Identification and partial characterization of a domain in CFTR that may bind cyclic nucleotides directly.

BACKGROUND: The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel that is activated by cAMP-dependent phosphorylation. CFTR channel activity is also stimulated by cGMP-dependent protein kinase and protein kinase C. RESULTS: Here, we show that CFTR channel activation by cGMP may also occur directly. In oocytes from one-third of Xenopus donors, the activation of CFTR by cGMP averaged 87% of the level achieved by cAMP. The currents activated by either cyclic nucleotide displayed similar current-voltage relationships, kinetics, pharmacology and halide selectivity. Sequential stimulation by cAMP and cGMP was not additive, suggesting that both cyclic nucleotides activate the same channel; cGMP was one order of magnitude more potent than cAMP, and its action was insensitive to protein kinase inhibitors. Analysis of the amino-acid sequence of CFTR revealed a domain in the amino-terminal portion of the third cytoplasmic loop that resembles a class of cyclic-nucleotide-binding domains related to that of the catabolite-gene activator protein, CAP. Two CFTR residues in this domain--Val397 and Lys420--were identified which, when changed to alanine, altered the response to cGMP independently of the response to cAMP. CONCLUSIONS: We conclude that direct cyclic nucleotide binding may play a role in channel gating of CFTR. The cGMP-binding domain may provide a useful target for pharmacologic intervention in cystic fibrosis.

Dietary cholesterol increases transcription of the human cholesteryl ester transfer protein gene in transgenic mice. Dependence on natural flanking sequences.

To investigate the regulation of expression of the human cholesteryl ester transfer protein (CETP) gene, transgenic mice were prepared using a CETP minigene linked to the natural flanking sequences of the human CETP gene. By using a transgene containing 3.2 kb of upstream and 2.0 kb of downstream flanking sequence, five different lines of transgenic mice were generated. The abundance of CETP mRNA in various tissues was determined on standard laboratory diet or high fat, high cholesterol diets. In three lines of transgenic mice the tissues expressing the human CETP mRNA were similar to those in humans (liver, spleen, small intestine, kidney, and adipose tissue); in two lines expression was more restricted. There was a marked (4-10-fold) induction of liver CETP mRNA in response to a high fat, high cholesterol diet. The increase in hepatic CETP mRNA was accompanied by a fivefold increase in transcription rate of the CETP transgene, and a 2.5-fold increase in plasma CETP mass and activity. In contrast, CETP transgenic mice, in which the CETP minigene was linked to a metallothionein promoter rather than to its own flanking sequences, showed no change in liver CETP mRNA in response to a high cholesterol diet. Thus (a) the CETP minigene or natural flanking sequences contain elements directing authentic tissue-specific expression; (b) a high cholesterol diet induces CETP transgene transcription, causing increased hepatic CETP mRNA and plasma CETP; (c) this cholesterol response requires DNA sequences contained in the natural flanking regions of the human CETP gene.

An interaction between the human cholesteryl ester transfer protein (CETP) and apolipoprotein A-I genes in transgenic mice results in a profound CETP-mediated depression of high density lipoprotein cholesterol levels.

