Apolipoprotein B48 glycosylation in abetalipoproteinemia and Anderson's disease.

BACKGROUND & AIMS: Abetalipoproteinemia and Anderson's disease are hereditary lipid malabsorption syndromes. In abetalipoproteinemia, lipoprotein assembly is defective because of mutations in the microsomal triglyceride transfer protein. Here, we evaluated the intracellular transport of apolipoprotein B48 to localize the defect in Anderson's disease. METHODS: Asparagine-linked oligosaccharide processing of apolipoprotein B48 in normal and affected individuals was determined by the endoglycosidase H and F sensitivities of the protein after metabolic labeling of intestinal explants in organ culture. Cell ultrastructure was evaluated with electron microscopy. RESULTS: In Anderson's disease as in normal individuals, there was a time-dependent transformation of high mannose endoglycosidase H-sensitive oligosaccharides, of endoplasmic reticulum origin, to complex endoglycosidase H-resistant oligosaccharides, added in the Golgi network. In contrast, despite the translocation of apolipoprotein B48 into the endoplasmic reticulum in patients with abetalipoproteinemia and in biopsies treated with Brefeldin A, which blocks anterograde transport between the endoplasmic reticulum and the Golgi network, there was no transformation of endoglycosidase H-sensitive oligosaccharides. CONCLUSIONS: In abetalipoproteinemia and Anderson's disease, apolipoprotein B48 is completely translocated into the endoplasmic reticulum, but only in Anderson's disease is the protein transported to the Golgi apparatus. This suggests that Anderson's disease is caused by a post-Golgi cargo-specific secretion defect.

Effects of bile acids on the humoral immune response: a mechanistic approach.

Whereas bile acids in excess depress the cell-mediated immune response, their effects on the humoral response have been little investigated. The aim of this study was to investigate the effects of bile acids on immunoglobulin production. Human peripheral blood mononuclear cells were stimulated for 5 days by Staphylococcus aureus Cowan I (SAC-I). Immunoglobulins were measured in the supernatants and cell lysates using ELISA. We found that bile acids inhibited IgM production in a dose-dependent manner. The inhibitory effects of 50 microM chenodeoxycholic acid (CDCA) and its glyco- and tauro-conjugates (62, 53 and 51%, respectively) were stronger than those of ursodeoxycholic acid (UDCA) and its conjugates (45, 40 and 34%, respectively). The inhibition of IgG production by CDCA and UDCA was weak (23 and 12%, respectively, at 50 microM). IgA production was not modified. The inhibition of intracellular IgM concentration paralleled that observed in the secreted compartment. By contrast, CDCA enhanced intracellular concentration of IgG. In the absence of significant necrosis or apoptosis, CDCA-mediated inhibition of SAC-I-induced IgM production was significantly correlated to the ability of the bile acid to inhibit cell proliferation (r=0.98; p<0.05). In conclusion, we showed that hydrophobic bile acids strongly depress the primary humoral response. This effect resulted from both an inhibition of cell proliferation, and to a lesser extent from a deficient exocytosis of immunoglobulins.

Anderson's disease: exclusion of apolipoprotein and intracellular lipid transport genes.

Anderson's disease is a rare, hereditary hypocholesterolemic syndrome characterized by chronic diarrhea, steatorrhea, and failure to thrive associated with the absence of apo B48-containing lipoproteins. To further define the molecular basis of the disease, we studied 8 affected subjects in 7 unrelated families of North African origin after treatment with a low-fat diet. Lipid loading of intestinal biopsies persisted, but the pattern and extent of loading was variable among the patients. Electron microscopy showed lipoprotein-like particles in membrane-bound compartments, the densities (0.65 to 7.5 particles/mu(2)) and the mean diameters (169 to 580 nm) of which were, in general, significantly larger than in a normal fed subject (0.66 particles/mu(2), 209 nm mean diameter). There were also large lipid particles having diameters up to 7043 nm (average diameters from 368 to 2127 nm) that were not surrounded by a membrane. Rarely, lipoprotein-like particles 50 to 150 nm in diameter were observed in the intercellular spaces. Intestinal organ culture showed that apo B and apo AIV were synthesized with apparently normal molecular weights and that small amounts were secreted in lipid-bound forms (density <1.006 g/mL). Normal microsomal triglyceride transfer protein (MTP) and activity were also detected in intestinal biopsies. Segregation analyses of 4 families excluded, as a cause of the disease, significant regions of the genome surrounding the genes for apo AI, AIV, B, CI, CII, CIII, and E, as were the genes encoding 3 proteins involved in intracellular lipid transport, MTP, and fatty acid binding proteins 1 and 2. The results suggest that a factor other than apoproteins and MTP are important for human intestinal chylomicron assembly and secretion.

