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PubMed1995

Translocation of ricin A-chain into proteoliposomes reconstituted from Golgi and endoplasmic reticulum.

Translocation to the cytosol is an essential and rate-limiting step in the cytotoxicity of the potent plant toxin ricin. In an attempt to study the mechanism of ricin A-chain translocation in a cell-free assay, we have partially purified Golgi and endoplasmic reticulum from Jurkat cells by discontinuous sucrose gradient fractionation. The membranes of the organelle fractions were solubilized by the addition of sodium cholate and reconstituted into proteoliposomes by dialyzing out the detergent. The resulting vesicles supported cell-free translocation of RTA (as assessed by an enzyme protection assay) at a rate which was linearly dependent on the concentration of the vesicle preparation. Ricin B-chain (RTB) neither translocated into the vesicles, nor increased the efficiency of RTA translocation. Liposomes prepared from purified phospholipids were not capable of supporting RTA translocation. Furthermore, protease treatment of concanavalin A adsorption of proteins from lysates prior to vesicle reconstitution resulted in abrogation of the translocation process, suggesting that the protein components of organelle membranes are required for RTA translocation. Reconstitution of translocation-competent proteoliposomes from detergent-solubilized membranes of endoplasmic reticulum- and Golgi-enriched fractions provides a convenient cell-free system to study the mechanism of RTA translocation.

Cell Fractionation↗

PubMed1996

Purification of a 3beta-hydroxy-delta5-C27-steroid dehydrogenase from pig liver microsomes active in major and alternative pathways of bile acid biosynthesis.

A 3beta-hydroxy-Delta5-C27-steroid dehydrogenase active in bile acid biosynthesis was purified from pig liver microsomes by solubilization with sodium cholate and by chromatography on DEAE-Sepharose, aminohexyl-Sepharose, and blue Sepharose. The last step in the purification procedure was preparative isoelectric focusing in a Rotofor cell. The final enzyme preparation showed only one protein band upon SDS-polyacrylamide gel electrophoresis. The isoelectric point was estimated to about 7.0 and the apparent Mr was 36,000. The purified enzyme catalyzed the conversion of 7alpha-hydroxycholesterol, 7alpha,25-dihydroxycholesterol, 7alpha, 27-dihydroxycholesterol, and 3beta,7alpha-dihydroxy-5-cholestenoic acid into the corresponding 3-oxo-Delta4 compounds. The enzyme was inactive with C19 and C21 steroids as substrates. The enzyme was also inactive with C27 steroids having the 7-hydroxy group in beta- instead of alpha-position. The Km was found to be 0.30 and 0.32 microM with 7alpha-hydroxycholesterol and 7alpha, 27-dihydroxycholesterol as substrates, respectively. NAD+ was the preferred cofactor. A monoclonal antibody raised against the 3beta-hydroxy-delta5-C27-steroid dehydrogenase was prepared. After coupling to Sepharose, the antibody was able to bind the dehydrogenase and to decrease the conversion of 7alpha-hydroxycholesterol into 7alpha-hydroxy-4-cholest-3-one by more than 90%. The N-terminal amino acid sequence was determined and found to be similar but not identical with those of known 3beta-hydroxy-Delta5-steroid dehydrogenases active in steroid hormone biosynthesis. Thus, the purified enzyme active toward C27 steroids in bile acid biosynthesis appears to represent a novel type of 3beta-hydroxy-delta5-steroid dehydrogenase.

3-Hydroxysteroid Dehydrogenases↗

PubMed1997

The carboxyl-terminal hydrophobic residues of apolipoprotein A-I affect its rate of phospholipid binding and its association with high density lipoprotein.