We have previously described two transgenic mouse lines, one heterozygous for the human apo A-I gene and the other heterozygous for a human cholesteryl ester transfer protein (CETP) minigene driven by the mouse metallothionein-I gene promoter. In the current study, these two lines were crossed producing control, HuCETPTg, HuAITg, and HuAICETPTg mice to study the influence of CETP on HDL cholesterol levels, particle size distribution, and metabolism in animals with mouse and human-like HDL. In the HuCETPTg and HuAICETPTg animals, zinc induction approximately doubled plasma CETP activity, with no activity in plasma from the control and HuAITg animals. The only significant effect of CETP on lipoprotein subfraction cholesterol concentrations was for HDL-C. Compared to control animals, HuCETPTg animals had lower HDL-C, 20% before and 35% after Zn induction, and compared to HuAITg animals, HuAICETPTg animals had lower HDL-C, 35% before and 66% after Zn induction. Control and HuCETPTg HDL consist primarily of a single size population with a mean diameter of 10.00 +/- 0.10 nm and 9.71 +/- 0.05 nm, respectively. HuAITg HDL consists primarily of three distinct HDL size subpopulations with peak diameters of 10.35 +/- 0.08 nm, 8.80 +/- 0.06 nm, 7.40 +/- 0.10 nm, and HuAICETPTg HDL also consists primarily of three distinct HDL size subpopulations with peak diameters of 9.87 +/- 0.05 nm, 8.60 +/- 0.10 nm, 7.30 +/- 0.15 nm before, and 9.71 +/- 0.08 nm, 8.50 +/- 0.11 nm, 7.27 +/- 0.15 nm after zinc induction, respectively. Western blotting analysis of nondenaturing gradient gels of plasma with a monoclonal antibody to CETP indicated that in HuCETPTg and HuAICETPTg mice, 22 and 100%, respectively, of the CETP was HDL associated. Turnover studies with HDL doubly labeled with 125I apo A-I and 3H cholesteryl linoleate indicated that the CETP-induced fall in HDL-C was associated with increased HDL-cholesterol ester fractional catabolic rate in both the absence and presence of human apo A-I, suggesting CETP-mediated transfer of HDL-cholesterol ester to apo B-containing lipoproteins. In summary, these studies suggest that CETP has a much more profound effect on HDL cholesterol levels in transgenic animals expressing human apo A-I. This may be due to an enhanced interaction of CETP with human compared to mouse apo A-I or to the HDL particles they produce.

Atherogenic diet increases cholesteryl ester transfer protein messenger RNA levels in rabbit liver.

Cholesteryl ester transfer activity is increased in plasma of cholesterol-fed rabbits. To investigate the mechanisms leading to changes in activity, we measured cholesteryl ester transfer protein (CETP) mass by RIA and CETP mRNA abundance by Northern and slot blot analysis using a human CETP cDNA probe in control (n = 8) and cholesterol-fed rabbits (n = 10). Cholesterol feeding (chow plus 0.5% cholesterol, 10% corn oil) for 30 d increased CETP mass in plasma 3.2-fold in the cholesterol-fed rabbits (12.45 +/- 0.82 micrograms/ml) compared with controls (3.86 +/- 0.38 micrograms/ml). In the hypercholesterolemic rabbit, liver CETP mRNA levels were increased 2.8 times control mRNA levels. Actin, apo E, lecithin-cholesterol acyltransferase, and albumin mRNA abundances were unchanged. In contrast to the widespread tissue distribution in humans, CETP mRNA was not detected in extrahepatic tissues of either control or cholesterol-fed animals. Using a sensitive RNase protection assay, the increase in liver CETP mRNA was detectable within 3 d of beginning the high cholesterol diet. Thus, in response to the atherogenic diet there is an early increase in liver CETP mRNA, probably causing increased CETP synthesis and secretion, and increased plasma CETP. The results indicate that the CETP gene may be regulated by diet-induced changes in lipid metabolism.

Hypertriglyceridemia and cholesteryl ester transfer protein interact to dramatically alter high density lipoprotein levels, particle sizes, and metabolism. Studies in transgenic mice.

