Biochemical and Molecular Roles of Nutrients

Prickly Pear (Opuntia sp.) Pectin Alters Hepatic Cholesterol Metabolism without Affecting Cholesterol Absorption in Guinea Pigs Fed a Hypercholesterolemic Diet It2 MARIA LUZ FERNANDEZ,~EMME C. K. LIN, AUGUST0 TREJO* DONALD J. McNAMARA

AND

Department of Nutritional Sciences and Interdisciplinary Nutritional Sciences Program, University of Arizona, Tucson, AZ 85721 and *CIIDIR-IPN, Unidad Michoacan, Jiquilpan de Juarez, Michoacan, Mexico 59510

ABSTRACT Prickly pear pectin intake decreases plasma LDL concentrations by increasing hepatic apolipoprotein B/E receptor expression in guinea pigs fed a hypercholesterolemic diet. To investigate whether prickly pear pectin has an effect on cholesterol absorption and on enzymes responsible for hepatic cholesterol homeostasis, guinea pigs were fed one of three semipurified diets, each containing 15 g lard/100 g diet: 1) the lard-basal diet with no added cholesterol or prickly pear pectin (LB diet); 2) the LB diet with 0.25 g added cholestero1/100 g diet (LC diet); or 3) the LC diet containing 2.5 g prickly pear pectin/100 g diet, added at the expense of cellulose (LC-P diet). Animals fed the LB diet had the lowest plasma LDL and hepatic cholesterol concentrations, followed by animals fed the LC-P diet ( P < 0.001). Hepatic 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase activity was highest in the group fed the LB diet, with similar values for animals in the other two groups. A positive correlation existed between plasma LDL cholesterol concentration and hepatic acyl CoA:cholesterol acyltransferase activity (r = 0.87, P < 0.001). Cholesterol absorption was not different among the three dietary groups. These results indicate that the decreased plasma and hepatic cholesterol concentrations of animals fed prickly pear pectin are not explained by differences in cholesterol absorption but rather are due to mechanisms that alter hepatic cholesterol homeostasis, resulting in lower plasma LDL concentrations. J. Nutr. 124: 817-824, 1994. INDEXING KEY WORDS: acy 1 CoA:cholesterbl acy ltransferase 3-hydroxy-3-methylglutayl coenzyme A reductase cholesterol pectin guinea pigs

I

High dietary fiber intake has been shown to be Protective against ischemic heart hsease mortality (Khaw and Barrett-Connor 1987). Because various di-

etary soluble fibers, including pectin, guar gum, psyllium and oat bran, have been shown to decrease plasma cholesterol concentrations in humans and several animal species (Everson et al. 1992, Fernandez et al. 1990 and 1992a, Kelley and Tsai 1978, Spiller et al. 1991), a protective effect against ischemic heart disease could be expected from these soluble fibers. One of the proposed mechanisms for this hypocholesterolemic effect relates to the viscous properties of soluble fibers, which disrupt micelle formation, interfere with lipid absorption and decrease delivery of cholesterol to the liver (Ulrich 1987). Increased binding of bile acids by functional groups of soluble dietary fibers has also been proposed as a primary mechanism by which these compounds decrease plasma cholesterol concentrations [Story 1986). The specific effects of soluble fiber on cholesterol absorption and hepatic cholesterol homeostasis vary, depending on the experimental design, the animal model and the soluble fiber tested. Psyllium has been shown to decrease plasma LDL cholesterol in hypercholesterolemic men by decreasing cholesterol absorption [Everson et al. 1992). In contrast, studies using African green monkeys could not relate the hypocholesterolemic effects of psyllium to effects on cholesterol absorption, neutral steroid excretion or hepatic 3-hydroxy-3-methylglutaryl coenzyme A

l ~ u ~ ~ o rby t e ad grant-in-aid from the American Heart Association, Arizona Affiliate (IG-2-11-91). 2 ~ h costs e of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734 solely to indicate this fact. 3 ~ whom o correspondence and reprint requests should be addressed.