We performed a series of mutations in the human apolipoprotein A-I (apoA-I) gene designed to alter specific amino acid residues and domains implicated in lecithin:cholesterol acyltransferase (LCAT) activation or lipid binding. We used the mutant apoA-I forms to establish nine stable cell lines, and developed strategies for the large scale production and purification of the mutated apoA-I proteins from conditioned media. HDL and dimyristoyl phosphatidylcholine binding assays using the variant apoA-I forms have shown that replacement of specific carboxyl-terminal hydrophobic residues Leu222, Phe225, and Phe229 with lysines, as well as replacement of Leu211, Leu214, Leu218, and Leu219 with valines, diminished the ability of apoA-I to bind to HDL and to lyse dimyristoyl phosphatidylcholine liposomes. The findings indicate that Leu222, and Phe225, Phe229 located in the putative random coil region, and Leu211, Leu214, Leu218, and Leu219 located in the putative helix 8, are important for lipid binding. In contrast, substitutions of alanines for specific charged residues in putative helices 7, 8, or 9 as well as various point mutations in other regions of apoA-I, did not affect the ability of the variant apoA-I forms to bind to HDL or to lyse dimyristoyl phosphatidylcholine liposomes. Cross-linking experiments confirmed that the carboxyl-terminal domain of apoA-I participates in the self-association of the protein, as demonstrated by the inability of the carboxyl-terminal deletion mutants delta185-243 and delta209-243 to form higher order aggregates in solution. Lecithin:cholesterol acyltransferase analysis, using reconstituted HDL particles prepared by the sodium cholate dialysis method, has shown that mutants (Pro165-->Ala,Gln172-->Glu) (Leu211-->Val,Leu214-->Val, Leu218-->Val,Leu219-->Val), Leu222-->Lys,Phe225-->Lys, Phe229-->Lys) and delta209-243 reduced LCAT activation (38-68%). Mutant (Glu191-->Ala,His193-->Ala,Lys195-->Ala) enhanced LCAT activation (131%), and mutant (Ala152-->Leu, Leu159-->Trp) exhibited normal LCAT activation as compared with the wild type proapoA-I and plasma apoA-I forms [corrected]. The apparent catalytic efficiency (Vmax(app)/Km(app)) of the apoA-I mutants ranged from 17.8 to 107.2% of the control and was the result of variations in both the Km and the Vmax in the different mutants. These findings indicate that putative helices 6 and 7, and the carboxyl-terminal helices 8 and 9 contribute to the optimum activation of lecithin:cholesterol acyltransferase. In addition to their use in the present study, the variant apoA-I forms generated will serve as valuable reagents for the identification of the domains and residues of apoA-I involved in binding the scavenger receptor BI, and facilitating cholesterol efflux from cells as well as aid in the structural analysis of apoA-I.

Apolipoprotein A-I↗

PubMed1998

Regulation of GRP1-catalyzed ADP ribosylation factor guanine nucleotide exchange by phosphatidylinositol 3,4,5-trisphosphate.

Cellular levels of phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) are rapidly elevated in response to activation of growth factor receptor tyrosine kinases. This polyphosphoinositide binds the pleckstrin homology (PH) domain of GRP1, a protein that also contains 200 residues with high sequence similarity to a segment of the yeast Sec7 protein that functions as an ADP ribosylation exchange factor (ARF) (Klarlund, J., Guilherme, A., Holik, J. J., Virbasius, J. V., Chawla, A., and Czech, M. P. (1997) Science 275, 1927-1930). Here we show that dioctanoyl PtdIns(3,4,5)P3 binds the PH domain of GRP1 with a Kd = 0.5 microM, an affinity 2 orders of magnitude greater than dioctanoyl-PtdIns(4,5)P2. Further, the Sec7 domain of GRP1 is found to catalyze guanine nucleotide exchange of ARF1 and -5 but not ARF6. Importantly, PtdIns(3,4,5)P3, but not PtdIns(4,5)P2, markedly enhances the ARF exchange activity of GRP1 in a reaction mixture containing dimyristoylphosphatidylcholine micelles, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid, and a low concentration of sodium cholate. PtdIns(3,4,5)P3-mediated ARF nucleotide exchange through GRP1 is selectively blocked by 100 microM inositol 1,3,4,5-tetrakisphosphate, which also binds the PH domain of GRP1. Taken together, these data are consistent with the hypothesis that selective recruitment of GRP1 to PtdIns(3,4,5)P3 in membranes activates ARF1 and -5, known regulators of intracellular membrane trafficking.

ADP-Ribosylation Factor 1↗

PubMed1986

A mechanism for the hypocholesterolaemic activity of saponins.