Several types of transgenic mice were used to study the influence of hypertriglyceridemia and cholesteryl ester transfer protein (CETP) expression on high density lipoprotein (HDL) levels, particle sizes, and metabolism. The presence of the CETP transgene in hypertriglyceridemic human apo CIII transgenic mice lowered HDL-cholesterol (HDL-C) 48% and apolipoprotein (apo) A-I 40%, decreased HDL size (particle diameter from 9.8 to 8.8 nm), increased HDL cholesterol ester (CE) fractional catabolic rate (FCR) 65% with a small decrease in HDL CE transport rate (TR) and increased apo A-I FCR 15% and decreased apo A-I TR 29%. The presence of the CETP transgene in hypertriglyceridemic mice with human-like HDL, human apo A-I apo CIII transgenic mice, lowered HDL-C 61% and apo A-I 45%, caused a dramatic diminution of HDL particle size (particle diameters from 10.3 and 9.1 to 7.6 nm), increased HDL CE FCR by 107% without affecting HDL CE TR, and increased apo A-I FCR 35% and decreased apo A-I TR 48%. Moreover, unexpectedly, hypertriglyceridemia alone in the absence of CETP was also found to cause lower HDL-C and apo A-I levels primarily by decreasing TRs. Decreased apo A-I TR was confirmed by an in vivo labeling study and found to be associated with a decrease in intestinal but not hepatic apo A-I mRNA levels. In summary, the introduction of the human apo A-I, apo CIII, and CETP genes into transgenic mice produced a high-triglyceride, low-HDL-C lipoprotein phenotype. Human apo A-I gene overexpression caused a diminution of mouse apo A-I and a change from monodisperse to polydisperse HDL. Human apo CIII gene overexpression caused hypertriglyceridemia with a significant decrease in HDL-C and apo A-I levels primarily due to decreased HDL CE and apo A-I TR but without a profound change in HDL size. In the hypertriglyceridemic mice, human CETP gene expression further reduced HDL-C and apo A-I levels, primarily by increasing HDL CE and apo A-I FCR, while dramatically reducing HDL size. This study provides insights into the genes that may cause the high-triglyceride, low-HDL-C phenotype in humans and the metabolic mechanisms involved.

Intracellular transport of bile acids.

Bile acids originate from the liver and are transported via bile to the intestines where they perform an important role in the absorption of lipids and lipid-soluble nutrients. Most of the bile acids are reclaimed from the terminal ileum and returned to the liver via portal blood for reuse. The transport of bile acids is vectorial in both liver and intestinal cells, originating and terminating at opposite poles. Bile acids enter through the basolateral pole in liver cells, and through the apical pole in intestinal cells. During the past decade, much has been learned about the mechanisms by which bile acids enter and exit liver and intestinal cells. By contrast, the mechanisms by which bile acids are transported across cells remain poorly understood. The current body of evidence suggests that bile acids do not traverse the cell by vesicular transport. Although a carrier-mediated mechanism is a likely alternative, only a handful of intracellular proteins capable of binding bile acids have been described. The significance of these proteins in the intracellular transport of bile acids remains to be tested.

Active taurocholic acid flux through hepatoma cells increases the cellular pool of unesterified cholesterol derived from lipoproteins.

The effect of bile acid flux on the fate of lipoprotein-derived cholesterol was studied in bile acid-transporting McNtcp.18 hepatoma cells. The intracellular unesterified cholesterol (UC) concentration rose when McNtcp.18 cells grown in the presence of either high density lipoproteins (HDL) or low density lipoproteins (LDL) were incubated with taurocholic acid (TCA). This effect was more pronounced when the exogenous source of cholesterol was HDL. The presence of TCA in the culture medium of McNtcp.18 cells had no discernible effect on the uptake of cholesteryl esters (CE) from either lipoprotein. TCA treatment of cells preincubated with either lipoprotein did not affect cholesterol synthesis but antagonized the stimulation of cholesterol esterification in cells that were incubated with LDL. The CE concentration in cells treated with TCA was decreased, relative to cells not incubated with TCA, suggesting that cellular CE stores were also hydrolyzed. The TCA treatment reduced the amount of total cholesterol released into the medium by the lipoprotein-treated cells, which was coincident with the reduction in the amount of apolipoprotein B in the culture medium. However, the proportion of UC released into the medium by the lipoprotein-treated cells was increased in cells capable of active bile acid transport. The results indicate that active bile acid flux through hepatoma cells increases the cellular pool of UC derived from lipoproteins. The UC released by the cells into the culture medium under this condition may represent cholesterol destined for direct biliary secretion.

