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The Journal of Nutrition Vol. 128 No. 7 July 1998,
pp. 1199-1203
Department of Foods & Nutrition and * Department of Animal Sciences, Purdue University, West Lafayette, IN 47907
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ABSTRACT |
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Abstract
Introduction
Methods
Results
Discussion
References
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Psyllium, a source of dietary fiber rich in soluble components results in lower serum cholesterol concentration in several species. Suggested mechanisms for the hypocholesterolemic effect include a greater excretion of fecal bile acids and total steroids, and up-regulation of bile acid biosynthesis. The activity of cholesterol 7
-hydroxylase (7OHase), the rate limiting enzyme in bile acid biosynthesis, is higher in rats fed 5% psyllium. Whether this higher activity corresponds to an increase in mRNA levels has not been determined. Four groups of 10 rats were fed a semipurified diet containing 5% cellulose (CEL; control), 5% cellulose plus 1% cholic acid (CCA), 5% cellulose plus 2% cholestyramine (CHY) or 5% psyllium hydrocolloid (PSY) for 3 wk. Liver cholesterol concentration, fecal bile acid and total steroid excretion, 7OHase activity and 7OHase mRNA levels were measured. Liver cholesterol content in rats fed CCA was significantly higher than in all other groups. Rats fed CHY and PSY had significantly lower liver cholesterol content than those fed CEL. Total fecal steroid and bile acid excretions were significantly greater in rats fed CCA, CHY and PSY than in those fed CEL. Activities and mRNA levels of 7OHase in rats fed CHY and PSY were significantly higher than in rats fed CEL or CCA. These data indicate that feeding psyllium to rats increases fecal bile acid and total steroid excretion as well as 7OHase activity and 7OHase mRNA levels.
Dietary fibers rich in soluble components result in reduced serum cholesterol concentrations in several species; however, the mechanisms involved are not clearly defined. One example is psyllium hydrocolloid, a gel-forming polymer derived from the seeds of Plantago ovata (Sandhu et al. 1981 Suggested mechanisms for this hypocholesterolemic effect have focused on greater excretion of bile acids and total steroids leading to an up-regulation of bile acid biosynthesis. Experiments with dietary fiber sources that result in lower cholesterol levels tend to support this increased excretion/increased synthesis hypothesis (Arjmandi et al. 1992a Synthesis of bile acids from cholesterol is regulated by feedback inhibition of the rate limiting enzyme, cholesterol 7 The purpose of this study was to understand more fully the role of these two potential mechanisms in the hypocholesterolemic action of dietary psyllium in rats. This was done by comparing fecal bile acid excretion, total steroid excretion and regulation of bile acid biosynthesis in rats fed diets containing components known to regulate these processes (cholic acid and cholestyramine) with their effects in rats fed psyllium.
Animals and diets.
Four groups of 10 male Wistar rats (90 g; Harlan Sprague Dawley, Indianapolis, IN) were housed individually in a temperature (24°C) and light (dark, 0600-1800 h) controlled room. Rats were fed nonpurified diet (Rodent Laboratory Chow, Ralston Purina, St. Louis, MO) for a 1-wk stabilization period after which modifications of the AIN93M (AIN 1993) purified rodent diet (Dyets, Bethlehem, PA) were fed for 3 wk. Diet groups included the following: 5% cellulose (CEL, control); 5% cellulose plus 2% cholestyramine resin (CHY) (Sigma, St. Louis, MO); 5% cellulose plus 1% sodium cholate (CCA) (Sigma); and 5% psyllium hydrocolloid (PSY) (Proctor & Gamble, Cincinnati, OH). Rats were given free access to food and water. Food intake was monitored on alternate days for a 6-d period during the final week of the study.
Cholesterol 7 Cholesterol 7
cDNA probes.
The plasmid pSK-7
Fecal steroid analysis.
Fecal steroids were quantified by the method of Chezem and Story (1997) Statistical analysis.