Steatohepatitis-inducing drugs cause mitochondrial dysfunction and lipid peroxidation in rat hepatocytes.

BACKGROUND & AIMS: 4,4'-Diethylaminoethoxyhexestrol (DEAEH), amiodarone, and perhexiline cause steatohepatitis in humans. The mechanisms of these effects are unknown for DEAEH and have not been completely elucidated for amiodarone and perhexiline. The aim of this study was to determine these mechanisms. METHODS: Rat liver mitochondria, cultured rat hepatocytes, or rats were treated with these drugs, and the effects on mitochondrial respiration, beta-oxidation, reactive oxygen species formation, and lipid peroxidation were determined. RESULTS: DEAEH accumulated in mitochondria and inhibited carnitine palmitoyl transferase I and acyl-coenzyme A dehydrogenases; it decreased beta-oxidation and caused lipid deposits in hepatocytes. DEAEH also inhibited mitochondrial respiration and decreased adenosine triphosphate (ATP) levels in hepatocytes. DEAEH, amiodarone, and perhexiline augmented the mitochondrial formation of reactive oxygen species and caused lipid peroxidation in rats. CONCLUSIONS: Like amiodarone and perhexiline, DEAEH accumulates in mitochondria, where it inhibits both beta-oxidation (causing steatosis) and respiration. Inhibition of respiration decreases ATP and also increases the mitochondrial formation of reactive oxygen species. The latter oxidize fat deposits, causing lipid peroxidation. We suggest that ATP depletion and lipid peroxidation may cause cell death and that lipid peroxidation products may account, in part, for other steatohepatitis lesions.

Ultrastructural immunogold labeling of lipid-laden enterocytes from patients with genetic malabsorption syndromes.

Intestinal biopsies from patients having genetic disorders of lipoprotein assembly and secretion, such as abetalipoproteinemia (ABL) or Anderson's disease (AD), contain large amounts of lipids which are accumulated in the enterocytes. Determination of the intracellular sites in which the lipids accumulate and to which apolipoproteins the lipids are bound would help to identify the defects in these diseases and further elucidate the mechanisms by which lipoprotein assembly and secretion occur normally. Ultrastructural immunogold labeling, however, is hampered by the poor preservation of the lipids accumulated in the enterocytes of these patients. We have used routine electron microscopy (fixation and ultra-thin sectioning) along with three methods for immunogold labeling of lipid-laden enterocytes: ultrathin cryosectioning, low temperature freeze substitution with embedding in Lowicryl K4M, and ultra-low temperature freeze substitution with embedding in Lowicryl HM20, to establish a protocol for investigating the intestinal tissue from these patients. Ultracryosectioning, while preserving the overall morphology of the lipid laden enterocytes, did not preserve the lipid content and the immunogold labeling of apolipoprotein B (ApoB) appeared dislocated. Freeze substitution and low temperature embedding in Lowicryl K4M, in contrast, appeared to better preserve the lipid and lipoprotein structures; however, the antigenicity of both apoAI and apoB appeared to be lost and no specific labeling could be obtained. Freeze substitution and embedding in Lowicryl HM20 best preserved the lipid and lipoprotein structures while maintaining apoprotein antigenicity. In conclusion, immunogold labeling of apolipoproteins on lipid structures in the lipid-laden enterocytes of patients with ABL and AD is best obtained by freeze substitution and embedding in Lowicryl HM20.

