Differential effects of simple vs. complex carbohydrates on VLDL secretion rates and HDL metabolism in the guinea pig

Biochi~ic~a ELSEVIER

et Biophysica A~ta Biochimica et Biophysica Acta 1256 (1995) 31-38

Differential effects of simple vs. complex carbohydrates on VLDL secretion rates and HDL metabolism in the guinea pig Maria Luz Fernandez *, Ghada Abdel-Fattah, Donald J. McNamara Lipid Metabolism Laboratory, Department of Nutritional Sciences and Interdisciplinary Nutritional Sciences Program, Shantz Building 309, Unie,ersity of Arizona, Tucson, AZ 85721, USA Received 29 July 1994; accepted 16 December 1994

Abstract

Guinea pigs were fed isocaloric diets containing 52% (w/w) carbohydrate, either sucrose or starch, to investigate effects of simple vs. complex carbohydrates on plasma VLDL and HDL metabolism. Plasma cholesterol concentrations were not different between dietary groups while plasma triacylglycerol (TAG) and VLDL cholesterol levels were significantly increased in animals fed the sucrose diet (P < 0.05). Hepatic VLDL TAG secretion rates measured following intravenous injection of Triton WR-1339 were not affected by carbohydrate type whereas the rate of apo B secretion was 1.9-fold higher in sucrose fed animals (P < 0.02). Nascent VLDL from the sucrose group contained less TAG per apo B suggesting that the higher plasma TAG in animals fed simple carbohydrates results from increased secretion of VLDL particles with lower TAG content. Sucrose fed animals exhibited higher concentrations of hepatic free cholesterol (P < 0.01) while hepatic TAG levels and acyl CoA:cholesterol acyltransferase (ACAT) activity were not different between groups. Plasma HDL cholesterol concentrations and composition, and plasma lecithin cholesterol acyltransferase (LCAT) activity were not affected by diet yet there was a positive correlation between HDL cholesteryl ester content and LCAT activities (r = 0.70, P < 0.05). Hepatic membranes from the sucrose group had a higher hepatic HDL binding protein number (Bma x) with no changes in the dissociation constant (Ka). These results suggest that at the same carbohydrate energy intake, simple sugars induce modest changes in HDL metabolism while VLDL metabolism is affected at multiple sites, as indicated by the higher concentrations of hepatic cholesterol, dissociation in the synthesis rates of VLDL components, and compositional changes in nascent and mature VLDL. Keywords: VLDL secretion rate; VLDL composition; HDL binding ; Sucrose; Starch; (Guinea pig)

I. Introduction

High carbohydrate diets have been negatively correlated with plasma H D L concentrations in population studies [1,2]. In addition, the substitution of complex carbohydrate in the diet by simple sugars has been shown to increase plasma triacylglycerol (TAG) concentrations in clinical [3,4] and animals studies [5,6]. Intake of high carbohydrate diets (60% of total calories) has resulted in significant decreases in plasma H D L concentrations [3,7]; however, an increase in plasma T A G was observed only when subjects were fed sucrose in a liquid formula [3]. A high fat-sucrose diet has been found to be hypertriglyceridemic in rats compared to a low fat-complex carbohydrate diet although no changes in plasma H D L concentrations were

* Corresponding author. Fax: + 1 (602) 621 9446.

0005-2760/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0 0 0 5 - 2 7 6 0 ( 9 5 ) 0 0 0 0 7 - 0

observed [6]. The results of these studies suggest that both the type and amount of dietary carbohydrate can alter plasma lipoprotein metabolism and modify cardiovascular disease risk [1-7]. Evidence from clinical and animal studies indicates that the observed higher concentration of plasma TAG associated with sucrose intake might be due to either increased secretion of T A G from the liver [6,8], decreased TAG removal from plasma [9,10], or both. Studies in rats suggest that fructose, and not glucose, is involved in the hypertriglyceridemic effect of simple sugars [9,10] and this response is attributed to reduced clearance of plasma V L D L - T A G rather than an increase in V L D L synthesis. In contrast, an increase in rat hepatic V L D L - T A G secretion mediated by dietary sucrose was found in one study [5]; however, interpretation of these results is complicated due to a synergistic effect between the sugar and the type of protein (casein or soybean). Rats had higher rates of