Data were analyzed with the general linear models procedure (SAS Version 6.07, SAS Institute, Cary, NC) using one-way ANOVA followed by Duncan's Multiple Range Test to determine the relationship between the means when P-values were <0.05 (Bruning and Kintz 1987 Body weight gain for rats fed CCA was significantly lower than for rats in all other groups (Table 1). The lower weight gain could be due to the significantly lower food intake, which may be related to the palatability of the cholic acid-containing diet. Total liver cholesterol was significantly lower in rats fed CHY or PSY than in those fed CEL. Liver cholesterol concentration and total content were significantly greater in rats fed CCA than in all other groups (Table 1).
Historically, the hypocholesterolemic effect of dietary fiber has been attributed to its ability to inhibit intestinal absorption of bile acids and neutral steroids, resulting in greater fecal bile acid and total steroid excretions. These effects vary with type of fiber and are species dependent. Feeding psyllium to humans also results in higher fecal bile acid and total steroid excretions (Abraham and Menta 1988
INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References
).
, Matheson and Story 1994, Matheson et al. 1995). Changes in fecal bile acid and steroid excretions in rats in response to psyllium feeding have been inconsistent (Arjmandi et al. 1992a, Vahouny et al. 1987). It is proposed that feeding psyllium causes greater viscosity in the intestine, thus preventing absorption of bile acids and neutral steroids, a phenomenon that has been observed for other viscous sources of dietary fiber (Carr et al. 1996, Gallaher et al. 1993a and 1993b).
-hydroxylase (7OHase:5 EC 1.14.13.17), by bile acids returning to the liver via the enterohepatic circulation. Hepatic 7OHase activity, protein mass, mRNA levels and the rate of transcription are all higher in rats fed cholestyramine, a bile acid sequestrant, and lower in rats fed bile acids (Chiang et al. 1990, Heuman et al. 1988, Jelinek et al. 1990, Li et al. 1990). Matheson et al. (1995) demonstrated that 7OHase activity and bile acid pool size were greater in rats fed a diet containing 5% psyllium compared with rats fed a diet containing 5% cellulose. In addition, psyllium has been shown to coordinately increase 7OHase activity and mRNA levels in hamsters (Horton et al. 1994). A bile acid response element has been identified in the promoter of the 7OHase gene, suggesting a molecular mechanism involved in transcriptional regulation of 7OHase (Chiang and Stroup 1994, Hoekman et al. 1993, Sundseth and Waxman 1990).
View this table:
Table 1.
Body weight gain and liver cholesterol concentrations in rats fed diets containing 5% cellulose (CEL), 5% cellulose plus 1% cholic acid (CCA), 5% psyllium (PSY) or 5% cellulose plus 2% cholestyramine (CHY) for 3 wk1
MATERIALS AND METHODS
Abstract
Introduction
Methods
Results
Discussion
References
View larger version (16K):
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Fig 1.
Daily fecal bile acid and total steroid excretions in rats fed semipurified diets containing 5% cellulose (CEL), 5% cellulose plus 1% cholic acid (CCA), 5% psyllium (PSY) or 5% cellulose plus 2% cholestyramine (CHY). Values are means ± SEM, n = 10. Data were log transformed before analysis by ANOVA. Bars with different letters are significantly different, P < 0.05.
20°C until analysis for cholesterol (Rudel and Morris 1973). Feces were collected for a 72-h period during the final week of the study, lyophilized and stored at 20°C. The experimental protocol was reviewed and approved by the Purdue University Animal Care and Use Committee.
-hydroxylase assay.
Microsomes were isolated by ultracentrifugation (100,000 × g, 45 min) and stored in liquid nitrogen. Activity of 7OHase was measured by incorporation of liposome-solubilized cholesterol isotope into labeled 7-hydroxycholesterol by microsomal preparations (Junker and Story 1985). The results are expressed as picomoles 7-hydroxycholesterol produced per minute per milligram microsomal protein. Microsomal protein was determined by using the BCA protein assay (Pierce, Rockford, IL).
-hydroxylase mRNA assay.
