©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Regulation of Hepatic 7-Hydroxylase Expression and Response to Dietary Cholesterol in the Rat and Hamster (*)

(Received for publication, September 13, 1994; and in revised form, December 16, 1994)

Jay D. Horton (§) Jennifer A. Cuthbert David K. Spady (¶)

From the Department of Internal Medicine, The University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75235

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Although dietary cholesterol raises plasma total and low density lipoprotein (LDL) cholesterol concentrations, the response to a given intake of cholesterol varies enormously among different species and even among individuals of the same species. The mechanisms responsible for differing sensitivity to dietary cholesterol were examined by comparing the rat, which is able to adapt to large fluctuations in sterol intake or loss with little change in plasma LDL levels, with the hamster, where changes in sterol balance strongly influence plasma LDL concentrations. When fed the same cholesterol-free diet, hepatic 7alpha-hydroxylase activity was 16-fold higher in the rat than in the hamster. As a consequence, rates of hepatic cholesterol synthesis were 20-fold higher in the rat than in the hamster. In both species, hepatic cholesterol synthesis was suppressed >90% in response to increasing loads of dietary cholesterol. However, the quantitative importance of this adaptive mechanism was much greater in the rat since the absolute reduction in hepatic cholesterol synthesis in the rat (2,110 nmol/h/g) was much larger than in the hamster (103 nmol/h/g). In the rat, the high basal level of 7alpha-hydroxylase expression was further induced by substrate (cholesterol) allowing these animals to convert excess dietary cholesterol to bile acids efficiently. In contrast, the low basal level of enzyme expression in the hamster was not induced by dietary cholesterol. Thus, the low basal rates of bile acid and cholesterol synthesis coupled with a lack of 7alpha-hydroxylase induction by cholesterol render the hamster much more sensitive than the rat to the cholesterolemic effects of dietary cholesterol.


INTRODUCTION

Epidemiological studies in human populations show a strong correlation between dietary cholesterol intake and coronary heart disease(1) . Although some studies suggest that dietary cholesterol may play a role in atherosclerosis independent of its effects on plasma cholesterol(2, 3) , the link between dietary cholesterol and atherosclerotic lesions is thought to be primarily related to its effects on plasma low density lipoprotein (LDL) (^1)cholesterol. Dietary cholesterol is not incorporated directly into LDL but rather increases plasma LDL concentrations indirectly through effects on sterol metabolism in the liver(4, 5) . The liver is the key organ involved in adaptation to a high cholesterol diet since it is not only the recipient of dietary cholesterol but also the major site through which cholesterol is eliminated from the body, either directly or after conversion to bile acids. In response to changes in cholesterol intake, the liver compensates by reducing the rate of de novo cholesterol synthesis and, in some cases, the rate of receptor-dependent LDL cholesterol uptake. Since the liver is the principal site of LDL catabolism, changes in hepatic LDL receptor expression usually result in reciprocal changes in plasma LDL concentrations. Finally, in some species, 7alpha-hydroxylase, which catalyzes the initial and rate-limiting step in the bile acid biosynthetic pathway, is subject to induction by substrate (cholesterol), thereby making it possible for these animals to convert excess dietary cholesterol to bile acids(6) .

Although dietary cholesterol usually raises plasma total and LDL cholesterol levels, the response to a given intake of cholesterol varies remarkably among different species and even among individuals of the same species(7) . This marked heterogeneity in sensitivity to dietary cholesterol presumably represents polymorphisms at gene loci involved in the absorption of dietary cholesterol, the hepatic conversion of cholesterol to bile acids, the feedback inhibition of endogenous cholesterol synthesis, or the regulation of the LDL receptor pathway. Although heritable differences in several of these major pathways have been correlated with responsiveness to dietary cholesterol(8, 9, 10, 11) , the actual gene(s) involved and the polymorphisms responsible for variability are unknown.

