L 14c u amino acid mixture aa

After prawns were injected with 10 μg of siGLUT1 (with siGFP as a control), 36 prawns (12 prawns for each treatment group) were used to perform the metabolic tracking of ¹⁴C-labeled nutrient test, following a 24 h fast, with or without hypoxia treatment. The metabolic tracking test was determined by injecting [1-¹⁴C]-PA (PA^∗) or D-[1-¹⁴C]-glucose (Glu∗) or L-[¹⁴C(U)]-amino acid mixture (AA^∗) (PerkinElmer) on the base of the third pereiopod according to previous studies (71 (link), 72 ). Afterward, these injected prawns were divided into three groups to perform three different radioactivity retention assays (four prawns for one assay, n = 4). The retention radioactivity was assayed as described previously (73 (link)).

After the eight-week trial, Nile tilapia from control and high cholesterol (1.6%) treatments were fasted overnight and then used for metabolic tracking of ¹⁴C-labelled nutrient. Six fish from each treatment were subjected to ¹⁴C-labelled nutrient tracking, including D-[1-¹⁴C]-glucose (Glu^∗), [1-¹⁴C]-palmitic acid (PA^∗) and L-[¹⁴C(U)]-amino acid mixture (AA^∗) (PerkinElmer). The doses for intraperitoneal injection of ¹⁴C-labelled were based on our previous studies (Li et al. 2020c (link)). To measure the ¹⁴CO₂ released from the oxidation of different nutrients, the injected fish were immediately transferred to oxygen-saturated water in sealed bottles, which were connected to another glass bottle containing 1 mol/L potassium hydroxide (KOH) solution. The fish were digested with a strong lysate (HClO₄ /30% H₂O₂, 2:1, v/v; 1:5, w/v) at 60 °C in a water bath for 6 h to ensure total retention of ¹⁴C in the whole body. The radioactivities of strong lysate and KOH solution (500 μl) were measured using the Tri-Carb 4910TR Liquid Scintillation Analyzer (PerkinElmer) with 2 ml of scintillation fluid (Ultima Gold XR; Packard, Conroe, TX, USA).