Use of In Vitro Systems for Non-Alcoholic Fatty Liver Disease and Non-Alcoholic Steatohepatitis Studies

Non-alcoholic fatty liver disease (NAFLD) is a common liver pathology, characterized by hepatic steatosis. It can progress to non-alcoholic steatohepatitis (NASH) with the added presence of hepatocyte ballooning, lobular inflammation, and fibrosis that is not brought on through excess alcohol consumption.¹ Although NAFLD and NASH are common, with an approximate worldwide prevalence of 25% and 1.5-6.5% respectively, there are very few specific diagnostic criteria and no specialized therapeutics for either condition.^2,3 Currently, the therapeutic strategy for NAFLD and NASH relies on the off-label use of drugs that target some molecular outcomes of fatty liver disease, such as the use of select diabetes medications to treat underlying metabolic dysfunction, statins, antioxidants, anti-hypertensives (i.e. angiotensin receptor blockers) and anti-fibrotic agents.³ Many pharmaceutical companies have active drug discovery programs working to develop new modalities to treat these diseases and the underlying causation.

Excess fat accumulation in adipocytes induces inflammation, which in turn can lead to insulin resistance and the release of free fatty acids (FFA) from adipose tissue into the circulation¹. FFA can be delivered to multiple organs, including the liver, where they pathologically accumulate in hepatocytes.^1,4

^{Representative H&E images from BioIVT’s ASTERAND® Human Tissue Repository}

Inflammation is a crucial factor influencing NASH development and progression. Several immunologic pathways have been implicated in NASH pathology as a result of inflammatory signaling. For example, Kupffer cell (KC) activation has been demonstrated as critical in the development of NASH.⁴ Activated KCs release inflammatory mediators including interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α), which serve to recruit additional immune cells to further perpetuate the inflammatory environment⁴. Inflammation and reactive oxygen species (ROS) can also lead to fibrosis through activation of hepatic stellate cells (HSC), driving their conversion into myofibroblasts¹.

Effect of NAFLD and NASH on Drug Metabolism

Co-morbid conditions are common in NAFLD and NASH. For instance, fatty liver is reported in 40–80% of patients with type 2 diabetes and 30–90% of obese patients.¹ Additionally, NASH can promote the development of hepatocellular cancer (HCC).¹ Given the high prevalence of co-morbid conditions with NASH, it is likely that patients with fatty liver disease are taking one or more pharmaceuticals in order to treat these ailments. It is therefore important to discern whether NASH can influence drug metabolism in order to anticipate potential adverse drug reactions. Fatty acids have been demonstrated to alter the KC phenotype toward an M1 (inflammatory) type.⁸ In separate studies, inflammatory cytokines have been shown to down-regulate cytochrome P450 expression and activity.^9,10

In vitro predictions for in vivo drug metabolism are primarily carried out using primary hepatocytes. In a recent study at BioIVT, we sought to determine whether changes in drug metabolizing enzymes could be examined using primary human hepatocytes in suspension. Metabolic activity for both phase I and phase II enzymes was generated by assessing metabolite formation with prototypic substrates in suspension incubations. In parallel, formalin fixed paraffin embedded (FFPE) tissue samples from the same donor livers were blindly examined by a board-certified pathologist and given an NAFLD Activity Score (NAS), comprised of graded assessments of hepatic steatosis (0-3), lobular inflammation (0-3) and ballooning (0-2), with NAS ≥ 5 commonly used as a histologic diagnosis of NASH. Donors with NAS ≥ 5 had significantly lower activity of CYP1A2 and CYP2C19 and trending decreased activity of CYP3A4 and the multiple enzyme substrate 7-ethoxycoumarin (ECOD). Furthermore, we separately evaluated the contribution of different pathological features of NASH to alterations in the activity of drug metabolizing enzymes. Categorizing hepatocytes by donor steatosis scores alone uncovered a multitude of changes in metabolic enzyme activities, including some which were not seen when total NAS was evaluated. Hepatocytes from donors with a score of 3 had significantly reduced activity of CYP1A2, CYP2C8, CYP2C19, CYP3A4 and a trending reduction in CYP2C9 and UGT1A1 activity compared to those from donors with a steatosis score of 0. No changes in enzymatic activity were seen based on the inflammation score, while minimal changes were seen based on the ballooning score (reduced CYP2E1 activity). Metabolism based on the fibrosis score showed significant reductions in activity of CYP1A2, CYP2C19 and trending reductions in ECOD and CYP3A4. These findings largely replicate reported findings on drug metabolism changes in NASH and suggest the utility of primary hepatocytes as a tool for assessment of metabolic changes brought on by NASH.^11,12

How can BioIVT support your NAFLD/NASH research?