Total RNA was isolated from ~1 g of liver by using the guanidium thiocyanate-phenol-chloroform method of Chomczynski and Sacchi (1987). Northern blot analysis was performed with standard procedures (Sambrook et al. 1989) as modified by Tsang et al. (1993). Briefly, total RNA was loaded (20 µg/lane), separated by electrophoresis in a 1% agarose-formaldehyde gel, transferred by capillary action to a Gene Screen membrane (DuPont, NEN Boston, MA), UV crosslinked and baked at 80°C to remove formaldehyde. The membrane was then prehybridized, hybridized with 32P-labeled 7OHase and 18S rRNA cDNA probes and washed before it was exposed to Kodak X-OMAT AR imaging film (Rochester, NY) at 80°C (Donkin et al. 1996). The membrane was reprobed after stripping. Levels of mRNA were quantified by scanning autoradiograms with a Microtek ScanMaker E3 (Redondo Beach, CA) scanner, used in conjunction with Adobe Photoshop 4.0 (Adobe Systems, San Jose, CA) and Sigma Gel (Sigma), and expressed as units 7OHase mRNA per unit 18S rRNA. Jelinek and Russell (1990) demonstrated in blot hybridization experiments that the multiple mRNAs detected arise as a consequence of differential polyadenylation in the 3'-end of the 7OHase gene and not in different 5' start sites or multiple genes. Multiple mRNAs detected in this experiment were coordinately regulated in response to the experimental treatments and, as a result, were combined in quantification. Levels of 18s rRNA were used to normalize for equal loading and transfer efficiency.
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Fig 2.
Hepatic cholesterol 7 -hydroxylase (7OHase) activity (per milligram microsomal protein) in rats fed semipurified diets containing 5% cellulose (CEL), 5% cellulose plus 1% cholic acid (CCA), 5% psyllium (PSY) or 5% cellulose plus 2% cholestyramine (CHY). Values are means ± SEM, n = 10 for all groups except CEL where n = 9. Data were log transformed before analysis by ANOVA. Bars with different letters are significantly different, P < 0.05.
(Trawick et al. 1996), containing a 1.64-kb EcoR1 fragment that contained the entire coding region of the rat 7OHase gene, was kindly provided by John D. Trawick (San Diego State University). The plasmid pDF 8 (Donkin et al. 1996), containing a 1.06 kb BamH1-Eco R1 fragment corresponding to the central region of the rat 18S rRNA gene, was kindly provided by Richard Torzynski (Cytoclonal Pharmaceutics, Dallas, TX).
View larger version (55K):
[in a new window]
View larger version (13K):
[in a new window]
Fig 3.
Northern blot analysis of hepatic 7 -hydroxylase (7OHase) mRNA levels in rats fed semipurified diets containing 5% cellulose (CEL), 5% cellulose plus 1% cholic acid (CCA), 5% psyllium (PSY) or 5% cellulose plus 2% cholestyramine (CHY). Panel A: total RNA was isolated from rat liver and 20 µg/lane was subjected to electrophoresis, blotting and hybridization with cDNA probes specific for rat 7OHase mRNA and 18S rRNA as described in Materials and Methods. Panel B: values are means ± SEM, n = 10 for all groups except CEL where n = 9. Data were log transformed before analysis by ANOVA. Bars with different letters are significantly different, P < 0.05.
. Briefly, sterols were extracted from a 0.5-g lyophilized fecal sample with 5
-cholanic acid and 5-cholestane as internal standards for acid steroids and neutral steroids, respectively. After deconjugation of bile acids with cholylglycine hydrolase, neutral steroids were extracted with petroleum ether and bile acids with diethyl ether and ethyl acetate. Quantification of trimethylsylyl ethers of neutral steroids involved gas liquid chromatography (Hewlett Packard, Cincinnati, OH) with a 30-m DB1701 capillary column (J&W Scientific, Folsom, CA). Bile acids were further purified by using octadecylsilane-bonded silica cartridges (Milipore, Milford, MA) and quantified as trimethylsylyl ethers with a 30-m DB5 capillary column (J&W Scientific).
). Data were log transformed before ANOVA when inequality of variance was observed. Linear regression analysis for 7OHase activity and mRNA was performed by using Fig. P for Windows (version 2.7, Biosoft, Cambridge, UK) (Bruning and Kintz 1987).