32

M.L. Fernandez et a l . / Biochimica et Biophysica Acta 1256 (1995) 31-38

VLDL-TAG synthesis only when they were fed sucrose in combination with casein [5]. Low HDL levels induced by a high carbohydrate diet have been attributed to a decrease in apo A-I synthesis [11,12], although an increase in apo A-I fractional catabolic rates has also been reported [12]. In this study [12] the decreases in plasma HDL cholesterol and apo A-I levels correlated with decreases in apo A-I synthesis, but not with an increase in apo A-I fractional catabolic rate. These data suggest that the mechanism of diet-mediated changes in plasma HDL differ from the mechanisms accounting for intra-individual differences in HDL levels [12]. Based on this information, the type of carbohydrate (simple vs. complex) is expected to alter plasma lipid levels which are important determinants of cardiovascular disease risk. The effects of carbohydrate type can be on VLDL and HDL synthesis rates and catabolism plus possibly influence lipoprotein intravascular processing. The present studies were undertaken to evaluate regulatory mechanisms determining plasma VLDL and HDL levels and metabolism when starch is substituted by sucrose in a high carbohydrate diet (52% w / w ) . We used the guinea pig model for our studies due to similarities to humans in their lipoprotein distribution (high L D L / l o w HDL) and metabolism [13] and in their response to dietary factors [14,15].

2. Materials and methods

Table 1 Composition of experimental diets Ingredient

Protein Fat Starch Sucrose Vitamins Minerals Fiber Cholesterol

Sucrose diet

Starch diet

wt(%)

Cal(%)

wt(%)

Cal(%)

20.5 7.5 51.7

23.2 19.1 58.5

20.5 7,5

23.2 19.1

51.7 7,5 1.0 12.5 0.04

58.5

7.5 1.0 12.5 0.04

The semipurified sucrose and starch diets were prepared by Research Diets (New Brunswick, N J). Vitamin and mineral mixes were formulated to meet NRC-specified dietary requirements of the guinea pig. The protein source consisted of casein/soybean in a 60:40 proportion. Fat was a mixture of saturated/monounsaturated/polyunsaturated fatty acids in the proportion 1:1:1. Fiber was a mixture of cellulose and guar gum (84:26). Energy content was equivalent for both diets to 3.56 kcal/g.

content was 1.0% ( w / w ) for both diets and the fat source was a mixture of oils giving a 1:1:1 ratio of polyunsaturated/monounsaturated/saturated fatty acids to mimic a Step I diet [17]. Sitosterol was added to equal the amount in a 15% corn oil based diet which we use as a baseline diet [13,14] to facilitate comparisons of diet effects. The fatty acid composition of the diets was: 12:0, 5.8%, 14:0, 2.5%, 16:0, 17.1%, 18:0, 6.1%, 18:1, 32.9% and 18:2, 33.3%. Vitamins and mineral mixes have been reported previously [18].

2.1. Materials 2.3. Animals

Tyloxapol (Triton WR-1339), sitosterol and triacylglycerol (NIT) 20 enzymatic kit were purchased from Sigma (St. Louis, MO). Enzymatic cholesterol/HP, cholesterol oxidase, cholesterol esterase and hydroperoxidase were purchased from Boehringer-Mannheim (Indianapolis, IN) and halothane from Halocarbon (Hackensack, NJ); [t25I]Na, oleoyl [1-14C]coenzyme A (1.8 GBq/mmol) from Amersham (Arlington Heights, IL) and cholesteryl [1,2,6,7-3H]oleate (370 GBq/mmol) from New England Nuclear (Boston, MA). 2.2. Diets

Diets were prepared and pelleted by Research Diets (New Brunswick, N J). Both diets had identical composition except for the type of carbohydrate which consisted of 51.7% ( w / w ) of sucrose or corn starch (Table 1). Carbohydrate constituted 58.5% of the total energy and fat 19%. Protein source was a mix 60:40 of casein/soybean to mimic the American mixture of animal/vegetable protein [15]. Cholesterol was added to a final concentration of 0.04% which is equivalent to 112 mg/1000 kcal or less than 300 m g / d a y the human equivalent [16]. Sitosterol

Male Hartley guinea pigs (Sasco Sprague-Dawley, Omaha, NE) weighing between 250 to 300 g were randomly assigned to the test diets. They were maintained under a controlled light cycle (light 0700-1900 h) with free access to the semipurified diet and water. After 4 weeks on the experimental diets, animals were fasted overnight (12 h) prior to killing to reduce post-prandial plasma TAG. Animals for the in vitro studies were anesthetized with halothane vapors and exsanguination by cardiac puncture to obtain liver for measurement of hepatic lipids, isolation of microsomes for analysis of ACAT activity, and hepatic membranes for determination of HDL binding; and plasma for analysis and isolation of lipoproteins. Animals for the in vivo experiments were also exsanguinated under halothane anesthesia to obtain plasma for isolation and characterization of nascent VLDL. Both dietary groups consumed equal amounts of food and had similar weight gains (Table 2). Animal experiments were conducted in accordance with US Public Health Service and US Department of Agriculture guidelines and experimental protocols were approved by the University of Arizona Institutional Animal Care and Use Committee.

