Localization of ABCG5 and ABCG8 proteins in human liver, gall bladder and intestine

BACKGROUND: The molecular mechanisms that regulate the entry of dietary sterols into the body and their removal via hepatobiliary secretion are now beginning to be defined. These processes are specifically disrupted in the rare autosomal recessive disease, Sitosterolemia (MIM 210250). Mutations in either, but not both, of two genes ABCG5 or ABCG8, comprising the STSL locus, are now known to cause this disease and their protein products are proposed to function as heterodimers. Under normal circumstances cholesterol, but not non-cholesterol sterols, is preferentially absorbed from the diet. Additionally, any small amounts of non-cholesterol sterols that are absorbed are rapidly taken up by the liver and preferentially excreted into bile. Based upon the defects in sitosterolemia, ABCG5 and ABCG8 serve specifically to exclude non-cholesterol sterol entry at the intestinal level and are involved in sterol excretion at the hepatobiliary level. METHODS: Here we report the biochemical and immuno-localization of ABCG5 and ABCG8 in human liver, gallbladder and intestine using cell fractionation and immunohistochemical analyses. RESULTS: We raised peptide antibodies against ABCG5 and ABCG8 proteins. Using human liver samples, cell fractionation studies showed both proteins are found in membrane fractions, but they did not co-localize with caveolin-rafts, ER, Golgi or mitochondrial markers. Although their distribution in the sub-fractions was similar, they were not completely contiguous. Immunohistochemical analyses showed that while both proteins were readily detectable in the liver, ABCG5 was found predominately lining canalicular membranes, whereas ABCG8 was found in association with bile duct epithelia. At the cellular level, ABCG5 appeared to be apically expressed, whereas ABCG8 had a more diffuse expression pattern. Both ABCG5 and ABCG8 appeared to localize apically as shown by co-localization with MRP2. The distribution patterns of ABCG5 and ABCG8 in the gallbladder were very similar to each other. In the small intestine both ABCG5 and ABCG8 appear to line the brush border. However, at the level of the enterocyte, the cellular distribution patterns of ABCG5 and ABCG8 differed, such that ABCG5 was more diffuse, but ABCG8 was principally apical. Using standard deglycosylation methods, ABCG5 and ABCG8 do not appear to be glycosylated, suggesting a difference between human and mouse proteins. CONCLUSION: We report the distribution patterns of ABCG5 and ABCG8 in human tissues. Cell fractionation studies showed that both proteins co-fractionated in general, but could also be found independent of each other. As predicted, they are expressed apically in both intestine and liver, although their intracellular expression patterns are not completely congruent. These studies support the concept of heterodimerization of ABCG5 and ABCG8, but also support the notion that these proteins may have an independent function

Znf202 Affects High Density Lipoprotein Cholesterol Levels and Promotes Hepatosteatosis in Hyperlipidemic Mice

Background: The zinc finger protein Znf202 is a transcriptional suppressor of lipid related genes and has been linked to hypoalphalipoproteinemia. A functional role of Znf202 in lipid metabolism in vivo still remains to be established. Methodology and Principal Findings: We generated mouse Znf202 expression vectors, the functionality of which was established in several in vitro systems. Next, effects of adenoviral znf202 overexpression in vivo were determined in normo- as well as hyperlipidemic mouse models. Znf202 overexpression in mouse hepatoma cells mhAT3F2 resulted in downregulation of members of the Apoe/c1/c2 and Apoa1/c3/a4 gene cluster. The repressive activity of Znf202 was firmly confirmed in an apoE reporter assay and Znf202 responsive elements within the ApoE promoter were identified. Adenoviral Znf202 transfer to Ldlr-/- mice resulted in downregulation of apoe, apoc1, apoa1, and apoc3 within 24 h after gene transfer. Interestingly, key genes in bile flux (abcg5/8 and bsep) and in bile acid synthesis (cyp7a1) were also downregulated. At 5 days post-infection, the expression of the aforementioned genes was normalized, but mice had developed severe hepatosteatosis accompanied by hypercholesterolemia and hypoalphalipoproteinemia. A much milder phenotype was observed in wildtype mice after 5 days of hepatic Znf202 overexpression. Interestingly and similar to Ldl-/- mice, HDL-cholesterol levels in wildtype mice were lowered after hepatic Znf202 overexpression. Conclusion/Significance: Znf202 overexpression in vivo reveals an important role of this transcriptional regulator in liver lipid homeostasis, while firmly establishing the proposed key role in the control of HDL levels

A Bovine Model of Respiratory Chlamydia psittaci Infection: Challenge Dose Titration