1. Saponins are steroid or triterpene glycosides which occur in a number of important food plants, including such staples as soya beans (Glycine max) and chickpeas (Cicer arietinum). They are known to be hypocholesterolaemic. 2. Some saponins form an insoluble complex with cholesterol which prevents its absorption from the small intestine. Others cause an increase in the faecal excretion of bile acids, an indirect route for elimination of cholesterol. 3. We have investigated the effects of different saponins on absorption of the bile salt sodium cholate from perfused loops of small intestine, in vivo, in the rat. Purified saponins from soapwort (Saponaria officinalis), soya beans and quillaia (Quillaia saponaria) reduced the rate of absorption of the bile salt; soya-bean and soapwort saponins substantially so but quillaia saponin to a much lesser extent. 4. These results were explained by the formation of large mixed micelles by bile acid and saponin molecules in aqueous solution. These aggregates can have molecular weights in excess of 10(6) daltons, consequently the bile acid molecules incorporated in them are not available for absorption. 5. Control of plasma cholesterol and nutrient absorption through dietary saponins could provide substantial health and nutritional benefits in humans.

Absorption↗

PubMed1992

Relationship between structure and function of dietary fibre: a comparative study of the effects of three galactomannans on cholesterol metabolism in the rat.

Male adult rats were fed on diets containing 80 g/kg galactomannans with different galactose (G): mannose (M) ratios/kg. The galactomannans were compared with purified cellulose (Solkaflok) and the animal were also fed on a basal diet free from fibre. All diets contained cholesterol (10 g/kg) and sodium cholate (2 g/kg). The three galactomannans were fenugreek gum (1G:1M), guar gum (1G:2M) and locust-bean gum (1G:4M). In comparison with the fibre-free and Solkaflok diets, all three galactomannans lowered the concentrations of cholesterol in both liver and blood plasma. The galactomannans also decreased the rate of hepatic synthesis of cholesterol. Dietary galactomannans increased caecal volatile fatty acids, particularly propionic, increased the weight of the caecum and its contents and increased the amount of water in the faeces. The increase in propionic acid production was significantly related to a decrease in caecal pH, but not to changes in plasma cholesterol or hepatic cholesterol synthesis. These effects were significantly influenced by chemical composition and structure of the galactomannan; they were most evident when the proportion of galactose in the galactomannan was highest (i.e. fenugreek gum). The three galactomannans also differed markedly in their effects on the viscosity of the digesta, but the galactomannan which gave the highest viscosity was least effective in lowering plasma cholesterol. A separate experiment with perfused loops of small intestine in vivo showed that the most effective galactomannan, fenugreek gum, had no direct effect on cholesterol absorption.

Animals↗

PubMedDate not supplied

The effect of oral absorption enhancers on the in vivo performance of insulin-loaded poly(ethylcyanoacrylate) nanospheres in diabetic rats.

Poly(ethylcyanoacrylate) (PECA) nanospheres have been employed as biodegradable polymeric carriers for oral (po) delivery of insulin. The main goal of this investigation was to screen various absorption enhancers, which were used to protect insulin-loaded PECA, by following their in vivo performance after oral administrations to streptozotocin-induced diabetic rats. The nanospheres were prepared by polymerization in a continuous aqueous phase at pH 2.5 and in the presence of Pluronic 68 (0.5%). This polymerization technique was able to hold 85 +/- 7.5% of insulin added, 30 min after initiation of polymerization. The drug loading was evaluated by HPLC. Insulin absorption after oral administration was evaluated by its hypoglycemic effect. The addition of protease inhibitor to insulin-loaded PECA nanospheres significantly reduced the blood glucose level after po administrations. Capric acid (0.5%) showed the maximum reduction in blood glucose level (bG max = 34.4 +/- 4.1 mg%), while cholic acid (0.5%) showed the fastest reduction in blood glucose level (Tmax, G = 6.0 +/- 0.2 h) among the tested oral absorption enhancers. Insulin associated with PECA nanospheres retained its biological activity up to 12 days in 50% of the rats in the presence of glycyrrhizic acid (1%). The per cent pharmacological availabilities (PA%) were in the order of capric acid (0.5%) > glycyrrhizic acid (1%) > deoxycholic acid (0.5%) > hydroxypropyl-beta-cyclodextrin (HPbetaCD) (1%) > cholic acid (0.5%). There was no significant difference in the PA% between capric acid (0.5%), glycyrrhizic acid (1%) and deoxycholic acid (0.5%). Although sodium cholate (0.5%) showed the lowest increase in PA% (3.1 +/- 3.11%), its Tmax G was the shortest (6 +/- 0.2 h) among the tested absorption enhancers. The reduction in blood glucose levels was maintained < 200 mg/dL in the order of glycyrrhizic acid (166 +/- 56 h) > capric acid (72 h) >; deoxycholic acid (66 +/- 6 h) > no enhancer (64 +/- 16 h) > HPbetaCI) (31 +/- 17.5) > cholic acid (22 +/- 5.7 h) > aprotenin (20 +/- 3.5 h). Therefore, capric acid and glycyrrhizic acid could be successfully used as oral absorption enhancers.

