Dietary lipids modify intestinal lipid-binding protein RNA abundance in diabetic and control rats.

BACKGROUND: Lipid-binding proteins have been identified in the enterocyte, including the cytosolic intestinal and liver fatty acid binding proteins (I-FABP and L-FABP, respectively) as well as the brush border membrane fatty acid transporter (FAT). It is unclear whether variations in the type of dietary lipids or diabetes modify the RNA abundance of these proteins. Diabetes is associated with an increased intestinal lipid uptake, and the lipid uptake is greater in rats fed a semisynthetic saturated fatty acid (SFA) as compared with a polyunsaturated fatty acid (PUFA) diet. METHODS: Male Sprague-Dawley rats were injected with streptozotocin or control vehicle and fed chow or either SFA or PUFA for 2 weeks. Northern blotting was performed on RNA isolated from jejunal and ileal tissues. RESULTS: In controls, feeding SFA as compared with PUFA reduced the jejunal abundance of I-FABP and L-FABP RNA. In diabetic rats, feeding SFA increased the ileal FAT RNA. Feeding PUFA reduced jejunal L-FABP and ileal FAT RNA in diabetic rats as compared with controls. CONCLUSIONS: The enhanced lipid uptakes reported with feeding an SFA diet or with diabetes were not associated with parallel alterations in lipid-binding proteins. We speculate that these lipid-binding proteins act as a storage mechanism for lipids in enterocytes and are not directly involved in lipid uptake.

Effect of steroids on intestinal lipid uptake in rats is enhanced by a saturated fatty acid diet. Cell and molecular biology collaborative network in gastrointestinal physiology.

Glucocorticosteroids enhance sugar digestive and absorptive functions of the intestine, but their effect on lipid uptake is unknown. Modifications in dietary lipids alter the nutrient transport properties of the intestine. The influence of 4 weeks' treatment with budesonide (BUD), prednisone (PRED), or control vehicle in weanling rats fed either an isocaloric semisynthetic saturated fatty acid diet (SFA) or a polyunsaturated fatty acid diet (PUFA), on the uptake of lipids was assessed using everted gut rings. PRED and BUD increased the uptake of several fatty acids, and this was higher when the animals were fed SFA rather than PUFA. Changes in expression of the mRNAs for L-FABP (liver fatty acid binding protein) and ILBP (ileal lipid binding protein) did not explain these alterations in lipid uptake. Dietary lipid signalling of this adaptive response may involve proglucagon, c-jun, TNF-alpha and IL-10, whereas steroid signalling may involve proglucagon. In summary, steroids increase the absorption of lipids by a process which can be enhanced by the substitution of saturated for polyunsaturated lipids in the diet, and which is not explained by alterations in the expression of the mRNAs of L-FABP or ILBP.

Intestinal resection- and steroid-associated alterations in gene expression were not accompanied by changes in lipid uptake.

BACKGROUND/AIMS: Glucocorticosteroids alter the morphology and transport function of the intestine of adult rats. This study was undertaken to assess the possible effect on intestinal lipid uptake of the locally acting steroid budesonide, or the systemically active prednisone or dexamethasone. METHODS: Sprague-Dawley rats underwent intestinal transection or 50% intestinal resection. Budesonide, prednisone, dexamethasone, or control vehicle was given for 2 weeks from the time of surgery. Uptake was measured using ring uptake technique. RESULTS: Resection had no effect on the mRNA expression for the early response genes, for proglucagon, or for the ileal lipid binding protein (ILBP), but was associated with reduced jejunal ornithine decarboxylase (ODC) mRNA and with reduced jejunal mRNA for the liver fatty acid binding protein (L-FABP). All three steroids reduced jejunal mRNA for proglucagon and c-jun, and did not affect the mRNA for L-FABP or for ILBP. These resection- and steroid-associated changes in gene expression were not associated with alterations in the intestinal uptake of long chain fatty acids or cholesterol. CONCLUSIONS: The resection-associated alterations in the RNA expression of ODC and L-FABP and the steroid-associated changes in mRNA expression of c-jun and proglucagon were not accompanied by variations in lipid uptake.