0022-3166194 $3.00 O 1994 American Institute of Nutrition. Manuscript received 30 September 1993. Initial review completed 24 November 1993. Revision accepted 31 January 1994. 817

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1

FERNANDEZ ET AL.

reductase [HMG-CoA reductase, EC 1.1.1.34J4 activity (McCall et al. 1992). The hypocholesterolemic effect of pectin in rats fed a hypercholesterolemic diet has been attributed to decreased cholesterol absorption and increased cholesterol turnover (Kelley and Tsai 19781, and chitosan and guar gum have also been shown to effectively decrease cholesterol absorption in rats (Ebihara and Schneeman 1989, Ikeda et al. 1989). Intake of prickly pear pectin, compared with a control diet, has been shown to decrease plasma cholesterol concentrations in guinea pigs by increasing hepatic apolipoprotein (apo]B/E receptor expression and receptor-mediated LDL turnover (Fernandez et al. 1990 and 1992a). This increase in LDL receptor expression is considered to be a secondary metabolic response induced by changes in hepatic cholesterol homeostasis due to prickly pear pectin intake. The present studres were designed to evaluate relationships between the regulation of hepatic cholesterol homeostasis and plasma LDL concentrations to determine potential regulatory sites in addition to the increase in LDL receptor expression, by w h c h prickly pear pectin intake is hypocholesterolemic in guinea pigs. Because animals fed the diet containing prickly pear pectin had lower hepatic cholesterol pools than animals fed a control diet (Fernandez et al. 1992a1, the activities of the key enzymes of cholesterol synthesis and esterification [HMG-CoA reductase and acyl CoA:cholesterol acyltransferase (ACAT, EC 2.3.2.261) and rates of cholesterol absorption were measured to further evaluate effects of prickly pear pectin on hepatic cholesterol homeostasis. Guinea pigs were chosen in this and previous studies (Fernandez et al. 1990 and 1992a) as the experimental animal model because of their similarities to humans in terms of the plasma lipoprotein profile (Fernandez et al. 1992b), the drstribution of the hepatic free and esterified cholesterol pools, and the relative activities of the hepatic enzymes (Angelin et al. 1992).

TABLE composition

Component

I;

01 +e

LB

LC

( Protein (soybean] Fat (lard) Carbohydrate (sucrose-comstarchj2 Cellulose Guar gum Prickly pear pectin Cholesterol Vitamin mix3 Mineral m d Energy, kJlg

22.4 15.1

1

: 3.1

I

0.0

i

-

1.1

diets

I'

8.2 i 15.9

LC-P

g1100 g diet 22.4 15.1

39.6 10.5 3.1

-

0.25 1.1

8.2 15.9

22.4 15.1 39.6 8.0 3.1 2.5 0.25 1.1 8.2 15.9

1

l ~ i e abbreviations: t LB, lard diet ith no added cholesterol; LC, lard-cholesterol diet; LC-P, lard-chol sterol-pectin diet. ' ~ a t i o of sucrose to starch was .43:1. 3 ~ i n e r a land vitamin mixes werk ,formulated to meet NRCspecified requirements for guinea pigs. Compositions of the mineral and vitamin mixes were preqiously reported (Femandez et al. 1992a).

and hydroperoxidase were pur hased from Boehnnger Mannheim [hdianapolis, IN and halothane from Halocarbon (Hackensack, NJ). pear pectin was isolated by the method of A. Trejo [patent pending]. Diets. Diets were prepareb and pelleted by Research Diets (New Brunswiclq, NJ]. The semipurified diets contained 15 g lard/100 g stearic, 4 1 g oleic and 11 g with no added cholesterol 0.25 g cholestero1/100 g control (LC)and the in composition except for ths fiber sources (Table 1). Cellulose in the LC diet wqs partially replaced by prickly pear pectin (2.5 gJ100 diet) in the LC-P diet. The LB diet had the same co position as the LC diet (but without the added choles erol) (Table 1) and contained a minimal amount of ciolesterol (0.012 gJ100 g diet) from the lard. a pigs, purchased from Animals. Male H MATERIALS AND METHODS NE) and weighmg 250 Sasco Sprague Dawl to 300 g, were random ed to one of the three Materials. ~ ~ - H ~ d r o x ~ - [ 3 - ~ ~ ~glutaryl ]meth~l in plastic cages, three dretary groups and wer coenzyme A (1.81 GBqJmmol), ~ ~ - [ 5 - ~ ~ ] m e v a l o nanimals ic per cage. Aft rumals were killed by acid 1370 GBqJmmol], cholesteryl-[1,2,6,7-3~]oleate anesthetization with vapors and exsangui(370 GBq/mmol], [ 1,2,3~(N)]cholesterol ( 1650.2 GBq/ nation by cardiac pu tain liver and plasma mmol), [4-14C]cholesterol (1.9 GBq/mmol), Aquasol and Liquifluor were purchased from Du Pont NEN Products [Boston, MA). ~leoyl-[l-14C]coenzymeA [ 1.8 GBqJmmol) and DL-3-hydroxy-3-methylglutaryl 4~bbreviationsused: ACAT, ac/l CoA:cholesterol acylt~mscoenzyme A were purchased from Amersham (Clearferase; apo, apolipoprotein; HMG-CoA reductase, 3-hydroxy-3brook, IL), and cholesteryl oleate was from Sigma methylglutaryl coenzyme A reductase. Diet abbreviations: LB diet, Chemical [St. Louis, MO]. Enzymatic cholesterol lard diet with no added cholesterol; LC diet, lard-cholesterol diet; assay kits, cholesterol oxidase, cholesterol esterase LC-P diet, lard-cholesterol-pectin diet.

