Vitamin A and lysosomal stability in rats fed an atherogenic diet

281

24 (1976) 281-291 @ Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands

Atherosclerosis,

VITAMIN A AND LYSOSOMAL STABILITY IN RATS FED AN ATHEROGENIC DIET

P.R. SUDHAKARAN Department

and P.A. KURUP

of Biochemistry,

University

of Kerala,

Trivandrum

695001

(India)

(Received 10th October, 1975) (Revised, received 14th January, 1976) (Accepted 6th February, 1976)

Summary The relation between vitamin A status and lysosomal stability was studied in rats fed a high fat--cholesterol diet. Increase in the total activity of lysosomal enzymes as well as that in the nuclear fraction, intact lysosomal fraction and free activity (activity present in the 15,000 X g supernatant) in the liver was observed in rats fed an atherogenic diet with adequate vit. A. Vitamin A deficiency and hypervitaminosis (10,000 IU) augmented this increase in the total enzyme activity as well as the activity in the subcellular fractions except in the case of intact lysosomes where the activity was not significantly altered. At 2,000 IU, there was no significant alteration in either the total activity or the activity of the subcellular fractions. An analysis of the ratio of soluble activity (released from the lysosomes) to the activity present in the intact lysosomes, showed that hepatic lysosomal stability was decreased in the rats fed an atherogenie diet with normal dose of vit. A. Vitamin A deficiency as well as hypervitaminosis decreased the lysosomal stability still further. At a dose of 2,000 IU, lysosomal stability increased as compared to the rats fed an adequate dose of vit. A, while total lysosomal activity remained not significantly altered. Studies on the rate of release of enzymes from the lysosomes revealed that there was significantly more release of the enzyme between 30 and 45 min in the liver and aorta in the rats fed a high fat-cholesterol diet with adequate vit. A. This release was still more in the rats fed a low dose of vit. A. At 2,000 IU, there was no significant difference in the enzyme release. But the pattern of change in the liver and aorta in the hypervitaminotic group was different. In the case of hepatic lysosomes, there was an increase in the enzymes released while in the aorta there was significant decrease. This has been attributed to the fact that lytic concentration of the vitamin A is not attained in the aorta.

282

Key words:

Aorta - Cathepsin - Cholesterol - /%Glucuronidase -Lipids somes -Stability - Subcellular fractions - Vitamin A

-Liver

- Lyso-

Introduction Vitamin A is believed to play a role in ensuring the stability of cell membranes and of membranes of subcellular particles, particularly lysosomes. There are a number of reports that both vitamin A deficiency and hypervitaminosis A reduce the stability of rat liver lysosomes and increase the release of lysosomal hydrolases, both in vitro and in vivo [l-18] . The work of Dingle et al. on the effect of vitamin A deficiency and hypervitaminosis A on the stability of lysosomes in vivo deserves particular mention in this respect [12]. On the basis of available evidence the suggestion has been put forward that the presence of vitamin A in optimum concentration may be required for the maintainance of normal lysosomal stability. In a previous communication from this laboratory on the normal rat, it was reported that maximum lysosomal stability, as judged by release of lysosomal enzymes, was observed in the liver when the vitamin A intake ranged from 100 to 2,000 IU [ 191. Within this range, the variation in lysosomal stability was not appreciable; but above and below this range of vitamin A intake, the stability progressively decreased. It was reported in another communication from this laboratory that the activity of lysosomal enzymes increased significantly in the liver and aorta of atheromatous rats [20]. Peters et al. also reported a marked increase in certain lysosomal enzymes in atheromatous cells [21] . Subcellular fractionation of cells from atheromatous tissues showed the presence of a second population of low density lysosomes rich in certain enzymes but low in others [22]. Helen Shio and De Duve reported that the change from normal smooth muscle cell to foam cell in cholesterol-fed rabbits was accompanied by marked physical and chemical changes in the lysosomes [23] . Increased number of lysosome-like structures has been shown to be present both in human and experimentallyinduced atherosclerosis [ 24-291. Thus the lysosomes may be implicated in the pathogenesis of atherosclerosis. Since vitamin A has been reported to be antiatherogenic [ 30-331, the effect of vitamin A deficiency and hypervitaminosis has been studied on lysosomal stability in rats fed an atherogenic diet. Materials and methods Male albino rats (Sprague-Dawley strain, average initial weight 75-80 g) were divided into 6 groups of 25 rats each as follows: Group I. Normal diet with vitamin A intake of 100 IU/rat/day. Group II. High fat-cholesterol diet with vitamin A intake of 100 IU/rat/day. Group III. High fat-cholesterol diet with vitamin A intake of 4 IU/rat/day. Group IV. High fat-cholesterol diet with vitamin A intake of 2,000 IU/rat/ day. Group V. High fat-cholesterol diet with vitamin A intake of 10,000 IU/rat/ day.

