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Glucosamine and experimental atherosclerosis Increased wet weight and changed composition of cholesterol fatty acids in aorta of rabbits fed a cholesterol-enriched diet with added glucosamine
205
Atherosclerosis, 26 (1977) 205-213 Biomedical 0 Elsevier/North-Holland
GLUCOSAMINE
Press, Amsterdam
AND EXPERIMENTAL
- Printed in The Netherlands
ATHEROSCLEROSIS
INCREASED WET WEIGHT AND CHANGED COMPOSITION OF CHOLESTEROL FATTY ACIDS IN AORTA OF RABBITS FED A CHOLESTEROL-ENRICHED DIET WITH ADDED GLUCOSAMINE
STEEN STENDER and POUL ASTRUP Department
of Clinical Chemistry,
Rigshospitalet,
DK-2100
Copenhagen
(Denmark)
(Received 13 May, 1976) (Revised, received 9 August, 1976) (Accepted 12 August, 1976)
Summary Three groups of each 12 rabbits were fed a cholesterol-enriched diet. Glucosamine was added in amounts of 0.5% and 2.0% (w/w) to the diet of two of the groups, while the third group served as a control group. The amount of cholesterol in the diet, was individually adjusted, so that all rabbits experienced nearly the same level of serum cholesterol throughout the whole experimental period. Glucosamine did not affect the concentration response of serum cholesterol to dietary cholesterol or the amount of free and esterified cholesterol in the inner aorta. It did, however, cause an increase in the wet weight of the inner aorta with a corresponding decrease in the concentration of aortic cholesterol. Furthermore a decrease in the ratio between mono-unsaturated and di-unsaturated fatty acids of the cholesterol esters of serum and inner aorta were observed in the animals Key words:
which
received
glucosamine.
Aorta - Cholesterol - Cholesterol angiopathy - Glucosamine -Rabbits
ester fatty
acid composition
- Diabetic
Introduction Heparin and several other glucosaminoglycans have the ability to form complexes in vitro with low density lipoproteins in the presence of certain cations [l]. It has been proposed that complex formation between low density lipoThis work was supported by grants from The Danish Medical Research Council.
206
proteins from plasma and glucosaminoglycans from the arterial wall also takes place in vivo, implicating a role in the deposition of cholesterol in the intimal tissue [2,3]. Glucosaminoglycans have also been related to the development of the vascular complications in diabetes [4]. Heparin - which is a polymer composed of D-glucosamine, D-glucuronic acid, and sulphate residues - is not absorbed from the intestinal lumen, contrary to its subunit glucosamine, which penetrates the intestinal cells by facilitated diffusion without modification of the molecular structure [5]. It was therefore of interest to investigate the development of experimental atherosclerosis in rabbits fed a cholesterol-enriched diet with added glucosamine. Methods Animals Thirty-six inbred 4-months-old male rabbits of the White Danish Country Strain castrated at an age of 2 months were used. Each animal was fed 150 g per day of standard pellets (Boserup rabbit pellets@: protein 15%, fat 3%, mainly from soybeans and linseeds, water 12%, crude fiber 13%). Water was given ad libitum. 0.5% and 2.0% (w/w) of D-glucosamine hydrochloride (Sigma@) were incorporated in the food by the manufacturers of the pellets. After an initial adaptation period of 3 weeks with ordinary rabbit pellets the animals were divided into 3 groups, which had the same average concentration of serum cholesterol and the same average body weight. Two of the groups were fed the diet containing glucosamine for the following 12 weeks. The third group served as a control group. All three groups were fed an individually adjusted amount of dietary cholesterol for the first 6 weeks. For the next 6 weeks the respective diets were fed without cholesterol addition (Table I). Individually adjusted cholesterol feeding Serum cholesterol was determined weekly during the first 6 weeks. Rabbits which after 1 week with 1% cholesterol (w/w) in the diet had a higher serum cholesterol level than the average, received a diet enriched with O.l%, 0.2%, or 0.3% (w/w) of cholesterol during the following week, depending on the previous response to dietary cholesterol, while on the other hand the rabbits with a lower concentration of serum cholesterol received a diet enriched with 0.5%, l.O%, or 2.0% of cholesterol depending on the previous response. This procedure was continued for 6 weeks. The amount of cholesterol ingested by each
TABLE
1
Group
Adaptation
period
Week
o-6
Week
6-12
(3 weeks)
G-O
F
F+C
G-
0.5%
F
F+0.5%G+C
F + 0.5%
G
G-
2.0%
F
F+2.0%G+C
F + 2.0%
G
F = (Boserup samine
(w/w).
rabbit
pellets?:
150
g/day;
C = an individually
F
adjusted
amount
of cholesterol;
G = gluco-
207
rabbit was registered. As for the preparation of the diet, cholesterol equivalent to a 1% (w/w) diet was dissolved in ether and poured over the pellets. Pellets from this 1% batch were then mixed with pellets without cholesterol in appropriate amounts to produce the wanted percentages. Tissue and blood samples Body weights were registered every week. The animals were bled from the marginal ear vein. After discontinuation of cholesterol feeding blood samples were taken 3 weeks, as well as immediately, before the rabbits were killed. The remaining procedures, including removal of liver and aorta, isolation of the inner aorta, homogenization of the tissue samples, and chemical determination of total cholesterol are described elsewhere [6]. Glucose in serum was measured by a hexokinase method. Determination of cholesterol ester fatty acids (CEFA) CEFA was determined with TLC by a modification of the method described by Alling et al. [7]. The lipids were extracted from serum with chloroform : methanol (1 : 1, vol/vol). Aliquots of the supernatant and of standards containing known amounts of cholesterol palmitate (16 : 0), oleate (18 : l), linoleate (18 : 2) and linolenate (18 : 3) were applicated on precoated silica gel TLC plates (Merck). The chromatogram was developed in toluene : heptane (38 : 90, vol/ vol), 3 X 20 min. After drying, the plates were dipped in a solution of ethanol : water : perchloric acid (100 : 113 : 9, vol/vol/vol) for 10 set, and placed in an oven at 100°C for 10 min. The fluorescences of the different cholesterol ester bands were quantified in situ using a Vitatron TLD 100 Universal Densitometer. The serum samples were kept at -20°C from the day of blood sampling until the CEFA analyses were performed. No other precautions were taken to stop the LCAT activity in vitro. The samples were determined within a few days in randomized order. The same procedure was used for determination of the aortic CEFA composition. In a number of serum and aortic samples the cholesterol ester bands were scraped from the thin layer plates and the lipids eluted with diethyl ether. The fatty acids in the different bands were determined by GLC. It was found that 16 : 0 represented more than 90% of the saturated band, 18 : 1 represented more than 90% of the monosaturated band, and 18 : 2 more than 95% of the disaturated band in the aortic as well as in the serum samples. Statistics Data are presented as means +S.E.M. The significance of differences between means was determined by the Student t-test. Linear regressions were calculated by the method of least squares. Results General condition of the animals All the animals were in good health throughout
the experimental
period. All
208
TABLE 2 BODY WEIGHT, TISSUE WEIGHT INNER AORTA (Z + SEM).
