Nutrient Gene Expression

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1 Nutrient Gene Expression Dietary Psyllium Increases Expression of Ileal Apical Sodium-Dependent Bile Acid Transporter mrna Coordinately with Dose-Responsive Changes in Bile Acid Metabolism in Rats 1,2 Kimberly K. Buhman,* 3 Emily J. Furumoto,* 4 Shawn S. Donkin** and Jon A. Story* 5 Departments of *Foods and Nutrition and **Animal Science, Purdue University, West Lafayette, IN ABSTRACT Psyllium (PSY), a type of dietary fiber containing mainly soluble components, has been shown to decrease serum cholesterol concentrations in several species; however, mechanisms involved are not clearly defined. Four groups of 10 rats were fed semipurified diets containing 10% dietary fiber from cellulose and/or PSY for 21 d. Increasing levels of PSY were fed (0,3.33, 6.67 and 10% PSY) with the remaining 10% made up with cellulose. Liver cholesterol, cholesterol 7 -hydroxylase (CYP7A) activity and mrna, 3-hydroxy-3-methylglutaryl CoA reductase (HMGR) mrna, ileal apical sodium-dependent bile acid transporter (ASBT) mrna, fecal bile acids and total steroids, and intestinal bile acid content were measured. All variables responded in a dose-dependent manner to PSY in the diet. Total liver cholesterol content was significantly reduced in all groups fed PSY compared to cellulose-fed controls [138 a, 105 b, 105 b and 93 c mol (SEM 4.2) for 0, 3.33, 6.67 and 10% PSY, respectively]. Activity of CYP7A was significantly greater in all groups fed PSY compared to the cellulose-fed controls [6.36 c, b, b and a pmol min 1 mg protein 1 (SEM 3.19) for 0, 3.33, 6.67 and 10% PSY, respectively]. These differences in CYP7A activity were similar to differences in CYP7A, HMGR and ASBT mrna levels. Fecal bile acid and total steroid excretion as well as total intestinal bile acids were significantly greater in rats fed PSYcontaining diets compared to 0% PSY-fed rats. These results suggest that the reduction in liver cholesterol involves modulating the size and composition of the bile acid pool via regulation of ileal ASBT, CYP7A and HMGR mrna levels. J. Nutr. 130: , KEY WORDS: Psyllium bile acid absorption synthesis and excretion rats Dietary fibers rich in soluble components reduce serum cholesterol concentrations in several species; however, mechanisms involved are not clearly defined. One example is psyllium (PSY) 6 hydrocolloid, a gel-forming polymer derived from the seeds of Plantago ovata (Sandhu et al. 1981). Because bile acids are the main excretory route for cholesterol from the body, changes in bile acid metabolism in response to certain dietary fibers have been implicated in their hypocholesterolemic action. Synthesis of bile acids from cholesterol is regulated by feedback inhibition of the rate-limiting enzyme, cholesterol 7 -hydroxylase (CYP7A) [EC ], by bile acids returning to the liver via the enterohepatic circulation. When fed to hamsters and rats, PSY has been 1 Supported in part by the Indiana Agricultural Research Programs (paper #16,127) and the Purdue Research Foundation. 2 Presented in part at Experimental Biology 99, April 18, 1999, Washington. [Buhman, K. K., Story, J. A. & Donkin, S. S. (1999) Dietary PSY increases expression of apical sodium bile acid transporter in rats. FASEB J. 13:A235(abs).] 3 Present address: Gladstone Institute of Cardiovascular Disease, P.O. Box , San Francisco, CA Present address: Department of Food Science, 0111 Borland Lab, Pennsylvania State University, University Park, PA To whom reprint requests should be addressed. 