Control of pancreatic bile-salt-dependent-lipase secretion by the glucose-regulated protein of 94 kDa (Grp94).

Bile-salt-dependent lipase (BSDL; EC 3.1.1.13) is an enzyme expressed by the pancreatic acinar cell and secreted as a component of the pancreatic juice. During its route towards secretion, BSDL is associated with intracellular membranes by means of a multiprotein folding complex, which includes the glucose-regulated protein of 94 kDa (Grp94). We have postulated that the association of BSDL with membranes is required for the complete O-glycosylation of the protein, which diverts BSDL from a degradation route and consequently allows its secretion. To further characterize the role of Grp94 in BSDL secretion, we have studied the effect of a ribozyme specifically targeted to Grp94 mRNA. This ribozyme has been transfected into AR4-2J cells, and we have shown that a decrease in Grp94 expression leads to a concomitant decrease in BSDL secretion and expression. Geldanamycin (GA), which alters Grp94 functions, also affects the release of BSDL into the culture medium of AR4-2J cells. BSDL expressed in GA-treated AR4-2J cells is unstable. Furthermore, under conditions that decrease the level of BSDL secretion, no intracellular accumulation of the enzyme was observed, suggesting that BSDL that cannot associate with (or be structured by) Grp94 could be rapidly degraded. We have further shown that this degradation probably occurs via the ubiquitin-dependent pathway. Altogether, these results indicate that Grp94 has a pivotal role in BSDL folding and in the sorting of this pancreatic enzyme.

Peritoneal colonization by human pancreatic cancer cells is inhibited by antisense FUT3 sequence.

Several alpha(1,3/1,4) fucosyltransferases expressed in human pancreatic cancer cells can participate in the biosynthesis of cell surface sialyl-Lewis a and sialyl-Lewis x antigens that contribute to hematogenous metastatis. Previously, we observed a significant increase of the alpha(1,4) fucosyltransferase activity in tumoral pancreatic cell lines, suggesting that FUT3 could be involved in the sialyl-Lewis antigen expression. Therefore, we invalidated the expression of FUT3 by expressing FUT3 antisense sequence in the human pancreatic tumor BxPC-3 cell line, which expresses the alpha(1,4) fucosyltransferase activity and harbors the cell surface sialyl-Lewis antigens. The decrease of FUT3 transcript after transfection of antisense cDNA of FUT3 in these cells results in a substantial reduction of sialyl-Lewis antigen expression on cell surface. This decreased antigen expression was associated with an inhibition of adhesive properties to E-selectin and a decrease of metastatic power of FUT3 antisense-transfected BxPC-3 cells as tested in nude mice. Our study provides evidence that the expression level of FUT3 may regulate the expression of sialyl-Lewis a and sialyl-Lewis x surface antigens and consequently could play an important role in metastatic properties of human pancreatic cancer cells.

alpha(1,2)-fucosylation prevents sialyl Lewis x expression and E-selectin-mediated adhesion of fucosyltransferase VII-transfected cells.

E-selectin is a cytokine-inducible, calcium-dependent endothelial cell adhesion molecule that plays a critical role in the leucocyte-endothelium interaction during inflammation and is thought to contribute to the metastatic dissemination of tumour cells. Like the other selectins, E-selectin binds to ligands carrying the tetrasaccharide sialyl-Lewis x (NeuAcalpha2,3Galbeta1,4[Fucalpha1, 3]GlcNAc)1 or its isomer sialyl-Lewis a (NeuAcalpha2, 3Galbeta1, 3[Fucalpha1,4]GlcNAc). We examined the effect of expressing the H-type alpha(1,2)-fucosyltransferase or the alpha(2, 6)-sialyltransferase on the synthesis of sialyl-Lewis x by alpha(1, 3)fucosyltransferase. We found that H-type alpha(1, 2)-fucosyltransferase but not alpha(2,6)-sialyltransferase, strongly inhibited sialyl-Lewis x expression and E-selectin adhesion. We assume that H-type alpha(1,2)-fucosyltransferase competes with the endogenous alpha(2,3)-sialyltransferase for the N-acetyllactosamine structures assigned to further serve as acceptors for alpha(1, 3)fucosyltransferase.

Molecular cloning of the bile salt-dependent lipase of ferret lactating mammary gland: an overview of functional residues.

