis
Unless otherwise specified, data are presented as mean ± SEM Before conducting statistical analysis, normal distribution of the results was assessed using the D’Agostino and Pearson normality test, Shapiro–Wilk normality test and Kolmogorov–Smirnov test. For data that exhibited a Gaussian distribution, unpaired two-tailed Student’s t-test or one-way analysis of variance (ANOVA) or two-way ANOVA was used. In the case of data with non-Gaussian distributions, the Mann–Whitney U test was used for two-group comparisons, while the Kruskal–Wallis test followed by Dunn’s post hoc test was used for comparisons involving multiple groups. Body weight curves and food intake were longitudinally analysed with type II ANOVA and pairwise comparisons using the TumGrowth application (https://kroemerlab.shinyapps.io/TumGrowth/). All other statistical analyses were performed using GraphPad Prism 9 or R software.
Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.