Apolipoprotein B-100 is the major form of this apolipoprotein secreted by human intestinal Caco-2 cells

Although the discovery of stop codon has explained the mechanism for the formation of the intestinal marker, apolipoprotein B-48, the dispute regarding the presence of apolipoprotein B-100 in the intestine is still unsettled. To further investigate the characteristics of intestinal apolipoprotein B, the newly developed human colonic adenocarcinoma Caco-2 cells which express functional properties of the differentiated enterocytes, were used. SDS-polyacrylamide gel electrophoresis analyses of the intact culture medium or its lipoproteins of d less than 1.23 g/ml showed the presence of only a single protein band of apolipoprotein B-100 with no detectable apolipoprotein B-48. After immunoblotting with oligoclonal antibodies to the amino-terminal peptide of apolipoprotein B, a trace amount of apolipoprotein B-48 was observed in the isolated lipoproteins, but not in the intact culture medium. These results suggest that apolipoprotein B-100 is the major form of apolipoprotein B secreted by human intestinal cells.     

Mechanisms of glucosamine-induced suppression of the hepatic assembly and secretion of apolipoprotein B-100-containing lipoproteins

Glucosamine-induced endoplasmic reticulum (ER) stress was recently shown to specifically reduce apolipoprotein B-100 (apoB-100) secretion by enhancing the proteasomal degradation of apoB-100. Here, we examined the mechanisms linking glucosamine-induced ER stress and apoB-lipoprotein biogenesis. Trypsin sensitivity studies suggested glucosamine-induced changes in apoB-100 conformation. Endoglycosidase H studies of newly synthesized apoB-100 revealed glucosamine induced N-linked glycosylation defects resulting in reduced apoB-100 secretion. We also examined glucosamine-induced changes in VLDL assembly and secretion. A dose-dependent (1-10 mM glucosamine) reduction was observed in VLDL-apoB-100 secretion in primary hepatocytes (24.2-67.3%) and rat McA-RH7777 cells (23.2-89.5%). Glucosamine also inhibited the assembly of larger VLDL-, LDL-, and intermediate density lipoprotein-apoB-100 but did not affect smaller HDL-sized apoB-100 particles. Glucosamine treatment during the chase period (posttranslational) led to a 24% reduction in apoB-100 secretion (P < 0.01; n = 4) and promoted post-ER apoB degradation. However, the contribution of post-ER apoB-100 degradation appeared to be quantitatively minor. Interestingly, the glucosamine-induced posttranslational reduction in apoB-100 secretion could be partially prevented by treatment with desferrioxamine or vitamin E. Together, these data suggest that cotranslational glucosamine treatment may cause defects in apoB-100 N-linked glycosylation and folding, resulting in enhanced proteasomal degradation. Posttranslationally, glucosamine may interfere with the assembly process of apoB lipoproteins, leading to post-ER degradation via nonproteasomal pathways.     

Degradation of apolipoprotein B-100 in human chylomicrons

The purpose of this study was to investigate the molecular forms of apolipoprotein B (ApoB) in human chylomicrons under well-preserved conditions. To this end, plasma and serum were collected from the same normal subjects after ingestion of a fatty meal. The samples were divided into three or four aliquots before the addition of various preservative mixtures, including antibiotics, antioxidants and proteinase inhibitors. The chylomicrons were isolated immediately, and all steps were carried out at or below 4 degrees C. Changes in the molecular weight of ApoB in chylomicrons were followed by a time study using 3.3% polyacrylamide gel electrophoresis containing SDS. ApoB from chylomicrons analyzed within 5 h of blood collection showed a single band with mobility identical to that of ApoB (ApoB-100) in low-density lipoproteins. When analyzed after 1-2 days, satellite bands smaller than ApoB-100 were observed, and a very faint band with Mr 200,000 appeared, which comigrated with intestinal ApoB (ApoB-48). Upon storage, the molecular weight of ApoB was smaller in chylomicrons subjected to a higher number of reflotations than those in chylomicrons washed less frequently, suggesting that purified chylomicrons degrade faster. A longer storage time at 4 degrees C (i.e., 7 or 14 days) revealed a stepwise degradation of ApoB, yielding Mr 200,000 band as the prominent form. The degradation of ApoB-100 was slower when both proteinase inhibitors, leupeptin and epsilon-amino caproic acid, were employed, and the appearance of Mr 200,000 band was quicker when the chylomicrons were processed at higher temperature (15-25 degrees C) in the absence of a proteinase inhibitor. Immunoblotting shows that the segment removed from ApoB-100 was the carboxyl-terminal portion. These results suggest the possible presence of a proteinase(s), which copurified with chylomicrons, and which converts ApoB-100 from a large to a smaller molecular form. Although the stop codon has been discovered recently in intestinal ApoB mRNA, which explains the mechanism for direct synthesis of ApoB-48, apparently ApoB-100 is also synthesized in the intestine of all eight subjects studied here, and the ApoB-100 degrades to a form which is ApoB-48-like.     