This study aimed to establish and evaluate a bovine respiratory model of experimentally induced acute C. psittaci infection. Calves are natural hosts and pathogenesis may resemble the situation in humans. Intrabronchial inoculation of C. psittaci strain DC15 was performed in calves aged 2–3 months via bronchoscope at four different challenge doses from 106 to 109 inclusion-forming units (ifu) per animal. Control groups received either UV-inactivated C. psittaci or cell culture medium. While 106 ifu/calf resulted in a mild respiratory infection only, the doses of 107 and 108 induced fever, tachypnea, dry cough, and tachycardia that became apparent 2–3 days post inoculation (dpi) and lasted for about one week. In calves exposed to 109 ifu C. psittaci, the respiratory disease was accompanied by severe systemic illness (apathy, tremor, markedly reduced appetite). At the time point of most pronounced clinical signs (3 dpi) the extent of lung lesions was below 10% of pulmonary tissue in calves inoculated with 106 and 107 ifu, about 15% in calves inoculated with 108 and more than 30% in calves inoculated with 109 ifu C. psittaci. Beside clinical signs and pathologic lesions, the bacterial load of lung tissue and markers of pulmonary inflammation (i.e., cell counts, concentration of proteins and eicosanoids in broncho-alveolar lavage fluid) were positively associated with ifu of viable C. psittaci. While any effect of endotoxin has been ruled out, all effects could be attributed to infection by the replicating bacteria. In conclusion, the calf represents a suitable model of respiratory chlamydial infection. Dose titration revealed that both clinically latent and clinically manifest infection can be reproduced experimentally by either 106 or 108 ifu/calf of C. psittaci DC15 while doses above 108 ifu C. psittaci cannot be recommended for further studies for ethical reasons. This defined model of different clinical expressions of chlamydial infection allows studying host-pathogen interactions

Intestinal excretion of unconjugated bilirubin in man and rats with inherited unconjugated hyperbilirubinemia

Patients with Crigler-Najjar syndrome and Gunn rats cannot form bilirubin glucuronides owing to a lack of bilirubin UDP-glucuronosyltransferase activity. Because increased serum and tissue bilirubin levels remain constant, an alternative excretory route has to substitute for this deficiency. Gunn rats excrete in bile only 2-13% of the bilirubins eliminated in Wistar rats. In contrast, the biliary excretion rate of urobilinogen in Gunn and Wistar rats is comparable. The sum of bilirubins and urobilinogen excreted in the bile of Gum rats amounts to 10-30% of pigments excreted in Wistar rats. Despite this low biliary excretion, the intestinal content and fecal excretion of bile pigments in Gunn and Wistar rats were similar. These data support an extrabiliary entrance of unconjugated bilirubin into the intestine. Additional proof for this was found in that the intestinal lumen of Gunn rats still contains a high amount of bilirubins and urobilinogen after 3 d of external biliary drainage. A similar procedure in Wistar rats resulted in the complete disappearance of bile pigments from the intestine. The direct transmural transport of bilirubin from blood to all parts of the intestinal lumen was demonstrated by injecting C-14-bilirubin i.v. into Gunn rats with isolated parts of small and large intestine. In Crigler-Najjar and Gilbert's syndrome patients, the biliary excretion of bile pigments has previously been shown to be strongly reduced. Their stools, however, contained approximately the same amount of bile pigments as in normal subjects. Although only traces of unconjugated bilirubin were detected in the stool of normal persons (4 +/- 3% of total bile pigments), higher amounts were found in patients with Crigler-Najjar disease (20 +/- 12&). These results suggest a direct intestinal permeation of unconjugated bilirubin in severe unconjugated hyperbilirubinemia both in man and rats

Intestinal excretion of unconjugated bilirubin in man and rats with inherited unconjugated hyperbilirubinemia

Patients with Crigler-Najjar syndrome and Gunn rats cannot form bilirubin glucuronides owing to a lack of bilirubin UDP-glucuronosyltransferase activity. Because increased serum and tissue bilirubin levels remain constant, an alternative excretory route has to substitute for this deficiency. Gunn rats excrete in bile only 2-13% of the bilirubins eliminated in Wistar rats. In contrast, the biliary excretion rate of urobilinogen in Gunn and Wistar rats is comparable. The sum of bilirubins and urobilinogen excreted in the bile of Gum rats amounts to 10-30% of pigments excreted in Wistar rats. Despite this low biliary excretion, the intestinal content and fecal excretion of bile pigments in Gunn and Wistar rats were similar. These data support an extrabiliary entrance of unconjugated bilirubin into the intestine. Additional proof for this was found in that the intestinal lumen of Gunn rats still contains a high amount of bilirubins and urobilinogen after 3 d of external biliary drainage. A similar procedure in Wistar rats resulted in the complete disappearance of bile pigments from the intestine. The direct transmural transport of bilirubin from blood to all parts of the intestinal lumen was demonstrated by injecting C-14-bilirubin i.v. into Gunn rats with isolated parts of small and large intestine. In Crigler-Najjar and Gilbert's syndrome patients, the biliary excretion of bile pigments has previously been shown to be strongly reduced. Their stools, however, contained approximately the same amount of bile pigments as in normal subjects. Although only traces of unconjugated bilirubin were detected in the stool of normal persons (4 +/- 3% of total bile pigments), higher amounts were found in patients with Crigler-Najjar disease (20 +/- 12&). These results suggest a direct intestinal permeation of unconjugated bilirubin in severe unconjugated hyperbilirubinemia both in man and rats