The two mechanisms most commonly implicated in resistance to dietary cholesterol are 1) a diminished efficiency of cholesterol absorption and 2) an enhanced capacity to convert cholesterol to bile acids. Both mechanisms would reduce liver cholesterol and potentially lead to up-regulation of the LDL receptor pathway. Thus, rhesus monkeys and baboons that are sensitive to dietary cholesterol appear to absorb cholesterol more efficiently than resistant animals(11, 12) . On the other hand, studies in squirrel monkeys (13) and in inbred strains of rabbits (9, 10) suggest that the ability of the liver to convert cholesterol to bile acids may be more important in determining responsiveness to dietary cholesterol. In humans, both mechanisms may contribute to individual differences in responsiveness to a high cholesterol diet(14, 15, 16, 17, 18, 19, 20) .

While heterogeneity of response to dietary cholesterol has been observed among individuals of all outbred species examined, differences between species are even more pronounced. In the present studies, the mechanisms responsible for differing sensitivity to dietary cholesterol were examined by comparing the rat, which is able to adapt to large fluctuations in sterol input or loss with little change in plasma LDL concentrations, with the hamster, where even modest changes in sterol balance lead to alterations in plasma LDL levels(21, 22) . The response to dietary cholesterol in these two species, which are commonly used in studies of cholesterol and bile acid metabolism, spans the range of individual responses observed in humans.


EXPERIMENTAL PROCEDURES

Animals and Diets

Sprague-Dawley rats and Golden Syrian hamsters (Sasco, Inc., Omaha, NE) were housed in colony cages and subjected to light cycling for at least 3 weeks prior to introduction of the experimental diets. When used in specific experiments, the rats and hamsters were in the 190-240- and 120-150-g-weight range, respectively. The control semisynthetic diet used in these studies contained (wt/wt) 20% soy protein, 58% corn starch, 2% corn oil, 0.3% DLmethionine, 8.5% salt mix, 1% vitamin mix, 0.2% choline bitartrate, and 10% cellulose. The experimental diets were prepared by supplementing the control semisynthetic diet with cholesterol at the 0.05% (148 mg/1,000 kcal), 0.1% (296 mg/1,000 kcal), 0.25% (740 mg/1,000 kcal), 0.5% (1,480 mg/1,000 kcal), and 1% (2,960 mg/1,000 kcal) levels. The experimental diets were fed ad libitum for 6 weeks, and all studies were carried out during the mid-dark phase of the light cycle.

Determination of Hepatic 7alpha-Hydroxylase Activity

Hepatic 7alphahydroxylase activity was measured using a high pressure liquid chromatography-spectrophotometric assay that quantifies the mass of 7alpha-hydroxycholesterol formed from endogenous microsomal cholesterol after enzymatic conversion to 7alpha-hydroxy-4-cholesten-3-one using cholesterol oxidase(23) .

Determination of Hepatic Cholesterol Synthesis Rates

Rates of hepatic cholesterol synthesis were measured in vivo using [^3H]water. As previously described(24) , the animals were administered 100 mCi of [^3H]water intravenously and then returned to individual cages under a fume hood. 1 h after the injection of [^3H]water, the animals were anesthetized and exsanguinated through the abdominal aorta. Aliquots of plasma were taken for the determination of body water specific activity, and samples of liver were taken for the isolation of digitonin-precipitable sterols. Rates of sterol synthesis are expressed as the nmoles of [^3H]water incorporated into digitonin-precipitable sterols per hour per gram of liver (nmol/h/g).

Determination of Hepatic LDL Uptake Rates in Vivo

Plasma was obtained from donor rats and hamsters maintained on standard rodent diet (Wayne Lab Blox, Allied Mills, Chicago, IL). LDL was isolated from plasma by preparative ultracentrifugation in the density range of 1.020-1.055 g/ml and labeled with I- or I-tyramine cellobiose (25) . Rates of hepatic LDL uptake were measured using primed infusions of I-tyramine cellobiose-labeled LDL as previously described(26) . Changes in hepatic receptor-dependent LDL transport (defined as the rate of receptor-dependent LDL uptake in an experimental animal relative to the rate of receptor-dependent LDL uptake in control animals having the same plasma LDL concentration) were determined as described(26, 27) .

