Reproducible lung protective effects of a TGFßR1/ALK5 inhibitor in a bleomycin-induced and spirometry-confirmed model of IPF in male mice.

This study comprehensively validated the bleomycin (BLEO) induced mouse model of IPF for utility in preclinical drug discovery. To this end, the model was rigorously evaluated for reproducible phenotype and TGFß-directed treatment outcomes. Lung disease was profiled longitudinally in male C57BL6/JRJ mice receiving a single intratracheal instillation of BLEO (n = 10-12 per group). A TGFßR1/ALK5 inhibitor (ALK5i) was profiled in six independent studies in BLEO-IPF mice, randomized/stratified to treatment according to baseline body weight and non-invasive whole-body plethysmography. ALK5i (60 mg/kg/day) or vehicle (n = 10-16 per study) was administered orally for 21 days, starting 7 days after intratracheal BLEO installation. BLEO-IPF mice recapitulated functional, histological and biochemical hallmarks of IPF, including declining expiratory/inspiratory capacity and inflammatory and fibrotic lung injury accompanied by markedly elevated TGFß levels in bronchoalveolar lavage fluid and lung tissue. Pulmonary transcriptome signatures of inflammation and fibrosis in BLEO-IPF mice were comparable to reported data in IPF patients. ALK5i promoted reproducible and robust therapeutic outcomes on lung functional, biochemical and histological endpoints in BLEO-IPF mice. The robust lung fibrotic disease phenotype, along with the consistent and reproducible lung protective effects of ALK5i treatment, makes the spirometry-confirmed BLEO-IPF mouse model highly applicable for profiling novel drug candidates for IPF.

Characterization of six clinical drugs and dietary intervention in the non-obese CDAA-HFD mouse model of MASH and progressive fibrosis.

The choline-deficient L-amino acid defined-high fat diet (CDAA-HFD) mouse model is widely used in preclinical metabolic dysfunction-associated steatohepatitis (MASH) research. To validate the CDAA-HFD mouse, we evaluated disease progression and responsiveness to dietary and pharmacological interventions with semaglutide, lanifibranor, elafibranor, obeticholic acid (OCA), firsocostat and resmetirom.Disease phenotyping was performed in C57BL/6J mice fed CDAA-HFD for 3-20 weeks and ranked using the MASLD Human Proximity Score (MHPS). Semaglutide, lanifibranor, elafibranor, OCA, firsocostat or resmetirom were profiled as treatment intervention for 8 weeks, starting after 6 weeks of CDAA-HFD feeding. Semaglutide and lanifibranor were further evaluated as early (preventive) therapy for 9 weeks, starting 3 weeks after CDAA-HFD diet feeding. Additionally, benefits of dietary intervention (chow reversal) for 8 weeks were characterized following 6 weeks of CDAA-HFD feeding. CDAA-HFD mice demonstrated a non-obese phenotype with fast onset and progression of MASH and fibrosis, high similarity to human MASH-fibrosis, and tumor development after 20 weeks of diet-induction. Semaglutide and lanifibranor partially reversed fibrosis when administered as prevention, but not as treatment intervention. Elafibranor was the only interventional drug to improve fibrosis. In comparison, chow-reversal resulted in complete steatosis regression with improved liver inflammation and fibrosis in CDAA-HFD mice. CDAA-HFD mice recapitulate histological hallmarks of advanced MASH with progressive severe fibrosis, however, in the absence of a clinical translational obese dysmetabolic phenotype. CDAA-HFD mice are suitable for profiling drug candidates directly targeting hepatic lipid metabolism, inflammation, and fibrosis. The timing of pharmacological intervention is critical for determining antifibrotic drug efficacy in the model.

Paraoxonase-2 agonist vutiglabridin promotes autophagy activation and mitochondrial function to alleviate non-alcoholic steatohepatitis.

BACKGROUND AND PURPOSE: Only limited therapeutic agents have been developed for non-alcoholic steatohepatitis (NASH). Glabridin, a promising anti-obesity candidate, has only limited druggability due to its low in vivo chemical stability and bioavailability. Therefore, we developed vutiglabridin (VUTI), which is based on a glabridin backbone, and investigated its mechanism of action in treating NASH in animal models. EXPERIMENTAL APPROACH: Anti-NASH effects of VUTI were determined in in vitro fatty liver models, spheroids of primary human hepatocytes and L02 normal liver cell lines. To identify VUTI possible cellular target/s, biotin-labelled VUTI was synthesized and underwent chemical proteomic analysis. Further, the evaluation of VUTI therapeutic efficacy was carried out using an amylin-NASH and high-fat (HF) diet-induced obese (DIO) mouse models. This was carried out using transcriptomic, lipidomic and proteomic analyses of the livers from the amylin-NASH mouse model. KEY RESULTS: VUTI treatment markedly reduces hepatic steatosis, fibrosis and inflammation by promoting lipid catabolism, activating autophagy and improving mitochondrial dysfunction, all of which are hallmarks of effective NASH treatment. The cellular target of VUTI was identified as paraoxonase 2 (PON2), a newly proposed protein target for the treatment of NASH, VUTI enhanced PON2 activity. The results using PON2 knockdown cells demonstrated that PON2 is important for VUTI- activation of autophagy, promoting mitochondrial function, decreasing oxidative stress and alleviating lipid accumulation under lipotoxic condition. CONCLUSION AND IMPLICATIONS: Our data demonstrated that VUTI is a promising therapeutic for NASH. Targeting PON2 may be important for improving liver function in various immune-metabolic diseases including NASH.

