Effect of High NEFA Concentration on Lipid Metabolism Disorders in Hepatocytes Based on lipidomics

Description

Fatty liver is a common nutritional metabolic disease in periparturient dairy cows, which seriously affects their reproductive performance. During periparturient period, dairy cows are in a state of negative energy balance because they cannot meet the nutritional requirements for maintenance and lactation. As a result, non-esterified fatty acids (NEFA) are mobilized from adipose tissue, leading to an increase in NEFA concentration in the blood. When the rate of NEFA esterification into triglycerides (TAG) exceeds the rate of NEFA processing, fatty liver will form. Existing studies suggest that NEFA is closely associated with lipid metabolism disorders in hepatocytes, but it is not clear through which metabolites or metabolic pathways NEFA affects lipid metabolism disorders in hepatocytes. Therefore, the aim of this study was to investigate the effect of high concentration of NEFA on lipid metabolism in hepatocytes through the lipidomic approach and molecular biology techniques. Here, we found that NEFA (0.6-2.4 mM) significantly reduced the cell viability in a concentration-dependent manner, indicating that high concentrations of NEFA have lipotoxicity on hepatocytes. In addition, NEFA promoted TAG accumulation, increased the mRNA expression of the lipogenic molecules SREBP and FASN, and decreased the mRNA expression of lipolytic molecules CPT1A and HSL in hepatocytes. Mechanistically, the results of lipidomics analysis further showed that NEFA induced lipid metabolism disorders in hepatocytes by regulating metabolic pathways such as glycerol phospholipid metabolism, glycosyl phosphatidylinositol anchored biosynthesis, triglyceride metabolism, sphingolipid metabolism, and inositol phosphate metabolism.

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1.Collection of samples:At the end of cell stimulation trypsin digestion and cell precipitate was placed in a centrifuge tube, this is the sample collected. All samples were placed at -80°C for storage prior to testing on the machine. 2.Sample Handling :Endogenous small molecule metabolites are easily influenced by surrounding temperature and environmental changes. Thawing of samples must be carried out slowly in an ice bath to avoid rapid recovery to room temperature, which can activate metabolic enzymes and cause changes in metabolite composition and concentration. Reagents used for extraction should be pre-cooled in a -20°C freezer to prevent heat release of organic solvents from causing degradation of small molecule metabolites in biological samples. The entire sample preparation process should be completed as quickly as possible. The specific procedure is as follows: (1) To minimize degradation, thaw the samples in an ice bath. (2) Add 10 grinding beads and 10 µL of deionized water to each tube of cell samples, then homogenize for 3 min. (3) Add 300 µL of lipid extraction solvent to each tube of cell samples, then homogenize for 3 min. (4) Vortex the cell samples at 1200 r/min for 20 min at 10°C. (5) Centrifuge the cell samples at 4000 g for 20 min at 4°C. (6) Transfer 20 µL of the supernatant to a 96-well plate and mix with 80 µL of lipid dilution solvent for subsequent analysis. This project utilized an ultra-high performance liquid chromatography-triple quadrupole mass spectrometry (UPLC-TQMS) instrument (TACQUITY UPLCXevo TQ- S, Waters Corp., Milford, MA, USA) to perform targeted lipidome assays. The instrument parameters and method settings were as The instrument parameters and method settings are shown in the table below. System optimization and maintenance were performed every 48 hours. 3.Liquid Phase Parameters:Using the ACQUITY UPLC BEH C18 analysis column for separation, with a particle size of 1.7 µM, a cross-sectional diameter of 2.1 mm, a length of 100 mm, a column temperature of 40℃, a sample manager temperature of 10℃, the mobile phase is composed of A and B. The A phase is acetonitrile/water (6:4, 5 mM ammonium formate + 0.1% formic acid), and the B phase is isopropanol/acetonitrile (9:1, 5 mM ammonium formate + 0.1% formic acid). 4.Mass spectrometry parameters are as follows: capillary voltage is 3.0 kV, ion source temperature is 150°C, desolvation temperature is 550°C, desolvation gas flow rate is 1000 L/h, and collision gas flow rate is 0.13 L/h. 5.

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