Determination of mRNA Levels

Hepatic 7alpha-hydroxylase, 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase, LDL receptor, and glyceraldehyde-3-phosphate dehydrogenase (used as an invariant control) mRNA levels were determined by nuclease protection as previously described(21) . Probes were synthesized using 0.5 µM [P]dCTP and 1-2 µM (7alpha-hydroxylase, HMG CoA reductase, and LDL receptor) or 300 µM (glyceraldehyde-3-phosphate dehydrogenase) unlabeled dCTP(21, 26, 28) .

Samples of rat and hamster liver were homogenized in guanidinium thiocyanate, and the RNA was isolated by the method of Chomczynski and Sacchi(29) . Total RNA (40 µg) was hybridized with the P-labeled cDNA probes simultaneously at 48 °C overnight. Unhybridized probe, present in excess relative to the amount of specific mRNA, was then digested with 40 units of mung bean nuclease (Life Technologies, Inc.). The mRNA-protected P-labeled probes were separated on 7 M urea, 6% polyacrylamide gels together with P-labeled MspI-digested pBR322 size standards. The radioactivity in each band, as well as background radioactivity, was quantified using an isotopic imaging system (Ambis, Inc., San Diego, CA). The level of glyceraldehyde-3-phosphate dehydrogenase mRNA did not vary among the various experimental groups and was used to correct for any procedural losses.

Determination of Liver and Plasma Cholesterol Distribution

Liver cholesterol was quantified by capillary gas-liquid chromatography. The cholesterol distribution in plasma was determined by gel filtration chromatography using a Superose 6 column (Sigma). 2-ml aliquots were collected and assayed for cholesterol using an enzymatic kit (Boehringer Mannheim).


RESULTS

Responsiveness to dietary cholesterol varies greatly among different animal species. The rat is notoriously resistant to the cholesterolemic effects of dietary cholesterol, whereas the hamster is modestly responsive. Fig. 1shows the changes in the cholesterol content of liver and plasma LDL in rats and hamsters fed a low-fat semisynthetic diet supplemented with varying amounts of cholesterol. As shown in the toppanel, liver cholesterol in animals fed the cholesterol-free diet equaled 2.9 and 2.2 mg/g in the hamster and rat, respectively. In the hamster, dietary cholesterol increased the cholesterol content of the liver in a dose-dependent fashion with levels reaching 90 mg/g in animals fed 1% cholesterol. In contrast, dietary cholesterol had little effect on liver cholesterol in the rat with levels rising to only 7 mg/g in animals fed 1% cholesterol. Changes in plasma LDL cholesterol levels paralleled the changes in liver cholesterol. As shown in Fig. 1, bottompanel, plasma LDL cholesterol concentrations in the hamster increased from 24 mg/dl to 88 mg/dl as the cholesterol content of the diet was increased from 0 to 1%. In contrast, plasma LDL cholesterol concentrations in the rat were only minimally affected by dietary cholesterol.


Figure 1: Liver and plasma LDL cholesterol levels in rats and hamsters fed various amounts of cholesterol. Animals were fed a low-fat semisynthetic diet supplemented with various amounts (0-1%) of cholesterol for 6 weeks. Each value represents the mean ± 1 S.D. for data obtained in six animals.