AND
CONTENT
OF TOTAL
Control group (n = 12)
CHOLESTEROL
0.5% Glucosamine (n = 12)
IN LIVER
AND
2.0% Glucosamine (n = 12)
__ Body weight (kg)
week 0 week 12
3.09 3.85
Orally added cholesterol from week O-6
(g)
Wet weight of liver (g)
+ 0.10 f 0.09
45.3
+ 5.7
131
f 7
3.21 3.91 51.8 131
t 0.10 t 0.12
3.36 3.84
? 8.2
56.6
f 10
0.168
+ 0.020
0.265
f 0.038
Total cholesterol in liver (mmol)
2.17
+ 0.39
1.42
f 0.27
f 2.3
9.8
? 2.4
10.8
f 6.4
130
Wet weight of inner aorta (g)
Total cholesterol in inner aorta (pmol)
t 0.10 * 0.09
a
f7
0.323
f 0.029
2.01
+ 0.48
12.5
b
?r3.4
a P < 0.05; b P < 0.001.
rabbits ate the daily amount of food irrespective of the addition of cholesterol and glucosamine. A steady increase in body weight took place with no significant differences between the experimental groups and the control group (Table 2). Cholesterol in the serum and in the diet Variation8 in serum cholesterol concentration within each group was minimized by the individual cholesterol feeding. The coefficient of variation was in the range of 15-25% in each group. The three groups had the same average serum cholesterol levels during the increasing as well as the decreasing period of serum cholesterol concentration (Fig. 1).
SERUM
CHOLESTEROL mmol
CHOLESTEROL
/I
mmol T
T
-
0
---
SERUM
-
/lOOg
wet
weight
CONTROL
fl
0.5%
GLUCOSAMINE
a
2.0%
GLUCOSAHINE
LIVER
INNER AORTA
Fig. 1. Levels of total cholesterol in serum (1 mmol/l = 38.7 mg/lOO ml) during the experiment and levels of total cholesterol in liver and inner aorta.
209
No significant difference was found between the average concentration of serum glucose in the glucosamine groups and in the control group. The total amount of cholesterol added to the diet differed enormously among the rabbits, even within the same group. One rabbit was fed a total of 20 g cholesterol during the 6 weeks. while another rabbit during the same period received 100 g, and both rabbits had the same increase in serum cholesterol concentration. The mean value for cholesterol intake was higher in the groups receiving glucosamine than in the control group. Due to the large variation this difference was not significant (Table 2). Liver No significant difference was observed in liver wet weight or in the amount of liver cholesterol between the groups receiving glucosamine and the control group (Table 2). Inner aorta A significant increase in wet weight of the inner aorta was observed in the two groups receiving glucosamine, but the amount of total cholesterol accumulated in the inner aorta was not affected (Table 2). Calculating the concentration of cholesterol in the inner aorta by dividing the amount of cholesterol by the respective wet weight, a significant lower concentration was found in the glucosamine groups (Fig. 1). Figure 2 shows the content of aortic cholesterol in pmol plotted against the wet weight of the same inner aorta for all the rabbits. Composition CEFA was weeks before groups in the
of cholesterol ester fatty acids (CEFA) determined in serum obtained from the animals in week 9 - three the animals were killed. There were no differences between the concentration of total cholesterol in serum or in the distribution
Wet weight of inner
aorta
mg
I 5
10
15
20
25 Total
30 cholesterol
35 urn01 in inner aorta
Fig. 2. Relationship between content of total cholesterol (wnol) and wet weight (me) of the same inner aorta in Group G-O (e), Group G-O. 5% (A) and in Group G-2.0% (0). The regression lines: y = 7.1 x + 90. r = 0.84 for Group G-O and Y = 9.4 x + 187. r = 0.79 for Group G-O. 5% and Group G-2.2.0%.
:1 :2
3.1
0.8 1.6 1.4 0.6
f 0.3
f 0.03
f f f f * 0.03
1.6 c * 0.1
0.73
22.2 * 0.7 44.7 c + 1.4 28.2 c f 1.2 4.9 r 0.4
0.5% glucosamine
a The TLC bands were named after their major component. b P < 0.005. cp<0.001.
18 18
0.72
21.6 54.3 18.5 5.5
16:08 18 : 1 18 : 2 18 : 34
Free cholesterol Total cholesterol
control
Fatty acid % of total cholesterol ester
Serum (week 9)
PERCENTAGE FATTY ACID COMPOSITION OF CHOLESTEROL WITHOUT GLUCOSAMINE ADDED TO THE DIET (X f. SEM).