EFFECTS OF PRICKLY PEAR PECTIN

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for isolation of hepatic microsomes and plasma lipoproteins. Animals used for the absorption studies were lulled by an excess of halothane vapors. All animals consumed equal amounts of diet, and there were no significant hfferences in weight gain or final body weight among dietary groups. All animal experiments were conducted in accordance with U.S. Public Health Service and U.S. Department of Agriculture guidelines, and experimental protocols were approved by the University of Arizona Institutional Animal Care and Use Committee. Plasma and liver l b i d s . Total plasma cholesterol was determined by enzymatic analysis [Allain et al. 1974) using commercial kits from Boehringer ~ a n n h e i m Hepatic . concentrations of total and free cholesterol were measured by the method of Sale et al. (1984), and esterified cholesterol concentrations were estimated by subtracting hepatic free from total cholesterol. iblicrosome isolation- Guinea pigs were hlled at the nadir of the diurnal rhythm (0900 h), livers were removed, and hepatic microsomes for HMG-COA reductase and ACAT assays were isolated by pressing liver tissue through a tissue grinder into 1:3 homogenization buffer (50 mmol/L KH2P04~ O.l L 50 mmO1/L KC1l 50 mmO1/L NaCk 30 mmO1/LEDTA and mmO1/LdithiOthreitO1, pH 7.2). r his preparation was further homogenized with a POtter-E1venjhem A microsOmal fraction was isolated by two 15-min centrifugations at 10,000 x g [JA-20rotor in a J2-21 centrifuge, Beckrnan Instruments, Palo Alto, CA) followed by a 1-h centrifugation at 100,000 x g in a Ti-50 rotor at 4°C. Microsomes were resuspended in the homogenization buffer and centrifuged an additional hour at 100,000 x g. After centrifugations, microsomal pellets were homogenized and stored at -70°C. Microsomal protein was determined according to the method of Markwell et al. (1978). Hepatic enzyme assays. Microsomal HMG-CoA reductase activity was measured by incubating 200 pg of microsomal protein with [3-14C]HMG-CoA as previously described (Fernandez et al. 1994). The HMG-CoA reductase activity is expressed as picomoles of [14C]mevalonate produced per minute per milligram of microsomal protein. Recoveries of [3~]mevalonatewere between 60 and 80%. The ACAT activity was determined by preincubating 0.8 to 1.0 mg per assay of microsomal protein with 84 g/L albumin in a final volume of 0.18 mL for 5 min at 37°C; then 20 pL (500 pmol/L) of oleoyl-[114~]coenzymeA (0.15 GBq/pmol) was added and reaction proceeded as previously described (Fernandez et al. 1994. Smith et al. 19861. Recoveries of the [3H']cholesteryl oleate were between 75 and 90%. Absorption studies. Cholesterol absorption was determined by the method of Zilversmit 11972). This method has been validated both in humans (Samuel

