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The Journal of Nutrition Vol. 128 No. 11 November 1998,
pp. 1937-1943
Institute of Human Nutrition and Food Science, University of Kiel, 24105 Kiel, Germany
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ABSTRACT |
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Abstract
Introduction
Methods
Results
Discussion
References
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The mechanisms by which inulin may elicit its lipid-lowering effect are not well elucidated. To examine the lipid-lowering potential of inulin and especially its effect on bile acid metabolism, male golden Syrian hamsters were fed semipurified diets containing 20 g/100 g fat, 0.12 g/100 g cholesterol and 0 (control), 8, 12 or 16% inulin for 5 wk. Plasma total cholesterol concentrations were significantly lowered by 18, 15 and 29% in hamsters fed 8, 12 and 16% inulin, respectively. Dietary inulin specifically decreased VLDL cholesterol, which was significantly lower in hamsters fed 16% inulin compared with controls (1.1 ± 0.3 vs. 2.9 ± 0.6 mmol/L). LDL and HDL cholesterol were not significantly affected by dietary inulin. Plasma triacylglycerol was significantly reduced by 40 and 63% in hamsters fed 12 and 16% inulin, respectively. Hepatic total cholesterol and particularly esterified cholesterol accumulation were significantly lower in hamsters fed 8% inulin compared with controls. All three levels of dietary inulin caused distinct alterations in the bile acid profile of gallbladder bile. Taurochenodeoxycholic acid was significantly lower, whereas glycocholic and glycodeoxycholic acid were greater in hamsters fed inulin. Daily fecal bile acid excretion (µmol/d) tended to be greater (P = 0.056) in inulin-fed hamsters compared with controls, whereas daily neutral sterol excretion was not affected. These data demonstrate that the lipid-lowering action of inulin is possibly due to several mechanisms, including altered hepatic triacylglycerol synthesis and VLDL secretion and impaired reabsorption of circulating bile acids.
KEY WORDS: inulin · lipoproteins · bile acid profile · fecal sterol excretion · hamsters
Fructooligosaccharides such as inulin and oligofructose are naturally occurring indigestible carbohydrates that are readily fermented in the cecum and colon (Roberfroid 1993). They are found in many plant foods such as chicory, onions, artichokes and asparagus. In addition, they are also used as a food ingredient in various food items. Although oligofructose and inulin are consumed regularly in appreciable amounts in typical Western diets (van Loo et al. 1995 In recent animal studies in rats, a lipid-lowering effect of dietary oligosaccharides and specifically a decrease in the plasma triacylglycerol (TG)5 concentration has been reported (Delzenne et al. 1993 In this study, the lipid-lowering potential of inulin and especially its effect on bile acid metabolism were investigated in Syrian golden hamsters, a common model to study lipid metabolism because of similarities to human cholesterol and bile acid metabolism (Spady and Dietschy 1983 Animals.
Male golden Syrian hamsters (SASCO, Omaha, NE) weighing 58 ± 4 g were randomly assigned to four diet groups (n = 10/group). Hamsters were housed in groups of 3-4 per cage in a temperature-controlled environment under a 12-h light:dark cycle (lights on at 1800 h) with free access to water and semipurified diets. All experimental protocols and procedures were approved by the Animal Care and Use Committee at the University of Kiel, Germany.
Diets and feeding procedures.
Hamsters were fed semipurified diets containing 20 g/100g fat and 0.12 g/100 g dietary cholesterol for 5 wk. The diets had the same composition except for the fiber and starch content as indicated in Table 1. In the supplemented diets, 8, 12 or 16% inulin (Raftiline HP, ORAFTI, Tienen, Belgium) was added at the expense of wheat starch and cellulose (5 g/100 g in inulin-supplemented diets vs. 10 g/100 g in control). Hamsters were given free access to food, and actual food consumption was recorded daily. Body weights were monitored on a weekly basis.
Plasma lipid and lipoprotein analysis.