An unbiased ranking of murine dietary models based on their proximity to human metabolic dysfunction-associated steatotic liver disease (MASLD).

Metabolic dysfunction-associated steatotic liver disease (MASLD), previously known as non-alcoholic fatty liver disease, encompasses steatosis and metabolic dysfunction-associated steatohepatitis (MASH), leading to cirrhosis and hepatocellular carcinoma. Preclinical MASLD research is mainly performed in rodents; however, the model that best recapitulates human disease is yet to be defined. We conducted a wide-ranging retrospective review (metabolic phenotype, liver histopathology, transcriptome benchmarked against humans) of murine models (mostly male) and ranked them using an unbiased MASLD 'human proximity score' to define their metabolic relevance and ability to induce MASH-fibrosis. Here, we show that Western diets align closely with human MASH; high cholesterol content, extended study duration and/or genetic manipulation of disease-promoting pathways are required to intensify liver damage and accelerate significant (F2+) fibrosis development. Choline-deficient models rapidly induce MASH-fibrosis while showing relatively poor translatability. Our ranking of commonly used MASLD models, based on their proximity to human MASLD, helps with the selection of appropriate in vivo models to accelerate preclinical research.

Increased hepatoprotective effects of the novel farnesoid X receptor agonist INT-787 versus obeticholic acid in a mouse model of nonalcoholic steatohepatitis.

The nuclear farnesoid X receptor (FXR), a master regulator of bile acid and metabolic homeostasis, is a key target for treatment of nonalcoholic steatohepatitis (NASH). This study compared efficacy of FXR agonists obeticholic acid (OCA) and INT-787 by liver histopathology, plasma biomarkers of liver damage, and hepatic gene expression profiles in the Amylin liver NASH (AMLN) diet-induced and biopsy-confirmed Lepob/ob mouse model of NASH. Lepob/ob mice were fed the AMLN diet for 12 weeks before liver biopsy and subsequent treatment with vehicle, OCA, or INT-787 for 8 weeks. Hepatic steatosis, inflammation, and fibrosis (liver lipids, galectin-3, and collagen 1a1 [Col1a1], respectively), as well as plasma alanine transaminase (ALT) and aspartate transaminase (AST) levels, were assessed. Hepatic gene expression was assessed in Lepob/ob mice that were fed the AMLN diet for 14 weeks then treated with vehicle, OCA, or INT-787 for 2 weeks. INT-787, which is equipotent to OCA but more hydrophilic, significantly reduced liver lipids, galectin-3, and Col1a1 compared with vehicle, and to a greater extent than OCA. INT-787 significantly reduced plasma ALT and AST levels, whereas OCA did not. INT-787 modulated a substantially greater number of genes associated with FXR signaling, lipid metabolism, and stellate cell activation relative to OCA in hepatic tissue. These findings demonstrate greater efficacy of INT-787 treatment compared with OCA in improving liver histopathology, decreasing liver enzyme levels, and enhancing gene regulation, suggesting superior clinical potential of INT-787 for the treatment of NASH and other chronic liver diseases.

Dietary intervention reverses molecular markers of hepatocellular senescence in the GAN diet-induced obese and biopsy-confirmed mouse model of NASH.

