Simple steatosis sensitizes cholestatic rats to liver injury and dysregulates bile salt synthesis and transport

Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disorder. It is uncertain if simple steatosis, the initial and prevailing form of NAFLD, sensitizes the liver to cholestasis. Here, we compared the effects of obstructive cholestasis in rats with a normal liver versus rats with simple steatosis induced by a methionine/choline-deficient diet. We found that plasma liver enzymes were higher and hepatic neutrophil influx, inflammation, and fibrosis were more pronounced in animals with combined steatosis and cholestasis compared to cholestasis alone. Circulating bile salt levels were markedly increased and hepatic bile salt composition shifted from hydrophilic tauro-β-muricholate to hydrophobic taurocholate. This shift was cytotoxic for HepG2 hepatoma cells. Gene expression analysis revealed induction of the rate-limiting enzyme in bile salt synthesis, cytochrome P450 7a1 (CYP7A1), and modulation of the hepatic bile salt transport system. In conclusion, simple steatosis sensitizes the liver to cholestatic injury, inflammation, and fibrosis in part due to a cytotoxic shift in bile salt composition. Plasma bile salt levels were elevated, linked to dysregulation of bile salt synthesis and enhanced trafficking of bile salts from the liver to the systemic circulation.


Surgical procedures and tissue processing
The animals were weighed before surgery. Surgery was performed under inhalation anesthesia comprising a mixture of oxygen:air (1:1 volume ratio, 2 L/min) and 2-4% isoflurane (Forene, Abbott Laboratories, Chicago, IL, USA). Animals received analgesia pre-operatively by subcutaneous administration of 250 µg/kg buprenorphine (Temgesic, Schering-Plough, Kenilworth, NJ, USA). Body temperature was maintained at 37.0 ± 0.2 °C. A midline laparotomy was performed and the common bile duct was mobilized.
In animals undergoing BDL, the common bile duct was ligated proximally and distally relative to the liver, and the lumen was disconnected in between. In animals undergoing sham surgery, the bile duct was manipulated but not ligated and not disconnected. The abdomen was closed with dual layer sutures.
After 7 days the animals were weighed, anesthetized as described, and euthanized by exsanguination from the vena cava. Blood samples were collected in heparin and EDTA. Liver tissue was snap-frozen in liquid nitrogen and stored at -80 °C for subsequent RNA extraction and HPLC (left lateral and median lobe), and MPO activity, TNF-alpha, and IL-6 measurements (right lateral and median lobe). Samples were also fixed in 10% buffered formalin for histological analysis. A sample from the caudal lobe was weighed, desiccated at 37° C for 4 weeks, and weighed again to determine hepatic water content. Intergroup differences in hepatic water content, calculated by [1 -(dry weight/wet weight)] × 100%, were used as a measure of edema.

Measurement of bile salt composition
Individual bile salt/acid species were separated and quantified by reverse-phase HPLC [1,2]. Twenty µL of plasma was mixed with 5 volumes of acetonitrile to precipitate protein. For liver samples, ~100 mg tissue was sonicated in 5 volumes of H 2 O for 30-90 s, followed by addition of 10 volumes of acetonitrile. Samples were centrifuged at 20,000 ×g for 10 min. Solvent was evaporated from supernatants, bile salts/acids were solubilized in 200 µL of 25% methanol, and 100 µL was applied to a Hypersil C18 HPLC column We achieve good separation of the 17 relevant bile salt species present in rodents using this protocol, as demonstrated by a chromatogram ( Supplementary Fig. S1). Total bile salt concentrations quantified using HPLC refer to the cumulative of all bile salts/acids, which were consistent with results from an enzyme cycling assay ( Supplementary Fig. S4). The mean TβMCA/TCA ratio was calculated by averaging ratios from individual animals.
Messenger RNA levels were quantified as described in the main article.  Supplementary Fig. S4. The hepatic total antioxidant capacity is similar in the liver of sham control rats, rats with steatosis, cholestasis, and combined steatosis and cholestasis. A total antioxidant capacity assay (Sigma) was performed as described previously [3]. There were no statistically significant differences detected between the 4 groups (p = 0.42).

NASH activity score (range)
Sham NAFLD activity scores demonstrate that none of the animals met the histological criteria for NASH diagnosis, which requires a total score ≥ 5. The validated scoring system as described by Kleiner et al. [4] was applied.
Scores are shown as median (range) for n = 5-6 animals per group. Sequences were designed using genomic and cDNA data obtained from GenBank (NCBI). Human CYP7A1 and SHP primers were from Qiagen (QuantiTect Primer Assay). Sequences that were not available from the manufacturer are designated as N.A.