6 Abbreviations used: ASBT, apical sodium-dependent bile acid transporter; CYP7A, cholesterol 7 -hydroxylase; FXR, farensoid X receptor; HI, hydrophobic index; HMGR, 3-hydroxy-3-methylglutaryl CoA reductase; PSY, psyllium. shown to coordinately increase CYP7A activity and mrna levels (Buhman et al. 1998,Horton et al. 1994). The composition and size of the bile acid pool play a role in regulating gene transcription of CYP7A and 3-hydroxy-3- methylglutaryl coenzyme A reductase (HMGR), the rate-limiting enzyme in cholesterol synthesis. Heuman et al. (1989) reported that in rats fed bile acids to alter the composition of their bile acid pool, there was a significant negative correlation between the hydrophobic index (HI) of biliary bile acids and CYP7A and HMGR activities and mrna levels. A more hydrophilic mixture of bile acids (low HI) was less able to feedback inhibit enzyme activities and mrna levels than a more hydrophobic mixture (high HI). Matheson and Story (1994) and later Matheson et al. (1995) demonstrated that feeding 5% PSY compared to 5% cellulose to rats resulted in a more hydrophilic biliary bile acid spectrum and greater CYP7A activity. This suggests the hypocholesterolemic action of PSY may result from alterations in the HI of the bile acid pool which ultimately regulates both the synthesis (HMGR) and metabolism (CYP7A) of cholesterol. How does PSY act to alter the spectrum of bile acids in the bile acid pool? Reabsorption of bile acids in the gut occurs by either passive diffusion, mainly in the jejunum and colon, or by active absorption in the ileum. The passive component reabsorbs the more hydrophobic, dihydroxylated, unconjugated bile acids while the active component reabsorbs the /00 $ American Society for Nutritional Sciences. Manuscript received 21 October Initial review completed 9 December Revision accepted 15 May

2 2138 BUHMAN ET AL. more hydrophilic, trihydroxylated, conjugated bile acids (Wilson 1990). Therefore, changes in transport of bile acids via these two pathways may be important in regulation of bile acid pool HI. Active absorption of bile acids is principally mediated by the ileal apical sodium-dependent bile acid transporter (ASBT) (Wong et al. 1994). PSY forms a highly viscous solution (gel) in the intestine which could entrap bile acids, resulting in the prevention and/or delay of their reabsorption. A delay in reabsorption as well as increased expression of ileal ASBT may result in more hydrophilic bile acids being taken up via ileal ASBT, resulting in a more hydrophilic total bile acid pool. Aims of this study were to determine the dose-response relationship for CYP7A and HMGR mrna in response to dietary PSY and to examine the relationship between ASBT mrna abundance and changes in bile acid metabolism. MATERIALS AND METHODS 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, h) controlled room. Rats were fed nonpurified diet (Rodent Laboratory Chow, Ralston Purina, St. Louis, MO) for a 1-wk stabilization period. Diets employed were a modification of the AIN93M (American Institute of Nutrition 1993) purified rodent diet (Dyets, Bethlehem, PA). The level of dietary fiber was increased from 5 to 10% in all diets with the addition replacing cornstarch. Each of the four diets employed contained a combination of cellulose and/or PSY hydrocolloid (Procter & Gamble, Cincinnati, OH) combined as follows: 10% cellulose, 3.33% PSY with 6.67% cellulose, 6.67% PSY with 3.33% cellulose and 10% PSY. All diets were cholesterol-free. Rats were given free access to food and water for 3 wk, and food intake and body weight gain were monitored on alternate days for a 6-d period during the final week of the study. At the end of the experiment, rats were anesthetized using Ketamine/xylazine (9:1, 100 mg/kg body weight), liver and intestinal samples collected, and then killed by exsanguination. Animals were killed over a 5-d period beginning at 1000 h each day and balanced across treatments in order to maximize enzyme activity and avoid biases introduced by diurnal variation. Liver samples were collected and stored at 20 C until analysis for cholesterol (Rudel and Morris 1973), CYP7A activity, and CYP7A and HMGR mrna levels. The terminal ileum, defined as the terminal 20% of the length of the small bowel, was flushed with ice-cold normal saline, mucosal cells scraped using a razor blade, frozen under liquid nitrogen, and stored at 20 C until analysis of ileal ASBT mrna. The remainder of the intestine [small intestine (after removal of terminal ileum), cecum and colon], including entire contents, was collected, lyophilized and stored at 20 C until analysis for bile acid concentration. Feces were collected