Dietary lipids alter the effect of steroids on the uptake of lipids following intestinal resection in rats.

Steroids alter the transport function of the intestine. This study was undertaken to assess the effect of glucocorticosteroids on lipid uptake in rats fed either a saturated (SFA) or a polyunsaturated fatty acid (PUFA) diet. Sprague-Dawley rats underwent transection or 50% resection of the small intestine. The steroids had no effect on the uptake of lipids. However, resection decreased the jejunal uptake of palmitic acid in animals fed SFA and increased the jejunal uptake of palmitic and linoleic acids in those fed PUFA. In animals undergoing intestinal resection, fed SFA, and given control vehicle, there was a reduction in jejunal proglucagon mRNA expression as compared to those fed chow or PUFA. Ornithine decarboxylase (ODC) mRNA expression in the jejunum of resected animals was reduced. In summary, dietary lipids modify the uptake of lipids in resected animals and ODC and proglucagon may be involved in this adaptive response.

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.

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.

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.

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.

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.

COUP-TF1 antagonizes Nkx2.5-mediated activation of the calreticulin gene during cardiac development.

Calreticulin, a Ca(2+) binding chaperone of the endoplasmic reticulum, is also highly expressed in the embryonic heart, and knockout of the calreticulin gene is lethal during embryogenesis because of impaired cardiac development. The protein is down-regulated after birth, and elevated expression of calreticulin in newborn hearts is associated with severe cardiac pathology and death. Here we show that the transcription factor Nkx2.5 activates expression of the calreticulin gene in the heart. Binding of chicken ovalbumin upstream promoter-transcription factor 1 to the Nkx2.5 binding site suppresses transcription from the calreticulin promoter. Nkx2.5 and chicken ovalbumin upstream promoter-transcription factor 1 play antagonistic roles in regulating the expression of calreticulin during cardiac development. These studies indicate that cardiac-specific transcription factor Nkx2.5 plays a central role in activating calreticulin expression and that there is a cooperation between chicken ovalbumin upstream promoter-transcription factor 1 and Nkx2.5 at the calreticulin promoter.

The intestinal fatty acid binding protein is not essential for dietary fat absorption in mice.

The intestinal fatty acid binding protein (I-FABP) belongs to a family of 15 kDa clamshell-like proteins that are found in many different tissues. So far, nine types have been identified. Their primary structures are highly conserved between species but somewhat less so among the different types. The function of these proteins, many of which are highly expressed, is not well understood. Their ability to bind lipid ligands suggests a role in lipid metabolism, but direct evidence for this idea is still lacking. We tested the hypothesis that I-FABP serves an essential role in the assimilation of dietary fatty acids by disrupting its gene (Fabpi) in the mouse. We discovered that Fabpi-/- mice are viable, but they display alterations in body weight and are hyperinsulinemic. Male Fabpi-/- mice had elevated plasma triacylglycerols and weighed more regardless of the dietary fat content. In contrast, female Fabpi-/- mice gained less weight in response to a high-fat diet. The results clearly demonstrate that I-FABP is not essential for dietary fat absorption. We propose that I-FABP functions as a lipid-sensing component of energy homeostasis that alters body weight gain in a gender-specific fashion.

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.

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.

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.

The unique acyl chain specificity of biliary phosphatidylcholines in mice is independent of their biosynthetic origin in the liver.

The liver synthesizes phosphatidylcholine (PC) de novo from choline via the CDP-choline pathway, and from phosphatidylethanolamine (PE) via the phosphatidylethanolamine N-methyltransferase (PEMT) pathway. Significant amounts of PC, which are highly specific in their acyl chain composition, are secreted into bile by the liver. To determine whether either of the 2 PC biosynthetic routes is sufficient to provide physiological PC concentrations in bile, or is responsible for the unique acyl chain composition of bile PC, we analyzed gallbladder bile composition in mice that synthesized PC either via the PEMT pathway (induced by feeding a choline-deficient diet) or the CDP-choline pathway (based on genetic PEMT-deficiency). The PC concentration in gallbladder bile of mice that synthesize PC mainly via the CDP-choline pathway was comparable with control mice that synthesize PC via both pathways, whereas it was reduced by approximately 40% in mice that synthesize PC via the PEMT pathway. The acyl chain composition of bile PC was similar irrespective of the active PC biosynthetic pathway in the liver. These data demonstrate that the CDP-choline pathway alone, but not the PEMT pathway alone, can account for physiological concentrations of PC in gallbladder bile. Moreover, the specificity of biliary PC fatty acyl composition is determined independently from the synthetic origin of PC.