283

Group VI. Pair-fed control of Group V which received the same amount as consumed by Group V of the high fat-cholesterol diet with 100 IU of vitamin A. In order to prevent sudden exposure to high dose of vitamin A, animals of group IV received 100 IU for the 1st week, 500 IU for the 2nd and 3rd week, 1,000 IU for the 4th to 6th week, and 2,000 IU for the remaining period of the experiment (24 weeks). In order to prevent great disparity in growth, the rats in groups V were given 100 IU of vitamin A for the 1st week, 500 IU for the 2nd and 3rd week, 1,000 IU for the 4th to 6th week, 2,000 IU for the 7th to 25th week and then 10,000 IU from the 26th to 30th week. The duration of this experiment was 30 weeks. The composition of the normal and high fatcholesterol diets was as follows: Normal diet. Dextrin - 65%, casein (vitamin-free and fat-free) - 20%, ground-nut oil - 5%, salt mixture * - 4%, vitamin mixture ** - 1% and cellulose - 5%. High fat-cholesterol diet. Dextrin - 54.5%, casein - 20%, coconut oil 15%, cholesterol - 5%, sodium taurocholate - 0.5%, salt mixture - 4% and vitamin mixture - 1%. The rats in the respective groups were killed at the end of 30 weeks and their tissues were removed quickly and placed in ice-cold containers. Estimation of vitamin A Vitamin A was estimated nick and Pader [34].

in the liver and aorta by the method

of Oser, Mel-

Es tima tion of lipids Total cholesterol was estimated in the tissues by the method of Carr Drekter [ 351, phospholipids by the method of Zilversmit and Davis [ 361 and triglycerides by the method of Van Handel and Zilversmit [ 371, with the modification that a Florisil column was used to remove phospholipids. Activity of fl-glucuronidase and cathepsin in the various subcellular fractions Liver was homogenised with 0.25 M sucrose at 0°C and the homogenate was centrifuged at 600 X g for 10 min in a refrigerated centrifuge. The sediment which contained nuclei, unbroken cells and plasma membrane (nuclear fraction) was separated, and the supernatant was again subjected to centrifugation at 15,000 X g for 30 min. The 15,000 X g sediment (lysosome-rich fraction) and supernatant were collected. The nuclear and lysosome-rich fractions were resuspended in acetate buffer (0.1 M, pH 4.5) containing 1% Brij 35. The 15,000 X g supernatant was diluted with an equal volume of 0.2 M acetate buffer. The

* The salt mixture used had the following composition: 105 g N&l, 120 g KCl, 310 g KH2P04, 149 g Cag(P04)z. 210 g CaC03. 0.20 8 anhydrous MgS04. 0.09 g K2A12(S04)4 .24HzO, 90 g anhydrous MgS04. 14.7 g FeP04 . 4H20, 9.39 g CuS04 . 5H2 0, 0.57 g NaF, 0.05 g Kl. In addition, the following trace elements were also added: 15 mg/kg diet ZnCl2 and 0.15 mg/kg diet CoCl2 . 6H2 0. ** The vitamin mixture used contained (in mg/lOO g diet): 0.8 mg thiamine, 0.8 mg riboflavin, 0.6 mg pyridoxine, 0.4 mg folic acid, 2 pg vitamin Bl2. 20 pg biotin. 150 IU ergocalciferol. 12 mg cu-tocopherol, 4.0 mg calcium pantothenate. 200 mg choline chloride, 20 mg inositol, and 0.3 mg menadione. Vitamin A acetate (2.9 million W/g) dissolved in ground nut oil was administered orally and daily to all groups.