Modification of the N-terminal cysteine of plasma cholesteryl ester transfer protein selectively inhibits triglyceride transfer activity.

An invariant cysteine residue is found at the N-terminus of cholesteryl ester transfer protein (CETP) isolated from plasma of humans, rabbits and cynomolgus monkeys. We previously reported the expression of recombinant rabbit cholesteryl ester transfer protein in yeast (Kotake et al., J. Lipid Res. 1996; 37: 599-605). The recombinant CETP secreted into the medium contains an altered N-terminal sequence but was fully capable of facilitating both cholesteryl ester (CE) and triglyceride (TG) transfer between lipoproteins. We investigated the importance of the conserved N-terminal cysteine of plasma CETP in the lipid transfer activity by chemical modification of the free sulfhydryl groups of the recombinant CETP and CETP from human and rabbit plasma. The unmodified forms of these CETPs had similar specific activities of CE and TG transfer. Neither 5,5'-dithiobis-(2-nitrobenzoate) nor N-ethyl maleimide altered the lipid transfer activity. In contrast, p-chloromercuriphenyl sulfonate selectively inhibited the TG transfer activity of both human and rabbit plasma CETP. The TG and CE transfer activities of the recombinant CETP, which lacks the N-terminal cysteine residue, was not affected. These results demonstrate that the N-terminal cysteine residue of both human and rabbit plasma CETP is free and is likely to be involved in the construction of a critical part of the active site of CETP that can determine the selectivity of the lipid molecule for the transfer reaction.

Structure, expression profile and alternative processing of the human phosphatidylethanolamine N-methyltransferase (PEMT) gene.

Phosphatidylethanolamine N-methyltransferase (PEMT) catalyzes the conversion of phosphatidylethanolamine (PE) to phosphatidylcholine (PC) in a series of three methylation reactions. Preliminary studies of PEMT in humans led to the cloning of three cDNAs each of which has a different 5' untranslated region (5'UTR). To determine the origin of PEMT splice variants and to investigate expression of the gene in human liver, we isolated a bacterial artificial chromosome (BAC) clone containing the full-length human gene. Each of the three unique untranslated first exons is present in a contiguous array in the gene, confirming the integrity of the cDNAs and alternative processing of PEMT transcripts. Human liver, heart and testis contain the highest levels of PEMT transcripts and of these, liver has the greatest PEMT expression. Furthermore, each of the three PEMT transcripts is present in varying abundance in liver whereas heart and testis contain only one and two transcripts, respectively. Thus, differential promoter usage in the human PEMT gene generates three unique transcripts and confers a tissue-specific expression pattern.

Bile acid-induced morphological changes in hepatoma cells with elevated sodium-dependent bile acid uptake capacity.

McNtcp.24 cells are rat hepatoma cells that were made competent to take up conjugated bile acids actively from the culture medium. Treatment of McNtcp.24 cells with certain species of bile acids caused significant changes in cell structure. Incubation of McNtcp.24 cells in medium containing 100 microM taurocholic acid induced a profound alteration of cellular morphology. Very larger vesicles, visible by phase contrast microscopy, were the most prominent feature of bile acid-treated McNtcp.24 cells. Staining of cells with Oil red O and filipin indicated that the vesicles did not contain neutral lipids or free cholesterol. The vesicles remained in the cells after efflux of radiolabeled taurocholic acid from bile acid loaded cells, indicating that these structures are not intracellular stores of bile acids. Electron microscopic analysis of bile acid-treated McNtcp.24 cells confirmed that the vesicles were localized within the cells. Taurine-conjugated bile acid species were generally potent inducers of the morphological changes, although tauroursodeoxycholic acid did not have a significant effect. Unconjugated bile acid species were ineffective or only mildly effective. Bile acid treatment also caused profound alteration of mitochondrial structure. Surprisingly, there was no significant effect on the ability of treated cells to oxidize fatty acids. The bile acid-treated cells remained viable and upon withdrawal of bile acids from the culture medium, the cells returned to normal morphology by 24 h. The morphological changes observed after treatment of McNtcp.24 with bile acids are reminiscent of the morphological changes observed in hepatocytes following induction of cholestasis.