M.L. Fernandez et al. / Biochimica et Biophysica Acta 1256 (1995) 31-38 Table 2 Plasma and lipoprotein cholesterol concentrations and plasma triacylglycerol of guinea pigs fed 52% (w/w) sucrose or starch-based diets Plasma lipid (mg/dl) Cholesterol Total VLDL IDL LDL HDL Triacylglycerol

Diet Sucrose

Starch

65 _+19 5+3 3+2 44_+ 17 9_+3 113 _+58 a

58 _+11 3±1 b 1__+1 42_+ 11 10_+2 78 _+29 b

a

Guinea pigs were fed the experimental diets described in Table 1 for a period of 4 weeks. Lipoproteins were isolated as indicated in Section 2. Data are presented as mean + S.D. for guinea pigs fed sucrose (n = 18) or starch (n = 17) for total plasma cholesterol and triacylglycerol and sucrose (n = 12) or starch (n = 11) for plasma lipoproteins. Values in a row with different superscripts are significantly different as determined by Students' t-test (P < 0.05). 2.4. Plasma and liver lipids Plasma total and lipoprotein cholesterol and T A G concentrations were determined by enzymatic analysis [19] using commercial kits. V L D L + IDL, LDL and H D L were separated by sequential ultracentrifugation in a L8-M ultracentrifuge (Beckman Instruments, Palo Alto, CA) at 125 000 × g at 15° C for 19 h in a Ti-50 rotor. Separations used the following density fractionations: d = 1.006 g / m l for nascent and mature VLDL, 1.006 to 1.019 g / m l for IDL, 1.019 to 1.09 g / m l for LDL, 1.09 to 1.21 g / m l for HDL. These separations were based on previous determinations of the distribution of lipoproteins in guinea pigs [20]. In addition, H D L cholesterol was determined using the precipitation method of Warnick et al. [21]. Hepatic concentrations of total and free cholesterol and TAG were measured following lipid extraction with chloroform/methanol (2:1) according to Sale et al. [22]. Hepatic cholesteryl ester concentrations were estimated by subtracting hepatic free from total cholesterol.

33

in incubated plasma samples. Freshly isolated plasma samples from each guinea pig were incubated at 37 ° C for 6 h and compared to a control sample containing 1.5 mM DTNB (dithionitrobenzoic acid) and incubated at 0 ° C. Free cholesterol concentrations were determined by enzymatic analysis for both control and incubated plasma samples using a Titertrek Multiscan Plus microtiter plate reader at 492 nm [27]. The rate of cholesterol esterification is expressed as / z g / m l per h. 2. 7. HDL binding assays Hepatic membranes used for binding assays were isolated as previously described [13,14]. Human apo E-free H D L was obtained by sequential ultracentrifugation at d 1.063-1.21 g / m l and separation of apo E containing HDL using a dextran-heparin column. Apo-E free H D L was radioiodinated with 125I by the iodine monochloride method [28] to give a specific activity between 150-400 c p m / n g . Previous experiments have demonstrated that the more readily available human apo E-free H D L can be used effectively as the labeled ligand and the unlabeled competitor for measuring H D L binding protein number (Omax) expression of guinea pig hepatic membranes [29]. Hepatic membranes from animals fed the sucrose and starch diets were incubated with the radiolabeled apo E-free H D L over a concentration range of 5 to 60 ~ g / m l in the presence of 1 m g / m l of the unlabeled H D L to determine non-specific and total binding respectively. Following 2 h incubation, the membranes were pelleted by ultracentrifugation at 125 000 × g for 1 h in a Ti42.2 rotor at 15° C, washed and centrifuged for an additional 30 min under the same conditions. The supernatant was aspirated and the pellet counted in a gamma counter for radioactivity. The dissociation constant K d and the number of H D L binding protein sites Bmax were determined from Woolf plots [30]. 2.8. Hepatic acyl CoA:cholesterol acyltransferase ( A C A D assay

