Effects of Nutritional Perturbations on the Metabolomic Homeostasis of HepG2 Cells

Abstract

Background: Normal body function is based on the metabolic processes going inside the body which require energy (ATP) as fuel for the formation of metabolic intermediates to enrich other metabolic pathways and perform cellular functions. This energy is provided by diet which includes carbohydrates, fats, proteins, fiber, and minerals. The diet consumed is digested in the small intestine and enters the liver via the portal vein. The liver plays a significant role in the metabolism of gut-derived dietary components. Metabolism of these components will lead to the production of metabolites conferring physiological roles. Consumption of these dietary elements in adequate amounts maintains metabolic homeostasis. But excess intake for prolonged periods may cause toxicity and affect the metabolic processes and metabolites composition in the human body. This can lead to the development of metabolic syndrome (MetS) which may cause several metabolic diseases. These metabolomic shifts may negatively affect the liver and its function as the liver is the first line of detoxification for gut-derived metabolites. Scientists have shown the effects of different dietary elements on liver function with gene expression studies but the metabolome call remains critically underexplored. Metabolomic studies would help in understanding the interaction between diet and biomolecules in the body and identifying disease-associated markers specific to certain dietary stress. Aim: The effects of high concentrations of each diet were evaluated using human hepatoma (liver tumor) cells, (in this study, HepG2 cells), in which various chemicals were used to mimic the dietary stress and untargeted metabolome analysis was done to understand the changes in metabolites caused by various dietary stress. 2 Method: HepG2 cells were cultured in standard conditions and used to mimic liver responses to dietary stress. A sub-lethal dose for each treatment was selected using a standard MTT assay. Cells were treated with five dietary stresses (high glucose, high palmitic acid, high salt (NaCl), high bovine serum albumin (BSA), and high butyrate) and later metabolomics study was conducted. GC-MS technique was used for untargeted metabolomic analysis and relevant metabolites were obtained for each nutritional stress. Later, metabolites were mapped using PubChem, KEGG, HMDB, and SMPDB databases. Metabolic pathway enrichment, disease signatures, and subclasses for the metabolite set of each treatment were evaluated using Metaboanalyst (5.0) tool, and corresponding results were recorded. Result: Different metabolites were identified after GC-MS analysis. 54 metabolites were identified in control, 137 after glucose treatment, 211 after palmitic acid treatment, 92 after NaCl treatment, 231 after BSA treatment, and 122 after butyrate treatment. Out of all metabolites, cholesterol, 2- propyl-1-pentanol, palmitic acid, benzoic acid, acetamide, and leucine were common among all treatments. Three disease signatures were common in all which are phenylketonuria, phosphoenolpyruvate carboxykinase (PEPCK) deficiency, and type VI hers disease, and seven classes of metabolites were also found to be common, which include straight chain fatty acids, benzoic acids, carboximidic acids, amino acids, hydrocarbons, saturated fatty acids, and alkanes. Conclusion: Palmitic acid showed more deleterious effects on HepG2 cells in comparison to all other treatments and butyrate revealed a negative association with liver dysfunction.