Nonalcoholic Steatohepatitis: Natural History, Pathogenesis and Therapy

National Institute Of Diabetes And Digestive And Kidney Diseases

Investigators

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Abstract

Nonalcoholic fatty liver disease (NAFLD) is marked by accumulation of fat in liver cells with accompanying inflammation and variable degrees of cell injury and fibrosis. When cell injury and fibrosis are present, the disease has a potential to progress and is referred to as nonalcoholic steatohepatitis (NASH), which can lead to cirrhosis, liver cancer, morbidity and mortality. The etiology of NASH is not clear nor is there an approved treatment modality for it. NAFLD has become an extremely common disorder, estimated to affect up to 30% of individuals in the US. Unfortunately, it commonly goes unrecognized, as demonstrated by our recent finding that over a 5-year period, 28% of subjects enrolled as healthy volunteers to studies at the NIH Clinical Center were likely to have underlying NAFLD. As such, there is a clear need to understand the pathophysiology of the disease and its treatment. Our focus on NAFLD is three-fold: first, we aim to identify and characterize key genes that play a role in the pathogenesis of NAFLD through the use of genetic studies and cell- or animal models and identify their suitability as therapeutic target. Second, we aim to understand the physiology of fat accumulation and injury in the liver, especially as it relates to handling of oral caloric load. Third, we hope to identify and refine effective treatments for the disorder. Genome wide association (GWA) studies identified single nucleotide polymorphisms (SNPs) that are associated with increased hepatic fat or elevated liver enzymes, presumably reflecting nonalcoholic fatty liver disease (NAFLD). We initiated a study to investigate whether these SNPs are associated with histological severity in a large cohort of NAFLD patients. We confirmed the association of the rs738409G allele in the PNPLA3 gene with steatosis and were first to describe its association with histological severity. In pediatric patients, the high-risk rs738409G allele was associated with an earlier presentation of disease. Similarly, we discovered an association of several SNPs near or in the gene for hydroxysteroid (17) dehydrogenase 13 (HSD17B13) with histological features of NAFLD. In-depth genotyping of the gene region demonstrated associations of coding and splice-site SNPs in the gene with NAFLD, confirming a possible role for the protein in the pathogenesis of NASH. HSD17B13 is an enzyme that we found to be predominantly expressed in the liver and to colocalize with lipid droplets. We identified that HSD17B13 is involved in retinoid metabolism and that genetic variants that lead to loss of its enzymatic function are genetically associated with decreased severity of NAFLD. We further characterized the structure of the protein and the domains in it that are key to its function. These findings generated remarkable interest in the pharmaceutical industry and several HSD17B13 inactivating agents are being studied in human subjects as potential therapies for NASH. Despite the promising genetic association and pharma interest, the actual physiological role of HSD17B13 in vivo and the mechanism by which its inactivation leads to protection from NASH-associated injury are still unknown. To improve our understanding of HSD17B13s role, we established an Hsd17b13 knock-out mouse model and are currently studying it under multiple condition, aiming to replicate the human phenotype. Our mouse and cell models allow us to pinpoint the mechanism by which loss of HSD17B13 protects the liver from injury, as well as its association with other metabolic features. As NAFLD is intricately related to food intake and energy metabolism, we are undertaking clinical trials to evaluate the handling and fate of nutrients by the fatty liver. We used the BreathID real-time breath test device in combination with a labeled fatty acid to demonstrate a decrease in the rate of fatty acid oxidation in subjects with NAFLD compared to controls. We recently studied NAFLD subjects after ingestion of a liquid mixed meal. We collected plasma samples at multiple time points and utilized a lipidomic approach and parallel mouse models to identify an increase in packaging of hepatic diacylglycerols (DAG) into very low density lipprotein (VLDL) particles after a meal, that is unique to NAFLD. Finally, we are currently performing an additional study, where liver samples from subjects with NAFLD are obtained before and after an oral carbohydrate load aimed to elucidate the hepatic transcriptomic and lipidomic response as well as effects on intra-hepatic insulin signaling. Vitamin E has been shown in a randomized placebo-controlled trials to be an effective therapy for NASH. Curiously, treatment with vitamin E resulted not only in a decrease in injury (thought to reflect its antioxidant effect) but was also associated with a decrease in liver fat, through an unknown mechanism. We combined a clinical mechanistic trial with in vitro experiments to determine the mechanism of action of vitamin E. We identified a key cellular pathway by which oxidative stress (a common feature of NAFLD) increases liver fat through upregulation of hepatic de novo lipogenesis and confirmed that vitamin E blocks the activation of this pathway through its antioxidant activity. We were able to establish an automated method to quantify hepatic 4-hydroxynonenal (4-HNE) adducts, a marker of oxidative stress-induced damage, and confirmed its applicability with samples from the vitamin E trial. In a current trial, subjects with NAFLD are treated with semaglutide, a glucagon like peptide 1 (GLP-1) receptor agonist with effectiveness against NASH previously shown. This study aims to elucidate the mechanism of action of semaglutide in treating NASH, its effects on the liver lipidome and identify early predictors of clinical response.