2.5. Characterization of VLDL, LDL and HDL Nascent and mature VLDL, L D L and H D L were analyzed for protein [23], free and esterified cholesterol [22], T A G and phospholipids as previously described [13]. Apo B in V L D L was determined by selective precipitation with isopropanol [24]. The number of component molecules of nascent V L D L was calculated assuming one apo B per V L D L with a molecular weight of 412000 as reported for guinea pig [25]. The molecular weight of TAG, cholesterol and phospholipids were calculated as 885.4, 386.6 and 734, respectively, as previously reported [26]. 2.6. Plasma L C A T L C A T (EC 2.3.1.43) activity was determined by measuring the decrease in the mass of unesterified cholesterol

Hepatic microsomes were isolated and A C A T (EC 2.3.2.26) activity determined as previously described [15]. Briefly, 0.8 to 1.0 mg of microsomal protein is preincubated with 84 g / l albumin [31] and buffer (50 m m o l / 1 KH2PO4, 1 m o l / l sucrose, 50 m m o l / 1 KC1, 30 m m o l / 1 EDTA and 50 m m o l / 1 NaF) to a final volume of 0.18 ml. After 5 min at 37 ° C, 20 /zl (500 /zmol/1) of oleoyl-[114C]coenzyme A (0.15 G B q / p m o l ) was added and the reaction continued for 15 min at the same temperature. The reaction w a s s t o p p e d by addition o f 2.5 ml chloroform/methanol (2:1). A [3H]cholesteryl oleate recovery standard (0.045 GBq per assay) was added, the sample was mixed and let stand overnight at room temperature. The aqueous phase was removed and after evaporation of the organic phase to dryness, samples were resuspended in 150 /zl of chloroform containing 30 /zg of

M.L. Fernandez et al. / Biochimica et Biophysica Acta 1256 (1995) 31-38

34

unlabeled cholesteryl oleate. Samples were applied to 20 × 20 cm silica-gel TLC plates (Alltech) and developed in h e x a n e / d i e t h y l ether (9:1, v / v ) . Cholesteryl oleate was visualized with iodine vapors, scraped from the TLC plates, 5 ml of liquifluor added and counted in a scintillation counter. Recoveries of the [3H]cholesteryl oleate were 80 ___4% for n = 12 assays.

2.9. Triacylglycerol secretion rates The rate of secretion of VLDL T A G and apo B were determined by blocking VLDL catabolism with Triton WR-1339 [32], a detergent which coats VLDL particles blocking the action of lipoprotein lipase (LPL) in vivo. Animals were fasted overnight prior to surgery and a catheter was inserted into the jugular vein for injection of Triton and continuous plasma sampling. Animals continued fasting through the 8 h duration of the experiment to ensure that the measured plasma T A G levels reflected VLDL secretion and not influx of dietary T A G as chylomicrons. A 20% Triton solution (100 m g / k g of body wt) was injected and plasma samples (500 /xl) were taken at 10, 15, 20, 50, 75, 120, 180, 300 and 480 min. Plasma was separated from red blood cells and T A G concentrations were measured for each time point. T A G secretion rates were calculated by regression analysis as mg T A G sec r e t e d / k g body weight per h. Apo B secretion rates were calculated by multiplying VLDL T A G secretion rates × apo B concentration (%) divided by VLDL T A G (%).

2.10. Statistical analysis Student's t-test (INSTAT, San Diego, CA) was used to analyze differences between dietary treatments unless otherwise specified in the measured variables of plasma cholesterol, lipoproteins and TAG; the composition of lipoproteins, A C A T and LCAT activities, T A G secretion rates and HDL binding to hepatic membranes. Two-way A N O V A was used to evaluate differences in composition between nascent and mature VLDL. Values were considered significant at P < 0.05. Linear regression analysis was used to determine correlation coefficients between variables.

3. Results 3.1. Carbohydrate lipoproteins

type effects on plasma

lipids and

No differences in final body weights were observed between dietary treatments (628 ___88 g, n = 18 and 656 + 51 g, n = 17) for animals fed sucrose and starch-based diets, respectively, indicating that animals consumed similar amounts of food throughout the experimental period. Plasma total cholesterol levels were not different between

Table 3 Composition of nascent and mature VLDL of guinea pigs fed 52% (w/w) sucrose or starch-based diets Lipoprotein

Diet Sucrose

Nascent VLDL composition(%) Triacylglycerol 76.4_+4.4 x Free cholesterol 6.0+0.7 y Cholesteryl ester ND Phospholipids 13.7 + 3.2 y Protein 3.8+0.8 a,x Apo B 2.5 -+0.9 a Mature VLDL composition(%) Triacylglycerol 66.7 + 1.6 y Free cholesterol 5.9 _+0.9 y Cholesteryl ester 3.7 -+1.0 a,y Phospholipids 13.6_+1.2 y Protein 10.3 +_0.9 y ApoB 4.8-+1.7 y