et al. 1978) and in guinea pigs [Traber and Ostwald 1978). Food was withdrawn from animals 12 h before they were given 370 GBq of [1,2,3~(~)]cholesterol orally by adding 10 pL of the radiolabeled isotope to one pellet of the corresponding diet. The [3~]cholesterol was readily absorbed by the pellet, and guinea pigs consumed it within 2 min, followed by appropriate amounts of water and food. Once the pellet was consumed, animals were injected with 5.63 GBq of [4-14C]cholesterol through an indwelling catheter inserted in the jugular vein. The [14C]cholesterol was mixed with guinea pig plasma, incubated for 15 min at 3 7°C and vortexed for 10 min prior to injection. Blood samples were taken every 12 h, plasma was separated from red blood cells, plasma cholesterol was measured, and an aliquot (0.05 to 0.1 mL) was mixed with 5 mL of Aquasol and counted for radioactivity over 4 d to ensure that the plasma isotopic ratio had reached equilibrium [Fig. 1). Cholesterol absorption was calculated from the ratio of 1 4 (injected ~ dose) to 3~ [oral dose) multiplied by the ratio of 313 to 1 4 in ~ plasma at the time of equilibrium. Statistical analysis. Analysis of variance and the least significance difference test were used to evaluate differences in the measured variables of plasma cholesterol, hepatic cholesterol, HMG-CoA reductase, ACAT and absorption of cholesterol among the three dietary groups. Regression analyses were performed to

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(D

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u

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0

c 0

t U)

to

1

3H (PO)

100: 6

0

0

*

.

.

.

I4C (IV) s . .

Time (h) FIGURE 1 Specific activity-time course curves of plasma cholesterol from 0 to 96 h after intravenous administration [4-~4C]cholesterol and oral of [1,2~~]cholesterol in a guinea pig fed the lard-cholesterol (LC) bet.

FERNANDEZ E T AL.

820

determine significant correlations between variables. Differences were considered significant at P c 0.05.

TABLE 3

Hepatic free and esterified cholesterol concentrations in guinea pigs fed the experimental diets for 4 wkl

RESULTS

Hepatic cholesterol

No significant differences in body weight were observed for animals from the three dietary groups (Table 2). Plasma total and LDL cholesterol concentrations were lowest in animals fed the LB diet, intermediate in the group fed the LC-P diet and highest in animals fed the LC &et (P < 0.0001). Plasma LDL cholesterol concentrations were 3 1% lower in the group fed the LC-P diet compared with values for the LC diet-fed group (Table 21. This plasma cholesterol lowering was specific to LDL, because plasma VLDL and HDL cholesterol concentrations were unaffected (data not shown). Hepatic free and esterified cholesterol concentrations were lowest in animals fed the LB diet. Intake of cholesterol (LC diet) resulted in higher hepatic cholesterol concentrations, and intake of prickly pear pectin partially lowered these values. Compared with values for animals fed the LC diet, hepatic free and esterified cholesterol concentrations were reduced in animals fed the LC-P diet by 18 and 57%, respectively (Table 31 (P < 0.001). The activity of hepatic HMG-CoA reductase was significantly lower in animals fed the LC and LC-P diets than in animals fed the LB diet (P< 0.001.).The HMG-CoA reductase activities did not differ between animals fed the LC diet and those fed the LC-P diet indicating that the suppression of hepatic HMG-CoA reductase activity in response to high dietary cholesterol (0.25 g/100 g diet] was not reversed by prickly pear pectin intake (Table 4).

-

Dietary group2 LB (71 LC (81 LC-P 18)

Free

Ester

-

p o l l g liver 5.9 f 0.7a 0.4 k 0.3a 10.9 f 1.3C 7.5 f 3.1' 8.9 f 2.3b 3.2 k 1 . 6 ~ -

'values are means f SD for the number of guinea pigs shown in parentheses. Values in a column with different superscripts are significantly different (P < 0.001). ' ~ i e tabbreviations: LB, lard dlet (15.1 g lard/100 g diet) with no added cholesterol; LC, the LB diet with added cholesterol (0.25 g cholestero1/100 g diet) (no pectin); LC-P, the LC diet with 2.5 g prickly pear pectin/100 g diet, added at the expense of cellulose.