BACKGROUND: Hepatocellular senescence may be a causal factor in the development and progression of non-alcoholic steatohepatitis (NASH). The most effective currently available treatment for NASH is lifestyle intervention, including dietary modification. This study aimed to evaluate the effects of dietary intervention on hallmarks of NASH and molecular signatures of hepatocellular senescence in the Gubra-Amylin NASH (GAN) diet-induced obese (DIO) and biopsy-confirmed mouse model of NASH. METHODS: GAN DIO-NASH mice with liver biopsy-confirmed NASH and fibrosis received dietary intervention by switching to chow feeding (chow reversal) for 8, 16 or 24 weeks. Untreated GAN DIO-NASH mice and chow-fed C57BL/6J mice served as controls. Pre-to-post liver biopsy histology was performed for within-subject evaluation of NAFLD Activity Score and fibrosis stage. Terminal endpoints included blood/liver biochemistry, quantitative liver histology, mitochondrial respiration and RNA sequencing. RESULTS: Chow-reversal promoted substantial benefits on metabolic outcomes and liver histology, as demonstrated by robust weight loss, complete resolution of hepatomegaly, hypercholesterolemia, elevated transaminase levels and hepatic steatosis in addition to attenuation of inflammatory markers. Notably, all DIO-NASH mice demonstrated ≥ 2 point significant improvement in NAFLD Activity Score following dietary intervention. While not improving fibrosis stage, chow-reversal reduced quantitative fibrosis markers (PSR, collagen 1a1, α-SMA), concurrent with improved liver mitochondrial respiration, complete reversal of p21 overexpression, lowered γ-H2AX levels and widespread suppression of gene expression markers of hepatocellular senescence. CONCLUSIONS: Dietary intervention (chow reversal) substantially improves metabolic, biochemical and histological hallmarks of NASH and fibrosis in GAN DIO-NASH mice. These benefits were reflected by progressive clearance of senescent hepatocellular cells, making the model suitable for profiling potential senotherapeutics in preclinical drug discovery for NASH.

Evaluation of long acting GLP1R/GCGR agonist in a DIO and biopsy-confirmed mouse model of NASH suggest a beneficial role of GLP-1/glucagon agonism in NASH patients.

OBJECTIVE: The metabolic benefits of GLP-1 receptor (GLP-1R) agonists on glycemic and weight control are well established as therapy for type 2 diabetes and obesity. Glucagon's ability to increase energy expenditure is well described, and the combination of these mechanisms-of-actions has the potential to further lower hepatic steatosis in metabolic disorders and could therefore be attractive for the treatment for non-alcoholic steatohepatitis (NASH). Here, we have investigated the effects of a dual GLP-1/glucagon receptor agonist NN1177 on hepatic steatosis, fibrosis, and inflammation in a preclinical mouse model of NASH. Having observed strong effects on body weight loss in a pilot study with NN1177, we hypothesized that direct engagement of the hepatic glucagon receptor (GCGR) would result in a superior effect on steatosis and other liver related parameters as compared to the GLP-1R agonist semaglutide at equal body weight. METHODS: Male C57Bl/6 mice were fed a diet high in trans-fat, fructose, and cholesterol (Diet-Induced Obese (DIO)-NASH) for 36 weeks. Following randomization based on the degree of fibrosis at baseline, mice were treated once daily with subcutaneous administration of a vehicle or three different doses of NN1177 or semaglutide for 8 weeks. Hepatic steatosis, inflammation and fibrosis were assessed by immunohistochemistry and morphometric analyses. Plasma levels of lipids and liver enzymes were determined, and hepatic gene expression was analyzed by RNA sequencing. RESULTS: NN1177 dose-dependently reduced body weight up to 22% compared to vehicle treatment. Plasma levels of ALT, a measure of liver injury, were reduced in all treatment groups with body weight loss. The dual agonist reduced hepatic steatosis to a greater extent than semaglutide at equal body weight loss, as demonstrated by three independent methods. Both the co-agonist and semaglutide significantly decreased histological markers of inflammation such as CD11b and Galectin-3, in addition to markers of hepatic stellate activation (αSMA) and fibrosis (Collagen I). Interestingly, the maximal beneficial effects on above mentioned clinically relevant endpoints of NN1177 treatment on hepatic health appear to be achieved with the middle dose tested. Administering the highest dose resulted in a further reduction of liver fat and accompanied by a massive induction in genes involved in oxidative phosphorylation and resulted in exaggerated body weight loss and a downregulation of a module of co-expressed genes involved in steroid hormone biology, bile secretion, and retinol and linoleic acid metabolism that are also downregulated due to NASH itself. CONCLUSIONS: These results indicate that, in a setting of overnutrition, the liver health benefits of activating the fasting-related metabolic pathways controlled by the glucagon receptor displays a bell-shaped curve. This observation is of interest to the scientific community, due to the high number of ongoing clinical trials attempting to leverage the positive effects of glucagon biology to improve metabolic health.

Semaglutide reduces tumor burden in the GAN diet-induced obese and biopsy-confirmed mouse model of NASH-HCC with advanced fibrosis.

