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Department of Nutritional Sciences, University of Connecticut, Storrs, CT 06269 and * Monsanto Company, St. Louis, MO 63167
2To whom correspondence should be addressed. E-mail: maria-luz.fernandez{at}uconn.edu.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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-hydroxylase (CYP7) activity was 
88% higher in guinea pigs fed the two higher dosages of CFO (P < 0.05). In parallel, CYP7 mRNA abundance was 88% higher in guinea pigs fed all three CFO diets. CFO treatment also induced hepatic LDLR mRNA by 66–150% with significant differences at the highest CFO dose. These results suggest that CFO, as a result of decreased bile acid absorption, increased mRNA abundance and activity of CYP7. Because hepatic cholesterol is the substrate for CYP7, a lowering of cholesterol concentrations in the total and microsomal pools was observed. As a response to the depleted microsomal free cholesterol pool, the LDL receptor was up-regulated, drawing more cholesterol from plasma, thus leading to the observed decrease in plasma LDL cholesterol concentrations.
KEY WORDS: • corn fiber oil • microsomal cholesterol • LDL receptor • cholesterol 7-hydroxylase • guinea pigs
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). Lowering plasma LDL-C has been advocated to prevent coronary events and morbidity from atherosclerosis (2
). Some nontraditional dietary sources such as plant sterols (3
,4
), rice bran oil (5
), lime-treated corn husks (6
,7
) and corn fiber oil (CFO) (8
,9
) have been shown to exert hypocholesterolemic effects. CFO is obtained from the husk of corn, and the fatty acid profile is similar to that of corn oil. However, CFO also contains 13.9 g/100 g phytosterols, which are responsible for some of the hypocholesterolemic properties of CFO. We demonstrated previously that CFO lowers plasma total and LDL-C by altering hepatic cholesterol and lipoprotein metabolism (8
).
Plasma LDL levels are determined by a balance between the rates of production and clearance from the circulation. The LDL receptor (LDLR) plays a major role in the regulation of plasma LDL-C by mediating nearly two thirds of LDL clearance (10
). The expression of the LDLR gene is regulated by a feedback mechanism involving cholesterol. Cholesterol, derived either from endogenous sources or from the uptake of circulating LDL, acts as a feedback repressor and decreases the transcription of the LDLR gene (10
). In contrast, hepatic cells respond to the decrement in cholesterol levels by up-regulating hepatic LDLR.
Fernandez et al. (11
) documented that guinea pigs fed psyllium, a source of soluble fiber have reduced apolipoprotein B secretion rate and increased LDLR-mediated catabolism. Horton et al. (12
) reported that dietary soluble fiber lowers LDL-C in hamsters by enhancing the conversion of cholesterol to bile acids, which depletes hepatic cholesterol pools and up-regulates LDLR. In addition, mushroom fiber and sugar beet fiber have been shown to decrease serum cholesterol in rats by increasing hepatic LDLR mRNA levels (13
). Furthermore, in vivo studies have clearly shown that dietary cholesterol suppresses hepatic LDLR protein and activity levels primarily at the level of gene transcription (14
). All of these studies demonstrate that dietary constituents play a major role in the regulation of LDLR and thereby plasma LDL-C levels.
The main objective of this study was to determine whether hepatic LDLR are up-regulated by CFO treatment. To elucidate this mechanism, we partially cloned and sequenced the guinea pig LDLR to use as a probe to measure LDLR mRNA abundance through use of nuclease protection assays. We hypothesized that guinea pigs fed CFO would up-regulate hepatic LDLR and this would be one of the major mechanisms by which CFO mediates its hypocholesterolemic effects. Guinea pigs were used as the animal model in this study because of their similarity to humans with respect to lipoprotein metabolism, especially the fact that they transport the majority of cholesterol in the LDL fraction. In addition, like humans, guinea pigs respond to dietary and drug interventions primarily by lowering LDL-C (15
).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Reagents were obtained from the following sources: enzymatic cholesterol kit, cholesterol oxidase, cholesterol esterase and peroxidase from Boehringer-Mannheim (Indianapolis, IN). Free cholesterol enzymatic kits were obtained from Wako Pure Chemical (Osaka, Japan). Halothane was obtained from Halocarbon (Hackensack, NJ). Aquasol, Liquiflor (toluene concentrate) and [14C] cholesterol were purchased from Dupont NEN (Boston, MA). [-32P] CTP (800 Ci/mmol) were purchased from Amersham (Arlington, IL). TRIZOL reagent, restriction endonucleases, T4 DNA polymerase and T7 RNA polymerase were obtained from Gibco BRL (Rockville, MD) and plasmid pBluescript II SK (+) was obtained from Stratagene (Austin, TX). A one-step reverse transcription-polymerase chain reaction (RT-PCR) kit was purchased from Qiagen (Valencia, CA). Glucose-6-phosphate, glucose-6-phosphate dehydrogenase, NADP, EDTA, Ribonuclease A, Ribonuclease T1, formamide, proteinase K and sucrose were obtained from Sigma Chemical (St. Louis, MO). Glass silica gel plates were purchased from EM Science (Gibbstown, NJ). 5- Cholestane was obtained from Steraloids (Wilton, NH). Corn fiber oil was provided by Monsanto Company (St. Louis, MO).
