Participant blood samples
We took samples from 25 individuals in the NAFLD group based on the diagnostic criteria of China from the Endocrine and Metabolism Department of Shanghai Tenth People’s Hospital. Individuals were recruited in this study based on the exclusion of patients diagnosed with other known chronic liver diseases, including chronic hepatitis B or C, autoimmune hepatitis, or who were excessive consumers of alcohol (> 140 g/week for men or > 70 g/week for women). Patients who had used any medication or alternative treatment that could affect liver steatosis or fibrosis and glucolipid metabolism within six months of this study were also excluded. Additionally, 25 normal controls matched for age, sex, and body mass index (BMI) were recruited from the Physical Examination Center of the Shanghai Tenth People’s Hospital. For each participant, measured height and body weight and assessed alanine aminotransferase (ALT), aspartate aminotransferase (AST), γ-transaminase, (γ-GT), total cholesterol (TC), triglyceride (TG), high-density lipoprotein (HDL), and low-density lipoprotein (LDL) levels. Serum FABP1 levels were measured using enzyme-linked immunosorbent assays (ELISA; Abcam, #ab218261). Every serum sample was replicated twice, and the average was used for analysis.
Human liver samples
From January 2021 to June 2022, five individuals with NAFLD and morbid obesity who underwent laparoscopic sleeve gastrectomy (LSG) at the Endocrine and Metabolism Department of Shanghai Tenth People’s Hospital were enrolled in this study. We examined liver biopsy samples from patients who may have had a biopsy before or during the LSG to stage and grade steatosis and steatohepatitis. Three adjacent normal tissues located 5 cm from benign lesions were resected as controls, and hepatitis, cirrhosis, drug-induced liver injury, and additional influencing factors were excluded. All individuals signed an informed consent form for the use of clinical specimens, and all procedures adhered to the principles of the Declaration of Helsinki.
Reagents, plasmid constructs, and antibodies
Antibodies against FABP1 were purchased from Proteintech (#13626-1-AP) and Santa Cruz Biotechnology (#sc-374537). Derlin-1 and ubiquitin (Ub) were purchased from Sigma-Aldrich (#D4443) and Cell Signaling Technology (#58395), respectively. Anti-α-tubulin, anti-b-actin, anti-HA-tag, anti-FLAG-tag, and anti-Trim25 antibodies were purchased from Proteintech (#11224-1-AP, 81115-1-RR, 51064-2-AP, #66008-4-Ig, and 12573-1-AP, respectively). Other antibodies, including IgG and secondary mouse or rabbit antibodies, were purchased from Thermo Fisher Scientific (Waltham, UK). MG132 and cycloheximide were purchased from MCE (#HY-13259 and #HY-12320, respectively). Protein A/G agarose beads were purchased from Thermo Fisher Scientific (#WF324075). FABP1 was amplified from an ultimate open reading frame clone by PCR and cloned into pcDNA3.1. Constructs of wild-type HA-tagged Derlin-1, HA-tagged Derlin-1, shRNA Derlin-1 CT, Trim25, shRNA Trim25, and shRNA Trim25 CT were designed and cloned into pcDNA3.1. Wild-type Ub and mutated Ub plasmids were amplified by PCR and cloned into pcDNA3.1. All the plasmid constructs were confirmed by DNA sequencing.
Animals
C57BL/6J (C57) mice (6–8 weeks old) were obtained from the Shanghai Laboratory Animal Center (Shanghai, China). All male mice used in our study were housed in standard cages and maintained under a 12-hour light/dark cycle at 23–26°C (Animal House, Shanghai Tenth People’s Hospital of Tongji University). Water was provided ad libitum, and the relative humidity was maintained at 40 ± 5%. The study protocol was approved by the Animal Care and Use Committee of Tongji University. To establish an animal model of NAFLD and to verify FABP1 expression, mice were fed a high-fat diet (HFD), and a control group was established by feeding with a control diet (CD). In addition, we injected 2 × 107 purified viral particles per gram of body weight of control (AAV-GFP) or experimental (AAV-Derlin-1) viruses for adeno-associated virus experiments (Genechem Ltd. Shanghai, China).