Hepatic ACAT activity was significantly higher in animals fed 0.25 g cholestero1/100 g diet (LC and LCP diets) than in animals fed the LB diet IP < 0.01) (Table 4). Prickly pear pectin intake reduced the higher hepatic ACAT activity induced by the hypercholesterolemic diet by 21%, although, due to the variability within animals, the value was not significantly different from that of animals fed the LC diet [P.=0.09). A significant positive correlation was found between plasma LDL cholesterol concentration and hepatic ACAT activity (I = 0.87, P < 0.001) (Fig. 2). Significant correlations were found between hepatic cholesterol concentration and the activities of hepatic HMG-CoA reductase and ACAT (Fig. 3) for animals fed the LB, LC and LC-P diets. HMG-CoA reductase was negatively correlated with total hepatic cholesterol concentration (r = -0.77, P < 0.001) (Fig. 3,

TABLE 2

Body weight nod plasma total and LDL cholesterol concentrations of guinea pigs fed the experimental diets for 4 wkl -

--

p ~ p ~

--

-

Plasma cholesterol Dietary group LB (10) LC (16) LC-P (17)

Body weight

Total

g 548 f 30 551 k 49 533 i 60

LDL

mmollL 2.15 f 0.39a 1.55 f 0.33a 8.30 f 2.97' 7.16 f 2.8ZC 5.69 f. 2 . 0 9 ~ 4.86 f 1 . 8 9 ~

]values are means i SD for the number of animals shown in parentheses. Values in a column with different superscripts are significantly different (P < 0.000 1 2 ~ i eabbreviations: t LB, lard diet (15.1 g lard/100 g diet) with no added cholesterol; LC, the LB diet with added cholesterol (0.25 g cholestero1/100 g diet) (no pectin); LC-P, the LC diet with 2.5 g prickly pear pectin/lOO g diet, added at the expense of cellulose.

I.

TABLE 4

Hepatic 3-hydroxy-3-methylglutaryl CoA (HMGCoA) reductase and acyl CoA:cholesteryl wansferase (ACAT) activities in guinea pigs fed the experimental diets for 4 wkl Dietary group2 LB 171 LC (8) LC-P (8)

HMG-CoA reductase

ACAT

pmoll(min.mg protein) 38 f 1 4 ~ 5 f la 6 f 4a 83 f 1 7 ~ 8 k 5a 66 k 21b

l ~ a l u e sare means f SD for the number of assays shown in parentheses. Values in a column with different superscripts are significantly different (P < 0.01). = ~ i eabbreviations: t LB, lard diet 115.1 g lard/100 g diet) with no added cholesterol; LC, the LB diet with added cholesterol (0.25 g cholestero1/100 g diet] (no pectin]; LC-P, the LC diet with 2.5 g prickly pear pectin/100 g diet, added at the expense of cellulose.

PLASMA LDL (mmol/L) FIGURE 2 Correlation between plasma LDL cholesterol concentrations and hepatic acyl CoA:cholesterol transferase (ACATJactivities of guinea pigs fed the lard-basal (LB),lardcholesterol [LC) or lard-cholesterol-pectin [LC-P)diet. Each point represents individual animals; r = 0.87, P < 0.001.

upper panel), and ACAT was positively correlated with hepatic cholesterol concentration (r = 0.84, P < 0.001) (Fig. 3, lower panel). The plasma isotopic ratio of oral to intravenous cholesterol was constant 48 h after the administration of the isotopes, and ratio curves were parallel for all animals tested from the different dietary groups. Figure 1 represents a typical plasma cholesterol radioactivity curve for an animal fed the LC diet. Neither dietary cholesterol nor dietary prickly pear pectin affected cholesterol absorption, and no dfferences were found among guinea pigs fed the LB, LC and LCP diets (Table 5). These data indicate that

HEPATIC CHOLESTEROL (ymollg)

FIGURE 3 Correlation between hepatic cholesterol concentrations and 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity (upper panel) and between hepatic cholesterol concentrations and acyl CoA:cholesterol transferase (ACAT)activity (lower panel) of animals fed the lard-basal (LB), lard-cholesterol [LC) or lard-cholesterolpectin (LC-P)diet. Each point represents individual animals; r = -0.77, P < 0.001 (upper panel) and r = 0.84, P < 0.001 [lower panel).