Ferret lactating mammary gland bile salt-dependent lipase (BSDL, EC 3.1.1.-) has been cloned by RT-PCR. The open reading frame consists of 1869 nucleotides which encode 623 amino acids of the functional enzyme. When compared to other species, the greatest homology is observed between residues 1 and 484, with little or no homology at the C-terminal end where seven repeated segments of similar sequence are located. Ferret mammary gland BSDL retains residues involved in the active site and the tentative heparin binding site at similar positions in comparison to other milk or pancreatic BSDL. Other important items, such as binding peptide to chaperone molecular, phosphorylation site(s) or bile salt binding sites, were also tentatively located in well conserved regions of seven available BSDL sequences.

Digestive lipases of the newborn ferret: compensatory role of milk bile salt-dependent lipase.

The amount of mRNA hybridizing to bile salt-dependent lipase and to colipase-dependent lipase probes as well as their translation into active proteins were quantified in the adult and newborn pancreas and lactating mammary gland from the ferret, a species whose milk, similar to that of the human, has bile salt-dependent lipase. The concentration of colipase-dependent lipase mRNA correlated with the amount of activity found in the adult and newborn pancreas, whereas neither mRNA nor activity of this enzyme was detected in the kit pancreas or in the lactating mammary gland. These data indicate that colipase-dependent lipase is actually expressed in adult pancreas and might represent the main lipolytic system in the adult. mRNA hybridizing to the bile salt-dependent lipase probe used in this study were detected in adult and in newborn ferret pancreas as well as in lactating mammary gland. However, the bile salt-dependent lipase activity expressed in the newborn pancreas was very low when compared with the activity expressed either in the mammary gland or in the adult pancreas. These data argue for a compensatory role of milk bile salt-dependent lipase in lipid digestion in the newborn. The hydrolysis of dietary fat might be initiated by preduodenal lipase, the activity of which is only two times lower in the gastric mucosa of the newborn than in the adult ferret. The high concentration of mRNA hybridizing to the bile salt-dependent lipase probe associated with a very poor bile salt-dependent lipase activity and protein suggests either that these mRNA are very unstable or that they are poorly translated into an active pancreatic bile salt-dependent lipase.

Differences in hypolipidaemic effects of two statins on Hep G2 cells or human hepatocytes in primary culture.

1. The objective of this study was to compare in cultured human hepatocytes or Hep G2 cells, changes in the fate of unesterified low density lipoprotein (LDL)-cholesterol induced by crilvastatin, a new cholesterol lowering drug and a reference statin, simvastatin. 2. The experiments were carried out for 20 h, each well contained 4.2 x 10(5)/cm2 Hep G2 cells or 0.5 x 10(5)/Cm2 human hepatocytes, 130 microM ursodeoxycholate, 0.68 microCi or 1.59 microCi unesterified human [14C]-LDL-cholesterol, crilvastatin or simvastatin at 0 or 50 microM (both cell types) or 300 microM (Hep-G2 cells). Incubation with the two drugs resulted in increased amounts of unesterified [14C]-LDL-cholesterol taken by the two cell types, compared to control. 3. Crilvastatin 50 microM led to significantly higher quantities of [14C]-glyco-tauro-conjugated bile salts, compared to simvastatin. Statins reduced the apo B100 level secreted by the two cell types (simvastatin) or human hepatocytes (crilvastatin). Crilvastatin enhanced both the level of apo A1 secreted by the Hep G2 cells and the level of APF, a high density lipoprotein (HDL) and biliary apoprotein. 4. Crilvastatin not only acts by stimulating LDL-cholesterol uptake by hepatocytes, but also by enhancing the catabolism of LDL-cholesterol in bile salts and probably by stimulating HDL and/or bile component secretion. Such a mechanism was not previously described for HMG CoA reductase inhibitors. Our results on APF show that this apoprotein could be considered also as an indicator of changes in bile and/or HDL compartments. 5. The human hepatocyte model appeared to be a suitable and relevant model in the pharmacological-metabolic experiments carried out in this study. It led to more consistent data than those obtained with Hep G2 cells.

Association of bile-salt-dependent lipase with membranes of human pancreatic microsomes.