Thus, at any level of cholesterol intake, the hamster accumulated much more cholesterol in the liver and plasma than did the rat. A number of potential mechanisms may account for these differences in response to dietary cholesterol. One possibility is that the hamster simply absorbs dietary cholesterol more efficiently than the rat. However, the hamster has an unusually short bowel, and preliminary studies showed that the rat absorbed dietary cholesterol as well as the hamster. (^2)A second possibility is that the rat is more efficient than the hamster at eliminating excess cholesterol from the body. In both species, conversion of cholesterol to bile acids is the major regulated pathway by which cholesterol is removed from the body. Thus, differences in the regulation of this pathway may strongly influence responsiveness to dietary cholesterol. The initial and rate-limiting step in the conversion of cholesterol to bile acids is catalyzed by hepatic 7alpha-hydroxylase, and further studies were undertaken to examine the regulation of this pathway by sterols in the rat and hamster.

The major form of regulation of 7alpha-hydroxylase is thought to be feedback repression of gene transcription by bile acids returning to the liver in the enterohepatic circulation. In addition, 7alpha-hydroxylase is subject to induction by substrate (cholesterol) in some species, including the rat. Fig. 2shows 7alpha-hydroxylase activity in rats and hamsters as a function of the cholesterol content of the liver in animals fed increasing amounts of cholesterol. As indicated by the opensymbols, basal 7alpha-hydroxylase activity in animals fed the same cholesterol-free, low-fat diet were significantly higher in the rat than in the hamster. Thus, at the mid-dark point of the light cycle, 7alpha-hydroxylase activity in the rat and hamster equaled 1,410 and 88 pmol/h/mg of microsomal protein, respectively. Moreover, the high basal level of 7alpha-hydroxylase activity in the rat was further increased when cholesterol was added to the diet, whereas the low basal activity in the hamster was not significantly altered by dietary cholesterol in amounts that markedly raised the cholesterol content of the liver. Aliquots of liver were taken from these same animals for the determination of 7alpha-hydroxylase mRNA levels by nuclease protection. Autoradiograms illustrating the effect of dietary cholesterol on 7alphahydroxylase mRNA levels in the rat and hamster are shown in Fig. 3and Fig. 4, respectively, and the mean changes in 7alphahydroxylase mRNA levels are summarized in Fig. 5. As shown in Fig. 5, dietary cholesterol increased 7alpha-hydroxylase mRNA levels in the rat at liver cholesterol levels that were only modestly (2-3-fold) elevated. In the hamster, on the other hand, dietary cholesterol, in amounts that increased liver cholesterol levels by up to 10-fold, had no effect on 7alpha-hydroxylase mRNA levels. Hepatic 7alpha-hydroxylase mRNA levels did tend to increase (though not significantly so) in hamsters fed cholesterol in amounts that raised liver cholesterol levels by 15-30-fold. Higher levels of cholesterol intake resulted in massive cholesterol accumulation in the liver and a trend toward lower 7alpha-hydroxylase mRNA levels. It was not possible to compare the absolute amount of 7alpha-hydroxylase mRNA in the rat and hamster directly; however, based on the length, nucleotide content, and specific activity of the P cDNA probes that were used in these studies, 7alpha-hydroxylase mRNA was severalfold more abundant in rat than in hamster liver. Thus, in terms of the regulation of 7alpha-hydroxylase expression, the rat differed from the hamster both with respect to basal levels of expression and with respect to the ability to up-regulate expression in response to increasing levels of substrate. In contrast, regulation of hepatic 7alpha-hydroxylase by bile acids appeared to be similar in the two species. As shown in Fig. 6, 7alpha-hydroxylase activity was suppressed by dietary cholate and derepressed by cholestyramine in both species. As summarized in Fig. 7(with representative autoradiograms in Fig. 3and Fig. 4), these changes in 7alpha-hydroxylase activity were accompanied by similar changes in mRNA levels.


Figure 2: Hepatic 7alpha-hydroxylase activity in the rat and hamster as a function of the cholesterol content of the liver. Animals were fed a low-fat semisynthetic diet supplemented with the indicated amount of cholesterol for 6 weeks. Each value represents the mean ± 1 S.D. for data obtained in six animals.