Non-alcoholic steatohepatitis (NASH) is emerging as a major cause of hepatocellular carcinoma (HCC), however, it is not resolved if compounds in late-stage clinical development for NASH may have additional therapeutic benefits in NASH-driven HCC (NASH-HCC). Here, we profiled monotherapy with semaglutide (glucagon-like-receptor-1 receptor agonist) and lanifibranor (pan-peroxisome proliferator-activated receptor agonist) in a diet-induced obese (DIO) mouse model of NASH-HCC. Disease progression was characterized in male C57BL/6 J mice fed the GAN (Gubra Amylin NASH) diet high in fat, fructose and cholesterol for 12-72 weeks (n = 15 per group). Other GAN DIO-NASH-HCC mice fed the GAN diet for 54 weeks and with biopsy-confirmed NASH (NAFLD Activity Score ≥ 5) and advanced fibrosis (stage F3) received vehicle (n = 16), semaglutide (30 nmol/kg, s.c., n = 15), or lanifibranor (30 mg/kg, p.o., n = 15) once daily for 14 weeks. GAN DIO-NASH-HCC mice demonstrated progressive NASH, fibrosis and HCC burden. Tumors presented with histological and molecular signatures of poor prognostic HCC. Consistent with clinical trial outcomes in NASH patients, both lanifibranor and semaglutide improved NASH while only lanifibranor reduced fibrosis in GAN DIO-NASH-HCC mice. Notably, only semaglutide reduced tumor burden in GAN DIO-NASH-HCC mice. In conclusion, the GAN DIO-NASH-HCC mouse is a clinical translational model of NASH-HCC. Semaglutide improves both NASH and tumor burden in GAN DIO-NASH-HCC mice, highlighting the suitability of this preclinical model for profiling novel drug therapies targeting NASH-HCC.

Hepatoprotective effects of the long-acting fibroblast growth factor 21 analog PF-05231023 in the GAN diet-induced obese and biopsy-confirmed mouse model of nonalcoholic steatohepatitis.

Fibroblast growth factor 21 (FGF21) plays a key role in hepatic lipid metabolism and long-acting FGF21 analogs have emerged as promising drug candidates for the treatment of nonalcoholic steatohepatitis (NASH). It remains to characterize this drug class in translational animal models that recapitulate the etiology and hallmarks of human disease. To this end, we evaluated the long-acting FGF21 analog PF-05231023 in the GAN (Gubra Amylin NASH) diet-induced obese (DIO) and biopsy-confirmed mouse model of NASH. Male C57BL/6J mice were fed the GAN diet high in fat, fructose, and cholesterol for 34 wk before the start of the study. GAN DIO-NASH mice with biopsy-confirmed NAFLD Activity Score (NAS ≥5) and fibrosis (stage ≥F1) were biweekly administered with PF-05231023 (10 mg/kg sc) or vehicle (sc) for 12 wk. Vehicle-dosed chow-fed C57BL/6J mice served as healthy controls. Pre-to-post liver biopsy histopathological scoring was performed for within-subject evaluation of NAFLD Activity Score (NAS) and fibrosis stage. Terminal endpoints included quantitative liver histology and transcriptome signatures as well as blood and liver biochemistry. PF-05231023 significantly reduced body weight, hepatomegaly, plasma transaminases, and plasma/liver lipids in GAN DIO-NASH mice. Notably, PF-05231023 reduced both NAS (≥2-point improvement) and fibrosis stage (1-point improvement). Improvements in NASH and fibrosis severity were supported by reduced quantitative histological markers of steatosis, inflammation, and fibrogenesis as well as improvements in disease-associated liver transcriptome signatures. In conclusion, PF-05231023 reduces NASH and fibrosis severity in a translational biopsy-confirmed mouse model of NASH, supporting development of FGF21 analogs for the treatment of NASH.NEW & NOTEWORTHY It is unclear if long-acting FGF21 analogs have antifibrotic efficacy in NASH. We therefore profiled the clinically relevant FGF21 analog PF-05231023 in a translational diet-induced obese and biopsy-confirmed mouse model of NASH. We found PF-05231023 to exert hepatoprotective effects as indicated by notable improvements in plasma markers and histological hallmarks of NASH, including improved fibrosis stage. Collectively, the present study supports the continued exploration of long-acting FGF21 analogs for the treatment of NASH and other fibrotic diseases.

Structurally-engineered fatty acid 1024 (SEFA-1024) improves diet-induced obesity, insulin resistance, and fatty liver disease.