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.

High plasma cholesterol in drug-induced cholestasis is associated with enhanced hepatic cholesterol synthesis.

In alpha-naphthylisothiocyanate-treated mice, plasma phospholipid (PL) levels were elevated 10- and 13-fold at 48 and 168 h, respectively, whereas free cholesterol (FC) levels increased between 48 h (17-fold) and 168 h (39-fold). Nearly all of these lipids were localized to lipoprotein X-like particles in the low-density lipoprotein density range. The PL fatty acyl composition was indicative of biliary origin. Liver cholesterol and PL content were near normal at all time points. Hepatic hydroxymethylglutaryl CoA reductase activity was increased sixfold at 48 h, and cholesterol 7alpha-hydroxylase activity was decreased by approximately 70% between 24 and 72 h. These findings suggest a metabolic basis for the appearance of abnormal plasma lipoproteins during cholestasis. Initially, PL and bile acids appear in plasma where they serve to promote the efflux of cholesterol from hepatic cell membranes. Hepatic cholesterol synthesis is then likely stimulated in the response to the depletion of hepatic cell membranes of cholesterol. We speculate that the enhanced synthesis of cholesterol and impaired conversion to bile acids, particularly during the early phase of drug response, contribute to the accumulation of FC in the plasma.

Dietary rhubarb (Rheum rhaponticum) stalk fibre stimulates cholesterol 7 alpha-hydroxylase gene expression and bile acid excretion in cholesterol-fed C57BL/6J mice.

Both experimental and clinical studies have indicated that a novel source of dietary fibre, produced from rhubarb (Rheum rhaponticum) stalks, is potentially hypolipidaemic. The present study, using C57BL/6J mice, was undertaken to examine if this fibre source affects cholesterol degradation. Mice were maintained on semi-purified diets containing 50 g rhubarb fibre or cellulose/kg with or without 5 g cholesterol/kg for 4 weeks. In cholesterol-supplemented mice, rhubarb fibre caused significant lowering of plasma cholesterol (-13%) and the hepatic concentrations of total cholesterol (-34%) and cholesteryl esters (-34%). In parallel to the reduction of hepatic cholesteryl ester content, animals fed on rhubarb fibre had significantly lower activity of acyl CoA: cholesterol acyltransferase (EC 2.3.1.26) than the mice maintained on a diet containing cellulose and cholesterol. Rhubarb-fibre feeding accelerated the faecal bile-acid loss and diminished the gall-bladder bile-acid pool in both the normal and the cholesterol-fed mice. The increase in the bile-acid excretion was positively correlated with an increased activity as well as mRNA abundance of cholesterol 7 alpha-hydroxylase (EC 1.14.13.17). The increased excretion of bile acids and induction of cholesterol 7 alpha-hydroxylase activity may account for the hypocholesterolaemic effect of rhubarb fibre.

Biochemical and evolutionary significance of phospholipid methylation.

All nucleated mammalian cells synthesize phosphatidylcholine from choline via the CDP-choline pathway. Hepatocytes have a second pathway for the synthesis of phosphatidylcholine, a stepwise methylation of phosphatidylethanolamine, catalyzed by phosphatidylethanolamine N-methyltransferase and encoded by the Pempt gene. We report that when Pempt-deficient mice were fed a choline-deficient diet for 3 days, severe liver pathology occurred apparently due to a lack of phosphatidylcholine biosynthesis. The hepatic concentration of phosphatidylcholine decreased by 50% compared with wild type mice on the diet. The levels of plasma triacylglycerols and cholesterol were decreased by greater than 90% in the Pempt-deficient mice. We suggest that the Pempt gene has been maintained during evolution to provide phosphatidylcholine when dietary choline is insufficient, as might occur during starvation or pregnancy.