Results
Figure 1b shows the change in body weight over the treatment period of eight weeks. Resmetirom had no effect on weight or food intake (figure 1c). Body weight remained stable throughout the treatment period; transient weight loss observed at day 39 in all three groups resulted from the four-hour fasting period before taking blood for glucose determination.
THR-ß agonism had a profound effect on plasma transaminase activities, leading to a reduction by about 50 % for ALT and 26 % for AST compared to DIO-NASH vehicle controls (figures 2a and 2b). Plasma total cholesterol levels that were strongly elevated in DIO-NASH mice compared to lean controls nearly normalized upon treatment with resmetirom (figure 2c). There was a trend towards lower plasma triglycerides that was, however, not statistically significant (p=0.052, figure 2d). Four-hour fasting blood glucose levels determined on treatment day 39 were significantly lower in resmetirom-treated animals (figure 2e) whereas resmetirom had no influence on plasma insulin concentration (figure 2f).
Liver hypertrophy observed in DIO-NASH mice was markedly reduced upon treatment with the THR-ß agonist (figure 3a), accompanied by a strong reduction in liver triglycerides, total cholesterol and hepatic total lipid content (figures 3b-d). Liver col1a1 and α-SMA contents were lower after resmetirom treatment (figures 3e and f). The difference to vehicle-treated DIO-NASH mice remained statistically significant upon normalization to liver weight for α-SMA but not for col1a1 (not shown).
Resmetirom treatment was associated with changes in liver morphology (figure 4a, b) and a significant improvement (post-treatment vs. pre-treatment biopsy) in the histological NAFLD activity score (NAS) relative to DIO-NASH mice not exposed to the drug (figure 4c). Whereas in DIO-NASH controls, two out of eleven mice had a lower NAS after the intervention period, ten out of twelve resmetirom-treated mice showed an improved NAS (figure 4c), in seven cases by more than one point. Fibrosis staging (pre-post) was lower in two out of twelve mice on resmetirom whereas no change was observed in vehicle-treated DIO-NASH control mice (difference not statistically significant, figure 4d).
Results of hepatic gene expression analysis by RNASeq are summarized in figure 5. In a principal component analysis (PCA) of global gene expression, DIO-NASH vehicle-treated animals separated from lean control animals along the first two principal components (PC1) and (PC2),with resmetirom treatment leading to a separation along PC2 towards values observed in lean control mice (figure 5a). Among 53431 transcripts assessed by RNA Sequencing, DIO-NASH vehicle-treated mice demonstrated a >1.5-fold upregulation of 4614 genes (supporting information, figure S1a) in comparison to lean control animals. Only a fraction of these up-regulated genes (112 of 4614) was significantly reversed by treatment with resmetirom (supporting information, figure S1b and table S1). Furthermore, DIO-NASH vehicle-treated mice demonstrated a differential downregulation (>1.5-fold) of 1750 genes in comparison with lean control mice, with 79 of these transcripts being significantly reversed by resmetirom treatment. Notably, pathway analysis confirmed that changes in gene expression upon resmetirom treatment are consistent with THR-β activation (figure 5b). Compared to DIO-NASH vehicle control animals, THR-β target genes such as deiodinase 1 (Dio1), malic enzyme 1 (Me1), cytochrome P450 7A1 (Cyp7a1) and glycerolphosphate dehydrogenase 2 (Gpd2) were found to be strongly upregulated (figure 5c). Lower expression of fibrosis marker genes collagen 1a1 (Col1a1), α-SMA (Acta2), lysyl oxidase-like 2 (Loxl2) and hydroxysteroid 17-beta dehydrogenase 13 (Hsd17b13, figure 5d) indicates a decrease in incident fibrogenesis which is also reflected in lower col1a1 and α-SMA protein levels (figures 3e, f), although not enough to significantly improve the histological fibrosis staging (figure 4d) within the observed treatment period. Other genes that were recently identified as markers of hepatic fibrosis or hepatocellular carcinoma such as Lipopolysaccharide-binding protein (Lbp; Nien, Sheu, et al, 2019), ATP-binding cassette C3 (Abcc3; Carrasco-Torres, Fattel-Fazenda, et al, 2016) and fatty-acid binding protein 4 (Fabp4; Thompson, Austin, et al, 2018) were markedly downregulated upon treatment with resmetirom (figure 4e). Of note, genes related to fatty acid synthesis (Acaca, Acacb, Fasn), lipolysis (Pnpla2, Lipe, Mgll) and fatty acid oxidation (Cpt1a, Pgc1a, Hadhb) were not found to be regulated upon resmetirom treatment compared to vehicle-treated DIO-NASH mice (supporting information, figure S2).