TABLE 5

se

-

Cholesterol absorption by guinea pigs fed the experimental diets for 4 wkl 1

Dietary group2

Cholesterol absorption3

I

- I in Lre I

'

g ' g

I

se. j

LB I31 LC (61 LC-P 141 'values are means f SD for the number of guinea plgs shown in parentheses. ' ~ i e tabbreviations: LB, lard diet (15.1 g lard/100 g diet] with no added cholesterol; LC, the LB diet with added cholesterol (0.25 g cholestero1/100 g diet] (no pectin]; LC-P, the LC diet with 2.5 g prickly pear pectin/100 g diet, added at the expense of cellulose. 3~holesterolabsorption was determined by the dual isotope method as described in Materials and Methods.

I

mechanisms other than major effects on cholesterol absorption are responsible for the decreased hepatic and plasma cholesterol concentrations observed in animals fed the LC-P diet compared with the LC diet.

DISCUSSION Viscous polysaccharides such as psyllium have been found to decrease cholesterol absorption and to increase the fractional turnover of chenodeoxycholic and cholic acids in humans (Everson et al. 1992). A reduction in cholesterol absorption and an increased

822

FERNANDEZ ET AL.

rate of catabolism of cholesterol to bile acids have also been observed in hamsters fed psyllium mucilloid (Turley et al. 1991). S t u l e s using pectin in humans and rats have found that, although pectin is of generally low viscosity, it reduces cholesterol absorption (Kay and Truswell 1977, Kelley and Tsai 1978, Vahouny et al. 1978). Those studies, however, used higher concentrations of pectin than we used in the present study. The interference of pectin and other sources of soluble fiber with bile acid reabsorption has been previously demonstrated (Kay and Truswell 1977, ~ i e t t i n e nand Tarpila 1977, Turley et al. 1991). One possible mechanism by which prickly pear pectin could have a hypocholesterolemic effect would be its ability to bind bile acids, which would reduce hepatic cholesterol concentration by increasing the demand for hepatic cholesterol. However, it is important to mention that the amount of dietary pectin used in these previous studies (Miettinen and Tarpila 1977) is much higher than the amount (2.5 g/100 g l e t ! used in the present investigation. Another possible mechanism by which prickly pear pectin could have a hypocholesterolemic effect is by production of shortchain fatty acids resulting from fermentation of the fiber in the colon. In experimental animals, shortchain fatty acids, mainly propionic and acetic, have been shown to stimulate insulin secretion and reduce plasma glucose concentrations (Brockman 1982). However, Koseki et al. (1991)reported that rats fed a lard l e t had a lower production of short-chain fatty acids in the colon and that addition of pectin did not reverse t h s reduction, suggesting negligible effects on short-chain fatty acid production with pectin intake. In these s t u l e s we have demonstrated that the hypocholesterolemic effects of prickly pear pectin on hepatic and plasma cholesterol concentrations do not result from a significant reduction in exogenous cholesterol absorption. However, intake of prickly pear pectin does lower hepatic cholesterol concentration by as yet undefined mechanisms, resulting in effects on hepatic cholesterol homeostasis and on plasma LDL concentrations. Traber and Ostwald (1978) did not find differences in cholesterol absorption in guinea pigs fed either a cholesterol-free diet or a diet containing 1 g cholestero1/100 g, and they concluded that guinea pigs lack an effective mechanism to limit absorption of cholesterol. Similaxly, we found no lfferences in cholesterol absorption in guinea pigs fed the LB diet (0.012 g cholestero1/100 g) or the LC l e t (0.25 g cholesterol/ 100 g] diet. Our values for cholesterol absorption are higher than that previously found in guinea pigs fed 5 g fat1100 g diet (Traber and Ostwald 19781; however, in the present study we fed 15 g fat1100 g det, and the higher concentration of dietary fat might have resulted in higher values for cholesterol absorption. Studies of the effects of citrus pectin intake on