284

activity of 2 typical lysosomal enzymes (P-glucuronidase and cathepsin) was determined in the nuclear and lysosome-rich fractions, as well as in the 15,000 X g supernatant. Rate of release of /3-glucuronidase from the lysosome-rich fraction Liver or aorta was homogenised in 0.25 M sucrose solution and the lysosome-rich fraction obtained as above. It was washed once with 0.25 M sucrose solution and resuspended in 0.25 M sucrose. The suspension was incubated at 37°C and aliquots were withdrawn at various intervals, immediately cooled to 0°C and centrifuged at 15,000 X g for 30 min. &Glucuronidase activity was determined in the 15,000 X g supernatant. Total activity in the lysosome-rich fraction was estimated in acetate buffer containing 1% Brij 35. Determination of enzyme activity @Glucuronidase activity was determined according to the procedure described by Kawai and Anno using p-nitrophenyl P-D-glucuronide as the substrate [ 381. Cathepsin activity was determined by using 4% haemoglobin in 0.1 M acetate buffer (pH 4.5) as the substrate, and determining the amount of tyrosine liberated by the method of Folin and Ciocalteu [ 391. Serum was used directly for estimating fl-glucuronidase activity. Results and Discussion (1) Vitamin A status and concentration of lipids The vitamin A concentration of the aorta and liver of the animals of the various groups is given in Table la. The concentration of total cholesterol, phospholipids and triglycerides of the serum, liver and aorta is also given in Table lb. (2) The activity of P-glucuronidase and cathepsin in the nuclear fraction, lysosome-rich fraction and 15,000 X g superna tan t The activity of P-glucuronidase and cathepsin in the different subcellular TABLE

la

VITAMIN Average with

A LEVELS of

the

Group

Group

OF

values

II and

from

Group

Vit

AORTA

AND

6 rats

V with

A (W/g

wet

+ SEM.

its pair-fed

206

II III

140?_c

IV

1816

f 100.2

V

8075

f 410

116

?

8.5

18.16 _c

7.58

‘Not

+

a 4.8

< P < 0.05. present

No symbol

in detectable = not

II has

been

Group

VI.

f

0.92

_c b

a P < 0.01. b 0.01

control

Aorta

I

VI

Group

tissue)

Liver

-

LIVER

amounts.

significant.

180.00 610.00? _c

+

7.1

a

21.5

a

compared

with

Group

I. Groups

III and

IV

AND SERUM

6800 +160a

4125 f 130

4021 f 152a

5330 + 165

III

IV

V

VI

249 f 6.8 a

240 ?: 7.9 a

310 f 8.9

150.8 * 5.5a

209.9 f 6.1

348 f 12.0b

308 f 10.5 a

229.6 + 5.8 b

4199 f 106 209.3 f 5.2

218.9 + 5.7b

4397 f 115a

4086 + 125

210.2 f 5.1

201.6 f 5.0 a

129.5 k 2.8

(me/ 100 ml)

SelUm

3877 * 95.0 b

4299 + 120a

2510 * 105.5

170 + 5.5

160.4 + 5.4a

281.2 f 5.2a

< 0.01. b 0.01 < P < 0.05. No symbol = not significant.

aP

5525 * 15.0a

II

226.1 f 4.8 a

69.5 f 1.8

399.2 f 16.5

I

(mzl 100 9)

Aorta

(mgl 100 g)

Liver

Serum

(mgl 100 ml)

(mtzl 100 9)

Phospholipids

Total cholesterol

Liver

Group

2710 + 70.5

1929 f 58.0 =

2016 f 56a

3020 ? 99

285.0 + 95.0 a

+ 27.0

906

(mg/ 100 g)

Aorta

1289 f 31.0

1106 f 36 a

1186 * 30a

1537 f 46.5 b

1368 f 45.0 a

490.6 f 21.5

(mgl 100 g)

Liver

12.01 * 0.30

9.60 * 0.29 a

9.81 + 0.28 a

12.91 f 0.40

12.15 f 0.41 a

7.15 f 0.25

(me/ 100 ml)

Serum

Triglycerides (as glycerol)

2310 f 66.0

1908 f 55a

2001 f 0.58 a

2021 + 72.0 a

2514 f 66.5 a

1090 f 36.0

(mgl 100 g)

Aorta

Average of the values from 6 rats k SEM. Group II has been compared with Group I, Groups III and IV with Group II, and Group V with its pair-fed control Group VI.