for a 72-h period during the final week of the study, lyophilized and stored at 20 C until analysis for bile acid concentration. The experimental protocol was reviewed and approved by the Purdue University Animal Care and Use Committee. Cholesterol 7 -hydroxylase assay. Microsomes were isolated by ultracentrifugation (100,000 g, 45 min) and stored in liquid nitrogen. Activity of CYP7A was measured by incorporation of liposomesolubilized [4-14 C]cholesterol into labeled 7 -hydroxycholesterol by microsomal preparations (Junker and Story 1985), and results are expressed as picomoles 7 -hydroxy[4-14 C]cholesterol produced per minute per milligram microsomal protein. Microsomal protein was determined by using the BCA protein assay (Pierce, Rockford, IL). Northern blot analysis. Total RNA was isolated from1gofliver and from ileal mucosal scrapings 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 g/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, hydbridized with 32 P-labeled CYP7A, HMGR, ASBT 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. Autoradiograms were digitized with a Hewlett-Packard ScanJet4C scanner (Cincinnati, OH) and RNA quantified with Kodak ds ID digital science v software (Eastman Kodak, New Haven, CT) and expressed as arbitrary units CYP7A, HMGR or ASBT mrna per arbitrary unit 18S rrna. Levels of 18S rrna were used to normalize for differences in loading and transfer efficiency. cdna probes. The plasmid psk-7 (Trawick et al. 1996), containing a 1.64-kb EcoR1 fragment that contained the entire coding region of the rat CYP7A gene, was kindly provided by John D. Trawick (San Diego State University). The plasmid ASBT (Shneider et al. 1995), containing a 1.2 kb Eco R1-Xho1 fragment that contains the entire coding region of the rat ASBT gene, was kindly provided by Benjamin Shneider (Mt. Sinai Medial Center). The plasmid pgem-hmgr, containing a 1.2 kb Eco R1 fragment that contains the entire coding region of the rat HMGR gene, was kindly provided by Sohaib Khan (University of Cincinnati Medical Center). The plasmid pdf8 (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). Steroid analysis. Bile acids and neutral steroids were quantified from feces and intestines using the method of Chezem and Story (1997). Briefly, steroids were extracted from a 0.5 g lyophilized fecal and intestinal samples 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. Trimethylsilyl ethers of neutral steroids from fecal and intestinal samples were quantified using gasliquid chromatography (Hewlett-Packard) using a 30-m DB1701 capillary column (J&W Scientific, Folsom, CA) while quantification of trimethylsilyl ethers of bile acids utilized a 30-m DB5 capillary column (J&W Scientific). Statistical analysis. Data were analyzed with the general linear models procedure of SAS (Version 6.12; SAS Institute, Cary, NC) using one-way ANOVA followed by the least significant difference (LSD) method to determine the relationship between the means when P 0.05 (Montgomery 1991). Data were log-transformed before ANOVA analysis when a heterogeneity of error variance was determined using Bartlett s Test (data in Fig. 1-3 and 6). To determine if the effect was dose-responsive in a linear, quadratic or cubic format, general linear models procedure using one-way ANOVA followed by contrasts was used (P 0.05). RESULTS Body weight gain and food intake did not differ among groups (Table 1). Liver cholesterol concentration and total content were significantly reduced in rats fed PSY compared to cellulose-fed controls (Table 1). Rats fed the highest amount of PSY (10% PSY) had significantly less total liver cholesterol compared with rats fed 3.33 or 6.67% PSY. This effect was linear (P ) and quadratic (P ) in response to PSY dose. Fecal bile acid excretion increased in a dose-dependent, linear manner (P ). All PSY-fed rats resulted in significantly greater bile acid excretion than cellulose-fed controls. Total steroid excretion also increased in a dose-dependent, linear manner (P ) (Fig. 1). Total intestinal bile acids increased in a dose-dependent, linear (P ) and quadratic (P ) fashion with the level of PSY in the diet. Total intestinal bile acids were significantly greater in rats fed PSY-containing diets than cellulose-fed controls (Fig. 2). Activity of CYP7A increased in a dose-dependent, linear (P ) and quadratic (P ) manner. All groups