Figure 3: Measurement of hepatic 7alpha-hydroxylase mRNA levels in the rat. Hepatic RNA was isolated from rats fed a low-fat semisynthetic diet supplemented with 0.25% cholic acid, 1% cholestyramine, or 1% cholesterol. Total RNA (40 µg) was hybridized with P-labeled single-stranded cDNA probes, and the protected bands resistant to mung bean nuclease digestion were separated by polyacrylamide gel electrophoresis and autoradiographed. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; FABP, fatty acid binding protein; nt, nucleotides.




Figure 4: Measurement of hepatic 7alpha-hydroxylase mRNA levels in the hamster. Hepatic RNA was isolated from hamsters fed a low-fat semisynthetic diet supplemented with 0.25% cholic acid, 1% cholestyramine, or the indicated amount of cholesterol. Total RNA (40 µg) was hybridized with P-labeled single-stranded cDNA probes, and the protected bands resistant to mung bean nuclease digestion were separated by polyacrylamide gel electrophoresis and autoradiographed. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; nt, nucleotides.




Figure 5: Hepatic 7alpha-hydroxylase mRNA in the rat and hamster as a function of the cholesterol content of the liver. Animals were fed a low-fat semisynthetic diet supplemented with the indicated amount of cholesterol for 6 weeks. Each value represents the mean ± 1 S.D. for data obtained in six animals.




Figure 6: Regulatory effects of cholic acid and cholestyramine on hepatic 7alpha-hydroxylase activity in the rat and hamster. Animals were fed a low-fat semisynthetic diet supplemented with 0.25% cholic acid or 1% cholestyramine for 3 weeks. Each value represents the mean ± 1 S.D. for data obtained in six animals.




Figure 7: Regulatory effects of cholic acid and cholestyramine on hepatic 7alpha-hydroxylase mRNA in the rat and hamster. Animals were fed a low-fat semisynthetic diet supplemented with 0.25% cholic acid or 1% cholestyramine for 3 weeks. Each value represents the mean ± 1 S.D. for data obtained in six animals.



In response to changes in cholesterol influx or efflux, the liver maintains cholesterol homeostasis by regulating the rates of de novo cholesterol synthesis and LDL cholesterol uptake. Fig. 8shows rates of hepatic cholesterol synthesis as a function of hepatic cholesterol levels in rats and hamsters fed increasing amounts of cholesterol. On the cholesterol-free diet, rates of hepatic cholesterol synthesis at the mid-dark point of the light cycle equaled 2,245 nmol/h/g in the rat and 111 nmol/h/g in the hamster. Hepatic cholesterol synthesis was suppressed 90-95% by dietary cholesterol in both species; however, the absolute reduction in sterol synthesis was much greater in the rat (2,245 to 135 nmol/h/g) than in the hamster (111 to 8 nmol/h/g). Notably, hepatic cholesterol synthesis was exquisitely sensitive to the cholesterol content of the liver in both species. Thus, as the flux of cholesterol to the liver was progressively increased, the cholesterol content of the liver began to rise only after de novo sterol synthesis was completely suppressed. As shown in Fig. 9, changes in HMG CoA reductase mRNA could account for only a small fraction of the change in cholesterol synthesis rates, suggesting that posttranscriptional regulation of HMG CoA reductase accounts for much of the change in cholesterol synthesis rates in both species.


Figure 8: Hepatic cholesterol synthesis in the rat and hamster as a function of the cholesterol content of the liver. Animals were fed a low-fat semisynthetic diet supplemented with the indicated amount of cholesterol for 6 weeks. Rates of hepatic cholesterol synthesis were quantified in vivo using [^3H]water. Each value represents the mean ± 1 S.D. for data obtained in six animals.




