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An Overview of Available Preclinical Models for Non-alcoholic Fatty Liver Disease

NASH is thought to affect 25% of the global population and is a primary risk factor for Hepatocellular carcinoma (HCC)  the second most common cause of cancer deaths worldwide. Understanding the aetiology of NAFLD progression, developing non-invasive diagnostic tests and developing treatments are urgent priorities.

A Growing Public Health Concern

Overweight and obesity are a growing public health concern. 

Metabolic syndrome (MetS), in which disturbed lipid homeostasis and metabolic inflammation take the lead, is strongly associated with obesity. Besides type 2 diabetes (T2D) and cardiovascular diseases, MetS increases the risk of developing non-alcoholic fatty liver disease (NAFLD), the most prevalent chronic liver disease worldwide. 1

The spectrum of NAFLD ranges from benign simple steatosis 2 to steatohepatitis (NASH) and end-stage liver diseases. If steatosis is not managed in time, resident liver cells (e.g., Kupffer and hepatic stellate cells) become activated and immune cells (mainly macrophages) infiltrate the liver, a condition defined as NASH. 

This progressive form of NAFLD 3 can further trigger hepatocyte damage with/without fibrosis and increase the risk of cirrhosis 4 and hepatocellular carcinoma (HCC)5. In contrast to alcoholic liver disease- and viral hepatitis-induced HCC, NASH-related HCC is currently the most rapid growing indication for liver transplant in HCC patients.6 

The Need for Reliable Models

There is an urgent need for a reliable ‘humanized’ model that displays a liver phenotype that is identical to human disease, with macrovesicular steatosis, lobular inflammation, hepatocellular ballooning (including Mallory-Denk bodies), and fibrosis as predominant features. 

An optimal NAFLD model should have the ability to further progress to advanced fibrosis, cirrhosis, and ultimately HCC. Moreover, it should encompass MetS-related characteristics, such as obesity, disturbed lipid, glucose and insulin metabolism, as well as systemic inflammation. 

Available Preclinical Models for Non-alcoholic Fatty Liver Disease 

Dietary Murine Models 

Diet-induced obesity is believed to be the most common risk factor for NAFLD in humans. The experimental NAFLD/NASH models are often based on overnutrition, a condition that can be induced by means of diets varying in macronutrient composition, amongst others. Models include:

  • Regular high-fat diet (HFD) – 60% fat, 20% proteins, 20% carbohydrates
  • High-fat atherogenic diet (HFC) – 60% fat , 1.25% cholesterol, plus 0.5% cholate
  • Amylin Liver NASH (AMLN) diet – 40% high-fat, 22% high-fructose, ~18% trans-fatty acids, 2% high-cholesterol
  • Gubra Amylin NASH (GAN) diet – 40% high-fat, 22% high-fructose, 2% high-cholesterol
  • Fast-food-like nutritional regime based on high-fat/high- fructose/high-cholesterol (41%/30%/2%)
  • High-caloric (43%) Western-type diet composed of soybean oil (high n-6-PUFA, 25g/100g) and 0.75% cholesterol
  • Methionine/choline-deficient diet (MCD)
  • The American lifestyle induced obesity syndrome (ALIOS) diet – high fat combined with fructose-containing drinking water
  • Diet-induced animal model of non-alcoholic fatty liver disease (DIAMOND)

These models have provided better insights into NAFLD/NASH pathogenesis. Nevertheless, it is noteworthy that these models failed to consistently achieve the full spectrum of human NASH, thereby limiting preclinical validity.

Genetic Murine Models 

Genetic animal models are essential for unravelling the underlying mechanisms related to the progression of NAFLD. Genetic factors include:

  • Apolipoprotein E2 Knick-in (APOE2)
  • ApoE deficiency (ApoE-/-)
  • Low-desity lipoprotein receptor deficiency (Ldlr-/-)
  • Microsomal prostaglandin E synthase 1 deficiency (mPGES-1)
  • Patatin-like phospholipase domain-containing 3 knock-in (PNPLA-3)
  • Transmembrane 6 superfamily member 2 knockdown (mTm6s2-shRNA8)
  • Gankyrin liver-specific knockout (GLKO)
  • Truncated mutation in Alström (Alms1) gene (foz/foz)
  • Fatty liver Shionogi (FLS)
  • Augmenter of liver regeneration knock-out (Alr-/-)
  • Melanocortin receptor knockout (Mc4r-/-)

Chemically-induced Murine Models 

Another way to explore the progression and/or regression of liver fibrosis and subsequent development of cirrhosis is by targeting the liver with chemotoxins:

  • CCL4 
  • Thioacetamide (TAA) 

The exact pathophysiological mechanism underlying chemically-induced hepatic fibrogenesis requires further investigation.

Figure 1. Overview of existing preclinical models to study NAFLD.