3 DOSE RESPONSE TO PSYLLIUM IN BILE ACID METABOLISM 2139 FIGURE 1 Daily fecal bile acid and total steroid excretion in rats fed semipurified diets containing psyllium (PSY). Values are means SEM, n 10 for groups fed 0 and 10% PSY and n 9 for remainder. Data were log-transformed prior to analysis by ANOVA. Within each variable, groups with different letters are significantly different (P 0.05). For bile acids and total steroids, lines fit a linear dose response (P ) fed PSY resulted in significantly increased CYP7A activities (Fig. 3). The level of CYP7A and HMGR mrna also increased in a linear (P for both) and quadratic (P for CYP7A and P for HMGR) dosedependent fashion (Fig. 4). Feeding 6.67 and 10% PSY resulted in significantly greater HMGR mrna levels than 0 and 3.33% PSY. The level of HMGR mrna in 3.33% PSYfed rats was also significantly greater than 0% PSY-fed rats (Fig. 6). The level of ileal ASBT mrna expression was significantly greater in 10%-PSY fed rats compared to 0% PSY-fed rats (Fig. 5) with 6.67%- and 3.33% PSY-fed rats having intermediate levels. This result also follows a dose-response, linear format (P ) (Fig. 6). This increase in mrna would also have been amplified by increased mass of intestine in the psyllium-fed rats. Dry weights of intestine (small intestine, cecum and colon) were 4.6, 5.0, 5.3, and 5.8 g for CEL, 3.33%-, 6.67%-, and 10%-PSY, respectively. DISCUSSION Consistent with previous reports, feeding PSY to rats resulted in a significantly reduced total liver cholesterol content FIGURE 2 Total intestinal bile acids from rats fed semipurified diets containing psyllium (PSY). Values are means SEM, n 10 for groups fed 0 and 10% PSY and n 9 for remainder. Data were log-transformed prior to analysis by ANOVA. Groups with different letters are significantly different (P 0.05). Response fit linear and quadratic dose response with P-values as indicated in figure. compared to cellulose-fed rats (Buhman et al. 1998). Increasing consumption of PSY resulted in a significantly greater reduction in total liver cholesterol, suggesting that consumption of even small levels of PSY may be sufficient to produce a significant hypocholesterolemic effect, and even greater levels of PSY produce a greater hypocholesterolemic effect. The bile acid pool is maintained by an efficient enterohepatic circulation; therefore changes in the bile acid pool size are the result of changes in fecal bile acid excretion and/or hepatic bile acid synthesis. Increasing PSY consumption by rats resulted in increased fecal bile acid and total steroid excretion in a dose-responsive manner. In previous studies, feeding PSY to rats, hamsters or guinea pigs resulted in greater CYP7A activity and mrna levels compared to cellulose-fed animals (Buhman et al. 1998, Fernandez 1995, Horton et al. 1994, Matheson et al. 1995). In the present study, the increasing of PSY consumption by rats also resulted in dose-dependent increases in CYP7A activity and mrna levels. Changes in bile acid excretion and synthesis are consistent with the dose-responsive reduction of total liver cholesterol content. This suggests that greater fecal steroid excretion and bile acid synthesis are part of the mechanisms responsible for reduction of liver cholesterol in response to PSY. Similar findings, along with observed changes in cholesterol absorption and LDL TABLE 1 Body weight gain and liver cholesterol concentrations in rats fed diets containing 0% psyllium (0% PSY), 3.33% PSY, 6.67% PSY or 10% PSY for 3 wk1 Psyllium level 0% 3.33% 6.67% 10% Pooled SEM Body weight gain, g/d Food intake, g/d Liver weight, g 15.0a 12.9b 12.9b 11.5c 0.3 Liver cholesterol, mol/g 10.3a 8.29b 8.29b 8.15b 0.24 Total liver cholesterol, mol 138a 105b 105b 93c Values are means, n 10. Within a row, values with different superscripts are significantly different from each other (P 0.05).