TABLE 3
+ 0.03
1.7 c f 0.2
0.73
21.7 f 0.5 45.5 b f 1.9 28.4 c f 1.8 4.6 * 0.2
2.0% gIucosamine
3.4
0.54
- 22.1 57.2 17.2 3.7
control
+ 0.1
+ 0.04
_+0.3 f 0.9 t 0.6 -t 0.4
* 0.06 2.0 c + 0.1
0.54
22.4 * 0.8 48.7 c * 1.8 25.2 c f 1..3 3.5 * 0.5
0.5% glucosamine
OF CHOLESTEROL-FED
Inner aorta (week 12)
ESTERS IN SERUM AND INNER AORTA
WITH AND
+ 0.09 2.2 c f 0.1
0.65
21.5 r 0.9 51.9 c f 1.6 23.7 c * 1.3 3.0 f 0.6
2.0% glucosamine
RABBITS
211
between esterified and free cholesterol. However, the rabbits receiving glucosamine had on average a 10% higher level of linoleic acid (18 : 2) and a 10% lower level of oleic acid (18 : 1) than the control group (Table 3). Almost the same difference was observed in the average composition of CEFA in inner aorta (Table 3). The level of oleic acid was a little higher and the level of linoleic acid a little lower than the respective levels in serum. This was the case for the control rabbits as well as for the glucosamine rabbits. The ratio between oleic acid and linoleic acid in the cholesterol esters of serum in the control group was 3.1 f 0.3, while the same ratio in the 2.0% glucosamine group was 1.7 1: 0.2 and 1.6 + 0.1 in the 0.5% glucosamine group. This difference in ratios is highly significant (P < 0.001). Discussion Dietary cholesterol From Table 2 it is concluded that glucosamine did not significantly affect the response of cholesterol concentration in serum to the intake of dietary cholesterol, although a considerable difference existed between the mean values of the cholesterol intake in the control group and the glucosamine-fed groups. The large inter-individual variation in serum cholesterol concentration in rabbits fed a fixed amount of cholesterol is well known [8]. In our experimental procedure we halved this variation by transferring it to the amount of dietary cholesterol, which varied between 20 and 100 g in total ingested cholesterol during the 6 weeks. The mechanism behind this large variation has recently been investigated by kinetic analyses of cholesterol metabolism in rabbits during normo- and hypercholesterolaemic steady state conditions. It was found that the variation in cholesterol intake only partly can be explained by differences in cholesterol excretion rates. Differences in cholesterol synthesis and cholesterol absorption do also interfere [ 91. Individually adjusted cholesterol feeding has certain advantages compared with the traditional method of adding a fixed amount of dietary cholesterol to the rabbits: (1) It reduces the usually large inter-individual variation in concentration of serum cholesterol. (2) It is possible to investigate whether a substance having a serum lipid-lowering action also affects the metabolism of cholesterol in inner aorta in more direct ways, since the cholesterol-lowering effect in serum is compensated by feeding more cholesterol to the experimental groups [ 101. Inner aorta Since cholesterol accumulation in arterial walls continues after cessation of cholesterol feeding, and until the serum cholesterol concentration has become nearly normal, as shown by several authors and recently confirmed by us [6], the rabbits in the present experiment were not killed until this occurred. Although the inter-individual variation in serum cholesterol concentration was considerably reduced by the individual feeding, the variation in the concentration of cholesterol in the inner aorta was the same (coefficient of variation 50%) as seen in experiments with a much larger variation in serum cholesterol [6]. This indicates that the concentration of total cholesterol in serum is
212
not the only parameter related to the atherogenic action of cholesterol feeding in rabbits. The distribution of cholesterol in the different lipoproteins may be more important. Brattsand [ll] has recently demonstrated that the degree of aortic lipid infiltration in the cholesterol-fed rabbit is better related to the levels of LDL than to the VLDL cholesterol or the total cholesterol. Composition
of cholesterol-ester
fatty acids (CEFA)
In rabbits of the same strain and fed the same basic diet (Boserup rabbit pellets@) the 18 : l/18 : 2 ratio in CEFA of serum increased from 0.9 to 2.7 when the concentration of serum cholesterol was elevated from 1 mmol/l to 17 mmol/l by adding cholesterol to the diet [12]. An increase in the 18 : l/ 18 : 2 ratio has also been found in the cholesterol esters of liver and aorta when cholesterol-fed rabbits were compared with rabbits fed a conventional diet
v31. Three weeks after the cholesterol feeding was discontinued in the present experiment with glucosamine the 18 : l/18 : 2 ratio in CEFA of serum still exhibited a cholesterol feeding pattern in the control group, since this ratio was 3.1. Glucosamine affected this ratio in the direction of a more noncholesterol feeding pattern (1.7), but without affecting the total amount of cholesterol ester in serum (Table 3). It is only possible to speculate about the mechanism underlying this change in the composition of CEFA in serum: Contrary to humans, the liver of the rabbit is the main site of cholesterol esterification [14,15]. Glucosamine may therefore interfere in the liver with the incorporation of the oleic acids in the cholesterol esters, or glucosamine may facilitate the breakdown of VLDL in which the major part of the cholesterol-oleate in hypercholesterolaemic rabbit serum seems to be located [ 161, The composition of CEFA in the inner aorta is almost the same as the composition of CEFA in the serum samples taken 3 weeks before the animals were killed. Glucosamine affected the CEFA pattern in the same direction in serum and inner aorta. The glucosamineinduced change in CEFA of the inner aorta is therefore considered secondary to the