Diets.
Diets were prepared and pelleted by Research Diets (New Brunswick, NJ). Formulation of the five diets is presented in Table 1
. All of the diets were identical in composition except for the type of fat and the amount of dietary cholesterol. Diet I, the control, had 0 g/100 g CFO, 15 g/100 g regular corn oil and 0.25 g/100 g cholesterol. Diets II, III and IV contained 5, 10 or 15 g/100 g corn fiber oil replacing corn oil and 0.25 g/100g dietary cholesterol. Diet V, a low cholesterol (LC) diet had 15 g/100 g regular corn oil and 0.04 g/100 g cholesterol, an amount equivalent to 300 mg/d in humans (16
). The nutrient to energy density was adjusted for all diets (Table 1). A detailed composition of the corn fiber oil was reported previously (8
).
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Male Hartley guinea pigs (n = 50; Harlan Sprague Dawley, Indianapolis, IN), weighing between 300 and 400 g were randomly assigned to one of the five dietary groups (10 per group). Two guinea pigs were kept per metal cage and were housed in a light cycle room (light from 0700 to 1900h) at a temperature of 21°C for 4 wk. The guinea pigs had free access to food and water. They were killed by cardiac puncture after halothane anesthesia and blood was collected to analyze plasma lipids. Liver was harvested to determine hepatic lipids and to isolate microsomes for the measurement of microsomal cholesterol and the activity of cholesterol 7-hydroxylase (CYP7). A portion of the liver was immediately frozen at -70°C for RNA isolation. Animal studies were conducted in accordance with U.S. Public Health Service guidelines. Experimental protocols were approved by the University of Connecticut Institutional Animal Care and Use Committee.
Plasma and hepatic lipids.
Plasma samples were analyzed for cholesterol and triglycerides by enzymatic methods (17
). LDL was isolated by sequential ultracentrifugation in a LE-80K ultracentrifuge (Beckman Instruments, Palo Alto, CA) at d = 1.019–1.09 kg/L in quick-seal tubes at 15°C for 3 h at 200,000 x g in a vertical Ti-65 rotor as previously reported (18
). Hepatic total and free cholesterol were determined according to Carr et al. (19
), after extraction of hepatic lipids with chloroform/methanol (2:1). Cholesteryl ester concentrations were calculated by subtracting free from total cholesterol.
Hepatic microsomal isolation.
Hepatic microsomes were isolated as previously described (20
). Briefly, liver tissues were pressed through a tissue grinder into cold homogenization buffer (1:2.5 wt/v; 50 mmol/L KH2PO4, 0.1 mol/L sucrose, 50 mmol/L KCI, 50 mmol/L NaCl, 30 mmol/L EDTA and 0.002 mmol/L dithiothreitol, pH 7.2) and mixed using a Potter-Elvehjem homogenizer. A microsomal fraction was isolated after two 15-min centrifugations at 10,000 x g followed by ultracentrifugation of the supernatant at 100,000 x g in a Ti-50 rotor at 4°C. The microsomal pellets were resuspended in the homogenization buffer and centrifuged for an additional hour at 100,000 x g and resuspended again before storage at -70°C. The protein content of microsomes was measured according to Markwell et al. (21
).
Hepatic microsomal cholesterol.
Microsomal cholesterol (20 µg protein) was extracted with 2 mL of chloroform/methanol (2:1) overnight. After filtration the next day, 1 mL of acidified water was added and phases were separated with a separating funnel. The lower phase was retained and the volume was adjusted to 2 mL with chloroform/methanol (2:1). An aliquot of 20 µL was dried under nitrogen and the lipids were solublized in 1 mL of Triton X-100. Free cholesterol content was determined by enzymatic methods (17
).
Cloning of cDNA for the guinea pig LDLR.