4 2140 BUHMAN ET AL. FIGURE 3 Hepatic cholesterol 7 -hydroxylase (CYP7A) activity (pmol min 1 mg protein 1 ) in rats fed semipurified diets containing psyllium. Values are means SEM, n 10 for all groups. Data were log-transformed prior to analysis by ANOVA. Groups with different letters are significantly different (P 0.05). Response fit linear and quadratic dose response with P-values as indicated in figure. clearance, have been reported in hypercholesterolemic hamsters (Turley and Dietschy 1995, Turley et al. 1996). Despite coordinate changes in bile acid excretion and synthesis, the bile acid pool size was significantly greater in PSY compared to cellulose-fed rats. In the current study, bile acid pool size was estimated as the total bile acids present in the intestines and intestinal contents of the rats. Because rats do not store bile acids in a gall bladder, the predominant location for bile acids is in the intestine with very little bile acid present in the intestine tissue itself. The total amount of intestinal bile acids significantly increased in a dose-dependent manner in rats fed increasing levels of PSY. Using the washout technique, Matheson and Story (1994) demonstrated that rats fed PSY exhibited a larger bile acid pool size which was more hydrophilic in composition compared to rats fed cellulose. Turley et al. (1991) reported a similar increase in the intestinal bile acid pool in hamsters fed PSY. Bile acid synthesis is regulated by a feedback mechanism of bile acids on transcription of CYP7A. Hepatic CYP7A activity, protein levels, mrna levels and the rate of transcription are all higher in rats fed cholestyramine, a bile acid sequestrant and cholesterol-reducing drug, and lower in rats fed bile acids (Chiang et al. 1990, Heuman et al. 1988, Jelinek et al. 1990, Li et al. 1990). Therefore it is predicted that the greater bile acid pool size observed in PSY-fed rats would result in feedback inhibition of CYP7A gene expression which did not occur in the present study and is most likely due to the composition of the bile acid pool. Studies performed in cell culture and in vivo have demonstrated the importance of the composition of bile acid profiles in regulating CYP7A and HMGR gene expression. In rat hepatocytes cultured under conditions in which CYP7A mrna abundance was maintained at in vivo levels, hydrophobic bile acids repressed this enzyme at the level of gene transcription, and hydrophilic bile acids had no effect (Stravitz et al. 1993). Heuman et al. (1989) fed rats individual bile acids to create bile acid pools with defined hydrophobicity. Rats fed hydrophilic bile acids resulted in a larger and more hydrophilic bile acid pool. As the HI of the bile acid pool became more FIGURE 4 Northern blot analysis of hepatic cholesterol 7 -hydroxylase (CYP7A) and hepatic 3-hydroxy-3- methylglutaryl CoA reductase (HMGR) mrna levels per 18S rrna levels in rats fed semipurified diets containing psyllium (PSY). Total RNA was isolated from rat liver and 20 g/lane was subjected to electrophoresis, blotting and hybridization with cdna probes specific for each gene as described in the Materials and Methods section.