Fluorescence properties of lipoprotein B and apolipoprotein B

The fluorescence properties of apolipoprotein B (ApoB) in various media, including aqueous solutions of three different pH, 6 m urea, 6 m guanidine-HCl and native lipoprotein B (LP-B) particles have been compared by measuring the accessibility of trytophan side chains to iodide ions. The modified Stern-Volmer plots (FΔF vs. 1/[KI]) for LP-B demonstrate heterogeneity of quenching rates at pH 9.0, with a total accessibility of fluorescence to iodide of 43%. At pH 7.3, the total accessibility of LP-B fluorescence to iodide is only 20%. Quenching at pH 2.7 follows a pure Stern-Volmer mechanism. A straight line at this pH intercepting y-axis at 1.0 indicates 100% accessibility of tryptophan residues in LP-B. These results suggest that there are at least three different groups of tryptophan residues present per intact LP-B particle and that each group is situated in a different environment. One group, showing an enhanced quenching rate, is probably near the charged domain; another group, showing a slower quenching rate, is in a relatively hindered environment, and a third group is probably buried in a more hydrophobic environment, inaccessible to iodide at neutral or high pH. But at pH 2.7, all tryptophan residues appear to become situated closer to the surface of the LP-B particle. For isolated ApoB at pH 7.3 and 9.0 in aqueous buffer, about 30% of the fluorescence is relatively easily accessible; another 40% is less easily accessible and the remaining 30% is inaccessible to iodide. These inaccessible tryptophan residues are most likely located in a more hydrophobic matrix and probably in the β-pleated sheet region of ApoB. Similarly to LP-B at pH 2.7, all of the tryptophan residues of ApoB are exposed to the aqueous surface except that one third of them are quenched at a faster rate than the rest. At pH 7.3, in the presence of urea or guanidine-HCl, all of the fluorescence of ApoB is exposed to the aqueous surface, suggesting the presence of random and nonrigid conformation in these media. These results suggest that the conformation of ApoB in aqueous media is pH sensitive. This is true whether the ApoB is present in intact LP-B or as the isolated apolipoprotein. Furthermore, upon removal of lipids from LP-B and passing the ApoB into a denaturing environment, the apolipoprotein loses its ordered structure. When passing ApoB from denaturing agents back to aqueous buffers of neutral or basic pH. ApoB is able to reorient itself to gain an ordered structure, not necessarily identical to that in LP-B, but parallel to it.     

Integral apolipoproteins increase surface-located triacylglycerol in intact native apoB-100-containing lipoproteins

High-resolution NMR was used to measure the presence and quantity of triacylglycerol (TAG) in the surface of intact native apolipoprotein B-100-containing lipoprotein particles that are made by chickens in response to estrogen treatment and that in hens are deposited in yolk follicles (VLDLy). Integration of 13C NMR resonances shows that intact VLDLy particles contain more surface TAG (5.1 +/- 0.6 mol%, 6.7 +/- 0.8 weight %) than predicted by apolipoprotein-free models using similarly acyl-heterogenous TAG. Change in downfield chemical shift values of surface to core TAG in VLDLy was 0.8 ppm compared with 1.3 ppm in vesicles prepared with purified egg phosphatidylcholine and TAG isolated from the VLDLy, indicating that reduced surface TAG hydration may contribute to the resistance to lipase hydrolysis characteristic of this lipoprotein species. Apolipoprotein-mediated changes in surface lipid composition and lipid hydration provide possible general mechanisms for selectivity in lipoprotein substrate characteristics.     