At BioIVT we are committed to helping our clients select the optimal primary hepatocyte lots for their studies. Donor livers are processed, with histological blocks created to represent sections of the liver. These blocks are utilized to create H&E slides that are scored for histological markers of NASH utilizing the NAS scoring system and reviewed by a board-certified pathologist. Additional donor information regarding BMI, alcohol use, and history of hypertension and diabetes is also available for our primary hepatocyte lots. We routinely screen hepatocyte lots for genetic variants in PNPLA3 and HSD17B13, which are known to influence NASH susceptibility.

BioIVT also offers primary human liver non-parenchymal cells (NPC), Kupffer cells and the capability to perform custom isolations in order to facilitate the development of more complex in vitro models of steatosis and NASH. We have an active research program dedicated to optimizing the isolation and characterization of these cells, as well as additional cell types relevant to NAFLD and NASH pathology. Here at BioIVT, we recognize the value of scientific partnership. Our technical advisors and research and development scientists are available to assist you in the selection and use of reagents for your NAFLD/NASH studies. Additionally, we welcome opportunities to initiate joint discussions regarding your NASH research. Contact us if you are interested in learning more about our product offerings.

References

1. Brunt E.M., Wong V.W., Nobili V., Day C.P., Sookoian S., Maher J.J. et al. (2015) Nonalcoholic fatty liver disease. Nat. Rev. Dis. Primers 1, 15080 10.1038/nrdp.2015.80
2. Tesfay M, Goldkamp WJ, Neuschwander‐Tetri BA. NASH: the emerging most common form of chronic liver disease. Mo Med. 2018;115(3):225–229.
3. Sumida, Y., & Yoneda, M. (2018). Current and future pharmacological therapies for NAFLD/NASH. Journal of gastroenterology, 53(3), 362–376. doi:10.1007/s00535-017-1415-1
4. Kutlu, O., Kaleli, H. N., & Ozer, E. (2018). Molecular Pathogenesis of Nonalcoholic Steatohepatitis- (NASH-) Related Hepatocellular Carcinoma. Canadian journal of gastroenterology & hepatology, 2018, 8543763. doi:10.1155/2018/8543763
5. Romeo, S., Kozlitina, J., Xing, C., Pertsemlidis, A., Cox, D., Pennacchio, L. A., … Hobbs, H. H. (2008). Genetic variation in PNPLA3 confers susceptibility to nonalcoholic fatty liver disease. Nature genetics, 40(12), 1461–1465. doi:10.1038/ng.257
6. Soumik BasuRay, Yang Wang, Eriks Smagris, Jonathan C. Cohen, Helen H. Hobbs (2019). Proceedings of the National Academy of Sciences May 2019, 116 (19) 9521-9526; DOI: 10.1073/pnas.1901974116
7. Ma Y, Belyaeva OV, Brown PM, Fujita K, Valles K, Karki S, et al. 17-beta hydroxysteroid dehydrogenase 13 is a hepatic retinol dehydrogenase associated with histological features of nonalcoholic fatty liver disease. Hepatology. (2019) 69:1504–19. 10.1002/hep.30350
8. Luo, W., Xu, Q., Wang, Q., Wu, H., & Hua, J. (2017). Effect of modulation of PPAR-γ activity on Kupffer cells M1/M2 polarization in the development of non-alcoholic fatty liver disease. Scientific reports, 7, 44612. doi:10.1038/srep44612
9. Morgan E.T., Goralski K.B., Piquette-Miller M., Renton K.W., Robertson G.R., Chaluvadi M.R. Regulation of drug-metabolizing enzymes and transporters in infection, inflammation, and cancer. Drug Metab Dispos. 2008; 36:205–216.
10. Aitken, A. E., Richardson, T. A., and Morgan, E. T. (2006) Regulation of drug-metabolizing enzymes and transporters in inflammation. Annu. Rev. Pharmacol. Toxicol. 46, 123–149
11. Donato, M T et al Potential impact of steatosis on cytochrome P 450 enzymes of human hepatocytes isolated from fatty liver grafts Drug Metab Dispos. 2006 34 1556 1562
12. Fisher C D et al Hepatic cytochrome P 450 enzyme alterations in humans with progressive stages of nonalcoholic fatty liver disease Drug Metab Dispos. 2009 37 2087 2094