Starch 79.6+ 1.7 x 5.3_+0.7 x ND 11.7 + 1.0 × 2.6_+0.6 b,x 1.9 + 0.4 b 67.8 + 2.7 y 5.9 -+0.6 y 2.3 + 1.2 b,y 13.3 +3.0 y 12.3 -+2.6 y 4.7-+1.1 y

Guinea pigs were fed the experimental diets described in Table 1 for a period of 4 weeks and the compositionsof lipoproteinswere measured as indicated in Section 2. Data are presented as mean+S.D, for n = 5 determinations except for mature VLDL from the sucrose group where n = 6. Values in the same row with different superscripts: ~,b are significantly different (P < 0.05). Values in the same column for the nascent and mature VLDL groups with different superscripts: x,y are significantly different (P < 0.05) as determined by two-way ANOVA. ND indicates not detectable.

groups; however, plasma T A G were 1.5-fold higher in animals fed the sucrose diet ( P < 0.05) (Table 2). VLDL cholesterol concentrations were higher in animals fed the sucrose diet while no differences in IDL, LDL or HDL cholesterol concentrations were observed for animals from the two dietary groups (Table 2). Nascent VLDL contained a higher proportion of T A G and phospholipids and lower percentage of cholesterol and protein than the mature plasma VLDL consistent with the compositional changes expected during the maturation process (Table 3). Nascent VLDL from the sucrose fed animals had a higher concentration of protein than nascent VLDL from the starch group. Apo B was selectively precipitated with isopropanol and found to be significantly higher in VLDL from guinea pigs in the sucrose group (Table 3). Although the number of cholesterol molecules per VLDL was not different for nascent VLDL from the sucrose or starch fed guinea pigs, the number of T A G and phospholipid molecules was higher in animals fed the starch-based diets (Fig. 1) indicating that guinea pigs fed the sucrose diet exhibited smaller nascent VLDL containing less T A G per particle. A 40% higher content of cholesteryl ester was observed in animals fed the sucrose diet while protein, free cholesterol, phospholipids and T A G relative proportions were not different in mature VLDL from the two dietary groups (Table 3). The number of cholesteryl ester (CE) molecules per VLDL was higher for animals fed the sucrose diet while TAG, free cholesterol (FC), and phospholipid (PL) molecules per particle were

M.L. Fernandez et al. / Biochimica et Biophysica Acta 1256 (1995) 31-38

#--

60

O

© r~ ,_1 >

35

1800

°

40

;~

1200

Y

20 0 0

< Nascent Mature SUCROSE

Nascent Mature STARCH

Fig. I. Number of phospholipid, triacylglycerol and free cholesterol molecules of nascent and mature VLDL from guinea pigs fed sucrose or starch-based diets. The number of TAG molecules in the nascent VLDL is significantly higher in the starch-fed guinea pigs while the number of cholesteryl ester molecules in the mature VLDL is higher in the sucrose fed guinea pigs ( P < 0,01). The number of TAG, phospholipid and free cholesterol molecules is lower in the mature VLDL ( P < 0.01) due to losses occurring during the maturation process.

not d i f f e r e n t in m a t u r e V L D L for g u i n e a pigs f r o m b o t h dietary g r o u p s (Fig. 1). D u r i n g the m a t u r a t i o n p r o c e s s V L D L a p p a r e n t l y h a s a loss in the n u m b e r o f T A G m o l e c u l e s a n d F C w i t h a c q u i s i t i o n o f C E (Fig. 1). P l a s m a L D L a n d H D L c o m p o s i t i o n w a s not a f f e c t e d b y f e e d i n g c o m p l e x vs. s i m p l e c a r b o h y d r a t e s ( T a b l e 4).

3.2. Dietary carbohydrate type effects on TAG secretion rates T A G s e c r e t i o n rates, m e a s u r e d as p l a s m a T A G a c c u m u lation following injection of Triton WR-1339, were not a f f e c t e d b y c a r b o h y d r a t e t y p e (Fig. 2) w h e r e a s a p o B

Table 4 HDL and LDL composition of guinea pigs fed 52% (w/w) sucrose or starch-based diets HDL composition (%) Protein Phospholipid Triacylglycerol Free cholesterol Cholesteryl ester LDL composition (%)