glucosamine-induced change in the CEFA pattern of serum. The increased wet weight of the inner aorta in the groups receiving glucosamine may be related to the change in the CEFA pattern of the accumulated cholesterol esters. This:would require a higher sclerogenic action of cholesterol linoleate than of cholesterol oleate in contrast to the sclerogenic order of subcutaneously injected cholesterol esters, as determined in the subcutaneous tissue of the rat [17]. Another possibility is that glucosamine had a direct action on the aortic tissue as also indicated in Fig. 2 where the wet weights of the inner aorta in the animals treated with glucosamine are about twice the wet weights of the control animals at all the observed levels of cholesterol content in the tissues. This aspect may be of interest since glucosamine is an-important component of’ the PAS-positive materials which accumulate in diabetic microangiopathy [4,18]. An increase in the.synthesis of glucosamine or its content in serum and tissues of diabetic patients and animals have been reported [19-221. The administration of glucosamine (1 g/kg body weight per OS) to the animals in our
213
experiment did not increase the level of serum glucose as has been demonstrated with a larger amount of glucosamine (4 g/kg) administered intra-peritoneally in rats [ 221. The increased wet weight of the inner aorta in the animals which had received glucosamine thus supports the hypothesis that an increased level of serum [ 231. glucosamine may be related to the development of diabetic angiopathy References 1 Iverius, P., Possible role of the glycosaminoglycans in the genesis of atherosclerosis. In: Atherogenesis: Initiating factors (Ciba Foundation Symposia, No. 12). Elsevier. Amsterdam, 1973, p. 185. 2 GerCi. S., Gergely. J., Devenyi. T., Jakab. L.. Szekely, J. and Vi&, S., Role of intimal mucoid substances in the pathogen&s of atherosclerosis, Part 1 (Complex formation in vitro between mucopolysaccharides from atherosclerotic aortic intimas and plasma P-lipoprotein and fibrinogen), J. Atheroscler. Res.. l(1961) 67. 3 Zilversmit, D.B.. A proposal linking atherogenesis to the interaction of endothelial lipoprotein lipase with triglyceride-rich lipoproteins, Circulat. Res., 33 (1973) 633. 4 Cohen, Margo P. And Foglla, V.G.. Aortic mucopolysaccharides in experimental diabetes, Diabetes, 19 (1970) 639. 5 Tesorlere, G., Dories. F.. Magi&o, D. and Castagnetta, L.. Intestinal absorption of glucosamine and N-acetyl-glucosamine. Experientia (Basel). 28 (1972) 770. 6 Stender. S., Turnover of cholesterol in aorta of rabbits previously fed a cholesterolenriched diet, Atherosclerosis, 23 (1976) 275. 7 Alling, C., Dencker, S.J., Svennerholm. L. and Tichy. J.. Serum fatty acid pattern in chronic alcoholics after acute abuse, Acta Med. Stand., 185 (1969) 99. 8 Roberts, D.C.K., West, C.E.. Redgrave, T.G. and Smith, J.B., Plasma cholesterol concentration in normal and cholesterol-fed rabbits -Its variation and heritability. Atherosclerosis, 19 (1974) 369. 9 Stender. S.. Inter-individual variations in metabolism of cholesterol studied in rabbits during normal and cholesterol-enriched diets, Proc. 4th Int. Symp. on Atherosclerosis, In press. 10 Brattsand, R.. Petersen, H. and Lundholm, L.. Action of niceritrol (pentaeythritaltetranicotinate) on lipid accumulation in aortas of cholesterol-fed rabbits independent of contemporary reduction of serum lipids, Atherosclerosis. 20 (1974) 469. 11 Brattaand, R.. Distribution of cholesterol and triglycerides among lipoprotein fractions in fat-fed rabbits at different levels of serum cholesterol, Atherosclerosis, 23 (1976) 97. 12 Stender. S. and A&up. P., Sex variations in aortic resistance to hypercholesterolemia in rabbits with same level of serum cholesterol, In preparation. 13 Chalvardjisn. AM. and Still, W.J.S.. Fatty acid composition of tissues in cholesterol-fed rabbits, J. Atheroscler. Res.. 4 (1964) 507-516. 14 Rose, H.G., Origin of cholesteryl esters in the blood of cholesterol-fed rabbits - Relative contributions of serum lecithin-cholesterol acyltransferase and hepatic ester synthesis, Biocbim. Biophys. Acta, 260 (1972) 312. 15 Barter. P.J.. Origin of esterlfied cholesterol transported in the very low density lipoproteins of human plasma, J. Lipid Res.. 15 (1974) 11. 16 Rodriquez, J.L., Ghiselli, G.C., Torreggiani, D. and Slrtorl, C.R., Very low density lipoproteins in normal and cholesterol-fed rabbits - Lipid and protein composition and metabolism. Part 1 (Chemical composition of very low density lipoproteins in rabbits). Atherosclerosis, 23 (1976) 73. 17 Abdulla. Y.H., Adams, C.W.M. and Morgan. R.S., Connective-tissue reactions to implantation of purified sterol. sterol esters, phosphoglycerldes, glycerides and free fatty acids, J. Pathol. Bacterial., 18 19 20 21 22 23
94 (1967) 63. Beisswenger, P.G. and Spiro, R.G., Human glomerular basement membrane in diabetes mellitus, Science, 168 (1970) 596. Fushimi, H., Ichlhara, K., Tarul. S. and Nishikawa. M.. Increase of urinary and tissue hexosamlne in streptozotocin diabetic rats, Proc. Sot. Exp. Biol.. 145 (1974) 302. Malathl, K. and Kurup. P.A.. Effect of insulin and some oral hypoglycaemic agents on in vitro synthesis of glucosamine by rat liver. Indian J. Biochem., 6 (1969) 231. Walker, F. and Patrick. R.S.. Effect of insulin on the hexosamine content of alloxan diabetic rabbit vitreous humor, Diabetes, 17 (1968) 105. Martin, J.M. and Bambers, G., Insulin secretion in glucosamine-induced hyperglycemia in rats. Amer. J. Physiol., 209 (1965) 797. Fushimi. H.. Ichiiara. W.. Shinji. T.. Tami. S. and Nishlkawa. M.. Effect of glucosamine admlnistration in normal rats in comparison with streptozotocin treatment, Proc. Sot. Exp. Biol.. 145 (1974) 305.
