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Non-alcoholic fatty liver disease (NAFLD) is a common liver lesion that is untreatable with medications. Glucagon-like peptide-1 receptor (GLP-1R) agonists have recently emerged as a potential NAFLD pharmacotherapy. However, the molecular mechanisms underlying these drugs’ beneficial effects are not fully understood. Using Fourier transform infrared (FTIR) spectroscopy, we sought to investigate the biochemical changes in a steatosis cell model treated or not with the GLP-1R agonist Exendin-4 (Ex-4). HepG2 cells were made steatotic with 400 µM of oleic acid and then treated with 200 nM Ex-4 in order to reduce lipid accumulation. We quantified steatosis using the Oil Red O staining method. We investigated the biochemical alterations induced by steatosis and Ex-4 treatment using Fourier transform infrared (FTIR) spectroscopy and chemometric analyses. Analysis of the Oil Red O staining showed that Ex-4 significantly reduces steatosis. This reduction was confirmed by FTIR analysis, as the phospholipid band (C=O) at 1740 cm−1 in Ex-4 treated cells is significantly decreased compared to steatotic cells. The principal component analysis score plots for both the lipid and protein regions showed that the untreated and Ex-4-treated samples, while still separated, are clustered close to each other, far from the steatotic cells. The biochemical and structural changes induced by OA-induced lipotoxicity are at least partially reversed upon Ex-4 treatment. FTIR and chemometric analyses revealed that Ex-4 significantly reduces OA-induced lipid accumulation, and Ex-4 also restored the lipid and protein biochemical alterations caused by lipotoxicity-induced oxidative stress. In combination with chemometric analyses, FTIR spectroscopy may offer new approaches for investigating the mechanisms underpinning NAFLD.

No therapy exists for non-alcoholic fatty liver disease (NAFLD). However, glucagon-like peptide receptor agonists (GLP-1RAs) showed a beneficial effect on NAFLD, although the underpinning mechanisms remain unclear due to their pleiotropic effects. We examined the implicated signaling pathways using comparative transcriptomics in a cell model of steatosis to overcome pleiotropy. We treated steatotic HepG2 cells with the GLP-1RA Exendin-4 (Ex-4). We compared the transcriptome profiles of untreated steatotic, and Ex-4-treated steatotic cells, and used Ingenuity Pathway Analysis (IPA) to identify the signaling pathways and associated genes involved in the protective effect of Ex-4. Ex-4 treatment significantly reduces steatosis. RNA-seq analysis revealed 209 differentially expressed genes (DEGs) between steatotic and untreated cells, with farnesoid X receptor/retinoid X receptor (FXR/RXR) (p = 8.9 × 10−7) activation being the top regulated canonical pathway identified by IPA. Furthermore, 1644 DEGs were identified between steatotic cells and Ex-4-treated cells, with liver X receptor/retinoid X receptor (LXR/RXR) (p = 2.02 × 10−7) and FXR/RXR (p = 3.28 × 10−7) activation being the two top canonical pathways. The top molecular and cellular functions between untreated and steatotic cells were lipid metabolism, molecular transport, and small molecular biochemistry, while organismal injury and abnormalities, endocrine system disorders, and gastrointestinal disease were the top three molecular and cellular functions between Ex-4-treated and steatotic cells. Genes overlapping steatotic cells and Ex-4-treated cells were associated with several lipid metabolism processes. Unique transcriptomic differences exist between steatotic cells and Ex-4-treated steatotic cells, providing an important resource for understanding the mechanisms that underpin the protective effect of GLP-1RAs on NAFLD and for the identification of novel therapeutic targets for NAFLD.