Plasma total cholesterol (TC) and triacylglycerol (TG) concentrations were determined by enzymatic assays (Sigma kit #352 and #336, respectively, Sigma Chemicals, Deisenhofen, Germany). Plasma lipoproteins were isolated by sequential ultracentrifugation (Havel et al. 1955 Hepatic cholesterol analysis.
Cholesterol concentrations were analyzed after extraction with chloroform:methanol following the procedure described in detail previously (Trautwein et al. 1993 Bile analysis.
Gallbladder bile lipids were isolated using a modified Folch extraction (Folch et al. 1957). Biliary cholesterol (BC) and PL were determined enzymatically in an aliquot of the chloroform phase (Wako Free Cholesterol C kit for BC and Wako Phospholipid B kit for PL ). Biliary bile acids were analyzed in an aliquot of the methanol/KCl phase as taurine- and glycine-conjugated bile acids using an isocratic HPLC method adapted from Rossi et al. (1987) Determination of fecal bile acids and neutral sterols.
Fecal samples were collected over a 3-d period during wk 4 from six randomly selected hamsters per diet group to measure fecal sterol excretion. Fecal bile acid and neutral sterol concentrations were analyzed in an oven-dried fecal sample using a modification of the method of Suckling et al. (1991) Statistical analysis.
Statistical differences were calculated by using one-way ANOVA. When significant F-ratios were found, individual means were further compared by Scheffé's test with the use of the SuperANOVA statistical software package (Version 1.11, Abacus Concepts, Berkeley, CA). Differences were considered significant at P < 0.05. Results were expressed as means and SD.
Food intake and body weight.
The three dietary levels of inulin did not affect food intake or body weight gain (Table 2). Although 8 and 12% inulin had no apparent effect on total cecal weight, a significant enlargement of the cecum was observed in hamsters fed 16% inulin. In parallel, there was a significant increase in the cecal wall weight. The pH of the cecal contents was not altered by dietary inulin, suggesting no acidic conditions in the cecum possibly because hamsters were deprived of food for 18h.
Plasma lipids and lipoproteins.
Plasma TC concentrations were significantly lower by 18, 15 and 29% in hamsters fed 8, 12 and 16% inulin, respectively (Fig. 1). Plasma TG concentrations were even more effectively reduced by 34% (P = 0.07), 40% (P = 0.03) and 63% (P = 0.0003) in hamsters fed 8, 12 and 16% inulin, respectively (Fig. 1). Dietary inulin specifically decreased VLDL cholesterol (VLDL-C) in hamsters fed 16% inulin, whereas LDL-C and HDL-C were not significantly affected by diet (Table 3). The LDL-C/HDL-C ratio did not differ among groups, but the VLDL-C/HDL-C ratio was significantly lower in hamsters fed 16% inulin than in controls. The composition of VLDL was partially affected by dietary inulin (Table 4). The relative proportion of esterified cholesterol was significantly higher in hamsters fed 12 and 16% inulin, whereas the percentages of free cholesterol and phospholipids were not affected. The relative protein content of VLDL was significantly higher in hamsters fed 16% inulin compared with controls. When the core/surface ratio was calculated by dividing the relative percentages of esterified cholesterol plus triacylglycerol by the relative percentage sum of protein, phospholipids and free cholesterol, no apparent difference in core/surface ratios was found, suggesting no difference in particle size.
Hepatic cholesterol and biliary lipids.
Liver weights of hamsters fed the inulin-supplemented diets did not differ from controls (Table 2). Hepatic TC and EC concentrations in particular were lower in livers of hamsters fed 8% inulin compared with control animals. The largest reduction in hepatic TC (26%) was found in hamsters fed 8% inulin (P = 0.0016); decreases in those fed 12 and 16% inulin were only 14% (P = 0.1824) and 16% (P = 0.0942), respectively (Table 5). Hepatic free cholesterol concentrations did not differ among groups. Dietary inulin did not alter the total lipid concentration of gallbladder bile, and the molar percentages of the biliary lipids, cholesterol, phospholipids and bile acids were not significantly affected by inulin administration (data not shown).
