Non esterified free fatty acid assay kit

The following reagents were used: Streptozotocin (Merck, Darmstadt, Germany); AdipoRon (Selleckchem, Houston, TX, USA); a triglyceride assay kit and a non-esterified free fatty acid assay kit (Jiancheng Bioengineering Institute, Nanjing, China); and dihydroethidium (Thermofisher, MA, USA). The following antibodies were used: Rabbit monoclonal to AdipoR1 (EPR6626,Abcam, Cambridge, UK); rabbit polyclonal to RNA-binding protein with multiple splicing (RBPMS; Abcam); rabbit monoclonal to glutamine synthetase (EPR13022(B),Abcam); chicken polyclonal to glial fibrillary acidic protein (GFAP; Abcam); rabbit polyclonal to EGR4 (Abcam); phospho-AMPKα rabbit monoclonal antibody (mAb) and AMPKα rabbit mAb (40H9/D5A2,Cell signaling, Boston, USA); phospho-acetyl CoA carboxylase (ACC) rabbit mAb and ACC rabbit mAb (D7D11/C83B10,Cell signaling); cleaved caspase-3 rabbit mAb (Cell signaling), β-actin (8H10D10,Cell signaling); donkey anti-rabbit AlexaFluor-594 and donkey anti-chicken Alexa 488 (Jackson ImmunoResearch, Philadelphia, USA). The following reagents were used: 5,6-chloromethyl-2ʹ,7ʹ-dichlorodihydrofluorescein diacetate (CM-H₂DCFDA; Thermofisher); RNAimax (Thermofisher); Opti-MEM I (Thermofisher); small interfering RNA (siRNA; GenePharma, Shanghai, China); Cell Counting Kit-8 (CCK-8; Dojindo, Tokyo, Japan); and 4ʹ,6-diamidino-2-phenylindole (DAPI; Absin, Shanghai, China).

Soybean oil (10 μl g⁻¹ of fish body weight) was administered by oral gavage to 6 fish in each treatment according to their body weight after starvation for 12 h. After 90 min, fish were euthanized with MS-222 (25 mg liter⁻¹) and blood samples were collected and centrifuged at 3,000 × g for 10 min to obtain the serum. The intestinal content of the whole gut of these fish was collected. The concentrations of triglycerides and free fatty acids in the intestinal content and serum were determined by triglyceride assay kit and nonesterified free fatty acid assay kit (Jiancheng, Nanjing, China), respectively. All measurements were performed in triplicates in 96-well plates.