Background <p>In chronic diseases, hepatocytes may dedifferentiate and form a liver progenitor cell (LPC)-like population in response to long-term stress stimuli. We investigated the hypothesis that hepatocytes may dedifferentiate upon chronic high insulin stimulation and subsequently form adipocytes and/or fibroblast-like cells as a result of long-term fatty acid exposure.</p> Methods <p>HepG2 cells were treated with prolonged high insulin followed by oleic acid (OA). The expression level of LPC, adipocyte and fibroblast marker genes was measured. Populations of cells carrying the LPC marker CD34, the fibroadipogenic progenitor cell marker PDGFR1α and the activated fibroblast marker FAPα were detected. Lipid accumulation and the existence of the adipocyte marker perilipin-A were shown. The relevant molecular mechanism was investigated by applying specific inhibitors and determining related protein levels.</p> Results <p>With high insulin exposure, the number of CD34⁺ or PDGFR1α⁺ cells and the gene expression levels of LPC markers increased, whereas the gene expression of hepatocyte markers decreased. Lipid accumulation, adipogenesis and adipocyte marker gene expression levels and the density of Perilipin-A increased in cells treated with OA following high insulin. On the other hand, in cells treated with OA alone or OA following insulin, the expression levels of fibroblast marker genes and the FAPα⁺ cell population were increased. TLR4 and GSK3β inhibition reduced lipid accumulation whereas TLR4 and β-catenin prevented the increase in the FAPα⁺ cell population. Additionally, GSK3β and β-catenin levels increased in the nucleus in cells exposed to OA following high insulin.</p> Conclusion <p>Long-term high insulin stimulation is driving dedifferentiation of hepatocytes and causes the formation of fibroadipogenic progenitor cells. Long-term exposure of these cells to high fatty acids leads to adipogenesis, mainly via TLR4/GSK3β and fibrogenesis, via TLR4/β-catenin pathways.</p> Graphical abstract <p>The findings and conclusions obtained from the present study are summarized below. Molecular and morphological changes detected in hepatocytes under (A) chronic OA stimulation, (B) chronic OA stimulation following chronic low-level insulin, and (C) chronic OA stimulation following chronic high-level insulin. (D) Signaling pathways in adipocyte-like and fibroblast-like cells formed by chronic OA stimulation following chronic high-level insulin administration.</p>