Effects of emulsified policosanols with different chain lengths on cholesterol metabolism in heterozygous LDL receptor-deficient mice

Policosanol is a mixture of long-chain primary aliphatic saturated alcohols. Previous studies in humans and animals have shown that these compounds improved lipoprotein profiles. However, more-recent placebo-controlled studies could not confirm these promising effects. Octacosanol (C28), the main component of sugarcane-derived policosanol, is assumed to be the bioactive component. This has, however, never been tested in an in vivo study that compared individual policosanol components side by side. Here we present that neither the individual policosanol components (C24, C26, C28, or C30) nor the natural policosanol mixture (all 30 mg/100 g diet) lowered serum cholesterol concentrations in LDL receptor knock-out (LDLr(+/-)) mice. Moreover, there was no effect on gene expression profiles of LDLr, ABCA1, HMG-CoA synthase 1, and apolipoprotein A-I (apoA-I) in hepatic and small intestinal tissue of female LDLr(+/-) mice after the 7 week intervention period. Finally, none of the individual policosanols or their respective long-chain fatty acids or aldehydes affected de novo apoA-I protein production in vitro in HepG2 and CaCo-2 cells. Therefore, we conclude that the evaluated individual policosanols, as well as the natural policosanol mixture, have no potential for reducing coronary heart disease risk through effects on serum lipoprotein concentrations.     

Apolipoprotein B-100: immunolocalization and synthesis in human intestinal mucosa

Despite the evidence that the human small intestine produces two separate species of apoB mRNA encoding for B-100 and B-48, there is a paucity of data concerning the expression of the latter form in this organ. Using a high resolution immunogold approach, with specific polyclonal antibodies and a panel of monoclonal antibodies (2D8, 3A10, 4G3), both forms of apoB (B-48 and B-100) were revealed over enterocytes of pediatric intestinal samples. Intense labeling was observed over microvilli, apical smooth membrane vesicles, multivesicular bodies, the basolateral membrane, as well as the trans Golgi region. Only low labeling was found over the rough endoplasmic reticulum (rER). Similar patterns of apoB distribution characterized both duodenal and jejunal regions. The presence of labeling over the Golgi apparatus and rER suggests a synthetic activity of both forms of apoB by the epithelial cells. To test this hypothesis, human intestine was incubated with [3H]leucine, homogenized, and subjected to immunoprecipitation for apoB. Immunoprecipitates contained radioactivity mainly in apoB-48 with relatively small amounts in apoB-100 when examined by NaDodSO4-polyacrylamide gel electrophoresis. These findings were further supported by the biochemical determination of apoB-100 and apoB-48 in chylomicron particles isolated from thoracic duct lymph of a human donor. Taken together, our data suggest that the human intestine is able to synthesize and to express the apoB-100.     

Apolipoprotein B synthesis in rat small intestine: regulation by dietary triglyceride and biliary lipid

Apolipoprotein B (apoB) synthesis rates have been determined, in vivo, in rat enterocytes. Following intralumenal administration of a pulse of [3H]leucine, newly synthesized apoB was quantitated by specific immunoprecipitation and compared to [3H]leucine incorporation into total, trichloroacetic acid-insoluble protein. ApoB synthesis rates were determined after acute administration of either 0.1 or 1 g of triglyceride to fasting animals. No differences were found at any time from 90 min to 6 hr after challenge and values were not different from the basal values established in fasted controls. Animals rechallenged with triglyceride after 8 days' intake of fat-free chow also failed to demonstrate a change in intestinal apoB synthesis rate. By contrast, enterocyte content of apoB appeared to fall, temporarily, with the onset of active triglyceride flux. Groups of animals were then subjected to external bile diversion for 48 hr, a maneuver designed to remove all lumenal sources of lipid. Jejunal apoB synthesis rates fell by 43% (from 0.76% +/- 0.14 to 0.43% +/- 0.12, P less than 0.001), a change that was completely prevented by continuous replacement with 10 mM Na taurocholate. The suppression of jejunal apoB synthesis, induced by prolonged bile diversion, was reversed after 14 hr, but not 8 hr, of intralumenal perfusion with 10 mM Na taurocholate. The addition of micellar fatty acid-monoolein to the perfusate for 4 hr produced no further change in apoB synthesis. Ileal apoB synthesis rates fell by 70% (from 0.61% +/- 0.15 to 0.18% +/- 0.10, P less than 0.001) following 48 hr external bile diversion, a change that was only partially prevented by continuous bile salt replacement. These results suggest that jejunal apoB synthesis demonstrates bile salt dependence but not regulation by acute triglyceride flux. The data further suggest that key aspects of the regulation of apoB synthesis by cellular lipid flux may be mediated independently in jejunal and ileal enterocytes.     

