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  • ARCA Cy5 EGFP mRNA Hepatic inflammation and fibrosis are hal

    2023-01-31

    Hepatic ARCA Cy5 EGFP mRNA and fibrosis are hallmarks of the NASH phenotype and are thought to occur in a subset of patients with NAFLD. While it is hypothesized that therapeutic lowering of hepatic lipids will be sufficient to improve hepatic fibrosis and inflammation secondarily, this remains controversial and requires more evidence. Here we show that the hepatic actions of PF-06409577, presumably via the large reductions in hepatic lipids, are sufficient to reduce molecular indications of hepatic inflammation and fibrosis in a model with developed disease. While it is important to recognize the relatively mild fibrotic and inflammatory liver phenotype of the high fat diet fed DIO mouse, these data are encouraging and suggest that reduced hepatic lipid content can indeed impact this non-hepatocyte cell derived structural liver phenotype secondarily. More work is required to demonstrate this in models of NASH that have more significant degrees of fibrosis. In vivo, where PF-06409577 was present in the plasma at 20nM 4h after being dosed, the compounds effects on hepatic triglycerides and cholesterol levels was completely dependent on the presence of hepatocyte AMPK. In contrast, control and HepKO hepatocytes treated with PF-06409577 at 10μM were still capable of significantly lowering acetate incorporation into non-saponifiable lipids, a measure of cholesterol synthesis. From the summation of this work we conclude that the in vivo actions of PF-06409577 are dependent on liver AMPK, but it is possible that some off-target effects may impact cholesterol synthesis at high concentrations.
    Conflicts of Interest
    Author Contributions RME designed and performed mouse studies. CTS designed and performed rat studies. JD and YS performed hepatocyte studies. BA, EC, MP, and EB performed in vivo studies. AR and JW designed and performed in vitro pharmacology studies. RM performed bioinformatics studies of RNAseq data. MM, CN, and KFG designed and performed proteomic studies. JB performed measures of mevaolnic acid. BS, EAD, and GRS designed and performed studies in ACC DKI hepatocytes. JMK and AO performed histological analyses. ASK and DT evaluated drug metabolism. LB and NS designed and evaluated primate studes. TFO provided key reagents. MJB, KOC, and RAM designed and conceived of studies. RAM wrote the manuscript. All authors provided feedback and edited the manuscript. A. Phosphorylation of ACC1 S79 and ACC2 S212 (rat) or ACC2 S221 (monkey, human) in rat, monkey and human hepatocytes with increasing amounts of PF-06409577 was quantified by plate based MesoScale Diagnostics (MSD) ELISA-like assays. B. 14C-acetate incorporation into lipids of rat, monkey and human hepatocytes with increasing concentrations of PF-06409577. Assay points are in duplicate and a representative of three experiments is shown. A. Immunoblots for phospho-AMPK, phospho-ACC, total AMPK, total ACC and β-actin from primary hepatocytes isolated derived from wild-type and liver-specific AMPK deficient (HepKO) mice. Mouse primary hepatocytes were treated with vehicle (DMSO) and PF-06409577 (10μM) for 1h. 3H-acetate incorporation into (B) saponified and (C) non-sapanofied lipid fractions within primary hepatocytes derived from wild-type and AMPK HepKO mice. D. 3H-acetate incorporation into fatty acid fraction within primary hepatocytes derived from wild-type and ACC-DKI mice. E. 14C-acetate incorporation into hepatic lipids in rats that were orally dosed with vehicle or PF-06409577. 7–8 animals were used in each dose group. F. Plasma mevalonic acid production from rats that were orally dosed with vehicle, PF-06409577 or rosuvastatin. 8–10 animals were used in each dose group. 1-way ANOVA was used to evaluate statistical significance, where * denotes p<0.05, ** denotes p<0.01, *** denotes p<0.001, and **** denotes p<0.0001. A. Immunoblots for phospho-AMPK, phospho-ACC, phosho-SREBP-1, total AMPK, total ACC, precursor form of SREBP-1 and β-actin from liver lysates of control (GFP) and liver-specific AMPK deficient (HepKO) mice that were orally dosed vehicle or PF-06409577 (100mg/kg) 4h after dosing (acute study). B. Normalized phospho-AMPK levels in GFP mice dosed vehicle or PF-06409577 (PF-577) from single dose acute study measured by MSD assay. C. Normalized phospho-ACC levels in GFP and HepKO dosed vehicle or PF-06409577 from single dose acute study measured by MSD assay. D. Normalized phospho-AMPK levels in GFP mice dosed vehicle or PF-06409577 from 42day chronic study measured by MSD assay. E. Normalized phospho-ACC levels in GFP and HepKO dosed vehicle or PF-06409577 from 42day chronic study. 8–11 animals were in each dose group in the studies measured by MSD assay. F. Liver triglycerides of GFP and HepKO mice chronically dosed vehicle or PF-06409577 (100mg/kg) from 42day chronic study. G. Liver cholesterol of GFP and HepKO mice dosed vehicle or PF-06409577 (100mg/kg) from 42day chronic study. H. Plasma ALT levels of GFP and HepKO mice from 42day chronic study. (I) Plasma triglyceride,(J) plasma cholesterol and (K) plasma β-hydroxybutyrate of GFP and HepKO mice from 42day chronic study. L. Plasma β-hydroxybutyrate of mice from acute study. 10–11 animals were in each dose group. 1-way ANOVA was used to evaluate statistical significance, where * denotes p<0.05, ** denotes p<0.01, *** denotes p<0.001, and **** denotes p<0.0001.