Histopathological analysis, oil red staining, and immunofluorescence staining
The retrieved mouse liver tissues were fixed overnight with 4% paraformaldehyde. Paraffin-embedded and OCT-embedded liver sections were used for hematoxylin/eosin(H&E), immunohistochemistry (IHC), immunofluorescence (IF), and oil red staining (Sigma-Aldrich, #O0625), respectively. IHC and IF staining were followed by antigen retrieval and immunostaining with anti-FABP1 and Derlin-1. All images were obtained using an Olympus confocal microscope and processed using the Olympus FV1000 software.
Blood glucose and serum insulin analysis
Blood glucose levels were determined with a Gluco-meter Elite monitor (B.BRAUN, Germany), and serum insulin was measured using an insulin ELISA kit (Cusabio, #CSB-E05071m). After overnight fasting, glucose tolerance tests (GTTs) were completed by intraperitoneal injection of d-glucose (2 g/kg, Sigma, USA, #SLCK0748). Insulin tolerance tests (ITTs) were performed by i.p. injection of 1 unit/kg insulin (NovoMix 30; Bagsvaerd, Denmark) after 4 h of fasting. GTTs and ITTs were performed 15 weeks after being treated with a HFD.
Lipid analysis
Lipid metabolizing markers in the serum and hepatic tissues (TC, TG, and LDL) were measured using commercial kits (Nanjing Jiancheng, #A111-1-1, A110-1-1, and A113-1-1, respectively), according to the manufacturer’s instructions.
Co-immunoprecipitation assays and mass spectra
Protein lysates were prepared from mouse liver tissue using radioimmunoprecipitation assay buffer containing protease inhibitors for co-immunoprecipitation (CO–IP) assays, cleared by centrifugation, and the protein concentration was estimated. The lysates were washed with 100 µL of protein A/G agarose beads in 1 mL lysis buffer. The lysates were then incubated overnight with anti-FABP1 antibodies, anti-Derlin-1 antibodies, or control IgG with protein A/G agarose beads. The complexes were washed with 1 mL RIPA lysis buffer, followed by centrifugation (repeated 3–5 times), and resuspended in 2 × SDS loading buffer. The immunoprecipitated proteins were eluted from the beads by incubation at 95°C for 5 min. The eluted proteins were detected by immunoblotting after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
The gel pieces were dehydrated with acetonitrile and digested with trypsin for mass spectrometry-based proteomic analysis. The peptides were subjected to a nanospray source, followed by tandem mass spectrometry (MS/MS) on a Q Exactive™ Plus (Thermo) instrument coupled online to an ultra-performance liquid chromatographer. The resulting MS/MS data were processed using Proteome Discover 1.3. Liquid chromatography and tandem mass spectrometry (LC-MS/MS) analyses were performed at PTM Bio-lab (Hangzhou, Zhejiang, China).
Cell culture and transfection
Hek293t and HepG2 cells were cultured in DMEM supplemented with 10% FBS at 37 ℃ and 5% CO2. First, the constructed vectors were transfected into HEK293t cells for cell transfection using Lipo3000 (Invitrogen; #L3000015). Then, 4–6 h after transfection, the medium was complemented with blood serum, and the cells were maintained in an incubator at 37 ℃ and 5% CO2. Gene expression or knockdown was examined by Western blot 24–48 h after gene transfection in Hek293t cells.
