Animal experiments

Seven-week-old male C57BL/6 J mice were purchased from GemPharmatech (Nanjing, China). Mice were housed in a standard specific pathogen-free facility with free access to food and water. After a 1-week acclimatization period, eight-week-old mice were used in two experiments. In the first experiment, mice were divided randomly into three groups (n = 5 per group), which were exposed to a chow diet (CD, 11% fat [kcal%]; Guangdong Medical Laboratory Animal Center, Guangzhou, China), an HFD (58% fat [kcal%]; D12331; Research Diets, New Brunswick, NJ, USA), or an HFD and a ketogenic diet (KD, 90.5% fat [kcal%], TD.160153, Envigo, USA) alternating every 2 weeks for a total period of 16 weeks. In the second experiment, adeno-associated virus (AAV) expressing short-hairpin RNAs targeting β-klotho (shKlb) and negative control (shCtrl) were administered to generate liver-specific KLB knockdown mice and control mice, and then mice were subjected to 16-week diet intervention on the next day. Body weight, blood glucose, and food intake were recorded every 2 weeks. Blood and tissue samples were collected after overnight fasting. All animal experiments complied with the ARRIVE guidelines and were approved by the Institutional Animal Care and Use Committee of Sun Yat-sen University.

AAV9-mediated gene knockdown

AAVs expressing shKlb (5′-GCAATCTGTCCAAAGTTAACA-3′) and shCtrl were purchased from Genechem Co., Ltd (Shanghai, China). The sequence was validated in a previous study to effectively knock down Klb expression in mouse liver [20]. AAV9 (1.0 × 1011 active viral particles/mouse) was injected via the tail vein to suppress Klb expression in the liver.

Body composition measurement

After the dietary exposure, body composition was measured by a quantitative magnetic resonance EchoMRI™-100 (EchoMRI LLC, Houston, TX, USA) consciously according to the manufacturer’s instructions. Mice were carefully fixed in tubes and then inserted into the EchoMRI™-100 machine one at a time, and fat and lean masses were analyzed and recorded by the machine within 1 min. Fat mass is presented as the ratio to body weight.

Glucose and insulin tolerance tests

Glucose tolerance test was performed in overnight-fasted mice by intraperitoneally injecting with glucose (2.0 g/kg body weight). Blood samples were collected for serum insulin measurement. For the insulin tolerance test, the mice were intraperitoneally injected with insulin (0.65 units/kg body weight, Novolin R, Novo Nordisk Inc., Denmark) after 6 h of fasting. Blood glucose levels were recorded at various time points after the injection.

Biochemical and immunological analyses

Triglycerides, total cholesterol, and free fatty acids were quantified using commercial kits (triglycerides, K622-100; cholesterol, K603-100; free fatty acids, K612-100; Biovision, Milpitas, CA, USA). Serum insulin concentration was measured with an ELISA kit (#10-1247-01; Mercodia, Uppsala, Sweden), and another commercial kit (#32180; Immunodiagnostics Limited, Science Park, Hong Kong, China) was used in FGF21 measurement. Every sample was assayed in duplicate.

Hematoxylin and eosin staining

Hematoxylin and eosin staining was performed according to standard procedures [28]. Fresh tissues were fixed in 4% paraformaldehyde solution (GBCBIO, Guangzhou, China) at 4°C overnight. Then, tissues were embedded in paraffin and sectioned at 3–5 μm at room temperature. The sections were deparaffinized twice with fresh xylene and dehydrated with ethanol at a gradient concentration. Next, the nucleus was stained with hematoxylin, and the cytoplasm was stained with eosin. Photomicrographs were obtained using a DMi8 inverted microscope (Leica Microsystems, Wetzlar, Germany).

Oil Red O staining

Oil Red O staining was performed according to standard procedures [28, 29]. Briefly, fresh liver tissues were embedded in OCT compound (Sakura Finetek, Torrance, CA, USA) and stored at −80°C. Liver tissues were next sectioned at 6–8 μm at −18 °C and washed with 60% isopropanol. A stock solution was prepared using Oil Red O powder (O0625; Sigma Aldrich, St Louis, MO, USA) dissolved in isopropanol and protected from light; a working concentration of 60% was diluted with ddH2O. The sections were stained with Oil Red O working solution for 1 h and washed under running tap water for 20 min. After Oil Red O staining, tissue sections were counterstained with hematoxylin for 1 min. The Oil Red O staining procedure was performed in the dark. Photomicrographs were obtained using a DMi8 inverted microscope (Leica Microsystems).

