Ab234111

The mice's left atrium was homogenized in RIPA lysis buffer (P0013 B, Beyotime Biotechnology, China) containing protease inhibitor cocktail (1 : 100, Sigma). Total protein (50 μg) was separated by 10% SDS-PAGE gels and then separated protein was transferred onto a PVDF membrane. The membrane was blocked with 5% skimmed milk and subjected to primary antibodies overnight at 4°C against ANGPTL4 (1 : 200; ab196746, Abcam), PPARα (1 : 200; sc-398394, Santa Cruz), PPARγ (1 : 200; sc-7273, Santa Cruz); CPT-1 (1 : 500; ab234111, Abcam), SIRT3 (1 : 200; ab246522, Abcam), and ß-actin (1 : 1000; ab8226, Abcam), followed by incubation with HRP-conjugated secondary antibody. Protein bands were visualized using Enhanced Chemiluminescence Plus (Millipore, MA, USA). Relative expression of proteins was normalized to ß-actin.

The frozen tissues were homogenized in a lysis buffer (20 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton-X100, and protease inhibitor, pH 7.4) and then centrifuged for 20 min at 10,000 g to discard cell debris. The total protein concentrations were determined using a Bio-Rad kit. The proteins were subjected to Western blot analysis with the desired antibodies. Antibodies against carnitine palmitoyltransferase 1A (CPT1A) (ab234111), HMGCS2 (ab137043), FAS (ab128870), HMGCR (ab174830), liver glycogen phosphorylase (PYGL) (ab198268) and PYGL (phospho S15, ab227043) were obtained from Abcam. Antibodies against COX4 (A21348) was obtained from Invitrogen. Antibody against phosphoenolpyruvate carboxykinase 1 (PCK1) (A2036) and GCK (A6293) was purchased from ABclonal. Antibodies against AMPKα (2532), phospho-AMPKα (2535), CREB (9197), Phospho-CREB (9198), and SCD1 (2438) were purchased from Cell Signaling Technology. Antibody against G6PC (22169-1-AP) was purchased from Proteintech. Antibody against TUBULIN (T0198) was obtained from Sigma-Aldrich.

Small intestinal tissue was fixed with 4% paraformaldehyde, then embedded in paraffin and sectioned (about 3-4 μm thick). The paraffin sections were deparaffinized with xylene, dehydrated in an ethanol gradient, followed by hydration and antigen repair in a citrate high-temperature water bath, and blocked with BSA. Primary antibodies were incubated overnight at 4 °C ((CPT1A (1:1000, Abcam, ab234111), (LGR5 (1:400, Abcam, ab219107), Ki67(1:50, Abcam, ab279653)). The secondary antibodies were incubated at room temperature for 1 h, followed by microscopic observation.

A Western blot was carried out to detect the expression of carnitine palmityl transferase 1 (CPT1α, ab234111, Abcam, UK), pyruvate kinase isozymes M2 (PKM2, 4053S-100UL, Cell Signaling Technology, CST, USA), 3-hydroxy-3-methylglutaryl- Coenzyme A synthase 2 (HMGCS2, ab137043, Abcam, UK), Na+-coupled monocarboxylate transporter (SMCT1, orb101289, Biorbyt, UK), succinyl-CoA:3-ketoacid CoA transferase (SCOT, ab224250, Abcam, UK), peroxisome proliferator activated receptor co-activator-1α (PGC1α, NBP1-04676, Novus, USA) (PGC1α, ab191838, Abcam, UK), mitochondrial fusion protein 1 (Mfn1, 14739S, Cell Signaling Technology, CST, USA), mitochondrial dynamin-related protein 1 (Drp1, NB110-55288, Novus, USA), Bcl-2 (A19693, Abclonal, Wuhan, China), Bax (A12009, Abclonal, Wuhan, China), and silent information regulation 2 homolog 3 (SIRT3, 2627S, Cell Signaling Technology, CST, USA) proteins. Among them, CPT1α, PKM2, HMGCS2, SMCT1, SCOT, PGC1α, Mfn1, Drp1, Bcl2, Bax, and SIRT3 were all rabbit polyclonal antibodies with a dilution of 1:1000, while GAPDH was a mouse monoclonal antibody with a dilution of 1:3000.

Total protein was extracted from cultured cells and liver tissues by using ice cold RIPA lysis buffer. The protein concentration was determined by BCA protein assay kit (Solarbio, Beijing, China). 20 μg of protein was ran on a 10% SDS-PAGE gel and transferred to a PVDF (Millipore, USA) membrane. GAPDH was used as a control and non-specific bindings were blocked by 5% BSA for 40 min at room temperature. Incubates the specific primary antibodies against p-IKKβ (Abcam, ab59195, 1:1000), IKKβ (Abcam, ab124957, 1:1000), p-p65 (Abcam, ab86299, 1:1000 dilution), p65 (Abcam, ab16502, 1:1000 dilution), TNF-α (Abcam, ab183218, 1:1000 dilution), IL-6 (Abcam, ab259341, 1:1000 dilution), ABCA1 (Abcam, ab66217, 1:1000), CPT1 (Abcam, ab234111, 1:1000) at 4 ℃ overnight. After extensively washing with 100 µM PBST, peroxidase-conjugated secondary antibody (Abcam, ab205718, 1:5000) was applied to the membranes and incubated for 2 h at room temperature. Protein bands were detected by using ECL substrates (Thermal Fisher, Rockford, USA) and visualized under a ChemiDoc XRS + imager (Bio-Rad, Hercules, USA).

Liver tissues and epididymal adipose tissues fixed in 4% paraformaldehyde were embedded in paraffin, cut into 4 μm thick slices using a microtome, and then stained with hematoxylin and eosin (H&E). Immunohistochemical (IHC) staining of liver tissues was used to examine protein expression. Primary antibodies against SREBP1 (ab191857; Abcam, Cambridge, UK) and CPT1A (ab234111; Abcam) were used, followed by incubation with horseradish peroxidase- (HRP-) labeled anti-rabbit IgG secondary antibody for 20 min. The tissues were then stained with 3,3′-diaminobenzidine (DAB) and counterstained with hematoxylin. The IHC staining images were analyzed based on the mean optical density using ImageJ software (National Institutes of Health, Bethesda, MD, USA).

The proteins in tissues were lysed using lysis buffer, and same amount of samples was separated by 8% SDS-PAGE. After electrophoresis, the protein was transferred to nitrocellulose membrane (Millipore, USA). After blocking with TBST, the membranes were incubated with primary antibody (1:1000) at 4° C overnight. After washing twice with PBS, the membranes were incubated with secondary antibody (1:2000) for 2 h at room temperature. Membranes were visualized using an enhanced chemiluminescence kit (#34096, Thermo Fisher, Waltham, MA, USA) under the chemiluminescence imaging system (Bio-Rad, Hercules, CA, USA). The antibodies used in this study were listed as follows: Mouse monoclonal to GSK-3β (#ab93926, abcam, Cambridge, UK), Rabbit monoclonal to p-GSK-3β (##9323, CST, Danvers, CO, USA,), Mouse monoclonal to HK-2 (#ab209847, abcam, Cambridge, UK), Rabbit monoclonal to SREBP-1c (#ab28481, abcam, Cambridge, UK), Rabbit monoclonal to CPT-1 (#ab234111, abcam, Cambridge, UK), Mouse monoclonal to β-actin (#ab8226, abcam, Cambridge, UK), Goat anti-mouse antibody (#ab205719, abcam, Cambridge, UK), Goat anti-rabbit antibody (#ab205718, abcam, Cambridge, UK). The experiment was performed 3 times independently.