Administration, Oral↗

PubMed1992

Amino- and carboxy-terminal deletion mutants of Gs alpha are localized to the particulate fraction of transfected COS cells.

To elucidate the structural basis for membrane attachment of the alpha subunit of the stimulatory G protein (Gs alpha), mutant Gs alpha cDNAs with deletions of amino acid residues in the amino and/or carboxy termini were transiently expressed in COS-7 cells. The particulate and soluble fractions prepared from these cells were analyzed by immunoblot using peptide specific antibodies to monitor distribution of the expressed proteins. Transfection of mutant forms of Gs alpha with either 26 amino terminal residues deleted (delta 3-28) or with 59 amino terminal residues deleted (delta 1-59) resulted in immunoreactive proteins which localized primarily to the particulate fraction. Similarly, mutants with 10 (delta 385-394), 32 (delta 353-384), or 42 (delta 353-394) amino acid residues deleted from the carboxy terminus also localized to the particulate fraction, as did a mutant form of Gs alpha lacking amino acid residues at both the amino and carboxy termini (delta 3-28)/(delta 353-384). Mutant and wild type forms of Gs alpha demonstrated a similar degree of tightness in their binding to membranes as demonstrated by treatment with 2.5 M NaCl or 6 M urea, but some mutant forms were relatively resistant compared with wild type Gs alpha to solubilization by 15 mM NaOH or 1% sodium cholate. We conclude that: (a) deletion of significant portions of the amino and/or carboxyl terminus of Gs alpha is still compatible with protein expression; (b) deletion of these regions is insufficient to cause cytosolic localization of the expressed protein. The basis of Gs alpha membrane targeting remains to be elucidated.

Amino Acid Sequence↗

PubMed1956

Experimental production of gross atherosclerosis in the rat.

Gross atherosclerosis was produced in the rat by feeding purified diets containing cholesterol, sodium cholate, and thiouracil for periods up to 363 days. In a few weeks a marked increase of the serum cholesterol and beta lipoproteins as well as of the liver lipides was observed. Lesions visible in the gross were found on the intimal surfaces of the vessels of all 46 animals examined. These were most prominent in the heart valves and aortic arch. The earliest lesions, which were seen at 31 days, required Sudan staining for gross demonstration. Older lesions were visible without staining. Microscopic coronary artery lesions were present and in one instance were accompanied by massive myocardial infarction. Vascular lesions were characterized by medial and intimal lipide infiltration and cellular intimal plaque formation. In a part of this study the protein level of the diet was altered at the expense of sucrose. The hypercholesteremic response among the rats varied according to the dietary protein level. The lowest response was observed among those animals receiving the highest level of dietary protein. A difference, however, in the severity and extent of the arterial lesions among these relatively small groups of rats could not be established under these experimental conditions. In all these experiments a close correlation existed between the serum cholesterol levels and beta lipoproteins of the S(f)20-100 range.

Animals↗

PubMed1962

Alteration of resistance of the rat to tuberculosis when maintained on an atherogenic diet.

Albino rats maintained for 50 to 60 days on rations containing cholesterol, sodium cholate, and thiouracil were found to be highly susceptible to the lethal effects of infection with Mycobacterium tuberculosis. Maximum loss of resistance occurred only when all three components were present in a single ration. A lesser degree of susceptibility resulted from the deletion of one or more of the factors from the diet. Animals maintained on control rations never died of the infection. Histopathological studies of stained sections of tissues of infected animals revealed that the reaction of the host to the bacillus was more extensive and less well contained in animals fed the complete atherogenic ration than in the controls.