Figure 9: Hepatic HMG CoA reductase mRNA in the rat and hamster as a function of the cholesterol content of the liver. Animals were fed a low-fat semisynthetic diet supplemented with the indicated amount of cholesterol for 6 weeks. Each value represents the mean ± 1 S.D. for data obtained in six animals.



The LDL receptor, like HMG CoA reductase, is regulated by cellular cholesterol levels, and in cultured cells, these two pathways are regulated in parallel in response to changes in cholesterol availability. Fig. 10shows rates of hepatic receptor-dependent LDL transport as a function of liver cholesterol levels in rats and hamsters fed increasing amounts of cholesterol. In the rat, receptor-dependent LDL uptake by the liver was not down-regulated even at the highest level of cholesterol intake (1%), despite the fact that hepatic cholesterol synthesis was suppressed by nearly 95% in these animals. Similarly, in the hamster, modest amounts of dietary cholesterol had little effect on hepatic LDL receptor activity, even though hepatic sterol synthesis was largely suppressed. Indeed, significant down-regulation of hepatic LDL receptor activity was only observed at dietary cholesterol intakes that caused massive accumulation of cholesterol in the liver. As shown in Fig. 11, sterol-dependent changes in receptor-dependent LDL transport in the hamster were accompanied by similar changes in LDL receptor mRNA levels.


Figure 10: Hepatic receptor-dependent LDL transport in the rat and hamster as a function of the cholesterol content of the liver. Animals were fed a low-fat semisynthetic diet supplemented with the indicated amount of cholesterol for 6 weeks. Rates of receptor-dependent LDL uptake were quantified in vivo using primed infusions of I-tyramine cellobiose-labeled LDL. Each value represents the mean ± 1 S.D. for data obtained in six animals.




Figure 11: Hepatic LDL receptor mRNA in the rat and hamster as a function of the cholesterol content of the liver. Animals were fed a low-fat semisynthetic diet supplemented with the indicated amount of cholesterol for 6 weeks. Each value represents the mean ± 1 S.D. for data obtained in six animals.




DISCUSSION

Dietary cholesterol raises total and LDL cholesterol levels; however, the response to a given level of dietary cholesterol varies greatly among different species and even among individuals of the same species(7) . In the present studies, the mechanisms responsible for differing sensitivity to dietary cholesterol were investigated by comparing the rat, which is able to adapt to large fluctuations in sterol input or loss with little change in plasma LDL concentrations, with the hamster, where changes in sterol balance usually lead to alterations in plasma LDL levels(5) . These studies suggest that differences in sensitivity to dietary cholesterol in the rat and hamster are due, in large part, to differences in the regulation of hepatic 7alpha-hydroxylase, the initial and rate-limiting enzyme in the bile acid biosynthetic pathway. Thus, a low basal level of 7alphahydroxylase expression coupled with a lack of enzyme induction by dietary cholesterol render the hamster more sensitive than the rat to the cholesterolemic effects of dietary cholesterol.

When fed the same cholesterol-free, low-fat diet, hepatic 7alpha-hydroxylase activity was 16-fold higher in the rat than in the hamster. Under steady-state conditions, the rapid conversion of cholesterol to bile acids in the rat must be balanced by a high rate of endogenous cholesterol synthesis, and, indeed, basal rates of hepatic cholesterol synthesis were 20-fold higher in the rat than in the hamster. The significance of a high basal rate of hepatic cholesterol synthesis is that a larger load of dietary cholesterol can be accommodated through feedback repression of endogenous synthesis. Dietary cholesterol suppressed hepatic sterol synthesis by >90% in both species; however, the quantitative importance of this adaptive mechanism was far greater in the rat since the absolute reduction in sterol synthesis in this species (2,110 nmol/h/g) was much larger than in the hamster (103 nmol/h/g). Hepatic cholesterol synthesis was exquisitely sensitive to cellular cholesterol levels in both species. Thus, as the flux of cholesterol to the liver was progressively increased by raising the cholesterol content of the diet, liver cholesterol levels began to rise only after de novo sterol synthesis was completely suppressed (Fig. 8). Feedback repression of endogenous cholesterol synthesis also appears to be an important adaptive mechanism for dealing with dietary cholesterol in humans(16, 17, 19) . In sterol balance studies carried out by Nestel and Poyser (19) , the greater the reduction in endogenous cholesterol synthesis, the less likely was plasma cholesterol to rise in response to a high cholesterol diet.