Bile acid profile.
All three inulin supplements caused distinct alterations in the bile acid profile of gallbladder bile (Table 6). The most evident changes were in the relative percentages of taurochenodeoxycholic, glycocholic and glycodeoxycholic acid. Taurochenodeoxycholic acid was significantly lower in hamsters fed 8, 12 and 16% inulin by 37, 46 and 55%, compared with controls, whereas glycocholic and glycodeoxycholic acid were significantly greater in hamsters fed 12 and 16% inulin. In particular, the secondary bile acid glycodeoxycholic acid was increased by 1.0- to 1.8-fold with dietary inulin, suggesting substantial bacterial degradation during the intestinal passage of the bile acids. As a result of these changes, the glycine to taurine conjugation ratio was 70-100% greater than that of controls in hamsters fed all three inulin doses, whereas the cholate to chenodeoxycholate ratio was not altered in hamsters fed 8 and 12% inulin but was significantly greater in those fed 16% inulin. Because glycodeoxycholic acid was higher in hamsters fed 8, 12 and 16% inulin compared with controls, the ratio of primary to secondary bile acids was significantly lower. The hydrophobicity of the bile acid profile calculated as the HI, the measure of the hydrophobic-hydrophilic balance of biliary bile acids, was slightly but significantly greater in hamsters fed inulin.
Fecal sterol excretion.
The 3-d fecal weight tended to be 10-20% lower (P = 0.35) in hamsters fed inulin compared with controls (Table 7). This slight reduction in fecal output was most likely due to the smaller amount of cellulose (5 vs. 10 g/100 g diet) in the inulin-supplemented diets. The moisture content of the fecal samples was not different (data not shown). Fecal total bile acid concentrations (µmol/g) were 56-73% higher in hamsters fed 8, 12 and 16% inulin compared with controls (P < 0.05). Daily fecal bile acid excretion (µmol/d) tended to be higher (P = 0.056) in inulin-fed hamsters.
We examined the lipid-lowering potential of dietary inulin and especially its effect on bile acid metabolism in hamsters. To our knowledge, this is the first study that used the Syrian hamster, the commonly used model for the study of lipid and cholesterol metabolism (Spady et al. 1985
INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References
), little attention has been paid until recently to their potential physiologic effects.
, Fiordaliso et al. 1995, Kok et al. 1996, Levrat et al. 1991 and 1994). The number of human studies on the cholesterol-lowering action of fructooligosaccharides is limited, and results concerning effects on plasma cholesterol and TG are not conclusive (Canzi et al. 1995, Davidson et al. 1998, Luo et al. 1996, Pedersen et al. 1997, Yamashita et al. 1984). Moreover, the mechanism(s) by which fructooligosaccharides may elicit their lipid-lowering effect have not been elucidated. Several mechanisms, including the modulation of hepatic cholesterol synthesis by fermentation products, e.g., propionate and an increased fecal excretion of bile acids, have been hypothesized (Levrat et al. 1994). In addition, it was postulated that the hypotriacylglycerolemic effect of oligofructose might be caused by a diminished capacity of hepatocytes to synthesize triacylglycerols (Fiordaliso et al. 1995). This hypothesis was further supported by a reduction in plasma VLDL particles. Although it was demonstrated in studies with rats that oligosaccharides are very effective substrates for propionic fermentation in the cecum, high concentrations of propionic acid seem not to be a major factor of the hypocholesterolemic effect (Levrat et al. 1991 and 1994). Moreover, in these studies, an increase in fecal bile acid excretion was found, suggesting that an interruption of the enterohepatic circulation of bile acids with an enhanced fecal excretion seemed to have a major impact on the hypolipidemic effect.
, Spady et al. 1985 and 1986).
