-1,61* 3,72* 2,29* 2,06* 1,83* -2,0 -1,0 0,0 1,0 2,0 3,0 4,0 Insulin induces de novo lipogenesis in human skin stem cell-derived hepatic cells Introduction Methods Results Conclusion Joost Boeckmans, Alessandra Natale, Karolien Buyl, Joery De Kock, Vera Rogiers, Robim M Rodrigues * and Tamara Vanhaecke * Department of In Vitro Toxicology & Dermato-Cosmetology (IVTD). Vrije Universiteit Brussel, Faculty of Medicine and Pharmacy, Laarbeeklaan 103, 1090 Brussels, Belgium. * Equally contributing senior authors. Non-alcoholic fatty liver disease (NAFLD) ranges from simple steatosis to hepatocellular carcinoma. Among 25% of the global population suffers from NAFLD. A strong correlation with the metabolic syndrome (insulin resistance, obesity, dyslipidemia...) is observed. Due to interspecies differences and ethical concerns, the use of laboratory animals to investigate this disorder is discouraged. Therefore, human-relevant in vitro NAFLD models are urgently needed by the pharmaceutical industry. Postnatal human skin precursor cells, differentiated towards hepatic cells (hSKP-HPC), proved already their potential to mimic in vivo toxicological responses to steatosis-inducing drugs. Here, we investigate whether insulin is able to induce triglyceride accumulation in hSKP-HPCs and mimic hepatic steatosis as observed in subjects suffering from the metabolic syndrome. The mechanisms by which insulin influences intracellular triglyceride accumulation are summarized in Figure 1. hSKP-HPCs were generated according to an earlier established in-house protocol [1]. Subsequently, the cells were exposed to 100 nM insulin for 24h. The expression of lipogenic (ACC1, FASN, PPAR-γ, SCD1 and SREBP-1c) and lipid metabolism-related genes (APOB, CD36, ACADSB, CPT1a, PPAR-α, DGAT1, DGAT2 and GPAT1) was evaluated by RT-qPCR. Additionally, 72h exposure was carried out to assess intracellular lipid accumulation. Hereto, the cells were stained with LipidTOX green for neutral lipids and examined by fluorescence microscopy . CPT1a Malonyl-CoA Acetyl-CoA Palmitic Acid Palmitoeic acid Glycerol-3-P GPAT 1-4 AGPAT 1 LPIN DGAT1 DGAT2 LXRα ChREBP Acetyl-CoA KREBS CYCLE FFA in blood stream SREBP-1c INSULIN FFA ACC1 Stearic Acid Pyruvate GLYCOLYSIS FAS SCD1 FAT (CD36) Oleic Acid SCD1 VLDL in blood stream APOB SCD1 FASN ACC1 DGAT2 APOB Control 100 nM insulin Increased intracellular lipid load was confirmed by fluorescence microscopy (Fig. 2). Upregulation of three de novo lipogenic genes (ACC1, FASN and SCD1), upregulation of DGAT2 which is responsible for the final esterification step and decreased triglyceride export by VLDL (very low-density lipoprotein) due to downregulated APOB (Fig. 3). Other tested genes (PPAR-γ, SREBP-1c, CD36, ACADSB, CPT1a, PPAR-α, DGAT1 and GPAT1) were not significantly modulated. * Student t-test; p < 0.05 Contact : Joost Boeckmans E: [email protected] T: 02/477.45.20 Reference: [1] R. M. Rodrigues et al., “In vitro assessment of drug-induced liver steatosis based on human dermal Sponsors: stem cell-derived hepatic cells.” Arch. Toxicol., vol. 90, no. 3, pp. 677–689, 2016. Insulin induces intracellular triglyceride accumulation in hSKP-HPCs by increasing de novo lipogenesis, increasing triglyceride esterification and decreasing triglyceride export Therefore, hSKP-HPCs may serve as a promising human-relevant in vitro model to study hepatic lipid-metabolism related disorders 72h ACADSB Figure 1 : Influence of insulin on intracellular hepatic triglyceride accumulation Figure 2 : hSKP-HPCs stained for neutral lipids Figure 3 : Gene expression modulation (fold change vs. control; n = 3)