Protein Phospholipid Triacylglycerol Free cholesterol Cholesteryl ester

Diet Sucrose

Starch

37.1 _+7.2 13.7 _+4.2 23.2 _+4.1 4.1 _+2.6 21.2 _+5.5 Diet

35.0 _+4.5 14.2 + 5.1 27.0 _+7.5 3.7 _+1,1 20.6 _+5.7

Sucrose

Starch

29.2 _+0.5 12.5 _+1.7 7.7 _+1.4 3.9 _+0.5 46.7 _+4.8

30.0 _+1.7 14.0 _+4.5 9.3 _+3.0 4.2 _+1.1 45.5 _+6.4

Guinea pigs were fed the experimental diets described in Table 1 for 4 weeks. The compositions of plasma LDL and HDL were measured as indicated in Section 2. Data are presented as mean_+ S.D. for guinea pigs fed sucrose (n = 12) or starch (n = 11) diets.

< .~

l

0

i

i

i

r

100

200

300

400

500

t~

TIME (MIN) Fig. 2. Triacylglycerol secretion rates in mg/kg per h of animals fed sucrose ( 0 ) and starch-based (O) diets. Each graph represents the mean of 5 determinations. Values were not significantly different between the sucrose (76.4 + 19.1 mg/kg per h) and starch (64.9 + 14.6 mg/kg per h) groups.

s e c r e t i o n w a s s i g n i f i c a n t l y h i g h e r in a n i m a l s fed the suc r o s e diet ( T a b l e 3). T h e s e data s u g g e s t that the h i g h e r p l a s m a T A G c o n c e n t r a t i o n s in a n i m a l s fed the s u c r o s e diet c o u l d b e e x p l a i n e d in part b y a n i n c r e a s e d s e c r e t i o n o f V L D L p a r t i c l e s c o n t a i n i n g less T A G / p a r t i c l e .

3.3. Dietary carbohydrate type effects on hepatic lipids and A C A T activity In o r d e r to e s t a b l i s h p o s s i b l e c o r r e l a t i o n s b e t w e e n h e p atic c h o l e s t e r o l h o m e o s t a s i s a n d p l a s m a l i p o p r o t e i n metabolism, hepatic cholesterol and TAG concentrations a n d A C A T activity w e r e d e t e r m i n e d . H e p a t i c c h o l e s t e r o l c o n c e n t r a t i o n s w e r e 1 2 % h i g h e r in a n i m a l s fed the s u c r o s e diet a n d this d i f f e r e n c e w a s s p e c i f i c for the free c h o l e s t e r o l p o o l ( T a b l e 6). H e p a t i c T A G a n d h e p a t i c A C A T a c t i v i t y w e r e not d i f f e r e n t b e t w e e n dietary g r o u p s ( T a b l e 6). A C A T activity t e n d e d to b e h i g h e r in a n i m a l s fed the s u c r o s e diet and there was a significant positive correlation between h e p a t i c c h o l e s t e r o l c o n c e n t r a t i o n s a n d A C A T activity ( r = 0.66, P < 0 . 0 2 ) ( d a t a n o t s h o w n ) .

Table 5 Triacylglycerol and apo B secretion rates of guinea pigs fed 52% (w/w) sucrose or starch-based diets Secretion rates (mg/kg per h)

Triacylglycerol Apo B

Diet Sucrose

Starch

76.4 ± 19.1 2.61_+1.53 a

64.9+ 14.6 1.38_+0.51 ~

Guinea pigs were fed the experimental diets described in Table 1 for a period of 4 weeks and triacylglycerol and apo B secretion rates were determined as described in Section 2. Values are presented as mean + S.D. for n = 5 guinea pigs per dietary group. Values in the same row with different superscripts are significantly different as determined by MannWhitney non-parametric t-test ( P < 0.02).

M.L. Fernandez et al. /Biochimica et Biophysica Acta 1256 (1995) 31-38

36

Table 6 Hepatic cholesterol and triacylglycerol of guinea pigs fed 52% sucrose or starch-based diets

4.1. Carbohydrate type effect on VLDL metabolism

Diet

Hepatic lipids ( m g / g ) Total cholesterol Free Esterified Triacylglycerol ACAT activity ( p m o l / m i n per mg)

Sucrose

Starch

4.3 ± 0.5 a 3.6 + 0.4 a 0.6 + 0.3 46.1 + 22.7 27.5 + 22.0

3.8 + 0.4 b 3.3 + 0.4 b 0.5 + 0.3 58.3 + 16.5 19.9 + 6.0

Guinea pigs were fed the experimental diets described in Table 1 for 4 weeks. Hepatic lipids were determined as indicated in Section 2. Data are presented as mean+S.D, for guinea pigs fed sucrose (n = 12) or starch (n = l l ) diets, except for ACAT where n = 6 for both dietary groups.