MATERIALS AND METHODS
Abstract
Introduction
Methods
Results
Discussion
References
View this table:
Table 1.
Composition of diets
40°C. After removal of the cecal contents, the tissues were rinsed with saline, blotted dry and weighed to determine cecal wall weight. Gallbladder bile was aspirated, weighed and analyzed for biliary lipids and bile acid composition.
) using a L7-65 ultracentrifuge and a 50.4 Ti rotor (Beckman Instruments, Munich, Germany). To obtain 2 mL of plasma, plasma from two hamsters was pooled if required. A preservative solution (final concentration in plasma: 1 mmol/L benzamidine, 0.04% EDTA, 0.005% gentamycin sulphate, 0.05% NaN3) was added to protect lipoproteins from enzymatic degradation. Three fractions were isolated based on the following densities: VLDL (d < 1.006 kg/L), LDL (1.006 < d < 1.055 kg/L), and HDL (1.055 < d < 1.21 kg/L). With the exception of VLDL, lipoprotein fractions were dialyzed against 0.15 mol/L NaCl, 0.04% EDTA and 0.05% NaN3 at 4°C for 24-36 h. TC, free cholesterol (FC), TG and phospholipid (PL) concentrations were determined using enzymatic assays (#352 for TC and #336 for TG, Sigma Chemicals, Wako Free Cholesterol C kit for FC and Phospholipid B kit for PL, Wako Chemicals, Düsseldorf, Germany). Protein concentration was determined by a modification of the Lowry procedure (Markwell et al. 1978). To verify the density cut-points and to check for cross-contaminations, LDL and HDL apolipoproteins were separated by gradient SDS-PAGE (4-20%), and stained with Coomassie Brilliant Blue. No traces of apolipoprotein B100 were present in the HDL fractions (data not shown).
). TC was determined enzymatically (Sigma kit #352) and FC was analyzed using HPLC (Kim and Chung 1984). Esterified cholesterol (EC) concentrations were calculated as the difference between TC and FC.
as previously described in detail (Trautwein et al. 1993). Total bile acid concentration was calculated as the sum of individual bile acids (taurine and glycine conjugates of cholate, chenodeoxycholate, deoxycholate and lithocholate) as measured by HPLC. The hydrophobicity index (HI), the measure of the hydrophobic-hydrophilic balance of biliary bile acids, was calculated as the sum of the molar fractions of individual bile acids multiplied by their individual HI values using the procedure of Heuman (1989).
as described in detail previously (Trautwein et al. 1993). Total bile acid concentration was determined using an enzymatic assay (Sigma bile acid kit #450, Sigma Chemicals) with some modifications. Neutral sterols were determined by gas chromatography as free sterols according to the method of Ausman et al. (1993) as described in detail previously (Trautwein et al. 1997).
RESULTS
Abstract
Introduction
Methods
Results
Discussion
References
View this table:
Table 2.
Food intake, body weight gain, weights of liver, small intestine and cecum and pH of the cecal contents in hamsters fed diets containing 0 (control), 8, 12 and 16% supplements of inulin1
View larger version (64K):
[in a new window]
Fig 1.
Plasma total cholesterol (TC) and triacylglycerol (TG) concentrations of hamsters fed diets containing 0 (control), 8, 12 or 16% supplement of inulin. Values are means ± SD, n = 10. Bars for each variable with different letters were significantly different (P < 0.05).
View this table:
Table 3.
Plasma lipoprotein cholesterol (C) and triacylglycerol (TG) concentrations of hamsters fed diets
containing 0 (control), 8, 12 and 16% supplements of inulin1
View this table:
Table 4.
Composition of VLDL particles of hamsters fed diets containing 0 (control), 8, 12 and 16% supplements of inulin1
View this table:
Table 5.
Hepatic cholesterol concentrations in hamster fed diets containing 0 (control), 8, 12 and 16% supplements of inulin1
View this table:
Table 6.
