Dual targeting of hepatocyte DGAT2 and stellate cell FASN alleviates nonalcoholic steatohepatitis in mice

Abstract

Nonalcoholic steatohepatitis (NASH) is a malady of multiple cell types associated with hepatocyte triglyceride (TG) accumulation, macrophage inflammation, and stellate cell-induced fibrosis, with no approved therapeutics yet available. Here, we report that stellate cell fatty acid synthase (FASN) in de novo lipogenesis drives the autophagic flux that is required for stellate cell activation and fibrotic collagen production. Further, we employ a dual targeting approach to NASH that selectively depletes collagen through selective stellate cell knockout of FASN (using AAV9-LRAT Cre in FASN^fl/fl mice), while lowering hepatocyte triglyceride by depleting DGAT2 with a GalNac-conjugated, fully chemically modified siRNA. DGAT2 silencing in hepatocytes alone or in combination with stellate cell FASNKO reduced liver TG accumulation in a choline-deficient NASH mouse model, while FASNKO in hepatocytes alone (using AAV8-TBG Cre in FASN^fl/fl mice) did not. Neither hepatocyte DGAT2 silencing alone nor FASNKO in stellate cells alone decreased fibrosis (total collagen), while loss of both DGAT2 plus FASN caused a highly significant attenuation of NASH. These data establish proof of concept that dual targeting of DGAT2 plus FASN alleviates NASH progression in mice far greater than targeting either gene product alone.

Figure 1:. FASN inhibition blunts human stellate cell activation and collagen production.

LX2 human stellate cell line was plated in 6 well plates and serum-starved overnight. After 4-hour treatment with 1nM or 100 nM FASN inhibitor (TVB-3664), stellate cell activation was initiated with the addition of recombinant human Tgf-β (5ng/ml final concentration). (A) Cell morphology difference upon FASN inhibition (B) Protein levels of collagen type-1 and phosphorylated Smad3 (C) Fibrotic gene expression changes (D) Oxidative stress and mitochondrial function related gene expression changes (ns: Not significant, *: p<0.05, **:p<0.005, ***:p<0.0005, ****:p<0.00005)

Figure 2:. Human FASN silencing blunts the activation of cultured human LX2 cells in association with inhibition of autophagic flux

(A) Representative cartoon of the chemically modified, cholesterol-conjugated siRNAs that were used for in vitro screenings. (B) LX2 human stellate cells were treated with siRNA compounds (1.5 µM) for 48 hrs prior to analysis of FASN mRNA levels to identify the most potent siRNA sequences (C) Dose-response relationships for the lead siRNAs that showed the strongest silencing. The IC50 values were determined by using 8-point serially diluted concentrations of the compounds starting from 1.5 µM. (D) TEM images, highlighting autolysosomes (black arrow) and autophagosomes (red arrows) of stellate cells which are Untreated, Tgf-β plus NTC treated, and Tgf-β plus cholesterol conjugated FASN-2314 treated (E) Immunoblotting analysis of fibrotic and autophagic pathway markers in stellate cells in a representative experiment with 3 replicates. (ns: Not significant, *: p<0.05, **:p<0.005, ***:p<0.0005, ****:p<0.00005)

Figure 3:. Stellate cell-specific deletion of FASN, in combination with DGAT2 silencing, ameliorates liver stiffness and plasma markers of NASH in CDAHFD-fed mice.

(A)10-week-old male Fasn fl/fl mice were injected with corresponding AAVs to obtain hepatocyte-specific KO (AAV8-TBG-Cre) and/or stellate cell-specific KO (AAV9-Lrat-Cre) via IV injection. Additionally, for the indicated groups, sdDgat2 (10mg/kg) was injected via a single subcutaneous injection. After one week of injections, mice were put on CDAHFD for 7 weeks. (B) Liver stiffness measurements by shear-wave elastography (C) Plasma ALT activity (D) Plasma total bilirubin (E) Plasma TNF alpha and (F) Plasma MCP1 protein levels. (ns: Not significant, *: p<0.05, **:p<0.005, ***:p<0.0005, ****:p<0.00005)

Figure 4:. Stellate cell-specific deletion of FASN, in combination with DGAT2 silencing, prevents NASH in CDAHFD-fed mice.

(A)mRNA levels of Dgat2 and Fasn (B) Total liver triglyceride measurement (C) Immunohistochemical analysis of type 1 collagen (D) Quantification of type 1 collagen positive area in IHC (E) Total collagen measurement via OH-Pro assay (F) Liver weights (ns: Not significant, *: p<0.05, **:p<0.005, ***:p<0.0005, ****:p<0.00005)