3.4. Dietary carbohydrate type effects on LCAT activity and HDL binding LCAT activities were not affected by the type of dietary carbohydrate. Values were 5.3 + 2.1 and 5.9 + 1.0 /zg/ml per h (n = 6) for the starch and sucrose groups respectively. LCAT activity and HDL cholesteryl ester content exhibited a positive correlation (r = 0.70, P < 0.02). Hepatic HDL binding was 1.3-fold higher in animals fed the sucrose-based diet ( P < 0.01) as shown by the saturation curves of HDL binding to hepatic membranes from guinea pigs fed the test diets (Fig. 3). The inset shows the Woolf plots to calculate hepatic HDL binding p r o t e i n Bma x. K a values calculated from the intercept were not different between diets while Bma x calculated from the slope were significantly higher in animals fed the sucrose diet (Fig. 3).

2.0

7

E

"~

1.5

(.9 z

1.o

z m

--

£3 "l-

0.5

0.0

i

i

i

20

40

60

HDL

4. Discussion

80

(pglml)

Fig. 3. Saturation curves of human apo E-free HDL binding to guinea pig hepatic membranes from the sucrose ( 0 ) and the starch (C)) groups. Each curve represents the mean of three determinations. The inset represents the Woolf plot from which the values of HDL binding protein Bmax are calculated from the slope and receptor dissociation constant K d from the intercept with the x axis. Bmax values were significantly higher in ,animals fed the sucrose diet (2.13___0.41 / x g / m g protein, n = 7 ) compared to those fed the starch diet (1,6+0.25 / x g / m g protein n = 7) ( P < 0.01). K d values were not different between the sucrose (25 + 10 / x g / m l ) and the starch (28_+ 1 7 / x g / m l ) groups.

Similar to previous reports on the effects of carbohydrate type on plasma lipids [2-4,8-10], we have demonstrated in these studies significant increases in plasma TAG with intake of sucrose compared to starch in guinea pigs fed diets where the caloric contribution of carbohydrate is relatively high (59%). Most of the changes in plasma TAG induced by sucrose intake have been attributed to fructose [33,34] due to the rapid utilization of this sugar by the liver and by the fact that fructose bypasses the phosphofructokinase regulatory step in glycolysis leading to significant effects on lipid metabolism [34]. The shift in balance from oxidation to esterification of nonesterified fatty acids is thought to result in an increased secretion of VLDL which leads to hypertriglyceridemia, decreased glucose tolerance and hyperinsulinemia [35,36]. The sucrose and fructose induced hypertriglyceridemia in the rat has been associated with increased insulin levels [37] and a higher secretion of VLDL-TAG has been reported in rats fed sucrose or fructose vs. glucose [5,38]. In the present study, although no changes in VLDL-TAG secretion rates were observed in guinea pigs from the two diet groups, the higher secretion rates of apo B and the lower T A G / a p o B ratio in nascent VLDL from animals fed the sucrose diet suggests that an increased secretion of smaller VLDL particles is probably responsible for the increased plasma TAG levels associated with simple carbohydrate intake. Dissociation of the effects of an intervention on VLDL TAG and apo B secretion rates have been previously reported. Melish et al. [8] found that subjects fed a high carbohydrate diet exhibited increased TAG secretion rates while apo B secretion did not change compared to a low carbohydrate intake. However, these investigators did not address the specific effects of simple sugars vs. complex carbohydrates on TAG and apo B secretion rates which were measured in the present investigation. Studies have demonstrated that surface lipids and protein are essential for the secretion of VLDL [39] and that cholesterol is required for the secretion of VLDL from rat liver [40]. Since guinea pigs fed the sucrose diet had higher concentrations of hepatic cholesterol, this could be related to increased secretion of VLDL particles in agreement with results from the in vivo studies. The modifications in VLDL composition induced by sucrose can also be a factor contributing to the higher plasma TAG observed in this group of animals. Lipoprotein size has also been shown to be a factor determining the ability of the particles to act as substrates for lipoprotein lipase [41] with smaller VLDL particles being poor substrates for the enzyme. Since sucrose fed guinea pigs secreted smaller nascent VLDL particles, the decreased