A cDNA probe for guinea pig LDLR was not available. cDNA was synthesized from hepatic RNA by RT. This 340-nt guinea pig LDLR cDNA fragment was amplified by PCR using primers derived from conserved regions of the published LDLR (22
) as follows: 5' primer 5'-ATTGGAATTCAAGCCCAGGGCCATCGTGGTG-3' and 3' primer, 5'-CACTGAAGATGGCTTCGTTGATG-3'.
PCR was carried out in a DNA thermal cycler with denaturation at 95°C for 45 s, annealing at 42°C for 1 min and primer extension at 72°C for 1 min for a total of 25 cycles followed by a final extension at 72°C for 10 min. This amplified 340-nt LDLR cDNA was purified by gel electrophoresis (Fig. 1
) blunt-ended with T4 DNA polymerase, digested with EcoR1 and subcloned into pBluescript II SK (+) (Stratagene) using standard recombinant DNA techniques. This insert was sequenced to confirm its identity. After linearization of the plasmid DNA with Eco R1, a [-32P] cRNA probe was transcribed from this plasmid using T7RNA polymerase.
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The nuclease protection assay was carried out by the method described by Pape et al. (23
). Total RNA was extracted from liver samples using TRIZOL (Gibco-BRL) reagent following the manufacturer’s instructions, modified by adding 250 µL of isopropyl alcohol and 250 µL of a high salt precipitation reagent (0.8 mol/L sodium citrate and 1.2 mol/L NaCl) to the aqueous phase for RNA precipitation. RNA (30 µg) was suspended in 30 µL of hybridization buffer containing 40% formamide, 600 mmol/L NaCl, 4 mmol/L EDTA, and 40 mmol/L Tris HCl (pH 7.5) along with 70,000 cpm of 32P-labeled LDLR and/or ß-actin cRNA probes. The tubes were incubated at 85°C for 5 min and hybridization was carried out overnight at 65°C. Digestion buffer (300 µL) [10 mmol/L Tris HCl (pH 7.5), 300 mmol/L NaCl, 5 mmol/L EDTA, 40 mg/L Ribonuclease A and 2 mg/L Ribonuclease T1) was added and the digestion reaction was carried out at 30°C for 45 min. The reaction was terminated by adding 20 µL of SDS and 2.5 µL of proteinase K with further incubation at 37°C for 15 min. Following phenol/chloroform extraction, the protected RNA hybrids were precipitated using 5 µg of tRNA as a carrier. The resultant pellets were dissolved in 7 µL loading buffer, heated for 5min at 85°C and loaded on a polyacrylamide sequencing gel. After electrophoresis, the gel was dried and then exposed to film for 1 wk at -70°C to visualize the bands. Parallel samples were used for quantification by trichloroacetic acid (TCA) precipitation. After RNAse digestion, 20 µg of tRNA and 1 mL of cold TCA were added and the samples were placed on ice for 30 min. Samples were then filtered through glass fiber circles and washed with 5% TCA followed by 95% ethanol. Filters were dried and quantified in a scintillation counter. Negative controls utilized tRNA in the hybridization reactions to determine background binding and precipitation of the probe.
Hepatic cholesterol 7-hydroxylase (CYP7).
CYP7 activity was assayed by the method modified by Jelinek et al. (24
) using [14C] cholesterol as a substrate. Cholesterol was delivered as cholesterol/phosphatidylcholine liposomes (1:8 wt/wt) prepared by sonication. A NADPH regenerating system (glucose-6-phosphate dehydrogenase, NADP and glucose-6-phosphate) was included in the assay. Glucose-6-phosphate dehydrogenase (0.3 IU) was added and the samples were incubated for an additional 30 min. The reaction was stopped by adding 5 mL of chloroform/methanol (2:1) and 1 mL of acidified water (5% sulfuric acid). The tubes were thoroughly mixed, the top layer was discarded and the samples were dried under nitrogen. Samples and 7- and 7ß-hydroxycholesterol standards each were dissolved in 100 µL of chloroform, applied to glass silica gel TLC plates and developed in ethyl acetate/toluene (3:2). The plates were exposed to iodine vapors to mark the 7- and 7ß-hydroxycholesterol standards and then placed on XAR-5 film with an intensifying screen over night. Using the film as a reference, the [14C] 7-hydroxycholesterol spots were located and scraped. Liquiflour (5 mL) was added and the radioactivity was quantified in a scintillation counter. The mRNA abundance of CYP7 was measured by RNAse protection assay using a guinea pig cDNA probe (25
).
Statistical analysis.