Hepatic 7alpha-hydroxylase activity in the rat is characterized by marked diurnal fluctuations with maximal activity occurring in the mid-dark phase of the light cycle(30, 31) . Diurnal changes in 7alpha-hydroxylase expression have not been systematically examined in the hamster. However, we have found similar levels of 7alpha-hydroxylase activity and mRNA at the mid-dark and mid-light points of the light cycle, suggesting that diurnal fluctuations in enzyme expression are much less pronounced in the hamster than in the rat(28) . This is consistent with the finding that hamsters consume approximately as much food during the two phases of the light cycle(32) , whereas rats eat exclusively during the dark phase of the light cycle. In any case, differences in integrated enzyme activity over the entire 24-h light cycle would certainly be less than the 16-fold difference in activity observed at the mid-dark point in the light cycle. In addition, it is possible that alternative pathways that circumvent 7alpha-hydroxylase (33, 34) might contribute to bile acid synthesis in the rat and/or hamster. Sterol balance studies suggest that rats synthesize 3-5-fold more bile acids over a 24-h period of time than hamsters, although direct comparisons have not been reported(35, 36, 37, 38) .

Why basal expression of 7alpha-hydroxylase is higher in rats than in hamsters is not known. In both species, 7alphahydroxylase expression is up-regulated severalfold by interventions that interrupt the enterohepatic cycling of bile acids(6, 28, 39) . The conventional interpretation of these data is that the enzyme is under feedback control by bile acids fluxing through the liver in the enterohepatic circulation. If this interpretation is true, one possible explanation for the unusually high basal level of 7alpha-hydroxylase expression in the rat is that they malabsorb bile acids. Under physiological conditions, bile acids are absorbed from the gut primarily by bile acid transporters located in the brush border membranes of ileal enterocytes (40, 41) . It is possible that differences in the expression of the ileal bile acid transporter could lead to differences in the conservation of bile acids in the enterohepatic circulation and differences in 7alpha-hydroxylase expression. However, the enterohepatic pool of bile acids is 3-fold larger in the rat (42, 43) than in the hamster, suggesting that the differences in basal 7alpha-hydroxylase expression are primary rather than secondary to bile acid malabsorption in the rat.

Not only was the basal level of 7alpha-hydroxylase expression very low in the hamster, enzyme expression was not induced by dietary cholesterol in this species. Induction of 7alpha-hydroxylase expression and bile acid synthesis by dietary cholesterol has been observed in several species, including the rat(37, 44) , mouse(45) , dog(46) , and certain species of nonhuman primates(13) , all of whom adapt to large amounts of dietary cholesterol with little change in plasma cholesterol. For the most part, dietary cholesterol does not lead to an increase in 7alpha-hydroxylase expression or bile acid synthesis in humans(16, 17, 18, 19, 20) . However, certain individuals maintain normal plasma cholesterol levels despite a high intake of cholesterol, and induction of bile acid synthesis appears to account, at least in part, for the resistance to dietary cholesterol observed in these individuals(14) .