Other Murine Models 

Besides a role for genetic and dietary factors in preclinical NAFLD development, recent focus has also discretely shifted towards the relevance of environment, thereby introducing a novel concept of thermoneutral housing (30–32 °C). Mice housed under thermoneutral conditions were not only shown to induce a pro-inflammatory immune response, but also to deteriorate HFD-induced NASH progression, compared to standard housing conditions. 

Additionally, mice displayed increased intestinal permeability and alterations in gut microbiome, features mimicking the human situation and suggesting a dietary stimulus is prerequisite for liver fibrosis development. 

It has also been recently suggested that chronic disruption of circadian rhythm may spontaneously induce the progression from NAFLD to NASH, fibrosis, and HCC, similar to the human situation, pointing towards its translational value. 

The Need for Robust Preclinical Models Remains

So far, considerable efforts have recently been made to better understand the pathogenesis of human NAFLD and/or related clinical questions using a wide variety of preclinical models. However, none of these models resemble the complete human NAFLD spectrum, including related metabolic features that recapitulate this chronic liver disease. 

Several models, exposed to various dietary compositions, have broadened our knowledge of NAFLD progression, in particular early-onset low-grade inflammation. 

Alternative models (genetic or chemically induced) have provided insights into fibrotic features of human NAFLD, one of the most important predictors of human NASH progression 7. Other models have been found suitable for testing therapeutic interventions in the context of NAFLD/NASH. 

Significant needs exist for non-invasive diagnostic methods, therapeutic target identification, and drug development. The urgent need for more robust preclinical models remains.8

This post has been adapted, with permission, from Yvonne Oligschlaeger and Ronit Shiri-Sverdlov’s article, ‘NAFLD Preclinical Models: More than a Handful, Less of a Concern?’, Biomedicines 2020, 8 (2), 28.

To view resources from the Global NASH Congress series click here.


References

1 – Younossi, Z.M.; Golabi, P.; de Avila, L.; Paik, J.M.; Srishord, M.; Fukui, N.; Qiu, Y.; Burns, L.; Afendy, A.; Nader, F. The global epidemiology of NAFLD and NASH in patients with type 2 diabetes: A systematic review and meta-analysis. Journal of Hepatology 2019, 71, 793–801, doi:10.1016/j.jhep.2019.06.021.

2 – Jensen, V.S.; Tveden-Nyborg, P.; Zacho-Rasmussen, C.; Quaade, M.L.; Ipsen, D.H.; Hvid, H.; Fledelius, C.; Wulff, E.M.; Lykkesfeldt, J. Variation in diagnostic NAFLD/NASH read-outs in paired liver samples from rodent models. Journal Pharmacological and Toxicological Methods 2019, 101, 106651, doi:10.1016/j.vascn.2019.106651. 

3 – Ganbold, M.; Owada, Y.; Ozawa, Y.; Shimamoto, Y.; Ferdousi, F.; Tominaga, K.; Zheng, Y.W.; Ohkohchi, N.; Isoda, H. Isorhamnetin Alleviates Steatosis and Fibrosis in Mice with Nonalcoholic Steatohepatitis. Scientific Reports 2019, 9, 16210, doi:10.1038/s41598-019-52736-y. 

4 – Li, B.; Zhang, C.; Zhan, Y.T. Nonalcoholic Fatty Liver Disease Cirrhosis: A Review of Its Epidemiology, Risk Factors, Clinical Presentation, Diagnosis, Management, and Prognosis. Canadian Journal of Gastroenterology and Hepatology 2018, 2018, 2784537, doi:10.1155/2018/2784537. 

5 – Anstee, Q.M.; Reeves, H.L.; Kotsiliti, E.; Govaere, O.; Heikenwalder, M. From NASH to HCC: current concepts and future challenges. Nature Reviews Gastroenterology & Hepatology 2019, 16, 411–428, doi:10.1038/s41575-019- 0145-7. 

6 – Wong, R.J.; Cheung, R.; Ahmed, A. Nonalcoholic steatohepatitis is the most rapidly growing indication for liver transplantation in patients with hepatocellular carcinoma in the U.S. Hepatology 2014, 59, 2188–2195, doi:10.1002/hep.26986. 

7 – Younossi, Z.M.; Loomba, R.; Anstee, Q.M.; Rinella, M.E.; Bugianesi, E.; Marchesini, G.; Neuschwander- Tetri, B.A.; Serfaty, L.; Negro, F.; Caldwell, S.H., et al. Diagnostic modalities for nonalcoholic fatty liver disease, nonalcoholic steatohepatitis, and associated fibrosis. Hepatology 2018, 68, 349–360, doi:10.1002/hep.29721. 

8 – Hansen, H.H.; Feigh, M.; Veidal, S.S.; Rigbolt, K.T.; Vrang, N.; Fosgerau, K. Mouse models of nonalcoholic steatohepatitis in preclinical drug development. Drug Discovery Today 2017, 10.1016/j.drudis.2017.06.007, doi:10.1016/j.drudis.2017.06.007. 

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