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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online January 18, 2006 as doi:10.1096/fj.05-5016fje. |
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* Clinic for Gastroenterology, Hepatology and Infectiology,
Department of Physiology,
Central Institute for Clinical Chemistry and Laboratory Medicine, Department of Biochemistry and Molecular Biology I,
Department of Medical Microbiology, Heinrich Heine University Düsseldorf, Germany;
|| Karolinska University Hospital Huddinge, Stockholm, Sweden;
¶ Centre for Molecular Biology and Neuroscience, University of Oslo, Norway; and
Department of Pathology, University of Cologne, Germany
1Correspondence: Clinic for Gastroenterology, Hepatology, and Infectiology, Heinrich Heine University, Moorenstrasse 5, D-40225 Düsseldorf, Germany. E-mail: haeussin{at}uni-duesseldorf.de
SPECIFIC AIMS
In view of the important role of taurine in liver physiology and in cell protection against various types of injury, the liver phenotype was examined in taurine transporter-deficient mice (taut–/–). The findings support the view that taurine deficiency can trigger hepatitis and liver fibrosis, probably due to a diminished antioxidant defense, up-regulation of inflammatory cytokines and mitochondrial dysfunction.
PRINCIPAL FINDINGS
1. Taurine levels are strongly decreased in Kupffer and sinusoidal endothelial cells, but not in parenchymal cells of (taut–/–) mice
Homozygous disruption of the taut gene resulted in a decrease in taurine levels in skeletal and heart muscles by 
99%, whereas hepatic taurine levels were diminished by 70% in adult (taut–/–) and by 30% in (taut+/–) mice. Immunoelectron microscopy revealed an almost complete depletion of taurine in Kupffer and sinusoidal endothelial cells, but not in hepatic parenchymal cells of (taut–/–) mice.
2. (taut–/–) and (taut+/–) mice develop moderate unspecific hepatitis and liver fibrosis with increased frequency of oval cell proliferation and neoplastic lesions
Compared with wild-type mice, (taut–/–) mice exhibited 3-times more often signs of acute and chronic liver inflammation as well as liver fibrosis, which both progressed with increasing age regarding frequency and severity. Hepatic lesions were quite variable. With disease progression more infiltrates of lymphocytes and plasma cells became involved, suggestive for a chronic evolution. However, more macrophages and neutrophils are found especially in the periportal area, indicating an acute inflammatory process. Severity of necroinflammatory activity was significantly associated with progression of fibrosis. Beyond 18 months, almost 85% of the (taut–/–) mice had liver fibrosis whereas this was observed in only 18% of the wild-type and 46% of the (taut+/–) mice.
Beyond 1 year of age, the number of oval cells was significantly enhanced paralleling severity of inflammation and hepatic damage. In addition, preneoplastic lesions and malignant areas were found more often in (taut–/–) mice than in wild-type mice. Steatosis was not a dominant feature of the liver pathology of (taut–/–) mice.
At the electron microscopic level, 18-month-old (taut–/–) mice showed conspicuous damage of hepatocytes with mitochondria displaying severe injury. Hepatic stellate cells were increased with a significant number of cells showing transition to myofibroblasts. The (taut+/–) mice displayed the same features of cell damage, but in a lower degree.
3. Liver disease in (taut–/–) and (taut–/–) mice is characterized by hepatocyte apoptosis and activation of the CD95 system
Livers from (taut–/–) mice at an age of 15–21 months showed compared with livers from wild-type animals a higher frequency of hepatocyte apoptosis and activation (i.e., tyrosine-phosphorylation) of the CD95 death receptor (Fig. 1
A). In line with this, an increased association of CD95 with FADD and caspase 8 was found, indicative for the presence of death-inducing signaling complexes (DISC). Activation of the CD95 system was found in livers from heterozygous (taut+/–) mice, however, to a lesser degree than in homozygous (taut–/–) mice.
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Livers from young (taut–/–) mice showed if at all only minor signs of CD95 activation when compared with livers from wild-type mice (Fig. 1B
). To test whether taut knockout predisposes to acute liver damage, 10-wk-old wild-type and (taut–/–) mice were subjected in vivo to an acute oral dose of ethanol (6 g/kg body weight). This ethanol challenge resulted in (taut–/–) compared with wild-type mice in a stronger activation of the CD95 system (Fig. 1B
), suggesting that livers from taut-deficient mice are more susceptible to CD95-dependent hepatocyte apoptosis in response to ethanol than livers from wild-type mice.