Immunolocalization studies indicated that, in contrast to other enzyme markers of human pancreatic secretion, bile-salt-dependent lipase (BSDL) was partly but specifically associated with endoplasmic reticulum membranes. In microsomes, temperature-induced phase separation using Triton X-114 elucidated the partition of BSDL between the aqueous phase and the detergent-rich phase containing hydrophilic and membrane proteins, respectively. The size of the membrane-associated BSDL (approx. 100 kDa) is compatible with that of the fully processed enzyme. Fucosylated O- and N-linked oligosaccharide structures were detected by means of specific lectins. The membrane-associated BSDL might therefore be released from membranes between the trans-Golgi compartment (where terminal fucose residues were added) and the zymogen granules where BSDL was mainly found in the soluble fraction. Even though BSDL associated with membranes was enzymically active, it appeared less efficient than the soluble form. The association of BSDL with membranes was pH-dependent and optimal association occurred between pH 5-6. The membrane-associated BSDL was released by KBr which suggests that the association of BSDL with microsomal membranes involves ionic interactions. Lipid-protein interactions are probably not involved in this association as BSDL did not associate with liver microsome membranes. We attempted to characterize the putative ligand and showed that BSDL and a 94-kDa protein, immunologically related to a glucose-regulated protein of 94 kDa (Grp94), were co-immunoprecipitated by specific antibodies directed against each individual species. It is suggested that the biogenesis of the human pancreatic BSDL involves an association with intracellular membranes and that its folding may be assisted by molecular chaperones.

Effect of crilvastatin, a new cholesterol lowering agent, on unesterified LDL-cholesterol metabolism into bile salts by rat isolated hepatocytes.

1. The aim of these experiments was to determine the effect of crilvastatin, a new cholesterol lowering agent, on the metabolism of unesterified low density lipoprotein (LDL)-cholesterol by rat freshly isolated hepatocytes. This preclinical model was developed as an alternative to in vivo experiments, to mimic the metabolic effects of a molecule on its target cells and to define optimal conditions for future experimentation on human hepatocytes. 2. Cells were obtained from normolipidaemic or hypercholesterolaemic rats, hypercholesterolaemia was nutritionally induced. Incubations were performed in a medium containing 600 microM taurocholate and 50 microM or 300 microM crilvastatin. 3. This molecule was shown in vitro to be carried by physiological transporters, i.e., albumin-bile salt micellar associations and LDL. Crilvastatin induced a significance increase in the synthesis and secretion by hepatocytes of bile salts resulting from the metabolism of unesterified LDL-cholesterol in both normolipidaemic and hypercholesterolaemic rats. Stimulation involved non-conjugated as well as tauro- and glyco-conjugated bile salts. These findings corroborate preliminary studies showing in vivo that crilvastatin enhances the secretion of bile acids by stimulating the uptake and incorporation of LDL-cholesterol by the liver.

Bile salt secretion by hepatocytes incubated with bile salts and liposomes or low density lipoproteins.

The purpose of this work was to determine the effect of exogenous unesterified cholesterol provided in either artificial liposomes or LDL on bile salt synthesis by isolated rat hepatocytes. Rates of de novo synthesis were determined in the presence of 300 or 600 microM taurocholate, 600 microM taurodehydrocholate, cholate, deoxycholate or chenodeoxycholate. There was no significant difference between the cholesterol uptake by hepatocytes when the degree of hydrophobicity of the bile salts changed (cholate vs deoxycholate or chenodeoxycholate). Compared to taurocholate, taurodehydrocholate lowered the hepatic incorporation of unesterified cholesterol for the first 60 minutes; compared to control, taurocholate stimulated the cholesterol incorporation for the first 20 minutes. A possible explanation for this finding would be an interaction between bile salts and exogenous cholesterol, depending on the kind of conjugated bile salt. Taurocholate increased the exchange of cholesterol between liposomes or LDL and hepatocyte membranes. It resulted in a significant increase of bile salt synthesis and secretion. This phenomenon was not observed with taurodehydrocholate.

Expression of a 46 kDa protein in human pancreatic tumors and its possible relationship with the bile salt-dependent lipase.

Bile salt-dependent lipase (BSDL), an enzyme normally found in human pancreatic secretions is a 100 kDa glycoprotein. A BSDL-specific 477 bp cDNA probe was prepared by performing polymerase chain reaction experiments. This cDNA was used to probe mRNAs extracted from human pancreatic tissue and tumoral cell lines. Two mRNAs were detected in normal human pancreas at 2.2 and 1.3 to 1.5 kb. In human pancreatic tumoral cells, mRNAs encoding for the BSDL were detected using in situ hybridization, and proteins with an M(r) of 46,000 to 48,000 were translated into an in vitro system using mRNAs extracted from these cells. Using an immunoprecipitation procedure, we observed here that the specific BSDL polyclonal antibodies recognized three proteins of 100 +/- 5 (p100), 46 +/- 2 (p46) and 22.7 +/- 1.2 (p23) kDa, respectively in the soluble extracts of normal adult human pancreas. The p100 protein was probably the glycosylated product resulting from the translation of the 2.2 kb transcript. The p46 protein, which electrophoresed as a doublet was the main component immunoprecipitated from extract of a differentiated human pancreatic adenocarcinoma as well as from the extracts of two pancreatic cell lines, BxPC-3 and SOJ-6. In addition, the p46 immunoform of the BSDL was detected in cell-free medium from SOJ-6 cell line and its expression was found to be correlated with the secretion of an esterolytic activity on 4-nitrophenyl caproate, whereas the BxPC-3 cell line neither secreted the p46 nor showed any esterolytic activity on this substrate. The p46 may be either a short variant of BSDL resulting from the translation of the 1.3 to 1.5 kb transcript or a protein structurally related to the enzyme. The p46 doublet immunoform was detected in the human pancreatic secretion.