Obesity is a global epidemic that drives morbidity and mortality through cardiovascular disease, diabetes, and non-alcoholic fatty liver disease (NAFLD). No definitive therapy has been approved to improve glycemic control and treat NAFLD in obese patients. Here, we investigated a semi-synthetic, long chain, structurally-engineered fatty acid-1024 (SEFA-1024), as a treatment for obesity-induced hyperglycemia, insulin-resistance, and fatty liver disease in rodent models. A single dose of SEFA-1024 was administered to evaluate glucose tolerance and active glucagon-like peptide 1 (GLP-1) in lean rats in the presence and absence of a DPP-4 inhibitor. The effects of SEFA-1024 on weight loss and glycemic control were assessed in genetic (ob/ob) and environmental (high-fat diet) murine models of obesity. Liver histology, serum liver enzymes, liver lipidomics, and hepatic gene expression were also assessed in the high-fat diet murine model. SEFA-1024 reversed obesity-associated insulin resistance and improved glycemic control. SEFA-1024 increased active GLP-1. In a long-term model of diet-induced obesity, SEFA-1024 reversed excessive weight gain, hepatic steatosis, elevated liver enzymes, hepatic lipotoxicity, and promoted fatty acid metabolism. SEFA-1024 is an enterohepatic-targeted, eicosapentaenoic acid derivative that reverses obesity-induced dysregulated glucose metabolism and hepatic lipotoxicity in genetic and dietary rodent models of obesity. The mechanism by which SEFA-1024 works may include increasing aGLP-1, promoting fatty acid oxidation, and inhibiting hepatic triglyceride formation. SEFA-1024 may serve as a potential treatment for obesity-related diabetes and NAFLD.

Effects of ALT-801, a GLP-1 and glucagon receptor dual agonist, in a translational mouse model of non-alcoholic steatohepatitis.

Body weight loss of ≥ 10% improves the metabolic derangements and liver disease in the majority of non-alcoholic steatohepatitis (NASH) patients, suggesting metabolic modulators may be effective in controlling disease. The pharmacodynamics of ALT-801, a GLP-1/glucagon receptor dual agonist optimized for NASH and weight loss, were compared to semaglutide (GLP-1 receptor agonist) and elafibranor (peroxisome proliferator-activated receptor, PPAR-α/δ, agonist) in a biopsy-confirmed, diet-induced obese (DIO) mouse model of NASH (DIO-NASH). Male C57BL/6J mice were fed Amylin Liver NASH (AMLN) diet for 32 weeks. Animals with biopsy-confirmed steatosis and fibrosis received ALT-801, semaglutide, elafibranor, or vehicle daily for 12 weeks while maintained on the AMLN diet. Study endpoints included body and liver weight, liver and plasma total cholesterol and triglycerides, plasma aminotransferases, histological analysis of liver steatosis, inflammation (galectin-3) and fibrosis (collagen type 1 alpha 1), and evaluation of individual animal changes in composite Non-alcoholic Fatty Liver Disease Activity Score (NAS), and fibrosis stage. ALT-801 demonstrated significant reductions in body weight (approx. 25%), plasma aminotransferases, plasma total cholesterol and liver triglycerides/total cholesterol in conjunction with improved liver steatosis, with greater reductions (p < 0.05) compared to semaglutide and elafibranor. ALT-801 significantly reduced the inflammation marker galectin-3 and the fibrosis marker collagen type 1 alpha 1 vs. vehicle (p < 0.05), with ALT-801 producing greater reductions in galectin-3 vs. elafibranor (p < 0.05). Importantly, all animals treated with ALT-801 significantly improved composite NAS compared to the active controls. This study provides evidence for a potential role for ALT-801 in the therapeutic treatment of NASH.

Hepatoprotective effects of semaglutide, lanifibranor and dietary intervention in the GAN diet-induced obese and biopsy-confirmed mouse model of NASH.

Non-alcoholic steatohepatitis (NASH) has emerged as a major challenge for public health because of high global prevalence and lack of evidence-based therapies. Most animal models of NASH lack sufficient validation regarding disease progression and pharmacological treatment. The Gubra-Amylin NASH (GAN) diet-induced obese (DIO) mouse demonstrate clinical translatability with respect to disease etiology and hallmarks of NASH. This study aimed to evaluate disease progression and responsiveness to clinically effective interventions in GAN DIO-NASH mice. Disease phenotyping was performed in male C57BL/6J mice fed the GAN diet high in fat, fructose, and cholesterol for 28-88 weeks. GAN DIO-NASH mice with biopsy-confirmed NASH and fibrosis received low-caloric dietary intervention, semaglutide (30 nmol/kg/day, s.c.) or lanifibranor (30 mg/kg/day, p.o.) for 8 and 12 weeks, respectively. Within-subject change in nonalcoholic fatty liver disease (NAFLD) Activity Score (NAS) and fibrosis stage was evaluated using automated deep learning-based image analysis. GAN DIO-NASH mice showed clear and reproducible progression in NASH, fibrosis stage, and tumor burden with high incidence of hepatocellular carcinoma. Consistent with clinical trial outcomes, semaglutide and lanifibranor improved NAS, whereas only lanifibranor induced regression in the fibrosis stage. Dietary intervention also demonstrated substantial benefits on metabolic outcomes and liver histology. Differential therapeutic efficacy of semaglutide, lanifibranor, and dietary intervention was supported by quantitative histology, RNA sequencing, and blood/liver biochemistry. In conclusion, the GAN DIO-NASH mouse model recapitulates various histological stages of NASH and faithfully reproduces histological efficacy profiles of compounds in advanced clinical development for NASH. Collectively, these features highlight the utility of GAN DIO-NASH mice in preclinical drug development.