M.L. Fernandez et al. / Biochimica et Biophysica Acta 1256 (1995) 31-38

interaction of these particles with lipoprotein lipase might also contribute to the higher plasma TAG in animals fed simple carbohydrates. Sucrose fed guinea pigs exhibited higher concentration of mature VLDL cholesterol with particles having a higher proportion of cholesteryl ester, and these compositional changes might affect the catabolism of VLDL and its conversion to LDL. Oschry et al. [42] reported that hypertriglyceridemic subjects have a sub-population of plasma VLDL with a higher proportion of cholesteryl ester and that this VLDL sub-population did not follow the delipidation cascade gradient and were not converted to LDL, contributing to the observed hypertriglyceridemia [42]. Fructose feeding in the rat has also been shown to produce modifications in VLDL composition with a lower ratio of apo E to C which makes the particle less susceptible to removal [43]. However, Verschoor et al. [44] have shown that changes in the composition of VLDL, specifically higher concentrations of TAG and total cholesterol in rats fed high glucose or fructose diets compared to chow-fed animals, were not associated with decreases in VLDL-TAG clearance. In contrast to these studies [44], Mamo et al. [9] reported increased VLDL TAG removal in rats fed simple compared to complex carbohydrates. In a clinical study measuring sucrose-induced apo B removal rates in hypertriglyceridemic patients, most of the subjects exhibited a decreased clearance of VLDL which correlated with concomitant increases in the VLDL pool size and this delayed clearance of VLDL could be associated with decreased conversion of VLDL to LDL [4]. Based on these published reports and our results in the present investigation, the increased plasma TAG of guinea pigs fed sucrose vs. starch-based diets could be explained by mechanisms affecting multiple sites of VLDL metabolism: (a) an increased secretion of the number of nascent VLDL particles containing less T A G / a p o B, a smaller particle likely to have a decreased interaction and catabolism by endothelial lipase [41]; (b) higher concentrations of hepatic cholesterol associated with and increased secretion of VLDL particles [40]; and (c) alterations in the intravascular processing of VLDL possibly associated with changes on cholesteryl ester transfer protein activity resulting in mature VLDL containing a higher proportion of cholesteryl ester, a compositional modification associated with decreased conversion of VLDL to LDL in hypertriglyceridemic patients [42]. 4.2. Carbohydrate type effect on H D L metabolism

Although no composition differences in HDL or plasma LCAT activity were found, HDL metabolism might have been affected to some extent by the type of carbohydrate since sucrose-fed guinea pigs exhibited an increased number for the hepatic HDL binding protein. Higher levels of HDL 2 have been found in cynomolgus monkeys fed highsucrose vs. high-starch diets [45] consistent with changes in the distribution of HDL sub-populations mediated by

37

the type of dietary carbohydrate. In addition, decreases in HDL 3 cholesteryl ester and protein have been observed in normal and hypertriglyceridemic subjects consuming a high carbohydrate diet while the HDL2-protein, phospholipid and cholesterol levels increased suggesting that the amount of dietary carbohydrates can modify the composition of HDL-subclasses as well as the subpopulation distribution [121. The increased expression of HDL binding sites in sucrose-fed animals could be related to either a change in HDL cholesteryl ester transfer to hepatic cells without catabolism of intact HDL [46] or to an increased hepatic uptake of HDL particles [47]. Previous reports of the effects of dietary factors on HDL binding have shown that dietary fat saturation [48], dietary cholesterol [49] and saturated fat composition [50] modify the number of hepatic HDL binding protein (Bmax); however, the physiological significance of these changes can be related [50] or be independent [48,49] of plasma changes in HDL cholesterol concentrations. Although no differences in HDL composition were observed, it is possible that the type of carbohydrate affected the distribution of HDL subclasses, as observed in clinical [12] and animals studies [45] and that this changes in subclass distribution is related to increased reversed cholesterol transport associated with increased hepatic HDL binding as found in the present investigation. From these studies we conclude that the type of dietary carbohydrate significantly alters VLDL metabolism in the guinea pig which results in alteration in plasma TAG levels in agreement with the reported effects of simple carbohydrate intake from the clinical trials [4,7,35]. In contrast, the effects of sucrose or starch intake on HDL metabolism were modest and not related to differences in plasma HDL levels or LCAT activity which emphasizes previous findings that the lower plasma HDL levels observed in certain populations [1,2] are a result of the amount rather than the type of dietary carbohydrates.

Acknowledgements We thank Mr. John Ebner and Mr. Dong-Ming Sun for the analysis of free and esterified cholesterol, tr!acylglycerol and A C A T activity in liver samples, and Mr. Carlos Montano for determining plasma LCAT activity. These studies were supported by a Grant-in-Aid from the American Heart Association, Arizona Affiliate (AZ-93GF-41).

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