Is bile salt-dependent lipase concentration in serum of any help in pancreatic cancer diagnosis?

The diagnostic value of bile salt-dependent lipase for pancreatic diseases was tested in sera of 187 patients. Of these patients, 76 suffered from pancreatic carcinoma, 43 from nonmalignant liver diseases (cirrhosis and chronic hepatitis), 18 from acute pancreatitis, and 20 from chronic pancreatitis. The remaining subjects were controls without pancreatic pathology. Bile salt-dependent lipase was determined by a sandwich enzyme-linked immunosorbent assay using polyclonal antibodies. Amylase and CA 19-9 antigen were also determined. In sera from control patients, the mean level of bile salt-dependent lipase was 1.5 micrograms/L. This level is quite similar to that of patients with benign liver diseases (1.1 micrograms/L) and with chronic pancreatitis (1.4 micrograms/L), but it was raised to 3.5 micrograms/L in patients with acute pancreatitis and decreased to 0.5 microgram/L in subjects with pancreatic adenocarcinoma. Thirty percent of control subjects and 73% of cancer patients had a bile salt-dependent lipase serum level below the cutoff value of 0.5 microgram/L. In acute pancreatitis, 11 of 16 subjects had levels above 1.5 micrograms/L. Amylase level largely increased in acute pancreatitis but was normal in all other groups. Concerning CA 19-9 antigen, 65% of control patients and > 80% of patients with nonmalignant pancreatic or liver diseases had normal levels. In sera from cancer patients, 80% presented with high levels. Accordingly, 36 of 38 patients with pancreatic cancer had either low serum levels of bile salt-dependent lipase (< 0.5 microgram/L) or high values of CA 19-9 antigen (> 37 U/ml; sensitivity 95%).(ABSTRACT TRUNCATED AT 250 WORDS)

Importance of high-density lipoprotein-phosphatidylcholine in secretion of phospholipid and cholesterol in bile.

The purpose of this work was to evaluate biliary phosphatidylcholine (PC) secretion after intravenous infusion of high density lipoprotein (HDL)-[3H]phosphatidylcholine (HDL-[3H]PC) in rats and to study the effect of infusion of dehydrocholic and cholic acids, which, respectively, inhibit and stimulate biliary secretion of PC. The data obtained in this study showed that, in the basal state, HDL-PC accounted for 38% of biliary PC. Dehydrocholic acid infusion caused only a "residual" secretion of HDL-PC in the bile; however, cholic acid infusion stimulated the secretion of HDL-PC as well as PC from intrahepatic microsomes. The low level of radioactivity of HDL-PC in intrahepatic compartments suggests that HDL-PC taken up by the liver is predestined for the bile secretion. The correlation between the kinetics of bile secretion of HDL-cholesterol and HDL-[3H]PC suggests the importance of HDL-PC in reverse transport of cholesterol to the liver and its transport to the bile. The differences between the effects of dehydrocholic acid and cholic acid infusions can be explained by the differences in bile salts binding to the surface of HDL.

Effects of cyclosporine and corticosteroids on bile secretion in the rat.

In order to study their effects on the bile secretion, cyclosporine and methylprednisolone were injected intravenously into rats at a dose of 10 mg/kg b.w. for 30 min. Methylprednisolone had no effect on bile secretion. Cyclosporine led to transient intrahepatic cholestasis characterized by decreased bile flow as well as a decrease of bile salts and cholesterol in bile. Phospholipid levels were not affected. Liver biopsy showed no particular anomaly. These findings suggest that the observed cholestatic reaction may be due to impairment of the metabolism of cholesterol into bile salts or of the conjugation of bile salts rather than to disturbances in bile secretion. After liver transplantation in humans, cholestasis associated with acute rejection or nonspecific cholestasis cannot be attributed directly to the effect of cyclosporine. Cholestasis can be offset by administering taurocholate at a dose of 10 mumol/min/kg b.w. in order to maintain bile salt and phospholipid levels high enough to ensure proper "vectorization" of cholesterol to bile.