One-way ANOVA was used to determine differences in total cholesterol, LDL-C, hepatic lipids, hepatic enzyme activity and mRNA abundance among the dietary groups. Newman-Keuls was used as a post-hoc test, to test differences among groups. Data are presented as means ± SD, and differences were considered significant at P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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There were no significant differences in body weight gain or food intake among the different groups (data not shown). Plasma total cholesterol was 30, 49 and 53% lower in the 5, 10 and 15/100 g CFO groups, respectively, compared with controls (0 g/100 g CFO) (P < 0.0005) (Table 2). With increasing doses of CFO, LDL-C was 32, 55 and 57% lower, respectively, compared with the controls (P < 0.0005). CFO intake by all of the groups resulted in plasma total and LDL-C not different from guinea pigs fed relatively low levels of dietary cholesterol (LC diet) (Table 2)
.
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CFO intake resulted in 32–43% lower hepatic total cholesterol compared with the control group (P < 0.001) (Table 3
). Guinea pigs fed the LC diet (0.04 g cholesterol/100 g) had 72% lower hepatic total cholesterol concentrations than the control group. Hepatic free cholesterol was not affected by increasing doses of CFO with the exception of the 5 g/100 g CFO group intake in which the free cholesterol was 41% lower than in the controls (Table 3)
. However, increasing doses of CFO resulted in decreased hepatic cholesteryl ester pools by 47, 70, and 72% compared with the control (Table 3)
.
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Similar to the results for hepatic cholesterol, microsomal free cholesterol concentrations were the lowest in guinea pigs fed the LC diet. Guinea pigs fed the LC diet had 52% lower microsomal cholesterol than the control (P < 0.0001). CFO lowered microsomal free cholesterol by 27–32% compared with the controls (P < 0.0001).
Guinea pig LDLR sequence.
A 340-nt guinea pig LDLR cDNA was cloned and sequenced. Comparison of the guinea pig LDLR partial sequence to the published rat and human LDLR sequences from GenBank using BLAST2 software is presented in Figure 2
. Sequence analysis revealed 87% (302/341) homology to the human cDNA sequence and 84% (285/337) homology to the rat LDLR sequence.
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Guinea pig LDLR mRNA was synthesized in vitro using T3 RNA polymerase to transcribe the opposite strand to verify the size of the fragment protected by guinea pig RNA. An autoradiograph showing the abundance of guinea pig hepatic LDLR (340 nt) and ß-actin (245 nt) riboprobes with different CFO treatment is shown in Figure 3
.
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. All values were normalized to ß-actin mRNA levels measured in parallel. In guinea pigs fed 5, 10 and 15 g/100 g CFO, the hepatic LDLR was up-regulated by 66 (P = 0.14), 88 (P = 0.063) and 150% (P < 0.01), respectively. In addition, guinea pigs fed the LC diet had 2.5-fold higher LDLR mRNA abundance than the controls (P < 0.001) (Table 4)
.
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CFO intake also increased hepatic CYP7 activity compared with the control, indicating an up-regulation of the regulatory enzyme of cholesterol catabolism. This up-regulation was significant only in guinea pigs fed the higher dosages (10 and 15 g/100 g) of CFO, which had CYP7 activities that were 88% greater than in the controls (P < 0.05) (Table 4)
. Guinea pigs fed the LC diet had CYP7 activity that was not different from the controls or the CFO-fed groups (Table 4)
. In parallel, the effect of CFO on CYP7 was also observed at the level of mRNA expression. All three CFO treatments resulted in 88% higher CYP7 mRNA abundance as measured by nuclease protection assays compared with the controls (P < 0.05) (Table 4)
.
In addition, relative amounts of hepatic LDLR mRNA were inversely correlated (r = -0.52, P < 0.05) with microsomal free cholesterol concentrations (Fig. 4
) suggesting a role for this pool of hepatic cholesterol in regulating LDLR expression.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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–28
). Nontraditional dietary sources such as rice bran, corn fiber oil, lime-treated corn husk and plant sterols and stanols have been shown to exert powerful hypocholesterolemic effects (4
–9
). It has been suggested that plant sterols and cholesterol share similarities in structure, which could be responsible for a decrease in cholesterol absorption by competitive inhibition (4
). Previous studies with CFO from our laboratory have shown that CFO lowers plasma total and LDL-C by a similar mechanism (8
). Results from this study suggest that an interruption of the enterohepatic circulation of bile acids is also involved in the hypocholesterolemic effects of CFO. The combination of these two mechanisms (decreased cholesterol and bile acid absorption) lowered the hepatic cholesterol concentrations, which up-regulated LDLR and decreased plasma LDL-C concentrations. CFO and CYP7
The initial and rate-limiting step in the biosynthesis of bile acids is catalyzed by CYP7 activity. This enzyme is subjected to feedback regulation by bile acids fluxing through the liver in the enterohepatic circulation (29
). Disruption of the enterohepatic circulation of bile acids has been shown to suppress the reabsorption of bile acids. This results in an up-regulation of CYP7, leading to an increase in bile acid synthesis from cholesterol (29
). ß-Cyclodextrin and cholestyramine have been shown to decrease cholesterol absorption by increasing fecal bile acid excretion in hamsters (30
). An inverse relationship between CYP7 activity and reabsorbed bile acids was documented in rats by Shefer et al. (31
). We determined that mRNA abundance and activity of CYP7 are increased by CFO intake, which suggests an interruption in the enterohepatic circulation of bile acids. It could be that in addition to increasing fecal neutral sterols, CFO treatment also increased fecal excretion of bile acids. Thus, as a response to the enhanced bile acid loss, bile acid biosynthesis was increased through the up-regulation of CYP7.