Empagliflozin Improves Metabolic and Hepatic Outcomes in a Non-Diabetic Obese Biopsy-Proven Mouse Model of Advanced NASH.

Empagliflozin, an established treatment for type 2 diabetes (T2DM), has shown beneficial effects on liver steatosis and fibrosis in animals and in humans with T2DM, non-alcoholic fatty liver disease (NAFLD) and steatohepatitis (NASH). However, little is known about the effects of empagliflozin on liver function in advanced NASH with liver fibrosis and without diabetes. This study aimed to assess the effects of empagliflozin on hepatic and metabolic outcomes in a diet-induced obese (DIO) and insulin-resistant but non-diabetic biopsy-confirmed mouse model of advanced NASH. Male C57BL/6JRj mice with a biopsy-confirmed steatosis and fibrosis on AMLN diet (high fat, fructose and cholesterol) for 36-weeks were randomized to receive for 12 weeks: (a) Empagliflozin (10 mg/kg/d p.o.), or (b) vehicle. Metabolic outcomes, liver pathology, markers of Kupffer and stellate cell activation and lipidomics were assessed at the treatment completion. Empagliflozin did not affect the body weight, body composition or insulin sensitivity (assessed by intraperitoneal insulin tolerance test), but significantly improved glucose homeostasis as assessed by oral glucose tolerance test in DIO-NASH mice. Empagliflozin improved modestly the NAFLD activity score compared with the vehicle, mainly by improving inflammation and without affecting steatosis, the fibrosis stage and markers of Kupffer and stellate cell activation. Empagliflozin reduced the hepatic concentrations of pro-inflammatory lactosylceramides and increased the concentrations of anti-inflammatory polyunsaturated triglycerides. Empagliflozin exerts beneficial metabolic and hepatic (mainly anti-inflammatory) effects in non-diabetic DIO-NASH mice and thus may be effective against NASH even in non-diabetic conditions.

Elafibranor and liraglutide improve differentially liver health and metabolism in a mouse model of non-alcoholic steatohepatitis.

BACKGROUND & AIMS: This study aimed to assess and compare the effects of the GLP-1 analog liraglutide and the PPARα/δ agonist elafibranor on liver histology and their impact on hepatic lipidome, metabolome, Kupffer and hepatic stellate cell activation in a model of advanced non-alcoholic fatty liver disease (NAFLD). METHODS: Male C57BL/6JRj mice with biopsy-confirmed hepatosteatosis and fibrosis induced by 36-week Amylin liver NASH (AMLN) diet (high-fat, fructose and cholesterol) were randomized to receive for 12 weeks: (a) liraglutide (0.4 mg/kg/day s.c.), (b) elafibranor (30 mg/kg/day p.o.) and (c) vehicle. Metabolic status, liver pathology, markers of inflammation, Kupffer and stellate cell activation, and metabolomics/lipidomics were assessed at study completion. RESULTS: Elafibranor and liraglutide improved weight, insulin sensitivity, glucose homeostasis and NAFLD activity score (pre-to-post biopsy). Elafibranor had a profound effect on hepatic lipidome, demonstrated by reductions in glycerides, increases in phospholipids, and by beneficial regulation of mediators of fatty acid oxidation, inflammation and oxidative stress. Liraglutide had a major impact on inflammatory and fibrogenic markers of Kupffer and hepatic stellate cell activation (Galectin-3, Collagen type I alpha 1, alpha-smooth muscle actin). Liraglutide exerted beneficial effects on bile acid and carbohydrate metabolism, demonstrated by restorations of the concentrations of bile acids, glycogen metabolism by-products and pentoses, thus facilitating glycogen utilization turnover and nucleic acid formation. CONCLUSIONS: Liraglutide and elafibranor robustly but through different pathways improve overall metabolic health and liver status in NAFLD. These data indicate important differences in the respective mechanisms of action and support the notion for their evaluation as combination therapies in the future.

Activation of thyroid hormone receptor-ß improved disease activity and metabolism independent of body weight in a mouse model of non-alcoholic steatohepatitis and fibrosis.