Total bile acid concentration and bile acid profile of gallbladder bile from hamsters fed diets
containing 0 (control), 8, 12 and 16% supplements of inulin1
View this table:
Table 7.
Fecal output, bile acid and neutral sterol excretion from hamsters fed diets containing
0 (control), 8, 12 and 16% supplements of inulin1
DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References
and 1986), to evaluate the cholesterol-lowering action of fructooligosaccharides such as inulin.
, Kok et al. 1996). In contrast, dietary inulin at levels of 5, 10 and 20% caused a significant dose-dependent hypocholesterolemic effect, whereas plasma TG concentrations were significantly lowered only in rats fed 20% inulin (Levrat et al. 1991). In another long-term study (16 wk) with rats, 10% dietary oligofructose led to a significant reduction of plasma TC (15% ) and TG (24%) (Fiordaliso et al. 1995). Because triacylglycerol-rich particles (VLDL) specifically were decreased, it was postulated that this decrease was associated with a reduction in VLDL production, thus indicating that oligofructose alters hepatic lipid metabolism (Fiordaliso et al. 1995). Further, it was demonstrated that the hypotriacylglycerolemic effect of oligofructose is due to a reduced hepatic de novo fatty acid and TG synthesis (Kok et al. 1996).
), whereas in healthy subjects, no lipid-lowering effect was found (Luo et al. 1996, Pedersen et al. 1997). In one study, a decrease in plasma TG by dietary inulin was reported (Canzi et al. 1995), whereas in another study a reduction in plasma TC was found in hypercholesterolemic subjects (Davidson et al., 1998).
). In the studies with rats (Delzenne et al. 1993, Fiordaliso et al. 1995, Kok et al. 1996) apparently cholesterol-free diets were fed, whereas a cholesterol-enriched diet was used in this study. In fact, 0.12% cholesterol is equivalent to an absorbed amount of cholesterol ~1.2 times the endogenous cholesterol synthesis rate in the hamster and should therefore down-regulate metabolic pathways related to cholesterol metabolism (Dietschy et al. 1993). Therefore, the hypocholesterolemic response in human subjects could possibly vary depending on whether individuals are normal or hypercholesterolemic.
, Levrat et al. 1991 and 1994). Most likely, the lack of an apparent decrease in cecal pH could be explained by the fact that the hamsters were deprived of food for 18 h and no active fermentation took place. It is also possible that acidic conditions occurring in the cecum were partially neutralized by various anions. Apparently, cecal fermentation and subsequent short-chain fatty acid production are accompanied by an increase in concentrations in the large intestine, calcium phosphates, which could counterbalance the decrease in cecal pH induced by organic acids (Levrat et al. 1991, Rémésy et al. 1993). The increases noted in cecal total weight and cecal wall weight, especially in hamsters fed 16% inulin, agree with similar findings in earlier studies after oligosaccharide consumption by rats (Campbell et al. 1997, Delzenne et al. 1995). Although short-chain fatty acids were not determined in this study, the increase in cecal tissue reflecting hypertrophy suggests increased intestinal activity, possibly resulting from short-chain fatty acid production. It is thought that short-chain fatty acids have beneficial effects such as normalizing cell proliferation in the mucosa and increasing bacterial mass especially of bifidobacteria (Campbell et al. 1997).
). Fecal concentration and daily excretion of neutral sterols were not affected by dietary inulin, suggesting that cholesterol digestibility was not impaired. Interestingly, fecal cholesterol concentration tended to be higher in hamsters fed inulin, whereas the concentration of coprostanol was lower. Coprostanol, the main breakdown product of cholesterol, is usually the predominant sterol excreted in feces. Therefore, these data suggest a reduced bacterial cholesterol degradation as a result of inulin ingestion. Although it was postulated that inulin does not bind bile acids in the intestinal tract, a considerable increase in fecal bile acid excretion has been observed in rats (Levrat et al. 1994). In this study, fecal bile acid concentrations were significantly higher in inulin-fed hamsters, and daily bile acid excretion tended to be enhanced. However, the sterol balance calculated as cholesterol intake minus total steroid excretion (neutral sterols and bile acids) was not affected by inulin, indicating that the rather modest effect on fecal bile acid excretion (25, 60 and 47% with 8, 12 and 16% inulin, respectively) cannot explain the cholesterol-lowering effect of inulin. Therefore, other mechanisms seem to be involved in the hypocholesterolemic action.
