Genetic metabolic liver disorders are a common cause of chronic liver disease and liver failure in infancy and childhood, accounting for over 30% of all liver transplants performed in children (27). Many metabolic disorders caused by inborn errors of metabolism, such as cystic fibrosis, galactosemia, hereditary tyrosinemia and mitochondrial hepatopathies, Wilson disease and congenital defects of glycosylation, share the histologic and biochemical features of micro- or macrovesicular steatosis in combination with cholestasis (28). We have called this combined liver injury "steatocholestasis" (29) and propose that mechanisms of cell injury may be shared among these disorders regardless of the underlying genetic etiology. Hepatic steatosis had been considered in the past to be a benign component of the pathophysiology of metabolic disorders, perhaps caused by malnutrition (30). It is now believed that steatosis in these disorders is caused, at least in part, by the accumulation of toxic intermediates or the absence of essential cofactors, leading to impaired transport or beta-oxidation of fatty acids in mitochondria or altered mitochondrial respiratory chain activity. In recent years, it has become evident that steatosis plays an important role in the biochemical pathogenesis of other liver disorders such as nonalcoholic steatohepatitis (31,32). Because simple steatosis may be well tolerated by hepatocytes, it is believed that a "second hit," most likely induction of oxidative stress, is necessary to trigger cellular injury in the fat-laden hepatocyte (31). Cholestasis in genetic metabolic disorders results from accumulated toxic metabolites or cytokines that down regulate or interfere with function of bile acid and phospholipid canalicular transport proteins, or that reduce mitochondrial oxidative phosphorylation and impair ATP-dependent bile acid secretion (33). It has been recently demonstrated that steatosis itself may perturb normal bile acid metabolism in the hepatocyte, further aggravating bile secretion (34). We propose that in steatocholestasis, hepatocellular retention of toxic bile acids (35) in fat-laden hepatocytes may provide a "second hit" through stimulation of ROS generation and signaling cascades that trigger hepatocellular injury (36-39). It should be emphasized that the "metabolic syndrome" associated with obesity and nonalcoholic steatohepatitis is not the condition described herein (40), but rather genetic metabolic liver diseases caused by inborn errors of metabolism.
To characterize the mechanisms underlying the hepatocyte response to concurrent bile acid toxicity and steatosis, we have developed a rodent model of steatocholestasis. In this model, freshly isolated rat hepatocytes in suspension from lean and obese Zucker rats were exposed to the hydrophobic bile acid, glycochenodeoxycholic acid (GCDC), in suspension for 4 hours. The results showed that oncotic necrosis was significantly increased and apoptosis was reduced in fat-laden hepatocytes compared with hepatocytes from lean Zucker rats. Necrosis was dependent on both ROS generation and the MPT and was abrogated by treatment of cells with antioxidants and MPT blockers. However, basal and dynamic ATP content and alpha-tocopherol concentrations did not differ between the fat-laden and lean hepatocytes. Furthermore, GCDC stimulated ROS generation, MPT and cytochrome c release to a similar extent in purified mitochondria from both obese and lean rats. Thus, the factors determining the different modes of cell death favored by the fat-laden cells in this model do not seem to include differences in mitochondrial function nor ROS generation by mitochondria. Investigation of other pathways and regulators of apoptosis and necrosis are currently underway in the fat-laden hepatocyte such as death receptor activation, Bcl-2 family proteins, MAP kinases and cell survival signals.
The chemical structure of GA shares structural features with molecules known to be ligands for nuclear hormone receptors (NHR). NHRs comprise a large family of ligand-activated transcription factors that control gene regulation in many biological processes (47). Several are activated by steroid and sterol molecules, including cholesterol, bile acids, tocopherols, retinoids and bile alcohols (52). Several class II NHRs regulate hepatic specific functions that may modulate hepatic injury in cholestasis. Particularly, CAR, FXR, LXR and PXR are transcription factors for genes that influence synthesis, metabolism, uptake and secretion of bile acids by the hepatocyte, CYP activities, and fatty acid and cholesterol synthesis. Thus, we propose that GA and GL may also function as ligands for one of the families of NRH leading to target gene regulation that is responsible, in part, for the effects of GA and GL on cholestatic (bile acid-induced) hepatic injury. Further exploration of these and related compounds as potential therapies is warranted.
The overall goals of our research program are to define the fundamental mechanisms by which retention of hydrophobic bile acids induce oxidative stress and cell death signaling pathways in cholestasis and steatocholestasis. Through a better understanding of these pathways, new targets for pharmaceutical intervention may be uncovered that may ultimately afford infants and children with cholestasis robust medical treatment to reduce hepatic injury and fibrosis.
The authors thank the post-doctoral fellows and trainees that have participated in the laboratory research studies.
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