Disclosures: Scott Friedman is a consultant to Galmed Aramchol Downregulates SCD1 and Induces PPARg in Hepatic Stellate Cells to Attenuate Cellular Activation and Fibrogenesis Brittany Allen 1 , Jose M. Mato 2 , Amanda Craig 1 , David Fernandez-Ramos 2 , Fernando Lopitz-Otsoa 2 , Liat Hayardeny 3 , Augusto Villanueva 1 and Scott L Friedman 1 , 1 Division of Liver Diseases, Icahn School of Medicine at Mount Sinai, New York, NY 2 CIC Biogune, Bilbao, Spain 3 Galmed Pharmaceuticals, Tel Aviv, Israel 1. INTRODUCTION • Activation of hepatic stellate cells (HSCs) drives hepatic fibrosis through a process that is inhibited by PPARg signaling. • Aramchol (arachidyl amido cholanoic acid) is a fatty acid-bile acid conjugate that reduces liver fat content in nonalcoholic fatty liver disease (NAFLD) and improved nonalcoholic steatohepatitis (NASH) without worsening fibrosis in a Phase 2b study (Abstract # LB-5). • Aramchol attenuates fibrosis in two distinct models (MCD diet and thioacetamide). In mice, Aramchol reduces liver fat by downregulating the fatty acid synthetic enzyme stearoyl Co-A desaturase 1 (SCD-1) in hepatocytes. • The mechanism for Aramchol’ s antifibrotic effect is not known; moreover, although HSCs also store lipids as retinyl esters. • The role of SCD-1 in HSCs and the impact of Aramchol on SCD-1 activity are unknown. . 3. METHODS • Serum-starved LX2 cells were treated with Aramchol (10 µM) for 24 or 48 hours. Fibrogenic gene expression and SCD-1 protein expression were measured by qPCR and Western, respectively. • RNAseq was performed at 24 and 48 h in duplicate. Differential gene expression was assessed with DSeq2, while gene set enrichment analysis (GSEA) and gene ontology (GO) analyses assessed functionality of gene expression changes. • Primary hepatocytes from C57BL/6 mice were harvested by perfusion and treated with Aramchol (10 µM) for 48 hours in culture. PPAR mRNA expression was measured by qPCR. Figure 1: Aramchol downregulates SCD-1 mRNA and fibrogenic genes, and upregulates PPAR mRNA in HSCs. Based on RT- PCR (N = 3) *p<0.05, **p<0.01, ***p<0.001. 2. AIM • To investigate the direct anti-fibrotic effect of Aramchol on HSCs using LX-2, a human hepatic stellate cell line • To define the mechanism of Aramchol’ s effects on HSCs. 4. RESULTS CONCLUSIONS • Aramchol elevates PPAR mRNA and down regulates SCD-1 mRNA and protein and in hepatic stellate cells. • By RNAseq and pathway analysis, Aramchol downregulates fibrogenic genes that are part of a clinically validated HSC activation signature, including COL1A1 and ⍺SMA, as well as pathways involved in cholesterol biosynthesis & homeostasis, and collagen formation. • Aramchol upregulates PPAR mRNA selectively in HSCs and not in hepatocytes. Figure 5: Aramchol downregulates cholesterol biosynthesis and collagen formation in HSCs. Gene Set Enrichment Analysis of LX-2 cells after 24 (A) or 48 (B) hours of treatment. Funding support: Galmed Pharmeceuticals Vehicle 10 uM 0.0 0.5 1.0 1.5 aSMA Relative Gene Expression *** Vehicle 10 uM 0.0 0.5 1.0 1.5 COL1A1 Relative Gene Expression **** Veh icle 10 uM 0.0 0.5 1.0 1.5 b-PDGFR Relative Gene Expression *** Vehicle 10 uM 0.0 0.5 1.0 1.5 MMP-2 Relative Gene Expression *** Veh i cle 10 uM 0 2 4 6 8 10 PPARg Relative Gene Expression *** Veh i cle 10 uM 0.0 0.5 1.0 1.5 SCD1 Relative Gene Expression ** Vehicle 10 uM 0.0 0.5 1.0 1.5 aSMA Relative Gene Expression *** Vehicle 10 u M 0.0 0.5 1.0 1.5 MMP-2 Relative Gene Expression * Vehicle 1 0 uM 0.0 0.5 1.0 1.5 COL1A1 Relative Gene Expression Vehicle 10 uM 0 2 4 6 8 PPARg Relative Gene Expression **** Veh icle 10 uM 0.0 0.5 1.0 1.5 b-PDGFR Relative Gene Expression *** Veh icle 10 u M 0.0 0.5 1.0 1.5 SCD1 Relative Gene Expression B A Figure 2: Aramchol downregulates SCD-1 in HSCs. Western Blot (A) and densitometry (B) of downregulated SCD-1 protein in HSCs treated with Aramchol for 24 or 48 hrs. (N=3) *p<0.05, **p<0.01, ***p<0.001 Figure 3: RNASeq confirms downregulation of fibrogenic genes and SCD-1 mRNA, and upregulation of PPAR mRNA after 48 hrs of treatment with Aramchol. The cholesterol efflux regulatory protein, ABCA1, is also upregulated. Figure 4: Aramchol does not affect PPAR mRNA expression in primary mouse hepatocytes after 48 hours of treatment. (N=3) *p<0.05, **p<0.01, ***p<0.001 V V V 10 10 10 V V V 10 10 10 24 hours 48 hours SCD-1 GAPDH A B Vehicle 10 µM 0 50 100 150 24 Hours Relative Protein Expression (% of vehicle control) * Vehicle 10 µM 0 50 100 150 48 Hours Relative Protein Expression (% of vehicle control) * A B Vehi c le 10 µ M Aramchol 0.0 0.5 1.0 1.5 2.0 PPARg Relative Gene Expression