Real-time qPCR

Total RNA was extracted from frozen tissues using Trizol Reagent (Sigma Aldrich) [30]. cDNA was synthesized from the RNA by reverse transcription using a Transcriptor First Strand cDNA Synthesis Kit (Roche Applied Science, Basel, Switzerland). Real-time PCR was performed using a LightCycler 480 System with LightCycler 480 SYBR Green Master Mix (Roche Applied Science). Target gene expression was normalized against β-actin, and the fold change in mRNA expression was determined using the 2-ΔΔCT method.

Western blotting

Protein was extracted from tissues using RIPA lysis and extraction buffer (89900; Thermo Fisher Scientific, Waltham, MA, USA) combined with Halt™ Protease and Phosphatase Inhibitor Cocktail (78440; Thermo Fisher Scientific). Next, an equal amount (30 μg) of protein was separated by 10% SDS-PAGE and transferred onto a polyvinylidene fluoride membrane (EMD Millipore, Burlington, MA, USA). Membranes were incubated with 0.1% (1:1000) target primary antibodies at 4°C overnight and then with 0.01% (1:10000) secondary antibodies at room temperature for 1 h protected from light. After washing thrice with TBS, the protein bands were visualized using the Odyssey Infrared Imaging System (LI-COR, Nebraska, USA). Antibodies against acetyl-CoA carboxylase (ACC) (3676 S), fatty acid synthase (FASN) (3180 S), stearyl-coenzyme A desaturase 1 (SCD1) (2794 S), and β-actin (4970 s) were purchased from Cell Signaling Technology (Danvers, MA, USA). Fibroblast growth factor receptor 1 (FGFR1) (60325-1-Ig) antibody was purchased from Proteintech (Wuhan, China). Carnitine palmitoyltransferase 1α (CPT1α) antibody (sc-31128) was purchased from Santa Cruz Biotechnology (Dallas, TX, USA). KLB antibody (AF-2619) was purchased from R&D System (Minneapolis, MN, USA).

Immunofluorescence staining

Immunofluorescence staining of mouse livers was performed as previously described [11]. Tissue sections were prepared for hematoxylin and eosin staining as previously described. Sections were immersed in preheated citrate buffer and blocked with 3% hydrogen peroxide. Primary antibodies (0.5%, 1:200) against ACC, FASN, CPT1α, FGFR1, and KLB were applied to the sections overnight at 4°C, followed by incubation with secondary antibodies (0.2%, 1:500). Finally, the nuclei were stained with DAPI for 10 min and washed with PBS. Photomicrographs were obtained using a DMi8 inverted microscope and Leica Qwin image analysis software (Leica Microsystems).

RNA sequencing

Total RNA was extracted from fresh mouse livers using an RNeasy Plus Mini kit (QIAGEN, Hilden, Germany) according to the manufacturer’s instructions. An Agilent 2100 Bioanalyzer System (Agilent Technologies, Palo Alto, CA, USA) and Agilent RNA 6000 Nano Kit (Agilent Technologies) were used to assess the integrity and concentration of total RNA. Samples with an RNA integrity number >8 were considered suitable for further analyses. RNA sequencing and data analysis (GEO: GSE226139) were conducted by Pan-Guarantee Biotechnology Co., Ltd (Guangzhou, China), as previously described [11].

Proteomic analysis

Proteins from mouse livers were isolated using a lysis buffer (8 mol/L urea, 1% protease inhibitor cocktail, 3 mmol/L trichostatin A, and 50 mmol/L nicotinamide), followed by sonication using a high-intensity ultrasonic processor (Scientz Biotechnology, Ningbo, China) and centrifugation. A BCA kit (Thermo Fisher Scientific) was used to measure the protein concentration according to the manufacturer’s instructions. Proteomic analysis (PRIDE: PXD040481) was performed using the Jingjie PTM Bio-Labs (Hangzhou, China) as previously described [11].

Statistical analyses

Data are presented as mean ± standard deviation. Statistical analysis was performed using SPSS 20.0 (IBM, USA). Unpaired two-tailed t-test was used in comparing differences between two groups, and one-way analysis of variance was used in calculating differences among the three groups with the least significant difference test. Statistical significance was set at P < 0.05.