5 DOSE RESPONSE TO PSYLLIUM IN BILE ACID METABOLISM 2141 FIGURE 5 Northern blot analysis of ileal apical sodium-dependent bile acid transporter (ASBT) mrna levels per 18S rrna levels in rats fed semipurified diets containing psyllium (PSY). Total RNA was isolated from rat ileum and 20 g/lane was subjected to electrophoresis, blotting and hybridization with cdna probes specific for each gene as described in the Materials and Methods section. hydrophilic, CYP7A was not feedback-inhibited as efficiently. Recently the farensoid X receptor (FXR) has been shown to bind bile acids and downregulate CYP7A transcription. This is interesting because FXR is activated by chenodeoxycholic acid, a relatively hydrophobic bile acid, and not cholic acid, a relatively hydrophilic bile acid (Makishima et al. 1999, Parks FIGURE 6 Quantification of northern blot analysis of hepatic cholesterol 7 -hydroxylase (CYP7A), hepatic 3-hydroxy-3-methylglutaryl CoA reductase (HMGR) and ileal apical sodium-dependent bile acid transporter (ASBT) mrna levels per 18S rrna levels in rats fed semipurified diets containing psyllium (PSY). Values are means SEM, n 10 for CYP7A and HMGR and n 10 for groups fed 0 and 3.33% PSY and n 8 for remainder of ASBT groups. Data were log-transformed prior to analysis by ANOVA. Within each variable, groups with different letters are significantly different (P 0.05). et al. 1999). These results are consistent with the observations seen in our laboratory that rats fed PSY result in a more hydrophilic bile acid pool and have significantly greater CYP7A activity and mrna levels compared to cellulose-fed rats (Buhman et al. 1998, Matheson and Story 1994, Matheson et al. 1995). Increased uptake of hydrophilic bile acids via ileal ASBT is one potential mechanism for changing the HI of the bile acid pool. In this study, feeding PSY to rats resulted in significantly greater ileal ASBT mrna levels. Previous studies have demonstrated that ASBT mrna expression levels correlate with protein levels and functional activity of ASBT (Torchia et al. 1996). Factors involved in regulating expression of ASBT in the ileum are not clearly defined. Our data suggest an increase in ASBT mrna per cell and, since PSY resulted in an increase in intestinal mass, the potential effect of ASBT on bile acid hydrophobicity would be amplified by this increase in mass. Our observation that HMGR mrna levels were increased in PSY-fed rats which exhibited a reduction in total liver cholesterol suggests a more complex regulation. Since PSY increased bile acid excretion, the observed increase in expression of HMGR may have been overcome by increased sterol excretion, as is the case with bile acid sequestrants. Additionally, Björkhem et al. (1993) have observed reduced mrna expression of both HMGR and CYP7A in lymph fistula rats fed bile acids but post-transcriptional regulation occurred only with HMGR, not CYP7A. The combination of increased steroid excretion and potential post-transcriptional downregulation of HMGR are factors which require further investigation. Taken together, these results suggest that mechanisms involved in the cholesterol-lowering action of PSY in rats include changing the composition of the bile acid pool to a more hydrophilic spectrum via increased expression of ileal ASBT mrna. This results in greater expression of CYP7A mrna

6 2142 BUHMAN ET AL. despite the larger bile acid pool. Higher levels of CYP7A results in a greater conversion of cholesterol to bile acids which are either excreted or maintained in the larger bile acid pool, both changes resulting in a loss of cholesterol from the liver. These conclusions are strengthened by the dose-responsive nature of all of these changes coordinately with the dose-responsive reduction in total liver cholesterol content. ACKNOWLEDGMENTS The authors thank Carol J. Spahr for her expert assistance with animal care and Jennifer Hartwell for her expert technical assistance with mrna assays. LITERATURE CITED American Institute of Nutrition. (1993) AIN-93 purified diets for laboratory rodents: final report of the American Institute of Nutrition ad hoc writing committee on the reformulation of the AIN-76A rodent diet. J. Nutr. 123: Björkhem, I., Andersson, U., Sudjana-Sugiaman, E., Eggertsen, G. & Hylemon, P. B. 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