Measuring VLDL-triglyceride turnover in humans using ex vivo-prepared VLDL tracer

There has been more interest in VLDL-triglyceride (TG) kinetics during the last decade. Unfortunately, robust measurement methods are elaborate and not readily available. Here, we describe a method using unique, ex vivo labeling of the fatty acid moiety of VLDL-TG followed by intravenous bolus infusion in the same person. We found that plasma disappearance of ex vivo-labeled VLDL-TG was comparable to that of in vivo-labeled VLDL-TG and that turnover rates can be safely estimated from the log linear decay of VLDL-TG specific activity. We found minor labeling of the plasma FFA (oleate) pool, which was largely attributable to coinfusion of free [14C]triolein; VLDL-TG did not contribute substantially to the plasma FFA pool. The plasma decay curve of VLDL-TG was not affected by the presence of tracer in the FFA pool, provided that the data from 2 h after the VLDL tracer bolus infusion was used. The FFA contamination problem was circumvented by minor modification of the VLDL-TG tracer preparation. The approach we describe should expand the opportunity to study processes that cannot be assessed if the FFA precursor pool is labeled. This method for VLDL-TG tracer preparation can allow measurement of VLDL turnover, tissue uptake of VLDL-TG, and oxidation of VLDL-TG.     

Rat monoclonal antibodies to rabbit and human serum low-density lipoprotein.

A total of 16 hybrid myeloma clones secreting monoclonal antibodies (McAb) to rabbit or human serum low-density lipoprotein (LDL) were derived from the fusion of spleen cells from LOU or DA rats immunized with rabbit or human LDL and the rat myeloma lines Y3 Ag1.2.3 or YB2/0. Anti-(rabbit LDL) McAb showed limited reactivity with LDL from human, rhesus-monkey, rat and mouse serum. Six out of seven anti-(human LDL) McAb reacted with rhesus-monkey LDL, and only one showed partial cross-reaction with rabbit LDL. Binding-competition experiments indicated that the epitopes recognized by the anti-(rabbit LDL) IgG could be grouped into two major clusters: McAb in the first cluster reacted either with apo-(lipoprotein B-100) (apoB-100) and apo-(lipoprotein B-74) (apoB-74) or with apoB-100 but not with apo-(lipoprotein B-48) (apoB-48), the lower-Mr form of apoB of intestinal origin; the McAb in the second cluster all reacted with apoB-48 in addition to apoB-100 or apoB-100 and apoB-74. The six anti-(human LDL) IgG bound to separate epitopes on LDL. Further data on the epitope specificity of these McAb were obtained by antibody blotting after partial proteolysis of apoB-100 with trypsin or staphylococcal V8 proteinase, and the data confirmed the results obtained with the binding-competition experiments. One McAb to rabbit LDL inhibited the binding of LDL to the fibroblast LDL receptor (50% inhibition at a McAb/LDL molar ratio of 10). A similar result was produced by two other McAb at higher concentrations of antibody.     

3H-cyclosporine internalization and secretion by human fetal pancreatic islets

Human fetal pancreatic islets were isolated from 16- to 20-week-old fetuses by a collagenase technique and cultured 48 hr in RPMI 1640 containing 10% human adult serum and unlabeled 0 to 5 micrograms cyclosporine A (CsA)/ml. Insulin secretory capacity of human fetal islets was expressed as a fractional stimulatory ratio FSR = F2/F1 of the fractional secretion rates during two successive 1 hr static incubations first with 2 mM glucose (F1) to stabilize secretion followed by maximal stimulus, i.e., 25 mM glucose plus 10 mM L-leucine and 10 mM L-arginine (F2). Unlabeled CsA at the above concentrations had no significant effects on the insulin secretory capacity expressed by FSR-values. Studies of net uptake of 3H-CsA by islets cultured for varying periods up to 40 hr and expressed as picomole 3H-CsA per picomole islet insulin content demonstrated that uptake rate was slow and did not reach isotopic equilibrium over the 40 hr of culture. When isolated fetal islets were cultured for 48 hr in the presence of 3H-CsA and varying concentrations of unlabeled CsA it was found during two successive 1 hr static incubations that fetal islets secrete insulin concomitantly with 3H-CsA following maximal stimulus for secretion. An optimal secretory molar ratio of 3H-CsA to insulin of 4.0 +/- 1.3 (n = 7) was found after islets were cultured 48 hr in the presence of a saturating 2.128 micrograms 3H-CsA per milliliter culture medium. In three successive 30-min static incubations of 3H-CsA loaded islets, first with low glucose, followed by high glucose plus L-arginine and L-leucine, and finally with high glucose plus L-arginine and L-leucine and 10 mM theophylline, the proportional fractional secretion rates of insulin and 3H-CsA were of the same magnitude. It is concluded that human fetal pancreatic islets during 48 hr of culture in the presence of pharmacologically relevant concentrations of CsA can internalize the drug, which is compartmentalized and concomitantly secreted with insulin following maximal stimuli. Transplanted human fetal islets utilized as delivering units for CsA could be beneficial for the induction of immunotolerance to allografted fetal islets.     