Many dietary interventions have shown that the accelerated conversion of cholesterol to bile acids due to alterations in bile acid homeostasis lowers LDL-C concentrations. For example, when bile acid–binding resins were administered to humans, there was a fivefold increase in CYP7 activity with a concomitant 15–20% decrease in plasma LDL-C concentration (32
). Roy et al. (25
) documented that feeding psyllium, a source of dietary soluble fiber, results in the up-regulation of CYP7 in guinea pigs. Spady and co-workers (33
) demonstrated that adenovirus-mediated overexpression of the CYP7 gene in hamsters results in an increase in CYP7 enzyme activity and a decrease in plasma total and LDL-C. In the present study CFO intake decreased the concentrations of hepatic microsomal free cholesterol and hepatic esterified cholesterol. CYP7 activity and mRNA abundance were almost doubled in the CFO groups compared with controls, confirming that CYP7 activity is regulated at the pretranslational level.
CFO and LDLR
Microsomal free cholesterol is the regulatory pool for LDLR expression (34
). In addition, free cholesterol is also a key regulator of cholesterol biosynthesis and esterification. In this study, we observed 30% lower microsomal free cholesterol with CFO treatment compared with the control group. In our preliminary study with CFO, we showed that it had no effect on 3-hydroxyl-3-methyl-glutaryl-CoA reductase, the regulatory enzyme of cholesterol synthesis (8
). In contrast, we observed that CFO feeding resulted in a dose-dependent decrease in the activity of acyl-CoA:cholesterol acyltransferase-1, the regulatory enzyme of cholesterol esterification (8
). This suggests that although hepatic cholesterol was lowered, the magnitude was not sufficient to induce cholesterol synthesis. We hypothesized then that in response to the depleted hepatic pool, the LDLR is up-regulated and this is indeed what we have demonstrated in the present study.
Dietary fatty acids and cholesterol have been shown to have an effect on LDLR expression. In hamsters, a polyunsaturated fat compared with a saturated fat diet has been shown to increase LDLR-mediated catabolism (34
). In guinea pigs, the intake of polyunsaturated fat results in a lowering of plasma LDL-C due to a greater number of hepatic LDLR and increased LDL turnover compared with saturated fat intake (35
). Increasing doses of dietary cholesterol have been shown to lower the number of hepatic LDLR in a dose-dependent manner in guinea pigs (36
). In addition, Spady et al. (37
) also showed that there is an increase in the cellular sterol regulatory pool when high amounts of cholesterol are presented to the liver through the diet. In the present study, we observed an inverse correlation between microsomal free cholesterol and hepatic LDLR mRNA abundance.
In this study, the guinea pig LDLR cDNA was partially cloned and sequenced for the first time. This is important because guinea pigs are good models with which to study lipoprotein metabolism and because they respond to dietary treatment similarly to humans (15
). It is also important to note that this partial sequence is well conserved among species because there is 87% homology to the human LDLR sequence. In summary, CFO intake depleted the hepatic and microsomal cholesterol pools, which led to the induction of hepatic LDLR as evidenced by the observed increase in LDLR mRNA abundance. This up-regulation of the hepatic LDLR resulted in an enhanced clearance of LDL particles, thus contributing to the observed lowering of plasma LDL cholesterol.
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FOOTNOTES |
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3 Abbreviations used: CFO, corn fiber oil; CYP7, cholesterol 7-hydroxylase; LC diet, low cholesterol diet; LDL-C, LDL cholesterol; LDLR, LDL receptor; RT-PCR, reverse transcription-polymerase chain reaction; TCA, trichloroacetic acid.
Manuscript received 14 September 2001. Initial review completed 18 October 2001. Revision accepted 5 December 2001.
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LITERATURE CITED |
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