Atherosclerosis, 26 (1977) 205-213 Biomedical 0 Elsevier/North-Holland
GLUCOSAMINE
Press, Amsterdam
AND EXPERIMENTAL
- Printed in The Netherlands
ATHEROSCLEROSIS
INCREASED WET WEIGHT AND CHANGED COMPOSITION OF CHOLESTEROL FATTY ACIDS IN AORTA OF RABBITS FED A CHOLESTEROL-ENRICHED DIET WITH ADDED GLUCOSAMINE
STEEN STENDER and POUL ASTRUP Department
of Clinical Chemistry,
Rigshospitalet,
DK-2100
Copenhagen
(Denmark)
(Received 13 May, 1976) (Revised, received 9 August, 1976) (Accepted 12 August, 1976)
Summary Three groups of each 12 rabbits were fed a cholesterol-enriched diet. Glucosamine was added in amounts of 0.5% and 2.0% (w/w) to the diet of two of the groups, while the third group served as a control group. The amount of cholesterol in the diet, was individually adjusted, so that all rabbits experienced nearly the same level of serum cholesterol throughout the whole experimental period. Glucosamine did not affect the concentration response of serum cholesterol to dietary cholesterol or the amount of free and esterified cholesterol in the inner aorta. It did, however, cause an increase in the wet weight of the inner aorta with a corresponding decrease in the concentration of aortic cholesterol. Furthermore a decrease in the ratio between mono-unsaturated and di-unsaturated fatty acids of the cholesterol esters of serum and inner aorta were observed in the animals Key words:
which
received
glucosamine.
Aorta - Cholesterol - Cholesterol angiopathy - Glucosamine -Rabbits
ester fatty
acid composition
- Diabetic
Introduction Heparin and several other glucosaminoglycans have the ability to form complexes in vitro with low density lipoproteins in the presence of certain cations [l]. It has been proposed that complex formation between low density lipoThis work was supported by grants from The Danish Medical Research Council.
206
proteins from plasma and glucosaminoglycans from the arterial wall also takes place in vivo, implicating a role in the deposition of cholesterol in the intimal tissue [2,3]. Glucosaminoglycans have also been related to the development of the vascular complications in diabetes [4]. Heparin - which is a polymer composed of D-glucosamine, D-glucuronic acid, and sulphate residues - is not absorbed from the intestinal lumen, contrary to its subunit glucosamine, which penetrates the intestinal cells by facilitated diffusion without modification of the molecular structure [5]. It was therefore of interest to investigate the development of experimental atherosclerosis in rabbits fed a cholesterol-enriched diet with added glucosamine. Methods Animals Thirty-six inbred 4-months-old male rabbits of the White Danish Country Strain castrated at an age of 2 months were used. Each animal was fed 150 g per day of standard pellets (Boserup rabbit pellets@: protein 15%, fat 3%, mainly from soybeans and linseeds, water 12%, crude fiber 13%). Water was given ad libitum. 0.5% and 2.0% (w/w) of D-glucosamine hydrochloride (Sigma@) were incorporated in the food by the manufacturers of the pellets. After an initial adaptation period of 3 weeks with ordinary rabbit pellets the animals were divided into 3 groups, which had the same average concentration of serum cholesterol and the same average body weight. Two of the groups were fed the diet containing glucosamine for the following 12 weeks. The third group served as a control group. All three groups were fed an individually adjusted amount of dietary cholesterol for the first 6 weeks. For the next 6 weeks the respective diets were fed without cholesterol addition (Table I). Individually adjusted cholesterol feeding Serum cholesterol was determined weekly during the first 6 weeks. Rabbits which after 1 week with 1% cholesterol (w/w) in the diet had a higher serum cholesterol level than the average, received a diet enriched with O.l%, 0.2%, or 0.3% (w/w) of cholesterol during the following week, depending on the previous response to dietary cholesterol, while on the other hand the rabbits with a lower concentration of serum cholesterol received a diet enriched with 0.5%, l.O%, or 2.0% of cholesterol depending on the previous response. This procedure was continued for 6 weeks. The amount of cholesterol ingested by each
TABLE
1
Group
Adaptation
period
Week
o-6
Week
6-12
(3 weeks)
G-O
F
F+C
G-
0.5%
F
F+0.5%G+C
F + 0.5%
G
G-
2.0%
F
F+2.0%G+C
F + 2.0%
G
F = (Boserup samine
(w/w).
rabbit
pellets?:
150
g/day;
C = an individually
F
adjusted
amount
of cholesterol;
G = gluco-
207
rabbit was registered. As for the preparation of the diet, cholesterol equivalent to a 1% (w/w) diet was dissolved in ether and poured over the pellets. Pellets from this 1% batch were then mixed with pellets without cholesterol in appropriate amounts to produce the wanted percentages. Tissue and blood samples Body weights were registered every week. The animals were bled from the marginal ear vein. After discontinuation of cholesterol feeding blood samples were taken 3 weeks, as well as immediately, before the rabbits were killed. The remaining procedures, including removal of liver and aorta, isolation of the inner aorta, homogenization of the tissue samples, and chemical determination of total cholesterol are described elsewhere [6]. Glucose in serum was measured by a hexokinase method. Determination of cholesterol ester fatty acids (CEFA) CEFA was determined with TLC by a modification of the method described by Alling et al. [7]. The lipids were extracted from serum with chloroform : methanol (1 : 1, vol/vol). Aliquots of the supernatant and of standards containing known amounts of cholesterol palmitate (16 : 0), oleate (18 : l), linoleate (18 : 2) and linolenate (18 : 3) were applicated on precoated silica gel TLC plates (Merck). The chromatogram was developed in toluene : heptane (38 : 90, vol/ vol), 3 X 20 min. After drying, the plates were dipped in a solution of ethanol : water : perchloric acid (100 : 113 : 9, vol/vol/vol) for 10 set, and placed in an oven at 100°C for 10 min. The fluorescences of the different cholesterol ester bands were quantified in situ using a Vitatron TLD 100 Universal Densitometer. The serum samples were kept at -20°C from the day of blood sampling until the CEFA analyses were performed. No other precautions were taken to stop the LCAT activity in vitro. The samples were determined within a few days in randomized order. The same procedure was used for determination of the aortic CEFA composition. In a number of serum and aortic samples the cholesterol ester bands were scraped from the thin layer plates and the lipids eluted with diethyl ether. The fatty acids in the different bands were determined by GLC. It was found that 16 : 0 represented more than 90% of the saturated band, 18 : 1 represented more than 90% of the monosaturated band, and 18 : 2 more than 95% of the disaturated band in the aortic as well as in the serum samples. Statistics Data are presented as means +S.E.M. The significance of differences between means was determined by the Student t-test. Linear regressions were calculated by the method of least squares. Results General condition of the animals All the animals were in good health throughout
the experimental
period. All
208
TABLE 2 BODY WEIGHT, TISSUE WEIGHT INNER AORTA (Z + SEM).