Microsomal triglyceride transfer protein expression in adipocytes: a new component in fat metabolism

Microsomal triglyceride transfer protein (MTP) is a carrier of triglyceride essential for the assembly of apolipoprotein (apo)B-containing lipoproteins by the liver and the small intestine. Its role in triglyceride transfer in tissues that do not secrete lipoproteins has not been explored. In particular, MTP would seem to be a candidate for a role in triglyceride metabolism within the adipocyte. To test this hypothesis, we probed adipocytes for the presence of MTP. Immunohistochemical and biochemical studies demonstrate MTP in adipocytes from brown and white fat depots of mice and human, as well as in 3T3-L1 cells. Confocal microscopy revealed MTP throughout 3T3 cells; however, MTP fluorescence was prominent in juxtanuclear areas. In differentiated 3T3 cells MTP fluorescence was very striking around lipid droplets. In vitro lipid transfer assays demonstrated the presence of triglyceride transfer activity within microsomal fractions isolated from rat adipose tissue. In addition, quantitative rtPCR studies showed that MTP expression in mouse white fat depots was approximately 1% of MTP expression in mouse liver. MTP mRNA in differentiated 3T3 cells was approximately 13% of liver expression. Our results provide unequivocal evidence for the presence of MTP in adipocytes and present new possibilities for defining the mechanisms by which triglyceride is stored and/or hydrolyzed and mobilized.     

Effects of the cholesteryl ester transfer protein inhibitor torcetrapib on VLDL apolipoprotein E metabolism

Cholesteryl ester transfer protein (CETP) inhibition leads to changes in lipoprotein metabolism. We studied the effect of the CETP inhibitor torcetrapib on VLDL apolipoprotein E (apoE) metabolism. Subjects, pretreated with atorvastatin (n = 9) or untreated (n = 10), received placebo followed by torcetrapib (4 weeks each). After each treatment, subjects underwent a primed-constant infusion of D(3)-leucine to determine the VLDL apoE production rate (PR) and fractional catabolic rate (FCR). Torcetrapib alone reduced the VLDL apoE pool size (PS) (-28%) by increasing the VLDL apoE FCR (77%) and leaving the VLDL apoE PR unchanged. In subjects pretreated with atorvastatin, torcetrapib increased the VLDL apoE FCR (25%) and PR (21%). This left the VLDL apoE PS unchanged but increased the VLDL apoE content, likely enhancing VLDL clearance and reducing LDL production in this group. Used alone, torcetrapib reduces the VLDL apoE PS by increasing the apoE FCR while leaving the VLDL apoE content unchanged. In contrast, torcetrapib added to atorvastatin treatment increases both the VLDL apoE FCR and PR, leaving the VLDL apoE PS unchanged. Adding torcetrapib to atorvastatin treatment increases the VLDL apoE content, likely leading to decreased conversion of VLDL to LDL, reduced LDL production, and lower levels of circulating VLDL and LDL.     

An intestinal galactose-specific lectin mediates the binding of murine IgE to mouse intestinal epithelial cells.

