Modeling the Complex Disorder of NASH

Megan M. MacBride, PhD
Thursday, August 19th, 2021
Healthy liver versus cirrhosis Changing diets and lifestyles across much of the world contribute to a wide range of diseases and health conditions, including heart disease, hypertension, and type 2 diabetes. Another looming health crisis related to lifestyle is now on the horizon: a surge in the incidence of non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH), a more severe form of NAFLD. As researchers aim to unravel this complex progression of disorders, new and evolving mouse models are contributing to their understanding.

An Increasingly Prevalent and Complex Disease to Study

About 25% of adults worldwide are estimated to have NAFLD, with higher rates in South America (about 30%) and the Middle East (over 31%)1. NASH, the more severe form, is estimated to occur in about 1.5% to 6.45% of the population, a figure medical experts expect to increase significantly in conjunction with rising rates of obesity and diabetes2. Both NAFLD and NASH impair liver function and are a leading reason for liver transplants.

NAFLD is a spectrum of disease, ranging from simple fatty liver disease, with little or no inflammation of the liver or the liver cells, to NASH, which is characterized by inflammation of the liver and damaged liver cells and which can include fibrosis. NAFLD can progress to cirrhosis, end-stage liver failure, and hepatocellular carcinoma. There are no FDA- or EMA-approved NASH therapeutics as of this writing; however, there is a robust pipeline of drug candidates against a wide range of targets.

The study of NASH is particularly complex for a variety of reasons. First, it is increasingly viewed not as a single disease but rather as a spectrum of conditions. Typically, NAFLD presents initially as fatty liver, which can, but does not always, proceed to an inflammatory state and more severe disease. NASH may occur concurrently with conditions like obesity, type 2 diabetes, insulin or glucose resistance, high cholesterol, or metabolic syndrome, though that is not always the case.

Additionally, researchers believe that disease development involves multiple pathways. In their review of NASH-related liver cancer development, Kutlu et al. noted that both clinical and epidemiological studies suggest multiple mechanisms are at work in the progression from simple fatty liver to NASH3. Factors implicated in NASH progression include lipotoxicity, inflammation, the microbiome, and more.

Further complicating matters, NASH may be largely asymptomatic in many instances, eluding both patients and physicians until it reaches a late stage. Most often a firm diagnosis is made only after a liver biopsy—a procedure that comes with risks, such as scarring, and the potential for inaccuracy, since it is based on a small tissue sample. Although various imaging and other diagnostic modalities are making great progress, liver biopsy remains the gold standard for NASH identification. Diagnostic difficulties and the lack of effective therapies for advanced NASH make this a challenging disease.

Multiple Mouse Models May Hold the Answer for NASH

These complexities have made preclinical research on NASH quite challenging. To study NASH effectively will likely require a range of research models, each focused on assessing different disease-related changes and relevant mechanisms. Various preclinical mouse models have been used and will continue to play a role in exploring both the mechanisms of NASH and potential therapeutics.

A number of strategies have been employed to induce fatty liver, inflammation, and fibrosis in a mouse model, including nutrient-deficient diets, high-fat diets, and chemical induction. These methods have been used in a variety of standard and mutant mouse strains. Chemical induction of liver fibrosis using carbon tetrachloride has been widely used but does not necessarily represent relevant mechanisms of metabolic syndrome-induced liver injury. Combining chemical induction with dietary manipulation is now becoming more commonly used as a strategy to combine rapid fibrosis development with relevant metabolic phenotypes.

Since diet is a known factor in NASH development and progression, the most common approach to modeling the disease in a mouse model has been diet modification through the administration of either a high-fat diet or a nutrient-deficient diet.

Diet-induced obese (DIO) mice generated through the application of standard high-fat diets have been used for NAFLD research, demonstrating that diet modification can help to drive phenotype progression. However, while this type of DIO mouse may develop steatosis, standard DIO models typically don't develop the inflammation and fibrosis seen in the more severe disease state of NASH. For example, Taconic Biosciences' DIO C57BL/6 model, conditioned on a 60% kcal high-fat diet, develops fatty liver consistently but does not progress to significant inflammation or fibrosis even after prolonged diet conditioning. Thus while standard DIO mice are useful for studying steatosis mechanisms and drugs targeted at this aspect of NAFLD, they are of limited use in studying other aspects of the disease.

High-fat diets incorporating fructose and cholesterol can induce the development of additional NASH manifestations such as inflammation and fibrosis in wild type C57BL/6 mice, but significant conditioning time is still required for disease development. This can pose a significant logistical barrier, delaying the start of a NASH study and tying up vivarium space and resources in the process.

To overcome these problems, Taconic developed an off-the-shelf diet-induced NASH model conditioned on a modified Amylin liver NASH diet, containing 40 kcal% fat, 20 kcal% fructose, and 2% cholesterol. Conditioned on this diet, the NASH B6 not only develops fatty liver in the early weeks; it also develops liver inflammation (including hepatic crown-like structures) and, after ~26 weeks on diet, fibrosis.

The ability to obtain a NASH model off the shelf greatly reduces conditioning time for investigators, enabling them to get studies underway sooner with a disease-specific model at the desired age. This approach also eliminates the need for investigators to tie up vivarium space waiting for research mice to age to the point that they develop NASH-associated phenotypes. Instead, a cohort of NASH mice can be delivered to the research facility for start on study after an acclimation period.

Taking NASH Research to the Next Level

Though no FDA- or EMA-approved NASH therapeutics exist, encouraging progress is being made on both the preclinical and clinical fronts. Drug targets for NASH fall into three categories: metabolic, anti-inflammatory, and anti-fibrotic. Most candidates are in the metabolic category, including FXR, FGF21, PPAR, and GLP-1 agonists. The anti-inflammatory field has not been as well developed, and anti-fibrotic therapeutics are a huge area of unmet need. There are a number of therapies currently in phase 3 clinical trials. Another promising area of research is combination therapy, and there are several phase 2 trials underway exploring this approach.

With the incidence of severe liver disease rising rapidly across the globe, NASH will remain an active area of investigation, both from a basic science and drug discovery perspective. By leveraging a variety of preclinical NASH models, the research community will continue to make strides toward understanding the pathogenesis of this complex disease and bringing effective therapeutics to market.

1. Younossi, Zobair M. MD, MPH1; Marchesini, Giulio MD2; Pinto-Cortez, Helena MD3; Petta, Salvatore MD4 Epidemiology of Nonalcoholic Fatty Liver Disease and Nonalcoholic Steatohepatitis: Implications for Liver Transplantation, Transplantation: January 2019 - Volume 103 - Issue 1 - p 22-27 doi: 10.1097/TP.0000000000002484.
2. Younossi ZM, Koenig AB, Abdelatif D, Fazel Y, Henry L, Wymer M. Global epidemiology of nonalcoholic fatty liver disease-Meta-analytic assessment of prevalence, incidence, and outcomes. Hepatology. 2016 Jul;64(1):73-84. doi: 10.1002/hep.28431. Epub 2016 Feb 22. PMID: 26707365.
3. Ozlem Kutlu, Humeyra Nur Kaleli, Ebru Ozer, "Molecular Pathogenesis of Nonalcoholic Steatohepatitis- (NASH-) Related Hepatocellular Carcinoma", Canadian Journal of Gastroenterology and Hepatology, vol. 2018, Article ID 8543763, 9 pages, 2018.

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