Animals↗

PubMed1978

Characterization of 7-alpha-dehydroxylase in Clostridium leptum.

7-alpha-Dehydroxylation of primary bile acids was demonstrated radiochromatographically in whole cells of Clostridium leptum but was not observed in intestinal Bacteroides species. Activity of 7-alpha-Dehydroxylase was detected within a pH range of 5 to 9 and was 8-fold higher in specific activity in cell cultures in the presence of 0.1 mM sodium cholate than in its absence. 7-alpha-Dehydroxylase activity in whole cells was markedly inhibitied by 2,4-dinitrophenol, carbonyl-cyanide-m-chlorophenylhydrazine, and dicyclohexylcarbodiimide. A hypothesis concerning the dietary regulation of 7-alpha-dehydroxylating intestinal anaerobic bacteria is presented.

Bacteroides↗

PubMed1986

Human milk kills Giardia lamblia by generating toxic lipolytic products.

This study supports our previous hypotheses that normal human milk kills Giardia lamblia trophozoites in vitro and that this killing is due to the release of free fatty acids (FFAs) from milk triglycerides by action of the bile salt-stimulated lipase (BSL) of human milk. Heat-stable killing of G. lamblia was generated when normal human milk was preincubated with sodium cholate, which activates BSL. Moreover, both the skim-milk (containing BSL) and cream (containing mainly triglycerides) fractions were required to kill G. lamblia. We measured the toxicity of FFA and other products of lipolysis to G. lamblia. cis-Unsaturated FFAs (LD50 less than 12 microM), three of four monoglycerides, and four of five lysophosphatidylcholines were toxic to the parasites (LD50 less than 100 microM). In contrast, the parasites were not harmed by the corresponding diglycerides, phosphatidylcholines, triolein, or glycerol. Thus, products of lipid hydrolysis in the normal digestive tract are toxic to G. lamblia. We also demonstrated that albumin and conjugated bile salts, which bind FFA, partially protected trophozoites from killing by oleic acid.

Bile Acids and Salts↗

PubMed1981

Effects of tomato pomace and mixed-vegetable pomace on serum and liver cholesterol in rats.

The effects of tomato pomace and mixed-vegetable pomace, vegetable processing by-products, on serum and liver cholesterol were compared against pectin, wheat bran, cellulose, and lignin. Male Sprague-Dawley rats (100 g) were fed high sucrose, semi-purified diets containing basal (0), 5% or 10% of the fiber source and either 0, 0.5% cholesterol or 0.5% cholesterol + 0.125% sodium cholate. After 28 days, there were highly significant differences (P less than 0.001) in serum and liver cholesterols due to fiber source, dietary cholesterol and the interaction. The effect of fiber source level was significant (P less than 0.05) for liver cholesterol only. Pectin groups had serum and liver cholesterol levels lower (P less than 0.05) than the others. At the 10% level, tomato pomace, wheat bran and lignin (Indulin AT) groups had serum cholesterols higher (P less than 0.05) than basal; the mixed-vegetable pomace and cellulose groups were not different from basal. Liver cholesterols of tomato pomace, mixed-vegetable pomace, wheat bran and lignin groups were not different at the 10% level, but were higher than the pectin, cellulose and basal groups (P less than 0.05). In a second experiment, serum cholesterols of rats fed pectin were lower (P less than 0.05) than for those fed cellulose or two types of lignin (Indulin AT, Reax 27).

Animals↗

PubMed1987

Factors influencing the level and interanimal variability of retinyl ester hydrolase activity in rat liver.