hepatic and plasma cholesterol concentrations in '. guinea pigs have demonstrated that doses of citrus . pectin in the range of 7.5 to 10 g/100 g diet (Fernandez et al. 1994) result in hepatic cholesterol con: ' centrations similar to those found in guinea pigs fed 2.5 g prickly pear pectin/100 g l e t . These data suggest that these two sources of dietary pectin result " in different dose-response effects on hepatic ' cholesterol homeostasis. In contrast to these observa. tions, studies with humans and rats using pectins varying in the extent of methylation have reported , similar hypocholesterolemic effects (Judd and ' Truswell 1982 and 1985), indcating that the chemical composition of the pectin is not necessarily a major determinant of the extent of the hypocholesterolemic response. From these reports testing other sources of I pectin and the data from the present study, we conclude that dietary pectin can significantly alter cholesterol absorption and bile acid metabolism, however, the dosage must be higher than the levels used in the present study. Prickly pear pectin intake I results in decreases in plasma LDL cholesterol and i hepatic cholesterol concentrations, as demonstrated in this and other studies (Fernandez et al. 1990 and 1992a); however, the primary mechanism by which , these metabolic changes occur is still unknown. Previous s t u l e s in guinea pigs fed the LC and LC-P 1 diets have shown that the hypocholesterolemic effect I of .prickly pear pectin was partly due to increased expression of hepatic apo B/E receptors and increased receptor-mediated LDL turnover (Fernandez et al. 1992a). Lower hepatic cholesterol concentrations seem to be related to increased expression of LDL receptors (Fernandez et al. 19941, possibly due to changes in a putative regulatory pool of hepatic free cholesterol, which modulates hepatic apo B/E receptor expression (Daumerie et al. 1992).Consistent with this concept, we have found that increasing : doses pi citrus pectin are positively correlated with decreased hepatic cholesterol concentrations and in- : creased expression of hepatic apo B/E receptors (Fernandez et al. 1994). Shinnick et al. (1988) reported similar decreases in hepatic cholesterol concentrations with increasing oat bran doses-in rats, and these : decreases correlated with decreased plasma cholesterol concentrations. It should be noted, however, that animals fed the LB diet have similar hepatic LDL receptor numbers as animals fed the LC-P diet (Fernandez et al. 1992a)but have significantly lower plasma LDL cholesterol concentrations than animals fed the hypercholesterolemic diet with prickly pear pectin (LC-P diet], a finding that indicates other alterations in hepatic cholesterol homeostasis and lipoprotein metabolism affecting plasma LDL concentrations independent of apo B/E receptor expression. Jenkins et al. (19781 demonstrated that dietary fiber intake decreased plasma glucose and insulin concentrations in healthy a

/'

I

'

t

'

EFFECTS O F PRICKLY PEAR PECTIN

humans, indicating that fiber intake modifies insulin: glucagon ratios. Recent studies using rats have demonstrated that, although both insulin and 2rglucagon increase LDL receptor expression, the Inmechanisms differ in that glucagon regulation is posted translational and more effective in decreasing plasma ~ta I LDL cholesterol concentrations (Ruding and Angelin llt 1993). The increases in LDL receptor binding and LDL :ic receptor-mediated turnover induced by prickly pear ra-. pectin in guinea pigs fed a hypercholesterolemic diet , (Femandez et al. 1992a) could be associated with ed modifications in insu1in:glucagon ratios which would nd alter LDL receptor expression. :a1 Hepatic HMG-CoA reductase activity was supor pressed by the hypercholesterolemic diet, and prickly ~ic pear pectin did not reverse this suppression. Although of animals fed the LC-P chet had lower hepatic ncholesterol concentrations than animals fed the LC er diet, these values were higher than those found in n; :Is , guinea pigs fed the LB diet. Hepatic HMG-CoA reductase suppression seems to be the first compenke satory mechanism by which guinea pigs respond to a ld hetary cholesterol challenge [Femandez et al. 1994, PA Lin et al. 1992), and it is n i t correlated with plasma , LDL concentrations. In contrast, hepatic ACAT ac3h tivity was positively correlated with plasma LDL concentrations, consistent with a role for ACAT in deter-p mining the rate of incorporation of cholesteryl ester into nascent VLDL particles, which, through the delipidation cascade, eventually determines plasma LDL concentrations [Carr et al. 19921. Studies using rats have shown that higher levels of dietary cholesterol results i n greater secretion of triglyceride and cholesteryl esters associated with increased concentrations of VLDL and LDL (Fungwe et al. 1992). These authors also found a decreased activity of mitochondria1 camitine palmitoyltransferase, the rate-limiting enzyme of fatty acid oxidation (Fungwe et al. 1993), which suggests that chetary cholesterol may reduce rates of fatty acid oxidation, possibly by direct stimulation of hepatic ACAT and increased cholesteryl ester incorporation into VLDL. Although animals fed the LC-P diet have greater receptor-mediated LDL turnover rates compared with animals fed the LC diet, both dietary groups have similar apo B LDL flux rates [Fernandez et al. 1992a), suggesting that the rate of conversion of VLDL to LDL is similar. In contrast, animals fed the LB diet have a receptor-mediated LDL turnover similar to that of animals fed the LC-P diet but lower apo B LDL flux rates (Fernandez et al. 1992b), suggesting either a decreased production of VLDL or a decreased conversion of VLDL into LDL. Results from this study are consistent with the hypothesis that in animals fed the hypercholesterolemic l e t s [LC and LC-P), compared with animals fed the LB diet, ACAT increases the rate of cholesteryl ester incorporation into VLDL, which in turn (via the delipidation cascade) increases in