Differential modulation of cellular death and survival pathways by conjugated bile acids.

BACKGROUND: The liver-derived McNtcp.24 cells transport bile acids and show distinctive responses to the two classes of conjugated bile acids. Whereas taurine-conjugated bile acids are non-toxic, glycine-conjugated bile acids efficiently induce apoptosis. The aim of this study was to determine if the differential sensitivity is limited to cells that normally transport bile acids and if bile acid binding proteins could reduce bile acid-mediated apoptosis. The apical sodium/bile acid co-transporter (asbt) was expressed in Chinese hamster ovary (CHO) cells to establish active bile acid transport in a non-liver-derived cell model (CHO.asbt). A high-affinity bile acid binder was expressed in McNtcp.24 cells. RESULTS: The tolerance of McNtcp.24 cells to taurine-conjugated bile acids was associated with the stimulation of phosphatidylinositol 3-kinase (PI3K) activity. Treatment of CHO.asbt cells with taurine- and glycine-conjugated bile acids resulted in apoptosis. Unlike in McNtcp.24 cells, PI3K activity was not increased in CHO.asbt cells treated with taurine-conjugated bile acids. High level expression of a bile acid binder did not attenuate bile acid-induced cytotoxicity in McNtcp.24 cells. CONCLUSION: The data suggest that McNtcp.24 cells possess a mechanism that can elaborate distinctive responses to the different classes of bile acids. Additionally, activation of a signaling pathway involving PI3K appears to be the dominant mechanism responsible for the tolerance of McNtcp.24 cells to taurine-conjugated bile acids.

Locally and systemically active glucocorticosteroids modify intestinal absorption of lipids in rats.

Orally administered systemically active steroids enhance the digestive and absorptive functions of the intestine, but their effect on lipid uptake is unknown. The effect of the locally acting steroid budesonide on intestinal absorptive function also is unknown. Accordingly, this study was undertaken to assess the influence of 4 wk of treatment of weaning male rats with a daily oral gavage of budesonide (BUD), prednisone (PRED), or control vehicle on the jejunal and ileal uptake of fatty acids and cholesterol. BUD enhanced jejunal uptake of oleic acid and ileal uptake of linoleic acid. PRED increased jejunal uptake of cholesterol and ileal uptake of lauric, palmitic, linoleic, and linolenic acids. Higher doses of BUD (up to 1 mg/kg) given to adult rats for 2 wk further increased the uptake of some lipids. The changes in the uptake of lipids were not due to variations in the weight of the intestinal mucosa or in the animals' food intake. Ileal ornithine decarboxylase mRNA expression was increased with PRED, but there were no steroid-associated changes in the expression of the mRNA of the early response genes c-myc, c-jun, or c-fos or of proglucagon, the liver fatty acid-binding protein (FABP), the ileal lipid-binding protein, tumor necrosis factor alpha, interleukin 2 (IL-2), IL-6, or IL-10. In summary, treatment of weanling rats with BUD and PRED enhances the uptake of some lipids by a process that is independent of the effects of early response genes and genes encoding cytokines, proglucagon, and FABP.

Partial transfection of liver with a synthetic cholesterol 7 alpha-hydroxylase transgene is sufficient to stimulate the reduction of cholesterol in the plasma of hypercholesterolemic mice.