AND
CONTENT
OF TOTAL
Control group (n = 12)
CHOLESTEROL
0.5% Glucosamine (n = 12)
IN LIVER
AND
2.0% Glucosamine (n = 12)
__ Body weight (kg)
week 0 week 12
3.09 3.85
Orally added cholesterol from week O-6
(g)
Wet weight of liver (g)
+ 0.10 f 0.09
45.3
+ 5.7
131
f 7
3.21 3.91 51.8 131
t 0.10 t 0.12
3.36 3.84
? 8.2
56.6
f 10
0.168
+ 0.020
0.265
f 0.038
Total cholesterol in liver (mmol)
2.17
+ 0.39
1.42
f 0.27
f 2.3
9.8
? 2.4
10.8
f 6.4
130
Wet weight of inner aorta (g)
Total cholesterol in inner aorta (pmol)
t 0.10 * 0.09
a
f7
0.323
f 0.029
2.01
+ 0.48
12.5
b
?r3.4
a P < 0.05; b P < 0.001.
rabbits ate the daily amount of food irrespective of the addition of cholesterol and glucosamine. A steady increase in body weight took place with no significant differences between the experimental groups and the control group (Table 2). Cholesterol in the serum and in the diet Variation8 in serum cholesterol concentration within each group was minimized by the individual cholesterol feeding. The coefficient of variation was in the range of 15-25% in each group. The three groups had the same average serum cholesterol levels during the increasing as well as the decreasing period of serum cholesterol concentration (Fig. 1).
SERUM
CHOLESTEROL mmol
CHOLESTEROL
/I
mmol T
T
-
0
---
SERUM
-
/lOOg
wet
weight
CONTROL
fl
0.5%
GLUCOSAMINE
a
2.0%
GLUCOSAHINE
LIVER
INNER AORTA
Fig. 1. Levels of total cholesterol in serum (1 mmol/l = 38.7 mg/lOO ml) during the experiment and levels of total cholesterol in liver and inner aorta.
209
No significant difference was found between the average concentration of serum glucose in the glucosamine groups and in the control group. The total amount of cholesterol added to the diet differed enormously among the rabbits, even within the same group. One rabbit was fed a total of 20 g cholesterol during the 6 weeks. while another rabbit during the same period received 100 g, and both rabbits had the same increase in serum cholesterol concentration. The mean value for cholesterol intake was higher in the groups receiving glucosamine than in the control group. Due to the large variation this difference was not significant (Table 2). Liver No significant difference was observed in liver wet weight or in the amount of liver cholesterol between the groups receiving glucosamine and the control group (Table 2). Inner aorta A significant increase in wet weight of the inner aorta was observed in the two groups receiving glucosamine, but the amount of total cholesterol accumulated in the inner aorta was not affected (Table 2). Calculating the concentration of cholesterol in the inner aorta by dividing the amount of cholesterol by the respective wet weight, a significant lower concentration was found in the glucosamine groups (Fig. 1). Figure 2 shows the content of aortic cholesterol in pmol plotted against the wet weight of the same inner aorta for all the rabbits. Composition CEFA was weeks before groups in the
of cholesterol ester fatty acids (CEFA) determined in serum obtained from the animals in week 9 - three the animals were killed. There were no differences between the concentration of total cholesterol in serum or in the distribution
Wet weight of inner
aorta
mg
I 5
10
15
20
25 Total
30 cholesterol
35 urn01 in inner aorta
Fig. 2. Relationship between content of total cholesterol (wnol) and wet weight (me) of the same inner aorta in Group G-O (e), Group G-O. 5% (A) and in Group G-2.0% (0). The regression lines: y = 7.1 x + 90. r = 0.84 for Group G-O and Y = 9.4 x + 187. r = 0.79 for Group G-O. 5% and Group G-2.2.0%.
:1 :2
3.1
0.8 1.6 1.4 0.6
f 0.3
f 0.03
f f f f * 0.03
1.6 c * 0.1
0.73
22.2 * 0.7 44.7 c + 1.4 28.2 c f 1.2 4.9 r 0.4
0.5% glucosamine
a The TLC bands were named after their major component. b P < 0.005. cp<0.001.
18 18
0.72
21.6 54.3 18.5 5.5
16:08 18 : 1 18 : 2 18 : 34
Free cholesterol Total cholesterol
control
Fatty acid % of total cholesterol ester
Serum (week 9)
PERCENTAGE FATTY ACID COMPOSITION OF CHOLESTEROL WITHOUT GLUCOSAMINE ADDED TO THE DIET (X f. SEM).