1

8

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plasma LDL concentrations. Because receptor expression is induced by prickly pear pectin, plasma LDL concentrations are lower in animals fed the LC-P diet than in those fed the LC diet. However, because of increased rates of formation of VLDL associated with hepatic ACAT activity, plasma LDL cholesterol concentrations are higher in animals fed the LC-P chet than in guinea pigs fed the LB diet. We conclude from this study that the plasma cholesterol lowering induced by prickly pear pectin intake is related to decreases in hepatic cholesterol concentrations in guinea pigs fed a hypercholesterolemic diet. These decreases in hepatic cholesterol increase apo B/E receptor expression, tend to lower hepatic ACAT activity and do not change hepatic HMG-CoA reductase, and these changes in hepatic cholesterol homeostasis have a major effect on plasma LDL cholesterol concentrations.

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cholesterol, fecal bile acids and biliary lipids in normolipidemic and hyperlipidemic inhviduals. Clin. Chim. Acta 77: 471-477. Rudling, M. & Angelin, B. (1993) Stimulation of rat hepatic low density lipoprotein receptors by glucagon. Evidence of a novel regulatory mechanism in vivo. J. Clin. Invest. 91: 2796-2805: Sale, F. O., Marchesini, S., Fishman, P. H. & Berra, B. (1984) A sensitive enzymatic assay for determination of cholesterol in lipid extracts. Anal. Biochem. 142: 347350. Samuel, P., Crouse, J. R. & Ahrens, E. H., Jr. (1978)Validation of an isotope ratio method for measurement of cholesterol absorption in man. J. Lipid Res. 19: 82-94. Shinnick, F. L., Longacre, M. J., Ink, S. L. & Marlett, J. A. (1988)Oat fiber: composition versus physiological function in rats. J. Nutr. 118: 144-151. Smith, J. L., de Jersey, J., Pillay, S. P. & Hardie, I. R. (19861Hepatic acyl-CoA:cholesterol acyltransferase. Development of a standard assay and determination in patients with cholesterol gallstones. Clin. Chim. Acta 158: 271-282. Spiller, G. A., Farquhar, J. W., Gates, J. E. & Nichols, S. F. (1991) Guar gum and plasma cholesterol. Effect of guar gum and oat fiber on plasma lipoproteins in hypercholesterolemic adults. Arterioscler. Thromb. 11: 1204-1208. Story, J. A. (1986)Modification of steroid excretion in response to dietary fiber. In: Dietary Fiber, Basic and Clinical Aspects (Vahouny,. G. V. & Kntchevsky, D., eds.), pp. 253-264. Plenum Press, New York, NY. Traber, M. G. & Ostwald, R. (1978) Cholesterol absorption and steroid excretion in cholesterol fed p n e a pigs. J. Lipid Res. 19: 448-456. Turley, S. D., Daggy, B. P. & Dietschy, J. M. (1991) Cholesterollowering action of psyllium mucilloid in the hamster: sites and possible mechanisms of action. Metabolism 40: 1063-1073. Ulrich, I. H. (1987) Evaluation of a hgh-fiber diet in hyperlipidemia: a review. J. Am. Coll. Nutr. 6: 19-25. Vahouny, G. V., Roy, T., Gallo, L. L., Story, J. A., Kritchevsky, D., Cassidy, M., Grund, B. M. & Treadwell, C. R. (1978). Dietary fiber and lymphatic absorption of cholesterol in the rat. Am. J. Clin. Nutr. 31: S208-S212. Zilversmit, D. B. (1972)A single blood sample dual isotope method for the measurement of cholesterol absorption in rats. Proc. Soc. Exp. Biol. Med. 140: 862465.

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