The effect of administering a synthetic transgene encoding cholesterol 7 alpha-hydroxylase (cyp7) on plasma cholesterol metabolism of intact mice was investigated. The synthetic cyp7 transgene (Tg1) was constructed by placing the cDNA sequence encoding the full-length cyp7 polypeptide under the control of a heavy metal inducible metallothionein promoter. The transgene was complexed with asialoorosomucoid-polylysine conjugate and introduced into mice via the tail vein. Cell marking experiments using a beta-galactosidase (lacZ) transgene as a tag showed that 5-10% of the liver can be transfected by this procedure. Administration of the Tg1 transgene to older hypercholesterolemic chow-fed mice resulted in about a 50% reduction of plasma cholesterol, regardless of whether or not transgene expression was induced by zinc treatment. In diet-induced hypercholesterolemic mice, the reduction (20%) in total plasma cholesterol was seen only when transgene expression was induced, and this reduction was due primarily to a decrease in non-high-density lipoprotein cholesterol. The maximum reduction was evident at 6 days after the introduction of the transgene and was no longer evident after 9 days. Introduction of the Tg1 transgene into young chow-fed mice had no effect on the already low levels of plasma cholesterol. However, compared with the no-transgene and lacZ transgene controls, the gallbladder bile acid content of Tg1-treated mice was increased. The results show that non-viral-mediated delivery of a synthetic transgene encoding cyp7 to a subpopulation of hepatocytes in the liver of intact hypercholesterolemic mice is sufficient to facilitate the temporary reduction of plasma cholesterol content.

Rainbow trout growth hormone: molecular cloning of cDNA and expression in Escherichia coli.

We have isolated several recombinant clones carrying the complementary DNA (cDNA) sequence of the rainbow trout (rt) Salmo gairdneri growth hormone (GH) mRNA by immunoblot screening using an antiserum to chum salmon (Oncorhyncus keta) GH. The nucleotide sequence of one of the rtGH cDNA clones (pAF51) was determined. The rt cDNA sequence in pAF51 encodes a hybrid polypeptide of 199 amino acid residues containing 9 amino acid residues of the bacterial beta-galactosidase, one residue from the codon at the junction of the beta-galactosidase gene, and the rtGH cDNA sequence, an additional residue from the presumptive signal peptide of the pre-rtGH and the entire sequence of the mature rtGH (188 amino acid residues). Pairwise matrix comparisons of the hydropathy profiles of bovine, human, rat, and rainbow trout GH polypeptides indicate that regions of similarity exist between the rtGH and mammalian GH. In particular, there are two major regions of similarity found near the amino-terminal region and at the carboxy-terminal region. These regions correspond to hydrophilic domains of the GH molecules. The possible significance of these domains is discussed.

Metabolism of cholesterol is altered in the liver of C3H mice fed fats enriched with different C-18 fatty acids.

We examined whether the degree of saturation of C-18 fatty acids influenced hepatic cholesterol metabolism in C3H mice. The mice were fed diets containing 20 g/100 g fat, enriched in stearic (18:0), oleic (18:1) or linoleic acid (18:2) with or without 1 g/100 g cholesterol. Plasma total cholesterol concentration was lower in mice fed the 18:0 diet relative to those fed the 18:1- or 18:2-enriched diets (P < 0.05) regardless of dietary cholesterol supplementation. Dietary cholesterol significantly raised hepatic total cholesterol concentration (P < 0.05) in those fed the 18:1- and 18:2-enriched diets, but not in mice fed the 18:0-enriched diet. Dietary cholesterol raised biliary cholesterol concentration (P < 0. 05) in mice fed the 18:1- and 18:2-enriched diets, but not in mice fed the 18:0-enriched diet. The cholesterol saturation index was variably affected by the fat diets. Feeding diets containing cholesterol suppressed the hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) activity and induced acyl coenzyme A:cholesterol acyl transferase (ACAT) activity compared with feeding diets without cholesterol (P < 0.05), indicating that the liver was exposed to dietary cholesterol. Hepatic ACAT activity was lower in mice fed the 18:0-enriched diet compared with those fed the 18:1- or 18:2-enriched diets (P < 0.05). Addition of cholesterol to the 18:1 diet induced the largest increase of hepatic ACAT activity, and this was associated with the enrichment of VLDL with cholesterol. Varying the degree of saturation of C-18 fatty acids influences the metabolism and disposition of hepatic cholesterol.