Retinyl ester hydrolase activity was studied in 25 7-wk-old rats that had been previously fed no vitamin A for 1 wk and then were fed 0, 5, 24, 60 or 240 micrograms retinol/d for 2 wk. This treatment produced rats with vitamin A reserves from depleted to normal (0.03 to 184 micrograms/g) and serum retinol concentrations of 4-67 micrograms/dl. Hydrolase activity in liver homogenates was optimal when assayed with 275 mM CHAPS (3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate), 2 mg/ml Triton X-100 and 5 mM retinyl palmitate at pH 7.0. By use of these concentrations and conditions, retinyl ester hydrolase activity displayed relatively low interanimal variability (fivefold). Furthermore, activity was independent of serum or liver vitamin A concentrations. When CHAPS in the assay was replaced with equimolar sodium cholate, or with reduced concentrations of CHAPS and retinyl palmitate, retinyl ester hydrolase activity was more variable, i.e., 37- and 23-fold, respectively. Furthermore, in the latter case, hydrolase activity was threefold higher in rats with liver vitamin A reserves less than 10 micrograms/g. Thus, the type and concentration of bile salt used to assay retinyl ester hydrolase affect its interanimal variability. Furthermore, hydrolase activity measured in reduced concentrations of substrate and detergent is elevated in rats with low liver reserves of vitamin A.

Animals↗

PubMed1990

Cholesterol-lowering activity of various undigested fractions of soybean protein in rats.

The undigested high-molecular-weight fraction (HMF) of soybean protein prepared after exhaustive digestion by microbial proteases significantly decreased serum cholesterol levels to approximately 45% (p less than 0.05) of those observed with the parent protein in rats fed diets containing cholesterol (0.5%) and sodium cholate (0.125%). HMF bound conjugated bile salts in vitro and significantly increased fecal excretion of both neutral and acidic steroids by 65-95% and 80-170% more, respectively (p less than 0.05), than did the intact protein. Extraction of HMF with methanol slightly decreased the activity, but the methanol-soluble fraction was not regarded as a principal determinant. Soybean saponin at the dietary level equivalent to that contained in HMF did not effectively lower serum cholesterol. The activity was not necessarily duplicated even when methanol-treated fractions were recombined. Further degradation of the methanol-extracted HMF by various proteases resulted in loss of activity. HMF obtained after pepsin digestion exerted a potential similar to that of HMF prepared after digestion by microbial proteases.

Animals↗

PubMed1997

Consumption of buckwheat protein lowers plasma cholesterol and raises fecal neutral sterols in cholesterol-Fed rats because of its low digestibility.

Buckwheat protein product (BWP) has a strong hypocholesterolemic activity in rats fed a cholesterol-enriched diet. In this study, we examined the influence of BWP on fecal excretion of sterols and nitrogen in rats fed a diet containing 5 g/kg cholesterol and 1.25 g/kg sodium cholate, and we examined whether the cholesterol-lowering activity of BWP is due to its low digestibility. In Experiment 1, rats fed BWP for 3 wk had significantly lower concentrations of plasma cholesterol and enhanced excretion of fecal total neutral sterols and nitrogen compared with rats fed casein. There was a significant correlation between fecal total neutral sterols and nitrogen (r = 0.89, P < 0.01). Fecal excretion of acidic sterols was unaffected by BWP. In Experiment 2, plasma cholesterol in rats fed trypsin-digested BWP for 2 wk was significantly higher than that in rats fed intact BWP. In Experiment 3, rats were fed BWP, low-molecular-weight fraction of the digest of BWP (LMF ) or high-molecular-weight fraction of the digest of BWP (HMF ) for 3 wk. Plasma cholesterol was lower in the BWP group than in the LMF group (P < 0.05), whereas that in the HMF group was intermediate. The in vitro digestibility of BWP with pepsin and pancreatin was significantly lower than that of casein. The results suggest that the cholesterol-lowering effect of BWP is mediated by higher fecal excretion of neutral sterols and that lower digestibility of BWP is at least partially responsible for the effect.

Animals↗

PubMed2001

Dietary gamma-linolenic acid suppresses aortic smooth muscle cell proliferation and modifies atherosclerotic lesions in apolipoprotein E knockout mice.