, Kok et al. 1996). This hypothesis is further supported by a reduction in plasma VLDL particles, indicating a decreased production and secretion of VLDL (Fiordaliso et al. 1995). In this study, plasma TG and VLDL-C were markedly lower in inulin-fed hamsters, which is supportive of this hypothesis. Therefore, changes in hepatic de novo lipogenesis, especially affecting VLDL production and secretion, seem to be the principal mechanism for the lipid-lowering action of inulin.
). One possible explanation could be that taurine conjugates are more stable at low pH and less apt to be deconjugated during intestinal transit (Zhang et al. 1992). At an acidic pH, a result of short-chain fatty acid production, it is plausible that bile acids may bind to bacteria or insoluble calcium salts (Levrat et al. 1994). In fact, it was shown in vitro as well as in rats that acidic pH is very effective in lowering the concentration of soluble bile acids and especially of dihydroxy bile acids such as chenodeoxycholate by binding to calcium salts (Rémésy et al. 1993).
). In general, dihydroxy bile acids, e.g. taurochenodeoxycholate, are more effective in terms of feedback inhibition on bile acid synthesis than trihydroxy bile acids (cholate) (Vlahcevic et al. 1991). Therefore alterations in the bile acid profile as induced by dietary inulin, especially at the level of 16%, could possibly lead to a stimulation of hepatic bile acid synthesis. In fact, it has been demonstrated that changes in the composition of bile acids (less dihydroxy bile acids) returning to the liver may stimulate cholesterol 7
-hydroxylase activity by interfering with a bile acid-responsive element (Chiang and Stroup 1994).
) but may also stimulate bile acid synthesis (Imaizumi et al. 1992). However, the possibility that a propionic acid fermentation by dietary inulin can interfere with alterations in bile acid and cholesterol metabolism is not fully established and requires further investigation.
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FOOTNOTES |
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Technology and Health Interaction, March 5-7,
1998, Lisbon, Portugal [Trautwein, E. A., Radüuz, E., Rieckhoff, D. & Erbersdobler, H. F. Effects of increasing doses of dietary inulin of cholesterol and bile acid metabolism in hamsters.].
Manuscript received 30 March 1998. Initial reviews completed 8 May 1998. Revision accepted 22 June 1998.
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ACKNOWLEDGMENTS |
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The authors thank Eike Radünz, Angelika Kunath-Rau, Karin Forgbert and Swantje Möller for their excellent technical assistance and Veronika Faist for helpful comments on the manuscript.
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LITERATURE CITED |
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Abstract
Introduction
Methods
Results
Discussion
References
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-hydroxylase gene promoter.
J. Biol. Chem.
1994;
269:17502-17507This article has been cited by other articles:
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  D. Letexier, F. Diraison, and M. Beylot Addition of inulin to a moderately high-carbohydrate diet reduces hepatic lipogenesis and plasma triacylglycerol concentrations in humans Am. J. Clinical Nutrition, March 1, 2003; 77(3): 559 - 564. [Abstract] [Full Text] [PDF]
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 C. A. Daubioul, H. S. Taper, L. D. De Wispelaere, and N. M. Delzenne Dietary Oligofructose Lessens Hepatic Steatosis, but Does Not Prevent Hypertriglyceridemia in Obese Zucker Rats J. Nutr., May 1, 2000; 130(5): 1314 - 1319. [Abstract] [Full Text]
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