Experimental non-alcoholic fatty liver disease results in decreased hepatic uptake transporter expression and function in rats

Abstract

Non-alcoholic fatty liver disease (NAFLD) encompasses a spectrum of diagnoses ranging from simple fatty liver (SFL), to non-alcoholic steatohepatitis (NASH). This study aimed to determine the effect of moderate and severe NAFLD on hepatic transporter expression and function in vivo. Rats were fed a high-fat diet (SFL model) or a methionine-choline-deficient diet (NASH model) for eight weeks. Hepatic uptake transporter function was determined by bromosulfophthalein (BSP) disposition. Transporter expression was determined by branched DNA signal amplification assay and western blotting; inflammation was identified by immunostaining of liver slices for interleukin 1 beta (IL-1beta). MC- rats showed significant retention of BSP in the plasma when compared to control rats. Hepatic NTCP, OATP1a1, 1a4, 1b2 and 2b1; and OAT 2 and 3 mRNA levels were significantly decreased in high-fat and MC- diet rats when compared to control. Protein expression of OATP1a1 was significantly decreased in high-fat animals, while OATP1a1 and OATP1b2 expressions were significantly lower in MC- rats when compared to control. Liver tissue from high-fat and MC- rats stained positive for IL-1beta, a pro-inflammatory cytokine known to decrease expression of NTCP, OATP and OAT transporters, suggesting a plausible mechanism for the observed transporter alterations. These data suggest that different stages of NAFLD result in altered hepatic uptake transporter expression that can lead to a functional impairment of xenobiotic uptake from the blood. Furthermore, NAFLD may alter the plasma retention time of clinically relevant drugs that are reliant on these transporters and may increase the potential drug toxicity.

Fig. 1. Liver histology

5 µm sections of normal, low-fat isocaloric, high-fat, MC+ and MC- diet livers were stained with hematoxylin and eosin. Images were generated using a standard light microscope at 20x magnification. Arrows indicate mild bridging fibrosis. Circle denotes inflammatory foci.

Fig. 2. BSP disposition in low-fat isocaloric, high-fat, MC+ and MC- rats

Animals were administered BSP (100 mg/kg, ip) and plasma and biliary BSP concentrations were determined over 1 h. BSP concentration was determined by light absorbance at 580 nm. BSP concentrations are expressed as mg/100 ml plasma and mg/ml bile ± standard error of mean, respectively. Bile flow is expressed as µl/min/kg. Asterisks indicate significance from MC+ control diet rats, while daggers indicate significant difference from normal diet rats (P < 0.05).

Fig. 3. Plasma chemistry

Plasma concentrations of alkaline phoshatase (ALP), alanine amino-transferase (ALT), glucose and cholesterol, were determined in control, high-fat and MC- rats. ALP and ALT levels expressed as U/L ± standard error of mean. Glucose and cholesterol levels expressed as mg/dL ± standard error of mean. Asterisk indicates significant difference from control (P < 0.05).

Fig. 4. Hepatic mRNA levels of OAT, OCT and NTCP uptake transporters

Total hepatic RNA from control, high-fat and MC- livers was analyzed for OAT, OCT and NTCP mRNA levels by branched DNA assay. Data is expressed as relative light units (RLU) ± standard error of mean. Asterisk indicates significant difference from control livers (P < 0.05).

Fig. 5. Hepatic mRNA levels of OATP uptake transporters

Total hepatic RNA from control, high-fat and MC- livers was analyzed for OATP mRNA levels by branched DNA assay. Data expressed as relative light units (RLU) ± standard error of mean. Asterisk indicates significant difference from control (P < 0.05).

Fig. 6. Hepatic protein levels of hepatic OATP1a1, 1a4, 1b2, Mrp2 and GAPDH

30 µg of control, high-fat and MC- liver whole cell lysate protein was added to each well and resolved using SDS-PAGE. Antibodies specific for rat OATP1a1, 1a4, 1b2, Mrp2 and GAPDH were used to determine protein expression levels of each diet group. Graphs indicate relative protein expression normalized to respective GAPDH expression as determined by densitometry. Asterisks indicates significant difference from control rats and dagger indicates significant difference from high-fat rats (P < 0.05)

Fig. 7. Hepatic glutathione concentrations in control, high-fat and MC- rats

Hepatic glutathione (GSH) levels were determined using an enzymatic method for quantification of total GSH. GSH levels are expressed as µM GSH/ gram liver tissue (wet weight).

Fig. 8. Immunohistochemical staining of hepatic IL-1β

Immunohistochemical staining was performed on paraffin sections of control, high-fat and MC- livers. Tissues were counterstained with hematoxylin and images of each group were taken at 20x and 40x magnification.