In the rat, 7alpha-hydroxylase expression is inhibited by bile acids fluxing through the liver in the enterohepatic circulation and is subject to induction by substrate (cholesterol). Presumably, the 7alpha-hydroxylase promoter contains sequences that mediate regulation by bile acids and cholesterol, i.e. sterol and bile acid response elements(47, 48, 49) . It has been suggested that expression of the enzyme may be titrated by these positive and negative response elements that somehow sense the levels of substrate and end products, respectively(6) . Hepatic 7alphahydroxylase expression was not induced by cholesterol in the hamster, suggesting that either the sterol response element or the proteins that interact with it may be nonfunctional in this species. If the basal level of 7alpha-hydroxylase expression is indeed titrated by negative and positive response elements in the rat, absence of the positive response mechanism could also explain the very low level of basal expression in the hamster. We previously showed in the rat that dietary cholesterol completely blocked the suppressive effects of bile acids on 7alpha-hydroxylase expression(21) . Indeed the highest levels of expression were observed in animals fed cholesterol plus cholic acid. Since cholic acid promotes cholesterol absorption (50) and greatly increased the cholesterol content of the liver(21) , these findings suggested that the putative sterol regulatory element contributed more to the regulation of gene expression than the putative bile acid response element. Although cholesterol regulates 7alpha-hydroxylase expression in the rat primarily at the level of the hepatocyte(44, 48, 51) , it has been suggested that large amounts of dietary cholesterol may also interfere with bile acid absorption, thereby leading to derepression of hepatic 7alpha-hydroxylase (52) . If true, events in the gut may also contribute to induction of hepatic 7alpha-hydroxylase expression by dietary cholesterol in the rat.

It was recently reported that dietary cholesterol actually down-regulated 7alpha-hydroxylase activity in African green monkeys(53) . However, the cholesterol-enriched diets used in these studies also contained large amounts of triglyceride. Dietary triglyceride has been shown to suppress 7alpha-hydroxylase activity in the rat(54) , and diverting dietary triglyceride away from the liver by lymph fistulization reportedly up-regulates enzyme activity(55) . While the production of bile acids is important for the maintenance of cholesterol homeostasis, bile acids serve an equally important role in the gut where they emulsify lipids, thereby promoting fat digestion and absorption. Thus, it would make mechanistic sense if one or more products of lipolysis regulated the rate of bile acid synthesis. Oleic acid has been shown to suppress bile acid secretion in the isolated perfused rat liver, suggesting that fatty acids may directly inhibit hepatic 7alpha-hydroxylase(56) .

Understanding the mechanisms responsible for resistance to dietary cholesterol may aid in the development of strategies aimed at inducing resistance to, or reversing, the cholesterolemic effect of Western diets. Cholesterol absorption efficiency correlates with plasma cholesterol levels in some human populations(15) , and agents that interfere with cholesterol absorption have modest cholesterol-lowering activity(57) . Hepatic 7alpha-hydroxylase represents another promising target. In recent studies, adenovirus-mediated transfer of a gene encoding cholesterol 7alpha-hydroxylase into hamsters increased hepatic 7alpha-hydroxylase activity by 10-15-fold (to levels seen in the rat) and rendered these animals highly resistant to the cholesterolemic effects of a high cholesterol diet(58) . Currently available bile acid sequestrants increase hepatic 7alpha-hydroxylase expression by only 2-3-fold and, as a consequence, have only modest cholesterol lowering activity. More effective strategies for increasing 7alpha-hydroxylase expression should prove proportionately more active in lowering plasma cholesterol levels.


FOOTNOTES

*
This research was supported by National Institutes of Health Grants HL-38049 and HL-47551. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported in part by U. S. Public Health Service Training Grant AM-07100.

To whom correspondence should be addressed: Dept. of Internal Medicine, UT Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75235-8887. Fax: 214-648-8290.

(^1)
The abbreviations used are: LDL, low density lipoprotein(s); HMG CoA, 3-hydroxy-3-methylglutaryl coenzyme A.

(^2)
D. K. Spady, unpublished data.


ACKNOWLEDGEMENTS

We thank Drs. David Russell, Diane Jelinek, Sohaib Khan, Richard Tanaka, and Ray Wu for making the cDNAs available and Jody Houston, Wendy Williams, and Matthew Zimmerman for excellent technical assistance.


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