BACKGROUND AND PURPOSE: Activation of hepatic thyroid hormone receptor ß (THR-ß) is associated with systemic lipid lowering, increased bile acid synthesis, and fat oxidation. In patients with non-alcoholic steatohepatitis (NASH), treatment with THR-ß agonists decreased hepatic steatosis and circulating lipids, and induced resolution of NASH. We chose resmetirom (MGL-3196), a liver-directed, selective THR-ß agonist, as a prototype to investigate the effects of THR-ß activation in mice with diet-induced obesity (DIO) and biopsy-confirmed advanced NASH with fibrosis. EXPERIMENTAL APPROACH: C57Bl/6J mice were fed a diet high in fat, fructose, and cholesterol for 34 weeks, and only biopsy-confirmed DIO-NASH mice with fibrosis were included. Resmetirom was administered at a daily dose of 3 mg·kg-1 p.o., for 8 weeks. Systemic and hepatic metabolic parameters, histological non-alcoholic fatty liver disease (NAFLD) activity and fibrosis scores, and liver RNA expression profiles were determined to assess the effect of THR-ß activation. KEY RESULTS: Treatment with resmetirom did not influence body weight but led to significant reduction in liver weight, hepatic steatosis, plasma alanine aminotransferase activity, liver and plasma cholesterol, and blood glucose. These metabolic effects translated into significant improvement in NAFLD activity score. Moreover, a lower content of α-smooth muscle actin and down-regulation of genes involved in fibrogenesis indicated a decrease in hepatic fibrosis. CONCLUSION AND IMPLICATIONS: Our model robustly reflected clinical observations of body weight-independent improvements in systemic and hepatic metabolism including anti-steatotic activity.

The Selective Peroxisome Proliferator-Activated Receptor Gamma Modulator CHS-131 Improves Liver Histopathology and Metabolism in a Mouse Model of Obesity and Nonalcoholic Steatohepatitis.

CHS-131 is a selective peroxisome proliferator-activated receptor gamma modulator with antidiabetic effects and less fluid retention and weight gain compared to thiazolidinediones in phase II clinical trials. We investigated the effects of CHS-131 on metabolic parameters and liver histopathology in a diet-induced obese (DIO) and biopsy-confirmed mouse model of nonalcoholic steatohepatitis (NASH). Male C57BL/6JRj mice were fed the amylin liver NASH diet (40% fat with trans-fat, 20% fructose, and 2% cholesterol). After 36 weeks, only animals with biopsy-confirmed steatosis and fibrosis were included and stratified into treatment groups (n = 12-13) to receive for the next 12 weeks (1) low-dose CHS-131 (10 mg/kg), (2) high-dose CHS-131 (30 mg/kg), or (3) vehicle. Metabolic parameters, liver pathology, metabolomics/lipidomics, markers of liver function and liver, and subcutaneous and visceral adipose tissue gene expression profiles were assessed. CHS-131 did not affect body weight, fat mass, lean mass, water mass, or food intake in DIO-NASH mice with fibrosis. CHS-131 improved fasting insulin levels and insulin sensitivity as assessed by the intraperitoneal insulin tolerance test. CHS-131 improved total plasma cholesterol, triglycerides, alanine aminotransferase, and aspartate aminotransferase and increased plasma adiponectin levels. CHS-131 (high dose) improved liver histology and markers of hepatic fibrosis. DIO-NASH mice treated with CHS-131 demonstrated a hepatic shift to diacylglycerols and triacylglycerols with a lower number of carbons, increased expression of genes stimulating fatty acid oxidation and browning, and decreased expression of genes promoting fatty acid synthesis, triglyceride synthesis, and inflammation in adipose tissue. Conclusion: CHS-131 improves liver histology in a DIO and biopsy-confirmed mouse model of NASH by altering the hepatic lipidome, reducing insulin resistance, and improving lipid metabolism and inflammation in adipose tissue.

Dual targeting of hepatic fibrosis and atherogenesis by icosabutate, an engineered eicosapentaenoic acid derivative.