The 3'-untranslated region of the mouse cholesterol 7alpha-hydroxylase mRNA contains elements responsive to post-transcriptional regulation by bile acids.

To investigate the importance of the 3'-untranslated region (UTR) of the mouse cholesterol 7alpha-hydroxylase (cyp7) mRNA in post-transcriptional regulation of expression of the cyp7 gene, chimaeric genes encoding mRNA containing the structural sequence of chloramphenicol acetyltransferase (CAT) linked to either the 3'-UTR of the mouse cyp7 mRNA or the SV40 early gene mRNA were constructed. The human cytomegalovirus (CMV) promoter was used to drive the expression of all the chimaeric genes. Thus the transgenes had identical sequences in the promoter, the regions encoding the 5'-UTR and translated sequence but differed in the region encoding the 3'-UTR of their respective mRNA species. The transgene containing the entire cyp7 3'-UTR (designated CMV.CAT.CYP7) gave rise to CAT activity in transfected hepatoma cells that was one-quarter of that obtained in cells transfected with the transgene containing the SV40 3'-UTR (designated CMV.CAT.SV40). The 3'-UTR of the cyp7 mRNA contains sequences resembling AU-rich elements (AREs). Deleting eight of nine putative AREs from the CYP7 3'-UTR sequence increased the CAT activity to a level greater than that observed for CMV.CAT. SV40, whereas deletion of the intron region had no effect. These results show that the AREs of the 3'-UTR of the cyp7 mRNA decrease transgene expression. Bile acids are known to repress the expression of the cyp7 gene. To test whether the 3'-UTR of the cyp7 mRNA has a role in this process, the expression of the chimaeric genes was evaluated in hepatoma cells competent for bile acid uptake. Conjugated bile acids, but not unconjugated bile acids, further decreased the expression of the CMV.CAT.CYP7 transgene. The same bile acids had no effect on the expression of the CMV.CAT.SV40 transgene. Deletion of the intron from the cyp7 sequence did not alter the CAT activity compared with the parental plasmid, and also did not alter the sensitivity of the transgene to the conjugated bile acids. Deletion of the AREs from the cyp7 3'-UTR, which increased the expression of the transgene, did not abolish the sensitivity of the transgene to repression by conjugated bile acids. Thus the 3'-UTR of the mouse cyp7 mRNA also contains elements that facilitate the further repression of transgene expression in the presence of conjugated bile acids. The results indicate that the 3'-UTR of the mouse cyp7 mRNA contains information specifying regulation at the post-transcriptional level.

The murine and human cholesterol 7alpha-hydroxylase gene promoters are differentially responsive to regulation by fatty acids mediated via peroxisome proliferator-activated receptor alpha.

We determined if fatty acids can regulate the murine Cyp7a1 and human CYP7A1 gene promoters via peroxisome proliferator-activated receptor alpha (PPARalpha)/9-cis-retinoic acid receptor alpha (RXRalpha). In transfected cells, the murine Cyp7a1 gene promoter displayed markedly lower basal activity, but greater sensitivity to fatty acid- or WY 14,643-activated PPARalpha/RXRalpha when compared with the human CYP7A1 gene promoter. PPARalpha/RXRalpha can bind to a site (Site II) located within the region at nucleotides -158 to -132 of both promoters. Mutagenesis of the human CYP7A1 Site II element abolished the response to activated PPARalpha/RXRalpha. The murine Cyp7a1 gene promoter contains an additional PPARalpha/RXRalpha-binding site (Site I) located within nucleotides -72 to -57. Replacement of a single residue in human CYP7A1 Site I with that found in the murine Cyp7a1 Site I sequence enabled PPARalpha/RXRalpha binding, and this mutation resulted in reduced basal activity, but substantially improved the response to activated PPARalpha/RXRalpha in transfected cells. We conclude that fatty acids can regulate the cyp7a gene promoter via PPARalpha/RXRalpha. The differential response of the murine Cyp7a1 and human CYP7A1 gene promoters to PPARalpha activators is attributable to the additional PPARalpha/RXRalpha-binding site in the murine Cyp7a1 gene promoter.

