Characterization and Pharmacological Validation of a Preclinical Model of NASH in Göttingen Minipigs.

Background: Nonalcoholic fatty liver disease (NAFLD) is the leading cause of chronic liver disease, which is associated with features of metabolic syndrome. NAFLD may progress in a subset of patients into nonalcoholic steatohepatitis (NASH) with liver injury resulting ultimately in cirrhosis and potentially hepatocellular carcinoma. Today, there is no approved treatment for NASH due to, at least in part, the lack of preclinical models recapitulating features of human disease. Here, we report the development of a dietary model of NASH in the Göttingen minipig. Methods: First, we performed a longitudinal characterization of diet-induced NASH and fibrosis using biochemical, histological, and transcriptional analyses. We then evaluated the pharmacological response to Obeticholic acid (OCA) treatment for 8 weeks at 2.5mg/kg/d, a dose matching its active clinical exposure. Results: Serial histological examinations revealed a rapid installation of NASH driven by massive steatosis and inflammation, including evidence of ballooning. Furthermore, we found the progressive development of both perisinusoidal and portal fibrosis reaching fibrotic septa after 6 months of diet. Histological changes were mechanistically supported by well-defined gene signatures identified by RNA Seq analysis. While treatment with OCA was well tolerated throughout the study, it did not improve liver dysfunction nor NASH progression. By contrast, OCA treatment resulted in a significant reduction in diet-induced fibrosis in this model. Conclusions: These results, taken together, indicate that the diet-induced NASH in the Göttingen minipig recapitulates most of the features of human NASH and may be a model with improved translational value to prioritize drug candidates toward clinical development.

Procollagen C-Proteinase Enhancer-1 (PCPE-1) deficiency in mice reduces liver fibrosis but not NASH progression.

BACKGROUND AND AIMS: Nonalcoholic Steatohepatitis (NASH) is a major cause of end-stage liver diseases such as cirrhosis and hepatocellular carcinoma resulting ultimately in increased liver-related mortality. Fibrosis is the main driver of mortality in NASH. Procollagen C-Proteinase Enhancer-1 (PCPE-1) plays a key role in procollagen maturation and collagen fibril formation. To assess its role in liver fibrosis and NASH progression, knock-out mice were evaluated in a dietary NASH model. METHODS: Global constitutive Pcolce-/- and WT male mice were fed with a Choline Deficient Amino acid defined High Fat Diet (CDA HFD) for 8 weeks. Liver triglycerides, steatosis, inflammation and fibrosis were assessed at histological, biochemical and gene expression levels. In addition, human liver samples from control and NASH patients were used to evaluate the expression of PCPE-1 at both mRNA and protein levels. RESULTS: Pcolce gene deficiency prevented diet-induced liver enlargement but not liver dysfunction. Furthermore, liver triglycerides, steatosis and inflammation were not modified in Pcolce-/- male mice compared to WT under CDA HFD. However, a significant decrease in liver fibrosis was observed in Pcolce-/- mice compared to WT under NASH diet, associated with a decrease in total and insoluble collagen content without any significant modifications in the expression of genes involved in fibrosis and extracellular matrix remodeling. Finally, PCPE-1 protein expression was increased in cirrhotic liver samples from both NASH and Hepatitis C patients. CONCLUSIONS: Pcolce deficiency limits fibrosis but not NASH progression in CDA HFD fed mice.

Mineralocorticoid receptor mutations differentially affect individual gene expression profiles in pseudohypoaldosteronism type 1.

CONTEXT: Type 1 pseudohypoaldosteronism (PHA1), a primary form of mineralocorticoid resistance, is due to inactivating mutations of the NR3C2 gene, coding for the mineralocorticoid receptor (MR). OBJECTIVE: The objective of the study was to assess whether different NR3C2 mutations have distinct effects on the pattern of MR-dependent transcriptional regulation of aldosterone-regulated genes. DESIGN AND METHODS: Four MR mutations affecting residues in the ligand binding domain, identified in families with PHA1, were tested. MR proteins generated by site-directed mutagenesis were analyzed for their binding to aldosterone and were transiently transfected into renal cells to explore the functional effects on the transcriptional activity of the receptors by cis-trans-cotransactivation assays and by measuring the induction of endogenous gene transcription. RESULTS: Binding assays showed very low or absent aldosterone binding for mutants MR(877Pro), MR(848Pro), and MR(947stop) and decreased affinity for aldosterone of MR(843Pro). Compared with wild-type MR, the mutations p.Leu843Pro and p.Leu877Pro displayed half-maximal aldosterone-dependent transactivation of reporter genes driven by mouse mammary tumor virus or glucocorticoid response element-2 dependent promoters, whereas MR(848Pro) and MR(947stop) nearly or completely lost transcriptional activity. Although MR(848Pro) and MR(947stop) were also incapable of inducing aldosterone-dependent gene expression of endogenous sgk1, GILZ, NDRG2, and SCNN1A, MR(843Pro) retained complete transcriptional activity on sgk1 and GILZ gene expression, and MR(877Pro) negatively affected the expression of sgk1, NDRG2, and SCNN1A. CONCLUSIONS: Our data demonstrate that MR mutations differentially affect individual gene expression in a promoter-dependent manner. Investigation of differential gene expression profiles in PHA1 may allow a better understanding of the molecular substrate of phenotypic variability and to elucidate pathogenic mechanisms underlying the disease.

Analysis of insulin sensitivity in adipose tissue of patients with primary aldosteronism.

OBJECTIVE: The objective of the study was to assess the effect of high aldosterone levels on insulin sensitivity of adipose tissue in humans. METHODS: Visceral adipose tissue (VAT) was obtained from patients with aldosterone-producing adenoma (APA; n=14) and, as controls, nonfunctioning adenoma (NFA; n=14) undergoing laparoscopic adrenalectomy. Homeostasis model assessment index was higher and potassium was lower in APA than NFA (P<0.05). Immunohistochemistry, Western blotting, and real-time PCR were used to detect and quantify mineralocorticoid receptor (MR) expression. Transcript levels of peroxisome proliferative-activated receptor-gamma, insulin receptor, glucose transporter 4, insulin receptor substrate-1 and -2, leptin, adiponectin, IL-6, monocyte chemoattractant protein-1, glucocorticoid receptor (GR)-alpha, 11beta-hydroxysteroid dehydrogenase (HSD11B) type 1, and HSD11B2 were quantified. The effect of increasing aldosterone concentrations on 2-deoxy-[3H]d-glucose uptake was tested in human sc abdominal adipocytes. RESULTS: Expression of MR was demonstrated in VAT, with no difference between APA and NFA as to mRNA levels of MR, GRalpha, HSD11B1, and glucose metabolism and inflammation factors. In cultured adipocytes, basal and insulin-stimulated glucose uptake were unaffected by 1-100 nM (normal/hyperaldosteronism) and impaired only by much higher, up to 10 microM, aldosterone concentrations. The impairment was prevented by RU486 but not by eplerenone. CONCLUSIONS: Gene expression of insulin signaling/inflammatory molecules was similar in VAT of APA and NFA patients, not supporting an effect of aldosterone excess on insulin sensitivity of adipose tissues. Only at pharmacological concentrations and through GR activation, aldosterone reduced glucose uptake in adipocytes. Insulin resistance in primary aldosteronism might occur in compartments other than fat and/or depend on concurrent environmental factors.