Utility of Diet Induced Rodent Models to Investigate Dysregulation of Bile Acids in NASH

Bile acids (BAs) are small detergent molecules that are primarily known for their ability to promote intestinal lipid absorption. While their primary function is associated with gut metabolism, they also are key players in other physiological signaling pathways related to microbiome maintenance, the regulation of glucose, lipid, and energy homeostasis, and cellular immunity.

One of the fundamental areas of investigation in BA metabolism is how BA mechanisms contribute to metabolic disease, particularly nonalcoholic steatohepatitis (NASH)¹. NASH is a chronic disease condition characterized by a spectrum of factors that includes BA and lipid primary toxicity, inflammation, cellular dysfunction, and liver damage. The liver is the site of BA synthesis, suggesting that BAs and BA homeostasis may be a contributing factor to NASH disease and onset.

Currently, BAs are considered clinical biomarkers for liver function and disease. However, not many NASH preclinical in vivo models highlight BAs as a biomarker or major contributing factor. However, more research is being done to show modulation of BA homeostasis as a potential area for treatment. Here we will examine the role of BAs in rodent models of diet induced NASH and determine if BAs can be used as translational preclinical markers for disease.

Comparison of BA profiles between diet induced rodent models of NASH and human NASH clinical phenotype

Dysregulation in BA homeostasis has become a hallmark of many metabolic diseases and has huge implications in NASH. Under physiological conditions, total levels of BAs tend to remain high in the enterohepatic tissues (liver, intestine, kidney, and BA pool) and low in serum/plasma. However, in pathological diseases such as NASH, there tends to be spillover into the systemic circulation which leads to an increase of BAs in this area^1,2.

When we examine the BA profiles in diet induced NASH models vs. human patients, there is some alignment in the data. Animals on high fat-cholesterol diets (HFC) tend to have significant increases in serum BAs, including most primary, secondary, conjugated, and unconjugated BA species³. Levels of chenodeoxycholic acid (CDCA) and deoxycholic acid (DCA), common BA markers for various cholestatic diseases, positively correlate to steatotis scores, serum alanine transaminase (ALT) levels, and fibrotic area scores^3-5. Rats fed an HFC or high fat diet (HFD) have severe disruption in BA homeostasis with many glycine-conjugate BA species present. Other dietary NASH models, such as mice or rats fed a methionine and choline deficient diet (MCD), have marked increases in serum levels of taurocholic acid (TCA) and beta-muricholic acid (βMCA). While we see increases in the presence of BAs in the serum and liver, there are variations in the species concentrations present. These differences could be due in part to dietary and species differences amongst the rodent models and humans⁶.

Overall, when we compare this data to human patients, we see increases in total BAs, including primary, secondary, conjugated, and unconjugated BA species in individuals with nonalcoholic fatty liver disease (NAFLD)/NASH³. Similarly, there is an increase in CDCA, cholic acid (CA), and DCA, as well as the conjugates of primary BAs with taurine and glycine. ALT and aspartate transaminase (AST) levels are also upregulated in these patients, suggesting a correlation between liver injury, NASH, and elevated BA levels. More conclusive data is needed to determine BA profiles in hepatic, fecal, and urine BAs between preclinical models and human patients.

Mechanisms leading to dysregulation of BA profiles in diet induced NASH models

Mechanisms that can lead to dysregulation of BA homeostasis and spillover of BAs into systemic circulation are usually a result of altered expression levels in BA synthesis and/or transport proteins. Levels of expression of BA synthesis enzymes such as Cyp7a1 and Cyp8b1 are inconsistent in diet induced models of NASH. Rats fed a high fat diet and treated with the pancreatic islet beta cell toxin streptozotocin (HFD-STZ) tend to have increased levels of Cyp7a1, Cyp27a1, and Cyp7b1³. Whereas in MCD and HFD-fed NAFLD mice, we see these levels are either decreased or unaltered compared to control animals. Animals fed a modified Amylin liver NASH (AMLN) diet also have mild increases in Cyp7a1 and decreases in Cyp8b1⁷.

