Insulin determines the effects of TGF-beta on HNF4alpha transcription and epithelial-to-mesenchymal transition in hepatocytes

To date, epithelial-to-mesenchymal transition (EMT) has been observed in cultured hepatocytes, but not in vivo. TGF-beta is supposed to initiate EMT in hepatocytes by inhibiting the hepatic master transcription factor HNF4alpha through the SMAD2/3 complex. However, we observe that the SMAD2/3 complex is required for HNF4alpha transcription. Besides SMAD2/3, C/EBPalpha is also essential for constitutive HNF4alpha expression in hepatocytes. In contrast to upregulating HNF4alpha transcription, SMAD2/3 represses C/EBPalpha transcription. Therefore, long-term TGF-beta incubation results in C/EBPalpha depletion, which inhibits HNF4alpha expression. Impressively, SMAD2/3 binding to the CEBPA promoter is inhibited by insulin. Maintaining a high insulin concentration in culture medium completely inhibits TGF-beta-induced hepatocyte EMT. Insulin inhibits TGF-beta-induced SMAD2/3 binding to the promoters of core EMT transcription factors e.g., SNAI1. SNAI1 transcription requires both SMAD2/3 and FOXO1 in nuclei. Insulin inhibits SNAI1 transcription through impeding SMAD2/3 binding to its promoter and inducing FOXO1 phosphorylation. Hence, insulin is the key factor that prevents TGF-beta-induced EMT in hepatocytes. ### Competing Interest Statement The authors have declared no competing interest.