Liver fibrosis in a patient with familial homozygous hypobetalipoproteinaemia: possible role of vitamin supplementation.

A case of apolipoprotein B-related disorder is reported in which liver fibrosis developed without long term administration of medium chain triglycerides, previously incriminated in the pathogenesis of this lesion. The patient was a young woman in whom the diagnosis of familial homozygous hypobetalipoproteinaemia was made at the age of 21. A first liver specimen taken at diagnosis revealed steatosis, hypertrophic Golgi apparatus and proliferating smooth endoplasmic reticulum. The patient was treated with vitamin A and E supplementation only. Two years later, a second liver biopsy, carried out because of increased serum alanine aminotransferase concentrations, showed fibrosis, mild cytolysis and marked mitochondrial alterations. Hepatic level of vitamin A was increased. This finding supports the hypothesis that liver disease observed in our patient might be an adverse effect of vitamin supplementation. Our observation underlines the importance of including liver function tests in the follow up of patients with apolipoprotein B-related disorders.

The isolated perfused rat spleen. An original method for studying the function of hepatocytes transplanted into the spleen.

The aim of this study was to assess directly the function of isolated hepatocytes 1 year after transplantation into the spleen, using an original model of isolated rat-spleen perfusion. Three specific liver functions, albumin synthesis, indocyanine-green clearance, and antipyrine oxidation, were studied. Five x 10(6) isolated hepatocytes were injected into the spleen of syngenic Wistar-Furth rats. One year later, splenectomy was performed, and the splenic pedicle was carefully isolated in order to allow a selective ex vivo perfusion for 3 hr. De novo albumin synthesis was studied by qualitatively using immunoelectrophoresis and autoradiography, and quantitatively using (35S)-methionine incorporation in albumin. De novo albumin synthesis was observed in spleens containing transplanted hepatocytes but not in controls (P less than 0.001); (35S)-methionine incorporation was significantly higher in spleens containing transplanted hepatocytes than in controls (132 +/- 67 cpm/spleen/hr vs. 14 +/- 6 cpm/spleen/hr, P less than 0.001). Antipyrine clearance was significantly higher in spleens with transplanted hepatocytes than in controls (67.4 +/- 4.9 microliters/min/g vs. 0.2 +/- 0.4 microliters/min/g, P less than 0.01). No statistically significant difference was observed with indocyanine-green clearance (4.2 +/- 6.0 microliters/min/g, vs. 5.2 +/- 5.1 microliters/min/g, P greater than 0.05); this was probably due to the absence of compartmentation between the sinusoid and biliary sectors in this model. In conclusion, using this original isolated rat-spleen perfusion model, it was directly observed that 1 year after transplantation, intrasplenic hepatocytes can perform two liver-specific functions, i.e., de novo albumin synthesis and antipyrine clearance.

Further cellular investigation of the human hepatoblastoma-derived cell line HepG2: morphology and immunocytochemical studies of hepatic-secreted proteins.

The hepatoblastoma cell line HepG2 has been a matter of many investigations; most of them include biochemical studies of lipoprotein and other hepatic protein metabolism. However, the accurate cellular features of these cells have not been emphasized. We studied the cellular histologic, histochemical, and ultrastructural characteristics of this cell line. In addition, we investigated by immunoenzymatic methods the cellular biosynthesis of several proteins: apolipoproteins-AI, -B, -D, and -E, albumin, alpha-fetoprotein, transferrin, alpha-1-antitrypsin, C-reactive protein, fibronectin, and collagens I, III and IV. The rates of accumulation, in the medium of HepG2 cells, of albumin, alpha-1-antitrypsin, transferrin, and alpha-fetoprotein were 13.2 +/- 1.9; 4.9 +/- 1.5; 3.2 +/- 0.4; and 10.7 +/- 1.7 micrograms/10(6) cells/24 h, respectively. Our results show that HepG2 cells exhibited most cellular features of normal human hepatocytes. Bile canaliculi as well as Golgi apparatus complexes were particularly developed. Except for the C-reactive protein, HepG2 cells have all retained the ability to synthesize hepatic proteins but with some variable intensity from cell to cell. This hepatoblastoma cell line seems to represent a useful tool in the understanding of hepatic protein biosynthesis, particularly for the investigation on the secretory pathway of plasma proteins.