TABLE 3
+ 0.03
1.7 c f 0.2
0.73
21.7 f 0.5 45.5 b f 1.9 28.4 c f 1.8 4.6 * 0.2
2.0% gIucosamine
3.4
0.54
- 22.1 57.2 17.2 3.7
control
+ 0.1
+ 0.04
_+0.3 f 0.9 t 0.6 -t 0.4
* 0.06 2.0 c + 0.1
0.54
22.4 * 0.8 48.7 c * 1.8 25.2 c f 1..3 3.5 * 0.5
0.5% glucosamine
OF CHOLESTEROL-FED
Inner aorta (week 12)
ESTERS IN SERUM AND INNER AORTA
WITH AND
+ 0.09 2.2 c f 0.1
0.65
21.5 r 0.9 51.9 c f 1.6 23.7 c * 1.3 3.0 f 0.6
2.0% glucosamine
RABBITS
211
between esterified and free cholesterol. However, the rabbits receiving glucosamine had on average a 10% higher level of linoleic acid (18 : 2) and a 10% lower level of oleic acid (18 : 1) than the control group (Table 3). Almost the same difference was observed in the average composition of CEFA in inner aorta (Table 3). The level of oleic acid was a little higher and the level of linoleic acid a little lower than the respective levels in serum. This was the case for the control rabbits as well as for the glucosamine rabbits. The ratio between oleic acid and linoleic acid in the cholesterol esters of serum in the control group was 3.1 f 0.3, while the same ratio in the 2.0% glucosamine group was 1.7 1: 0.2 and 1.6 + 0.1 in the 0.5% glucosamine group. This difference in ratios is highly significant (P < 0.001). Discussion Dietary cholesterol From Table 2 it is concluded that glucosamine did not significantly affect the response of cholesterol concentration in serum to the intake of dietary cholesterol, although a considerable difference existed between the mean values of the cholesterol intake in the control group and the glucosamine-fed groups. The large inter-individual variation in serum cholesterol concentration in rabbits fed a fixed amount of cholesterol is well known [8]. In our experimental procedure we halved this variation by transferring it to the amount of dietary cholesterol, which varied between 20 and 100 g in total ingested cholesterol during the 6 weeks. The mechanism behind this large variation has recently been investigated by kinetic analyses of cholesterol metabolism in rabbits during normo- and hypercholesterolaemic steady state conditions. It was found that the variation in cholesterol intake only partly can be explained by differences in cholesterol excretion rates. Differences in cholesterol synthesis and cholesterol absorption do also interfere [ 91. Individually adjusted cholesterol feeding has certain advantages compared with the traditional method of adding a fixed amount of dietary cholesterol to the rabbits: (1) It reduces the usually large inter-individual variation in concentration of serum cholesterol. (2) It is possible to investigate whether a substance having a serum lipid-lowering action also affects the metabolism of cholesterol in inner aorta in more direct ways, since the cholesterol-lowering effect in serum is compensated by feeding more cholesterol to the experimental groups [ 101. Inner aorta Since cholesterol accumulation in arterial walls continues after cessation of cholesterol feeding, and until the serum cholesterol concentration has become nearly normal, as shown by several authors and recently confirmed by us [6], the rabbits in the present experiment were not killed until this occurred. Although the inter-individual variation in serum cholesterol concentration was considerably reduced by the individual feeding, the variation in the concentration of cholesterol in the inner aorta was the same (coefficient of variation 50%) as seen in experiments with a much larger variation in serum cholesterol [6]. This indicates that the concentration of total cholesterol in serum is
212
not the only parameter related to the atherogenic action of cholesterol feeding in rabbits. The distribution of cholesterol in the different lipoproteins may be more important. Brattsand [ll] has recently demonstrated that the degree of aortic lipid infiltration in the cholesterol-fed rabbit is better related to the levels of LDL than to the VLDL cholesterol or the total cholesterol. Composition
of cholesterol-ester
fatty acids (CEFA)
In rabbits of the same strain and fed the same basic diet (Boserup rabbit pellets@) the 18 : l/18 : 2 ratio in CEFA of serum increased from 0.9 to 2.7 when the concentration of serum cholesterol was elevated from 1 mmol/l to 17 mmol/l by adding cholesterol to the diet [12]. An increase in the 18 : l/ 18 : 2 ratio has also been found in the cholesterol esters of liver and aorta when cholesterol-fed rabbits were compared with rabbits fed a conventional diet
v31. Three weeks after the cholesterol feeding was discontinued in the present experiment with glucosamine the 18 : l/18 : 2 ratio in CEFA of serum still exhibited a cholesterol feeding pattern in the control group, since this ratio was 3.1. Glucosamine affected this ratio in the direction of a more noncholesterol feeding pattern (1.7), but without affecting the total amount of cholesterol ester in serum (Table 3). It is only possible to speculate about the mechanism underlying this change in the composition of CEFA in serum: Contrary to humans, the liver of the rabbit is the main site of cholesterol esterification [14,15]. Glucosamine may therefore interfere in the liver with the incorporation of the oleic acids in the cholesterol esters, or glucosamine may facilitate the breakdown of VLDL in which the major part of the cholesterol-oleate in hypercholesterolaemic rabbit serum seems to be located [ 161, The composition of CEFA in the inner aorta is almost the same as the composition of CEFA in the serum samples taken 3 weeks before the animals were killed. Glucosamine affected the CEFA pattern in the same direction in serum and inner aorta. The glucosamineinduced change in CEFA of the inner aorta is therefore considered secondary to the