BACKGROUND & AIMS: While fibrosis stage predicts liver-associated mortality, cardiovascular disease (CVD) is still the major overall cause of mortality in patients with NASH. Novel NASH drugs should thus ideally reduce both liver fibrosis and CVD. Icosabutate is a semi-synthetic, liver-targeted eicosapentaenoic acid (EPA) derivative in clinical development for NASH. The primary aims of the current studies were to establish both the anti-fibrotic and anti-atherogenic efficacy of icosabutate in conjunction with changes in lipotoxic and atherogenic lipids in liver and plasma respectively. METHODS: The effects of icosabutate on fibrosis progression and lipotoxicity were investigated in amylin liver NASH (AMLN) diet (high fat, cholesterol and fructose) fed ob/ob mice with biopsy-confirmed steatohepatitis and fibrosis and compared with the activity of obeticholic acid. APOE*3Leiden.CETP mice, a translational model for hyperlipidaemia and atherosclerosis, were used to evaluate the mechanisms underlying the lipid-lowering effect of icosabutate and its effect on atherosclerosis. RESULTS: In AMLN ob/ob mice, icosabutate significantly reduced hepatic fibrosis and myofibroblast content in association with downregulation of the arachidonic acid cascade and a reduction in both hepatic oxidised phospholipids and apoptosis. In APOE*3Leiden.CETP mice, icosabutate reduced plasma cholesterol and TAG levels via increased hepatic uptake, upregulated hepatic lipid metabolism and downregulated inflammation pathways, and effectively decreased atherosclerosis development. CONCLUSIONS: Icosabutate, a structurally engineered EPA derivative, effectively attenuates both hepatic fibrosis and atherogenesis and offers an attractive therapeutic approach to both liver- and CV-related morbidity and mortality in NASH patients.

Activation of Adenosine Monophosphate-Activated Protein Kinase Reduces the Onset of Diet-Induced Hepatocellular Carcinoma in Mice.

The worldwide obesity and type 2 diabetes epidemics have led to an increase in nonalcoholic fatty liver disease (NAFLD). NAFLD covers a spectrum of hepatic pathologies ranging from simple steatosis to nonalcoholic steatohepatitis, characterized by fibrosis and hepatic inflammation. Nonalcoholic steatohepatitis predisposes to the onset of hepatocellular carcinoma (HCC). Here, we characterized the effect of a pharmacological activator of the intracellular energy sensor adenosine monophosphate-activated protein kinase (AMPK) on NAFLD progression in a mouse model. The compound stimulated fat oxidation by activating AMPK in both liver and skeletal muscle, as revealed by indirect calorimetry. This translated into an ameliorated hepatic steatosis and reduced fibrosis progression in mice fed a diet high in fat, cholesterol, and fructose for 20 weeks. Feeding mice this diet for 80 weeks caused the onset of HCC. The administration of the AMPK activator for 12 weeks significantly reduced tumor incidence and size. Conclusion: Pharmacological activation of AMPK reduces NAFLD progression to HCC in preclinical models.

Resolution of NASH and hepatic fibrosis by the GLP-1R/GcgR dual-agonist Cotadutide via modulating mitochondrial function and lipogenesis.

Non-alcoholic fatty liver disease and steatohepatitis are highly associated with obesity and type 2 diabetes mellitus. Cotadutide, a GLP-1R/GcgR agonist, was shown to reduce blood glycemia, body weight and hepatic steatosis in patients with T2DM. Here, we demonstrate that the effects of Cotadutide to reduce body weight, food intake and improve glucose control are predominantly mediated through the GLP-1 signaling, while, its action on the liver to reduce lipid content, drive glycogen flux and improve mitochondrial turnover and function are directly mediated through Gcg signaling. This was confirmed by the identification of phosphorylation sites on key lipogenic and glucose metabolism enzymes in liver of mice treated with Cotadutide. Complementary metabolomic and transcriptomic analyses implicated lipogenic, fibrotic and inflammatory pathways, which are consistent with a unique therapeutic contribution of GcgR agonism by Cotadutide in vivo. Significantly, Cotadutide also alleviated fibrosis to a greater extent than Liraglutide or Obeticholic acid (OCA), despite adjusting dose to achieve similar weight loss in 2 preclinical mouse models of NASH. Thus Cotadutide, via direct hepatic (GcgR) and extra-hepatic (GLP-1R) effects, exerts multi-factorial improvement in liver function and is a promising therapeutic option for the treatment of steatohepatitis.

Multi-omics characterization of a diet-induced obese model of non-alcoholic steatohepatitis.

To improve the understanding of the complex biological processes underlying the development of non-alcoholic steatohepatitis (NASH), a multi-omics approach combining bulk RNA-sequencing based transcriptomics, quantitative proteomics and single-cell RNA-sequencing was used to characterize tissue biopsies from histologically validated diet-induced obese (DIO) NASH mice compared to chow-fed controls. Bulk RNA-sequencing and proteomics showed a clear distinction between phenotypes and a good correspondence between mRNA and protein level regulations, apart from specific regulatory events discovered by each technology. Transcriptomics-based gene set enrichment analysis revealed changes associated with key clinical manifestations of NASH, including impaired lipid metabolism, increased extracellular matrix formation/remodeling and pro-inflammatory responses, whereas proteomics-based gene set enrichment analysis pinpointed metabolic pathway perturbations. Integration with single-cell RNA-sequencing data identified key regulated cell types involved in development of NASH demonstrating the cellular heterogeneity and complexity of NASH pathogenesis.