Immunoperoxidase localization of apolipoprotein D in human enterocytes and hepatocytes.

In this study we prepared a pure apolipoprotein D and obtained a specific antiserum to it. The purified apolipoprotein D migrated as a single band of Mr = 29,000 but appeared as five isoforms on isoelectrofocusing. The antiserum did not cross-react with other apolipoproteins. Immunoenzymatic staining revealed the presence of apolipoprotein D in the perinuclear area of the cytoplasm of isolated normal hepatocytes and HepG2 cells. Apolipoprotein D was also localized in intestinal epithelium and in liver cells. The intracellular distribution of apolipoprotein D was similar to that of apolipoprotein B. Our results indicated that apolipoprotein D, like many other circulating apolipoproteins, is synthesized in enterocytes and hepatocytes.

Can hepatocytes proliferate when transplanted into the spleen? Demonstration by autohistoradiography in the rat.

The aim of this work was to study hepatocyte multiplication after transplantation into the spleen, in order to apply this technique to the treatment of chronic liver disease. Hepatocytes isolated by an in situ collagenase perfusion technique in Wistar Furth rats were injected into the splenic parenchyma of three groups of syngeneic rats: controls with normal liver (group 1), 75% hepatectomies (group 2), and end-to-side portacaval shunts (group 3). The proliferation of transplanted hepatocytes was studied by autohistoradiography after the intraperitoneal administration of 0.6 microCi/g body weight of [3H]-thymidine, at 1, 3, 7 and 15 days after transplantation of hepatocytes. Significant incorporation of [3H]-thymidine by the transplanted hepatocytes during the study period was observed mostly in groups 2 and 3. The incorporation, although delayed was sustained and of greatest magnitude in the portacaval-shunted animals. The ability of transplanted hepatocytes to proliferate in the spleen, particularly after a portacaval shunt, indicates that this procedure may have therapeutic applications in the treatment of chronic liver disease.

High-yield preparation of porcine hepatocytes for long survival after transplantation in the spleen.

The creation of an auxiliary liver by autotransplantation of liver parenchymal cells into the spleen has mainly been studied in rats for the treatment of acute liver failure. In order to apply this procedure to humans with chronic liver insufficiency the aim of this work was: To demonstrate that hepatocytes can survive for long periods after autotransplantation into the spleen; to increase the yield of the isolation of hepatocytes obtained from pig livers since this animal has a more fibrous liver than rats or normal humans and consequently one which is more difficult to dissociate. In 21 pigs isolated hepatocytes were obtained with in collagenase dissociation technique, the yield being 1-3 X 10(7) cells per gram of liver and the viability 70-95%. The hepatocytes survived and maintained normal morphological and histochemical characteristics up to 7 months after transplantation, the date of sacrifice of the last animal.

[Histological and histoenzymological study of liver. II: In gallstone treated with chenodeoxycholic acid (author's transl)]. / Etude histologique et histoenzymologique du foie. II: Dans la lithiase vésiculaire traitée par l'acide chénodesoxycholique.

The histological and histochemical changes of liver from gallstones treated with ACDC are confronted with the same livers before treatment. There is few modification in histological patterns but the frequency of sinusoidal congestion increase. Numerous histoenzymological "injuries" are corrected by this treatment.

[Histological and histoenzymological study of liver. I: In gallstone without treatment (author's transl)]. / Etude histologique et histoenzymologique du foie. I: Dans la lithiase vésiculaire non traitée.

46 liver biopsies from gallstones and a control group have been studied by histological and histoenzymological technics. The most frequent injuries are in the portal tract, inflammatory infiltration and fibrosis; the most peculiar is a sinusoidal congestion. A brown pigment overload is seen in numerous livers. Lactic-, glyceric-, glutamic-, isocitric-, glucose-6-phosphate-deshydrogenase activities decrease but alkaline phosphatases-, transaminases activities are increased.