RESULTS
Abstract
Introduction
Methods
Results
Discussion
References
OHase were significantly greater in rats fed CHY and PSY compared with rats fed CCA or CEL. Activity of 7OHase was significantly higher in rats fed CHY than in rats fed PSY; however, mRNA levels of 7OHase did not differ between the two groups. 7OHase activity and mRNA levels were coordinately regulated for all diet groups (r2 = 0.57, P < 0.01, Fig. 4).
View larger version (18K):
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Fig 4.
Correlation between hepatic 7 -hydroxylase (7OHase) and mRNA levels in rats fed semipurified diets containing 5% cellulose (CEL), 5% cellulose plus 1% cholic acid (CCA), 5% psyllium (PSY) or 5% cellulose plus 2% cholestyramine (CHY); r2 = 0.57, P < 0.01.
DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References
, Gelisse et al. 1994, Stanley et al. 1973). Changes in fecal bile acid and steroid excretions in rats in response to psyllium feeding have been inconsistent (Arjmandi et al. 1992a, Vahouny et al. 1987).
), but has gel-forming ability (Sandhu et al. 1981), which may result in greater viscosity of intestinal contents, thus reducing absorption of bile acids from the small intestine. Cholestyramine, a bile acid-binding resin, results in higher fecal bile acid and total steroid excretions when fed to rats, a result also observed in this study. CCA-fed rats had significantly higher fecal bile acid and total steroid excretions than CEL-fed rats, primarily as a result of higher bile acid excretion. This results from exceeding the capacity for intestinal absorption and possibly from contamination of the feces with spilled diet.
OHase activity and mRNA levels than in control rats. Feeding cholestyramine interrupts the enterohepatic circulation of bile acids, thereby preventing feedback inhibition of 7OHase gene expression by bile acids. However, CCA-fed rats had 7OHase activity and mRNA levels that did not differ from rats fed cellulose despite the ability of cholic acid to feedback inhibit 7OHase gene expression, as demonstrated in other studies (Heuman et al. 1988, Shefer et al. 1992).
OHase activity and mRNA levels than rats fed CEL. Horton et al. (1994) demonstrated greater activity and mRNA levels in hamsters fed 7.5% psyllium. Our earlier work suggested a similar change in 7OHase activity (Matheson et al. 1995) and a change in the composition of the bile acid pool to one with a more hydrophilic nature (Matheson and Story 1994), favoring higher 7OHase activity and mRNA levels (Jelinek et al. 1990). However, further studies will be required to investigate adequately the effects of feeding psyllium on transcription of the 7OHase gene.
). In this study, rats fed CCA had significantly higher total liver cholesterol than all other treatment groups. Generally, feeding psyllium to rats does not cause a significant decrease in total liver cholesterol unless cholesterol is included in the diet (Arjmandi et al. 1992a and 1992b, Matheson et al. 1995). However, in this study, both CHY- and PSY-feeding resulted in significantly lower liver cholesterol content.
OHase activity and mRNA levels are involved in the hypocholesterolemic action of dietary psyllium. The identity of effectors of changes in mRNA levels and how these effectors are regulated by conditions in the intestine and/or intestinal absorption of bile acids remain to be elucidated.
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FOOTNOTES |
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-hydroxylase activity, hepatic mRNA levels and bile acid excretion in rats fed psyllium. FASEB J. 11: A3532(abs.)].
OHase, 7-hydroxylase; PSY, 5% psyllium diet.
Manuscript received 8 September 1997. Initial reviews completed 13 October 1997. Revision accepted 27 March 1998.
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ACKNOWLEDGMENTS |
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We thank The Procter and Gamble Company for supplying psyllium hydrocolloid and Carol Spahr for her assistance with animal care.
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LITERATURE CITED |
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Abstract
Introduction
Methods
Results
Discussion
References
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-hydroxylase in the liver: purification of cholesterol 7-hydroxylase and the immunochemical evidence for the induction of cholesterol 7-hydroxylase by cholestyramine and circadian rhythm.
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20:712-718[Medline] -hydroxylase in the liver: cloning, sequencing, and regulation of cholesterol 7-hydroxylase mRNA.
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