The present study was conducted to evaluate the antiatherogenic effects of dietary gamma-linolenic acid (GLA) (primrose oil) in apolipoprotein E (apoE) genetic knockout mice. Five-wk-old male mice were fed cholesterol-free diets containing 10 g/100 g lipid as corn oil (CO) [control diet, 0 mol/100 mol GLA and (n-3) polyunsaturated fatty acids (PUFA)], primrose oil (PO, 10 mol/100 mol GLA), fish oil-CO mix [FC; 9:1 wt/wt, 0 mol/100 mol GLA and 17 mol/100 mol (n-3) PUFA] or fish oil-PO mix [FP, 1:3 wt/wt, 8 mol/100 mol GLA and 5 mol/100 mol (n-3) PUFA] for 15 wk. Subsequently, diets were supplemented with cholesterol (1.25 g/100 g) and sodium cholate (0.5 g/100 g) and fed for an additional 10 and 16 wk. Plasma cholesterol and triglyceride levels generally did not differ among groups at 20, 30 and 36 wk of age. Mice fed GLA-containing diets (PO and FP) had significantly (P < 0.05) higher liver phospholipid levels of dihomo-gamma-linolenic acid, the elongated product of GLA, relative to CO and FC groups. Consumption of GLA (PO and FP diets) significantly reduced (P < 0.05) aortic vessel wall medial layer thickness at 20 and 30 wk. A parallel GLA-dependent suppression in the number of proliferating (proliferating cell nuclear antigen positive) aortic smooth muscle cells was also observed. Diets containing either GLA or (n-3) PUFA reduced (P < 0.05) atherosclerotic lesion size in 30-wk-old mice. These results indicate that dietary GLA can suppress smooth muscle cell proliferation in vivo and retard the development of diet-induced atherosclerosis in apoE knockout mice.

Animals↗

PubMed2001

Paradoxical increase in nitric oxide synthase activity in hypercholesterolaemic rats with impaired renal function and decreased activity of nitric oxide.

BACKGROUND: We have shown that acute exposure of oxidized low-density lipoprotein (OX-LDL) induces vasoconstriction in renal vessels and reduces glomerular filtration rate (GFR) in an isolated perfused rat kidney model by decreasing the activity of nitric oxide (NO). L-arginine has a protective role against OX-LDl-induced vasoconstriction. Micropuncture studies have demonstrated that short-term diet-induced hypercholesterolaemia is associated with decreased GFR and renal blood flow and increased glomerular capillary pressure. This may be mediated by decreased activity of NO. METHODS: Rats were made hypercholesterolaemic by supplementing the standard chow with 4% cholesterol and 1% sodium cholate. A group of rats on hypercholesterolaemic diet also received L-arginine in the drinking water. After 4 and 6 weeks, blood samples and 24-h urine samples were collected for the measurement of biochemical parameters. After 6 weeks, all rats were subjected to isolated perfusion of kidneys at a constant pressure of 100 mmHg. During isolated perfusion, the unused contralateral kidney was taken for morphological studies and for assessing the activity of nitric oxide synthase enzyme by beta-NADPH diaphorase histochemistry. RESULTS: Rats fed a high-cholesterol diet had LDL levels 3-6 times greater than the rats fed standard chow. Rats that received L-arginine in the drinking water had serum L-arginine levels 5-6 times greater than control rats. At 6 weeks, creatinine clearance was significantly lower in the rats on the high-cholesterol diet compared to the rats on standard chow and rats on high-cholesterol diet plus L-arginine. Twenty-four-hour urinary total nitrate and nitrite excretion in the hypercholesterolaemic rats was 1.5-2 times greater than that of control rats. Twenty-four-hour urinary cGMP excretion was significantly lower in the rats on a high-cholesterol diet, but in the rats on high-cholesterol diet and L-arginine, 24-h urinary cGMP excretion was not significantly different from that of control rats. During isolated perfusion of kidneys, renal perfusate flow was found to be significantly reduced in the kidneys taken from the rats on a high-fat diet compared to controls. L-arginine supplementation in the drinking water almost completely reversed the effect of a high-fat diet. Inulin clearance was also significantly reduced in kidneys on a high-fat diet in contrast to controls but not in kidneys on high fat-diet and L-arginine. Basal cGMP excretion in urine was significantly lower in the kidneys taken from the rats on a high-fat diet compared to controls. L-arginine supplementation restored the basal cGMP excretion in these kidneys. NO synthase (NOS) enzyme activity as assessed by NADPH diaphorase activity showed that kidney sections taken from the rats on a high-fat diet showed more intense staining, indicating increased activity compared to the kidney sections taken from the rats on a normal diet. CONCLUSION: Though activity of NO is diminished in hypercholesterolaemic rats with impaired renal function, there is a paradoxical increase in NO production and NOS activity. L-arginine reverses the effects of a high-fat diet.

Animals↗