A number of lactose-binding lectins have recently been identified in the rat and mouse intestine, one of which corresponds to the C-terminal domain of IgE-binding proteins, originally identified in rat basophilic leukemia (RBL) cells and mouse 3T3 fibroblasts. In the present report, we describe the affinity purification of a rat intestinal lactose-specific lectin which binds murine IgE antibodies. This binding most likely occurs via the immunoglobulin carbohydrate chains, as it is inhibited by lactose. This intestinal lectin molecule is also immunologically related to the previously described IgE-binding protein (epsilon BP) isolated from RBL cells, since it is recognized by antibodies raised against recombinant epsilon BP. This intestinal form of epsilon BP has a molecular mass of 17.5 kDa, which is much lower than that of its RBL cell analogue (31 kDa). The attachment of IgE to the mouse intestinal epithelium was demonstrated by immunohistochemistry, along with the presence of a corresponding mouse intestinal epsilon BP. The carbohydrate-dependent nature of this attachment was established by demonstrating that IgE binding to mouse epithelium was specifically abolished by lactose (4 mM) and by a blood-group-A-active tetrasaccharide (0.2 mM), but not by mannose (10 mM). Finally, the association of IgE with the mouse intestinal epithelium was prevented by competition with the purified IgE-binding lectin isolated from rat intestine. Although the physiological function of this intestinal protein is still unknown, the finding that IgE binds to a lectin in the intestinal epithelium pinpoints a possible novel mechanism for the regulation of IgE-mediated disorders, such as food allergy.     

Carboxyl terminal analysis of human B-48 protein confirms the novel mechanism proposed for chain termination

In this study we have confirmed the presence of a single base difference between intestinal mRNA coding for B-48 and hepatic mRNA coding for B-100, which results in the substitution of a stop codon (UAA) for a glutamine codon (CAA) at a point corresponding to amino acid residue 2153 in the B-100 sequence. Based on this finding, B-48 is predicted to terminate at residue 2152 with the sequence ... Met Ile. To confirm this finding at the protein level, B-48 and B-100 were each digested with cyanogen bromide and the digestion products were analysed for the presence of isoleucine. Isoleucine was found only in cyanogen bromide digests of B-48 confirming that only B-48 terminates with the predicted amino acid sequence ... Met Ile.     

Effects of chlorpromazine and methylprednisolone on perfusion preservation of rabbit livers

The isolated perfused rabbit liver was used to determine how continuous hypothermic perfusion affected liver function. Rabbit livers were perfused for 0, 24, 48, and 72 hr at 5 degrees C with the UW perfusate containing hydroxyethyl starch (5 g%) dissolved in a solution containing gluconate (80 mM), adenosine (5 mM), glutathione (3 mM), phosphate (25 mM), and additives as described previously, and they were used successfully for kidney preservation. At the end of preservation the livers were perfused in an isolated circuit with a Krebs-Henseleit solution with addition of 4 g% bovine serum albumin and 10 mM glucose at 38 degrees C for 120 min. Bile was collected from the cannulated common duct. Biliary excretions of indocyanine green and liver enzymes lactate dehydrogenase, aspartate aminotransferase, and alanine aminotransferase, were determined both in the cold perfusate and the normothermic perfusate. Livers were also studied after pretreatment of the donor with chlorpromazine (CPZ) and/or methylprednisolone (MP). Bile production (ml/120 min, 100 g liver) upon reperfusion produced the most interesting data and decreased from a control value of 10.3 +/- 2.6 to 9.3 +/- 1.0 (24 hr), 5.3 +/- 0.7 (48 hr), and 4.1 +/- 1.5 (72 hr). Enzyme release was not predictive of the degree of preservation-induced damage. Pretreatment of rabbits with a combination of CPZ/MP improved bile flow at 48 and 72 hr (8.3 +/- 3.0 and 7.0 +/- 1.3, P less than 0.05). Pretreatment with either drug alone also improved function after 72 hr of preservation (7.1 +/- 1.8, CPZ; 8.2 +/- 3.5, MP).(ABSTRACT TRUNCATED AT 250 WORDS)     

Receptor mediated yolk protein uptake in the crab Scylla serrata: crustacean vitellogenin receptor recognizes related mammalian serum lipoproteins