Separation and quantitation of bile acids using an isocratic solvent system for high performance liquid chromatography coupled to an evaporative light scattering detector.

We developed a quantitative method for the analysis of bile acids using a high performance liquid chromatograph coupled to an evaporative light scattering detector. An isocratic solvent system was used to resolve in a single run conjugated and unconjugated bile acid species relevant in human and rodent physiology. The detection of various bile acids was linear over a range of 0.08 to 10 nmol of injected molecules. The developed system is a convenient and cost-effective method for the routine analysis of a wide variety of bile acids.

Expression of cholesterol 7alpha-hydroxylase restores bile acid synthesis in McArdle RH7777 cells.

Bile acid synthesis involves several enzymes and occurs only in liver cells. The first and rate-determining step is catalyzed by cholesterol 7alpha-hydroxylase (cyp7a). McArdle RH7777 hepatoma cells do not synthesize bile acids and do not express the cyp7a gene. A synthetic cyp7a gene was stably expressed in this cell line to determine if restoration of cyp7a activity is sufficient to reconstitute the bile acid synthetic pathway. The transfected cells contained the recombinant cyp7a mRNA and the corresponding protein. Microsomes from recombinant cells converted cholesterol into 7alpha-hydroxycholesterol, indicating that the recombinant enzyme was active. Radiolabeled bile acids, originated from exogenously supplied radiolabeled cholesterol, were detected in the culture medium of recombinant cells. Thus, expression of cyp7a is sufficient in restoring bile acid synthesis in McArdle RH7777 cells. The results also show that the additional complement of enzymatic activities required to convert cholesterol into bile acids has remained active in this cell line.

Dietary fats modulate the regulatory potential of dietary cholesterol on cholesterol 7 alpha-hydroxylase gene expression.

Cholesterol 7 alpha-hydroxylase (cyp7) is the rate-limiting enzyme in bile acid biosynthesis. Previously, dietary cholesterol was shown to induce cyp7 gene expression. However, recent studies have produced data that are inconsistent with this observation, suggesting the possibility that other factors in the diet are also important in the regulation of cyp7 by dietary cholesterol. The effect of dietary fats on the ability of dietary cholesterol to regulate cyp7 activity and mRNA abundance was assessed. High fat diets composed primarily of polyunsaturated (PUFA), monounsaturated (MUFA), or saturated (SFA) fatty acids induced hypercholesterolemia regardless of whether cholesterol was present or not. However, the effects of each diet on bile composition and hepatic cholesterol content were variable. Microsomal fatty acid profiles reflected the fatty acid composition of the diets. Addition of cholesterol to the PUFA diet increased cyp7 mRNA abundance and activity, analogous with the results observed in mice fed a chow plus cholesterol diet. On the other hand, addition of cholesterol to diets high in MUFA or SFA caused a significant reduction of cyp7 mRNA abundance and activity. Addition of cholesterol to all the diets caused the expected changes in low density lipoprotein receptor and 3-hydroxy-3-methylglutaryl coenzyme A reductase mRNA abundance but was not correlated with the changes in cyp7 mRNA abundance. The relationship between cyp7 mRNA abundance and hepatic total cholesterol content or hepatic microsomal cholesterol content was evident, suggesting that cholesterol status does not necessarily determine cyp7 mRNA abundance. The results of this study illustrate that the type of dietary fat is important in elaborating the regulatory potential of dietary cholesterol on cyp7 gene expression and suggest that the regulation of cyp7 gene expression does not involve the classical sterol-mediated pathway.