4. (taut+/–) and (taut–/–) mice show increased plasma levels of tumor necrosis factor 
(TNF)
The inflammatory process in the liver of heterozygous and homozygous taut knockout mice was reflected by an 6-fold increase in the TNF plasma levels in 12- to 15-month-old (taut+/–) and (taut–/–) mice compared with wild-type mice.
5. Taut knockout had no effect on taurine-conjugated bile acids in bile
The possibility was addressed of whether increased hepatocyte apoptosis in (taut–/–) livers might result from an altered bile acid handling. As shown by electron spray-mass spectrometry, there was no difference between the bile acid conjugation pattern in bile from wild-type and (taut–/–) mice. Analysis of the bile acid composition showed lower amounts of cholic acid (CA) in wild-type mice and of 7-keto-CA in (taut–/–) mice, respectively. However, the sum of CA and 7-keto-CA did not differ in bile from wild-type and (taut–/–) mice. These findings suggest that bile salt-induced apoptosis may be a minor factor contributing to liver injury in taut-deficient mice.
6. (taut-/–) mice display mitochondrial dysfunction with a significantly lower respiratory control ratio
In view of the pronounced electron microscopic mitochondrial abnormalities in livers from Taut-deficient mice, respiratory control ratios were determined in liver mitochondria isolated from wild-type, (taut+/–) and (taut–/–) mice. Using either glutamate/malate or succinate as substrates, the respiratory control ratio found in liver mitochondria from (taut–/–) mice was significantly lower than that from wild-type mice. However, values of oxygen consumption during active and controlled respiration were not significantly different.
CONCLUSIONS
The present study shows that disruption of the taurine transporter gene taut leads to chronic hepatitis and liver fibrosis. The process of liver injury appears to progress slowly and major liver pathology is seen beyond the age of 1 year in (taut+/–) and (taut–/–) mice. It appears that both homo- and heterozygous taut knockout mice are prone to hepatitis and liver fibrosis, but the process may be slower in (taut+/–) mice than in (taut–/–) mice. Thus, a gene-dose effect may be anticipated.
Hepatocyte destruction and apoptosis were hallmarks of liver pathology in taut-deficient mice and the progressive loss of hepatocytes is expected to stimulate hepatocyte proliferation. In (taut–/–) mice this regenerative process apparently involves oval cell proliferation, which are abundantly found in livers from (taut–/–) mice with chronic hepatitis. In line with a supposed role of oval cells in liver carcinogenesis, livers from (taut–/–) mice showed significantly more frequent preneoplastic or neoplastic lesions than did livers from wild-type animals.
Although necrotic cell death may also occur, hepatocyte apoptosis is apparently the major mechanism contributing to liver injury in taurine transporter-deficient mice. Several mechanisms may augment the activation of the CD95 system in (taut–/–) and (taut+/–) mice. 1) Taurine depletion led to hepatocyte shrinkage, which was shown to be sufficient to activate the CD95 system. 2) Taurine deficiency may augment the formation of reactive oxygen species, which was shown to be an upstream event of CD95 activation. 3) Taut deficiency results in mitochondrial abnormalities, which may augment ROS formation. 4) Taurine deficiency in immune cells may lead to an up-regulation of proapoptotic cytokines after osmotic stress. In line with this, TNF levels were increased in the plasma of taut-deficient mice.
Liver mitochondria from (taut–/–) and (taut+/–) mice showed severe morphological abnormalities and signs of dysfunction. It is well conceivable that the decreased respiratory control ratio found in liver mitochondria from Taut-deficient mice reflects an increased formation of reactive oxygen species by these mitochondria due to a lower coupling of oxidative phosphorylation. Taurine deficiency may affect mitochondrial function in hepatocytes indirectly by interference with immune functions of Kupffer cells and circulating blood cells. Hepatic stellate cell (HSC) activation and proliferation, as found in livers from taut-deficient mice, may be the consequence of the inflammatory process in the liver, but may involve direct taurine effects on HSC proliferation. Taken together, it is likely that oxidative stress, resulting from mitochondrial dysfunction and up-regulation of inflammatory cytokines is a major cause of liver injury in taut-deficient mice (Fig. 2
).
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The relevance of mild taurine deficiency for the pathogenesis of liver disease is unknown and has not been investigated so far. This is the first report demonstrating that taurine transporter deficiency leads to liver disease at advanced age. Further, young taut-deficient mice, which had not yet developed liver pathology, respond to an acute ethanol challenge with a more pronounced CD95 activation in liver than do wild-type mice, indicating that taut-deficiency may predispose to alcoholic liver damage. Interestingly, the heterozygous animals were prone to liver damage, despite near normal taurine tissue levels. Thus, the possibility has to be investigate that Taut polymorphisms may predispose to liver disease.
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-5016fje;
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