Pulmonary, Gastrointestinal and Urogenital Pharmacology
Experimental non-alcoholic fatty liver disease results in decreased hepatic uptake transporter expression and function in rats

https://doi.org/10.1016/j.ejphar.2009.04.002Get rights and content

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-1β). 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-1β, 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.

Introduction

The liver plays a crucial role in the elimination of many clinically relevant drugs from the body. While drug-metabolizing enzymes are critical in hepatic drug clearance, often, hepatic transport is required to initiate and complete this process. Several basolateral transmembrane proteins are capable of transporting organic ions from plasma into the hepatocyte, including the organic anion transporting polypeptides (OATP), organic anion transporters (OAT); organic cation transporters (OCT); and the sodium-dependent bile salt uptake transporter Na+-taurocholate co-transporting polypeptide (NTCP). Coordinately, the overlapping affinities of these transporters provide an efficient means of extracting ionic xenobiotics from the sinusoidal blood (Kullak-Ublick and Meier, 2000).

Variations in patient response to drug therapy are a significant safety issue. Severe adverse drug effects among hospitalized patients in the United States may number as high as two million per year and as many as 100,000 of them prove fatal (Lazarou et al., 1998). Remarkably, the majority of these adverse reactions are in patients given the correct drug and proper dosage. It is estimated that less than 20% of these cases are due to genetic polymorphisms that result in altered metabolism: the vast majority of the adverse reactions are due to individual host or environmental factors such as age, nutrition, or disease state (Ingelman-Sundberg and Rodriguez-Antona, 2005). Both pre-clinical and clinical studies have indicated that hepatic disease may result in alterations in the pharmacokinetics and elimination of a drug (Williams and Mamelok, 1980, Westphal and Brogard, 1997). Alterations in drug disposition have been well documented for liver conditions such as sepsis (Pea et al., 2005, Kim et al., 2000) and primary biliary cirrhosis (Jorquera et al., 2001). In addition, altered expression of cytochrome P450 enzymes and hepatic transporters observed in viral and alcoholic hepatitis patients have been associated with variability in drug response, resulting in either drug toxicity or decreased therapeutic effectiveness (Morgan, 2001, Heinrich et al., 1990, Renton, 2005). In contrast, relatively little data has been reported with respect to the effect of non-alcoholic fatty liver disease (NAFLD) on hepatic drug transporter.

NAFLD encompasses a spectrum of symptoms ranging from steatosis (simple fatty liver, SFL), to steatohepatitis (fatty liver with liver cell damage, inflammation and progressive fibrosis) (Reynaert et al., 2005). The prevalence of NAFLD is estimated to be between 14% and 24% in the world population, and though once thought an adult disease, it is now known to afflict children as well (Browning and Horton, 2004). Simple steatosis is characterized by micro and macrovesicular steatosis and predisposes the liver to the more severe non-alcoholic steatohepatitis (NASH) (Koteish and Mae, 2002). NASH is histologically characterized by micro and macrovesicular steatosis, lobular inflammation, and hepatocellular damage, including accumulation of Mallory's hyaline, ballooning degeneration, necrosis, and fibrosis (zone 1 and zone 3) (Peters et al., 1975). NASH occurs in 2–3% of all patients with excess fat accumulation in the liver, and accounts for approximately 10% of all newly diagnosed cases of chronic liver disease.

The classic rodent model of NASH involves inducing steatohepatitis through a methionine-choline-deficient (MC−) diet. This model of “fibrosing steatohepatitis” is characterized by the development of fibrotic strands that surround hepatocytes in a fashion identical to those in human disorders of lipid-associated hepatic fibrosis. Additionally, steatosis, chronic hepatocyte injury, and hepatic inflammation precede activation of stellate cells and fibrosis by several weeks, a sequence of events that is analogous to that which occurs in NASH (George et al., 2003).

Previous studies have demonstrated that this model causes significant changes in hepatic efflux transporters resulting in a significant shift in the disposition of acetaminophen metabolites from the bile to the plasma (Lickteig et al., 2007a). In the current study, high-fat (SFL model) and MC− (NASH model) diets were used as models to determine the effect of SFL and NASH on hepatic uptake transporter expression and whether the resulting change in expression causes a functional alteration of xenobiotic pharmacokinetics.

Section snippets

Chemicals

Urethane, bromosulfophthalien (BSP), and NaOH were purchased from Sigma (St. Louis, MO). Heparin and sterile 0.9% sodium chloride solution (w/v) were obtained from Baxter Healthcare Corporation (Deerfield, IL). PE-10 and PE-50 polyethylene tubing were purchased from Braintree Scientific Inc. (Braintree, MA).

Animals

Male Sprague Dawley rats (8–9 weeks) were obtained from Harlan (Indianapolis, IN). All animals were acclimated in 12 h light and 12 h dark cycles in a University of Arizona AAALAC-certified

Liver histology

Control, low-fat isocaloric, and MC+ livers displayed no signs of steatosis or fibrosis (Fig. 1). High-fat livers showed primarily microvesicular steatosis with mild lobular inflammation, while MC− livers showed marked diffuse macrovesicular hepatic steatosis with mild lobular inflammation as well as early signs of bridging fibrosis. NAFLD Activity Scoring (NAS) for normal, low-fat isocaloric, high-fat, MC+ and MC− rat livers are summarized in Table 1. The histology and NAS scoring observed in

Discussion

Simple fatty liver (early stage NAFLD) is typically associated with obesity. In 1994, 22.5% of Americans were determined to be clinically obese, a statistic which is projected to reach almost 40% by 2025 (Kopelman, 2000). Due to this increase in obesity, information pertaining to liver function during the various stages of NAFLD is of increasing importance. In 2005, Geier et al. suggested that alterations in hepatic transporters may render fatty livers more vulnerable to various xenobiotics.

Acknowledgments

This work was supported by the National Institutes of Health grants ES007091 (C.D.F), ES011646 and DK068039 (to N.J.C). This work has been presented in part at the International Society for the Study of Xenobiotics meeting, Maui, Hawaii 2005.

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