glucosamine-induced change in the CEFA pattern of serum. The increased wet weight of the inner aorta in the groups receiving glucosamine may be related to the change in the CEFA pattern of the accumulated cholesterol esters. This:would require a higher sclerogenic action of cholesterol linoleate than of cholesterol oleate in contrast to the sclerogenic order of subcutaneously injected cholesterol esters, as determined in the subcutaneous tissue of the rat [17]. Another possibility is that glucosamine had a direct action on the aortic tissue as also indicated in Fig. 2 where the wet weights of the inner aorta in the animals treated with glucosamine are about twice the wet weights of the control animals at all the observed levels of cholesterol content in the tissues. This aspect may be of interest since glucosamine is an-important component of’ the PAS-positive materials which accumulate in diabetic microangiopathy [4,18]. An increase in the.synthesis of glucosamine or its content in serum and tissues of diabetic patients and animals have been reported [19-221. The administration of glucosamine (1 g/kg body weight per OS) to the animals in our
213
experiment did not increase the level of serum glucose as has been demonstrated with a larger amount of glucosamine (4 g/kg) administered intra-peritoneally in rats [ 221. The increased wet weight of the inner aorta in the animals which had received glucosamine thus supports the hypothesis that an increased level of serum [ 231. glucosamine may be related to the development of diabetic angiopathy References 1 Iverius, P., Possible role of the glycosaminoglycans in the genesis of atherosclerosis. In: Atherogenesis: Initiating factors (Ciba Foundation Symposia, No. 12). Elsevier. Amsterdam, 1973, p. 185. 2 GerCi. S., Gergely. J., Devenyi. T., Jakab. L.. Szekely, J. and Vi&, S., Role of intimal mucoid substances in the pathogen&s of atherosclerosis, Part 1 (Complex formation in vitro between mucopolysaccharides from atherosclerotic aortic intimas and plasma P-lipoprotein and fibrinogen), J. Atheroscler. Res.. l(1961) 67. 3 Zilversmit, D.B.. A proposal linking atherogenesis to the interaction of endothelial lipoprotein lipase with triglyceride-rich lipoproteins, Circulat. Res., 33 (1973) 633. 4 Cohen, Margo P. And Foglla, V.G.. Aortic mucopolysaccharides in experimental diabetes, Diabetes, 19 (1970) 639. 5 Tesorlere, G., Dories. F.. Magi&o, D. and Castagnetta, L.. Intestinal absorption of glucosamine and N-acetyl-glucosamine. Experientia (Basel). 28 (1972) 770. 6 Stender. S., Turnover of cholesterol in aorta of rabbits previously fed a cholesterolenriched diet, Atherosclerosis, 23 (1976) 275. 7 Alling, C., Dencker, S.J., Svennerholm. L. and Tichy. J.. Serum fatty acid pattern in chronic alcoholics after acute abuse, Acta Med. Stand., 185 (1969) 99. 8 Roberts, D.C.K., West, C.E.. Redgrave, T.G. and Smith, J.B., Plasma cholesterol concentration in normal and cholesterol-fed rabbits -Its variation and heritability. Atherosclerosis, 19 (1974) 369. 9 Stender. S.. Inter-individual variations in metabolism of cholesterol studied in rabbits during normal and cholesterol-enriched diets, Proc. 4th Int. Symp. on Atherosclerosis, In press. 10 Brattsand, R.. Petersen, H. and Lundholm, L.. Action of niceritrol (pentaeythritaltetranicotinate) on lipid accumulation in aortas of cholesterol-fed rabbits independent of contemporary reduction of serum lipids, Atherosclerosis. 20 (1974) 469. 11 Brattaand, R.. Distribution of cholesterol and triglycerides among lipoprotein fractions in fat-fed rabbits at different levels of serum cholesterol, Atherosclerosis, 23 (1976) 97. 12 Stender. S. and A&up. P., Sex variations in aortic resistance to hypercholesterolemia in rabbits with same level of serum cholesterol, In preparation. 13 Chalvardjisn. AM. and Still, W.J.S.. Fatty acid composition of tissues in cholesterol-fed rabbits, J. Atheroscler. Res.. 4 (1964) 507-516. 14 Rose, H.G., Origin of cholesteryl esters in the blood of cholesterol-fed rabbits - Relative contributions of serum lecithin-cholesterol acyltransferase and hepatic ester synthesis, Biocbim. Biophys. Acta, 260 (1972) 312. 15 Barter. P.J.. Origin of esterlfied cholesterol transported in the very low density lipoproteins of human plasma, J. Lipid Res.. 15 (1974) 11. 16 Rodriquez, J.L., Ghiselli, G.C., Torreggiani, D. and Slrtorl, C.R., Very low density lipoproteins in normal and cholesterol-fed rabbits - Lipid and protein composition and metabolism. Part 1 (Chemical composition of very low density lipoproteins in rabbits). Atherosclerosis, 23 (1976) 73. 17 Abdulla. Y.H., Adams, C.W.M. and Morgan. R.S., Connective-tissue reactions to implantation of purified sterol. sterol esters, phosphoglycerldes, glycerides and free fatty acids, J. Pathol. Bacterial., 18 19 20 21 22 23
94 (1967) 63. Beisswenger, P.G. and Spiro, R.G., Human glomerular basement membrane in diabetes mellitus, Science, 168 (1970) 596. Fushimi, H., Ichlhara, K., Tarul. S. and Nishikawa. M.. Increase of urinary and tissue hexosamlne in streptozotocin diabetic rats, Proc. Sot. Exp. Biol.. 145 (1974) 302. Malathl, K. and Kurup. P.A.. Effect of insulin and some oral hypoglycaemic agents on in vitro synthesis of glucosamine by rat liver. Indian J. Biochem., 6 (1969) 231. Walker, F. and Patrick. R.S.. Effect of insulin on the hexosamine content of alloxan diabetic rabbit vitreous humor, Diabetes, 17 (1968) 105. Martin, J.M. and Bambers, G., Insulin secretion in glucosamine-induced hyperglycemia in rats. Amer. J. Physiol., 209 (1965) 797. Fushimi. H.. Ichiiara. W.. Shinji. T.. Tami. S. and Nishlkawa. M.. Effect of glucosamine admlnistration in normal rats in comparison with streptozotocin treatment, Proc. Sot. Exp. Biol.. 145 (1974) 305.