Objective To investigate the changes in metabolic characteristics of human hepatocellular carcinoma HepG2 cells during metabolic stress⁃induced autophagy based on non⁃targeted metabolomics. Methods (1) HepG2 cells were divided into conventional culture group (cultured in DMEM complete medium) or 0⁃, 3⁃, and 6⁃hour starvation culture group (cultured in HBSS⁃based starvation medium for 0, 3, and 6 hours). The formation of autophagosomes in each group was detected using monodansylcadaverine (MDC) method. (2) HepG2 cells were divided into starvation group (cultured in HBSS⁃based starvation medium), chloroquine group (cultured in DMEM complete medium supplemented with 50 μmol/L chloroquine), or starvation+chloroquine group (cultured in HBSS⁃based starvation medium supplemented with 50 μmol/L chloroquine) for different time points (0, 3, and 6 hours). HepG2 cells cultured in DMEM complete medium served as the normal control group. The expressions of autophagy⁃related proteins (p62, LC3B) were detected by Western blot. (3) HepG2 cells were divided into metabolic stress⁃induced autophagy group (cultured in HBSS⁃based starvation medium for 6 hours), metabolic stress group (cultured in HBSS⁃based starvation medium for 3 hours), or control group (cultured in DMEM complete medium). Non⁃targeted metabolomics analysis was performed using ultra⁃high performance liquid chromatography⁃mass spectrometry. Differential metabolites were screened from various groups, and hierarchical clustering analysis and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis were conducted on the differential metabolites. Results (1) After 6 hours of starvation culture, dense fluorescent granules were observed around the nucleus, and autophagosomes began to increase in a cascade manner, indicating an activated autophagic state. Compared with the chloroquine group, the starvation+chloroquine group exhibited increased p62 protein expression and a higher LC3B⁃Ⅱ/LC3B⁃Ⅰ ratio. (2) A total of 442 differential metabolites were identified between the metabolic stress group and the control group, 535 between the metabolic stress⁃induced autophagy group and the control group, and 132 between the metabolic stress⁃induced autophagy group and the metabolic stress group. Hierarchical clustering analysis indicated that differential metabolites between the metabolic stress group and the control group were mainly involved in categories such as organic acids and their derivatives, glycerophospholipids, fatty acyl, and sphingolipid. Compared with the control group, in addition to the above categories, the metabolic stress⁃induced autophagy group exhibited group⁃specific differences in expressions of benzenoids and their derivatives, amino acids and their metabolites. Differential metabolites between the metabolic stress⁃induced autophagy group and the metabolic stress group were enriched in organic acids and their derivatives, glycerophospholipids, fatty acyl, and sphingolipid, etc. Differential metabolites between the metabolic stress group and the control group were mainly involved in metabolic pathways such as vitamin digestion and absorption, and fat digestion and absorption. Differential metabolites between the metabolic stress⁃induced autophagy group and the control group were mainly involved in metabolic pathways such as cholesterol metabolism, vitamin digestion and absorption, and glycine, serine, and threonine metabolism. Differential metabolites between the metabolic stress⁃induced autophagy group and the metabolic stress group were mainly involved in metabolic pathways such as glycerophospholipid metabolism and linoleic acid metabolism. Cholesterol metabolism, vitamin digestion and absorption, and the glycine⁃serine metabolism axis were identified as three key metabolic pathways for the differential metabolites. Conclusion Autophagy induced by metabolic stress in HepG2 cells is accompanied by significant metabolic reprogramming. Alterations in key metabolic pathways including cholesterol metabolism, vitamin digestion and absorption, and the glycine⁃serine metabolism axis are core metabolic characteristics during starvation⁃induced autophagy.

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