The receptor-mediated uptake of major yolk protein precursor, vitellogenin (Vg) is crucial for oocyte growth in egg laying animals. In the present study plasma membrane receptor for Vg was isolated from the oocyte of the red mud crab, Scylla serrata. Vitellogenin receptor (VgR) protein was visualized by ligand blotting using labeled crab Vg ((125)I-Vg) as well as labeled low density lipoprotein ((125)I -LDL) and very low density lipoprotein ((125)I-VLDL) isolated from rat. The endocytosis of Vg was visualized in the crab oocyte by ultrastructural immunolocalization of Vg. The Vg receptor was purified by gel filtration high performance liquid chromatography (HPLC) and its molecular weight was estimated to be 230 kDa. In direct binding studies, the receptor exhibited high affinity (dissociation constant K(d) 0.8x10(minus sign6) M) for crab Vg. Vitellogenin receptor was observed to have an increased affinity to crab Vg in the presence of Ca(2+) and the binding was inhibited by suramin, suggesting similarities between crab VgR and low density lipoprotein receptor (LDLR) superfamily of receptor protein. Furthermore, the crab VgR showed significant binding ability to mammalian atherogenic lipoproteins such as LDL and VLDL. This suggests that there is a tight conservation of receptor binding sites between invertebrate (crab) Vg and vertebrate (rat) LDL and VLDL.     

Hormonal regulation of 3-hydroxy-3-methylglutaryl coenzyme a reductase and alkaline phosphatase in cultured intestinal mucosa

The endocrine regulation of the key enzyme of cholesterol synthesis, 3-hydroxy-methylglutaryl-CoA reductase (EC 1.1.1.34) and of the brush border enzyme alkaline phosphatase (EC 3.1.3.1) was studied in short (2 h) and long tern (24 h) organ culture of rabbit ileum mucosa. In contrast to the hepatic enzyme, intestinal reductase is not subject to regulation by insulin or glucagon even at a pharmacological level. This applies to both ‘total’ and ‘active’ reductase, prepared in the absence or presence of sodium fluoride, respectively. During culture, there is a gradual, time-dependent increase in the active, dephosphorylated enzyme form. This endogenous activation was found to be unaffected by all hormones tested. Similarly, alkaline phosphatase was not influenced by both pancreatic hormones. In contrast, triamcinolone significantly (P < 0.05) suppressed reductase in a dose-dependent fashion to 38% of controls after 24 h, but not after 2 h culture. Alkaline phosphatase was induced after both periods, but the effect was more marked after 24 h. A parallel minor stimulation of both enzyme activities was noted in the presence of 10^?9 M triidothyronine (P < 0.05), lower and very high (10^?5 M) concentrations were ineffective. In view of the role of glucocorticoids as intestinal growth inhibitors and of thyroid hormones as growth stimulators, it is suggested that changes in reductase reflect alterations of crypt membrane cholesterol synthesis, whereas the induction of alkaline phosphatase is mediated through an enhanced enterocyte regeneration and/or maturation.     

Selective effects of hydrocortisone on intestinal lipoprotein and apolipoprotein synthesis in the human fetus

Studies employing human fetal intestine have yielded much interesting information on the role of polarized enterocytes in fat absorption and transport. Using the organ culture model, we examined the influence of hydrocortisone on the synthesis and secretion of lipids and lipoproteins. Human jejunal explants were cultured for 5 days at 37°C in serum-free medium containing either [¹⁴C]-oleic acid or [¹⁴C]-acetate, alone or supplemented with hydrocortisone (25 or 50 ng/ml). The uptake of [¹⁴C]-oleic acid was associated with the production of triglycerides, phospholipids, and cholesteryl esters, which were all affected by hydrocortisone. This hormonal agent (50 μg) led to the marked reduction of secreted triglycerides (43%, P < 0.01), phospholipids (39%, P < 0.01), and cholesteryl esters (36%, P < 0.05) without altering the characteristic distribution of tissue and medium lipid classes. Similarly, hydrocortisone significantly (P < 0.01) decreased (∼60%) the incorporation of [¹⁴C]-acetate into secreted free and esterified cholesterol in the medium. With [¹⁴C]-oleic acid as a precursor, hydrocortisone significantly diminished the delivery of chylomicrons and very low density lipoproteins to the medium while consistently enhancing the secretion of high density lipoproteins. In parallel, [³⁵S]-methionine pulse-labeling of jejunal explants revealed the concomitant inhibitory effect of hydrocortisone on apo B-100 synthesis and hydrocortisone's stimulatory effect on apo B-48 and apo A-I. These studies suggest that glucocorticoids play a critical role in lipoprotein processing during intestinal development. J. Cell. Biochem. 66:65–76 1997. © 1997 Wiley-Liss, Inc.