When we examine patients, we see dysregulation with increased mRNA levels of Cyp7a1 in NAFLD/NASH/steatosis patients suggesting increased synthesis. Although there are some discrepancies in mRNA and protein levels, overall, there is a trend of upregulation that correlates to changes in primary BA levels and spillover. However, there are inconclusive effects on other synthetic enzymes like CYP27a1 in NASH patients.

What could be the influential driving mechanism leading to these changes in diet induced NASH models and patients? Examining alterations in BA transport enzymes may direct researchers to the answer. Levels of bile salt export pump (BSEP), a major hepatic enzyme involved with biliary uptake and secretion, are significantly downregulated in HFD-STZ rats and mice fed a modified AMLN diet and unaltered in MCD-fed mice^3,7. Similarly, in patients, these levels are largely downregulated suggesting reduced hepatic biliary excretion and increased retention that could lead to liver damage. Alterations in Na+ taurocholate transporting polypeptide (NTCP) are not consistent between preclinical models and human patients and this pattern extends into the expression of compensatory BA transporters such as organic anion transporting polypeptides (OATP) and multidrug resistance associated proteins (MRP). However, MRP3/4 is significantly upregulated in the mouse model and in patients suggesting increased efflux and spillover of BAs into circulation^3,7. When we examine other regulatory mechanisms to determine their contribution to dysregulation of BAs in NASH, we see that farnesoid x receptor (FXR) signaling, which regulates hepatic glucose and lipid metabolism, appears to be impaired in HFD, HFD-STZ, and MCD diet induced NAFLD rats and NASH patients³. However, researchers should be cautious and consider that changes in these levels can vary and again could reflect dietary and species differences with the induction of NASH.

Beyond BA Profiles: Are BAs reliable preclinical biomarkers for NASH? Can diet induced NASH models be used to examine therapeutic efficacy of drugs targeted for improving BA homeostasis to treat NASH?

The frequency of changes in BA levels and profiles in NASH appears to be consistent throughout the data of diet induced models and patients, suggesting that one or more species could be used as a metabolite biomarker in the diagnosis of the disease. Utilizing CDCA or DCA as a biomarker in preclinical diet induced NASH models for translation to the clinic is an option. However, some studies have found that in humans, increased plasma BA levels in NASH are dependent on other metabolic factors such as insulin resistance⁸. More research is required to assess if these levels are consistent in both models and patients to determine the applicability of BAs as relevant markers for evaluation and diagnosis.

In addition to using diet induced models to determine if BA profiles or a specific BA species is reliable as a marker of disease, many of these models have been used to assess the efficacy of therapeutics targeted at improving BA homeostasis in NASH^9-12. Diet induced NASH models, specifically mice on a AMLN diet (similar to Taconic Biosciences' modified AMLN diet), have shown that BA-based therapies such as FXR agonist obetacholic acid (OCA) can reduce NAFLD Activity Scores (NAS) by lowering fibrosis and ALT levels¹³. Other HFD models have been used for investigating the efficacy of additional BA homeostatic therapeutics including fibroblast growth factor 21 (FGF21) agonists, fibroblast growth factor 19 (FGF19) agonists, G protein coupled BA receptor (GPBAR1/TGR5) agonists, ileal bile acid transporter (IBAT/ASBT) inhibitors as well as peroxisome proliferator activated receptor (PPAR) and liver x receptor (LXR) agonists and glucagon like peptide 1 (GLP-1) analogs⁹.

While modified-AMLN diet C57BL/6 mice such as Taconic's Diet Induced NASH B6 have demonstrated utility for efficacy assessment of therapeutics targeting steatosis, inflammation and fibrosis, more research is needed on the relevance of this model to study therapeutics targeting BA homeostasisin NASH. Data on BA metabolism and transport in the Diet Induced NASH B6 is limited, and Taconic Biosciences is actively seeking collaboration partners to obtain this data. Contact us if you are interested to discuss a pilot study or collaboration in this area.

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