HepG2 stable cell lines were also produced by lentiviral infection. The lentiviral empty vector and ORF expression vector (Derlin-1, shRAN Derlin-1, shRAN CT) were transfected into E. coli cells using Fugene HD transfection reagent (Roche) following the manufacturer’s instructions. Then, two days after transfection, cell culture supernatants were collected and centrifuged. Supernatants containing lentiviral particles were used to infect HepG2 cells. After lentiviral transduction, target HepG2 cells were selected for 10–14 days in 2.5 µg/mL puromycin. The efficiency of Derlin-1 and shRNA Derlin-1 transfection was determined using immunoblot analysis.
Ubiquitination assays
Hek293t cells were transfected with FABP1, Derlin-1, HA-ubiquitin, or empty vector plasmids using Lipo3000 for 48 h. Then, 20 µM MG132 proteasome inhibitor was added to the cells; 6 h later, they were washed with ice-cold PBS (repeated two times) and cleaved in RIPA lysis buffer. The lysates were also incubated with anti-FABP1 antibodies overnight and then with protein A/G agarose beads for 4 h at 4°C. The complexes were washed with 1 mL RIPA lysis buffer, and the proteins were dissolved in 2 × SDS loading buffer from the beads. We used an anti-ubiquitin antibody to analyze the proteins released by immunoblotting.
Quantitative real-time reverse-transcriptase polymerase chain reaction
Total RNA was isolated from the liver tissues and cells using TRIzol reagent (Invitrogen, Carlsbad, CA, #15596-026). Complimentary DNA (cDNA) was synthesized by reverse transcription of RNA (0.5 mg) using a PrimeScript RT Reagent Kit (TaKaRa, Japan, #A370). Quantitative real-time polymerase chain reaction (qRT-PCR) analyses were performed with SYBR Green Master Mix (Vazyme, #Q111-02) using a 7900HT real-time PCR system (ABI, CA, USA) to determine the expression of target genes. Expression levels were analyzed using the relative standard curve method and normalized to the housekeeping gene actin. The primer sequences for qRT-PCR were as follows. Human-Derlin-1: forwards 5’-TCGGACATCGGAGACTGGTT-3’, reverse 5’-GGCAGTGATTGGCCTCCAAA-3’; human-FABP1: forwards 5’-ATGAGTTTCTCC GGCAAGTACC-3’, reverse 5’-ATGAGTTTCTCCGGCAAGTA-3’; human-GAPDH: forwards 5’-TTGCTGTTG AAGTCGCAGGAG-3’, reverse 5’-TGTGTCCGTCGTG GATCTGA-3’; mouse-PPARa: forwards 5’-AACATCGAGT GTCGAATATGTGG-3’, reverse 5’-CCGAATAGTTCGCCGAAAGAA-3’; mouse-PPARb: forwards 5’-TCGCTGATGCACTGCCTATG-3’, reverse 5’-GAGAGGTCCACAGAGCTGATT-3’; mouse-PPARd: forwards 5’-TCCATCGTCAACAAAGACGGG-3’, reverse 5’-ACTTGGGCTCAATGATGTCAC-3’; mouse-acyl-CoA oxidase 1 (ACOX1): forwards 5’-TAACTTCCTCAC TCGAAGCCA-3’, reverse 5’-AGTTCCATGACCCA TCTCTGTC-3’; mouse- carnitine palmitoyl-transferase 1A(CPT1A): forwards 5’-TGGCATCATCACTGGTGTGTT-3’, reverse 5’-GTCTAGGGTCCGA TTGATCTTTG-3’; mouse-GAPDH: forwards 5’-AGGTCGGTGTGAACGGATTTG-3’, reverse 5’-TGTAGACCATGTAGTTGA GGTCA-3’.
Quantification and statistical analysis
All data were analyzed using appropriate statistical methods using SPSS software (v. 20.0, IBM, USA). Normally distributed continuous data are presented as means ± standard deviations (X ± SD). A P value of < 0.05 was considered statistically significant. We determined the sample size using the Power Analysis and Sample Size software (PASS; v. 11, NCSS, Kaysville, Utah, USA) to assess the test power of the current sample size. We collected data from animal studies in a blinded manner.