LIPID LEVELS OF LIVER, AORTA

TABLE lb

286

fractions of the liver in the animals of various groups is given in Table 2. Total enzyme activity in both cases was significantly increased in animals fed the high fat-cholesterol diet with an adequate dose (100 IU) of vitamin A when compared to the corresponding rats fed a normal diet. This increase is reflected in the activity of the nuclear fraction, lysosome-rich fraction and the 15,000 X g supernatant (soluble activity). A low dose of vitamin A (Group III) resulted in a further increase in the total enzyme activity. The activity in the nuclear fraction and soluble activity both increased, but, while cathepsin activity in the intact lysosomes was not significantly altered, the corresponding /3glucuronidase activity was significantly increased. In the animals receiving 2,000 IU, there was no significant alteration in the total activity of cathepsin or fl-glucuronidase or the activity in the subcellular fractions. In the rats fed the high fat-cholesterol diet with a massive dose of vitamin A, total cathepsin and /3-glucuronidase activity, nuclear and soluble enzyme activity increased, but intact lysosome activity was not significantly altered. The increase in total /3-glucuronidase and cathepsin activity in the liver of rats fed the high fat-cholesterol diet accords with results reported previously from this [20] and other [21,22,40-441 laboratories, but the change in the enzyme activity in different subcellular fractions does not seem to have been reported before. There is an increase in enzyme activity in the nuclear fraction, intact lysosomes and the soluble fraction in rats fed a high fat-cholesterol diet. Vitamin A deficiency and hypervitaminosis augment this increased and total fractional enzyme activity, except in the case of intact lysosomes where activity is not significantly altered. fl-Glucuronidase in vitamin A deficiency does, however, increase. The nuclear fraction also contains unbroken cells. The possibility has to be considered that the proportion of unbroken cells in this fraction may depend on cellular stability and in turn on vitamin A status. An analysis of enzyme activity in intact lysosomes and soluble activity reveals that the ratio of soluble activity (released from the lysosomes) to the activity present in the intact lysosomes is increased in rats fed the high fat-cholesterol diet with adequate vitamin A when compared with the corresponding animals fed a normal diet. This implies that lysosomal stability is decreased in rats fed the high fat-cholesterol diet. Further the total enzyme activity in the intact lysosomes and soluble fraction, which reflects total lysosomal activity, is also increased in the rats fed the atherogenic diet. Vitamin A deficiency, as well as hypervitaminosis, decreases lysosomal activity still further, as can be judged by the increased ratio of soluble activity to that in intact lysosomes. At a dose of 2,000 IU, lysosomal stability is increased since the ratio of soluble activity to the activity in the intact lysosomes is decreased while the total lysosomal activity remains not significantly altered. The results now obtained indicate that not only there is increase in total lysosomal enzyme activity but lysosomal stability itself is altered in the rats fed the atherogenic diet. (3) Rate of release of P-glucuronidase from hepatic and aortic lysosomes The results obtained with the lysosome-rich fraction from liver and aorta are shown in Figs. 1 and 2. The rate of release is expressed as percentage of total lysosomal activity.

137.9 f 8.4 a (8.775+ 0.26)

176.7 f 7.2 a (10.53 f 0.2)

134.4 + 9.5 (9.450 f 0.35)

185.7 f 5.3 a (11.27 f 0.21)

148.2 +_4.9 (8.992 f 0.16)

II

III

IV

V

VI

Activity in

317.4 + 16.1 (3.363 + 0.18)

lysosomes

647.1 f 26.5 (5.804 f 0.12)

78.5 ? 2.8 562.1 f 27.9 (0.8006 + 0.015)(5.509f 0.12)

116.1 f 3.2 a 607.7 f 19.5 (1.115 t 0.021) (5.611 + 0.14)

79.7 + 5.6 608.9 f 42.1 (0.8607 + 0.03)(6.062 f 0.16)

125.1 + 5.1 a (1.264 f 0.02)

75.36 f 4.5' 578.62 36a (0.782 + 0.025) (5.661 + 0.13)

30.5 * 1.5 (0.455 + 0.02)

Nuclear fraction

53.1 f 1.85 (2.191 r 0.05)

101.6 * 3.2 a (4.157 + 0.08)

41.33 t 2.9 (1.990 f 0.05)

78.52 * 3.1a (3.149 f.0.05)

49.75 f 3.2 a (2.106 f 0.04)

9.28 + 0.6 (0.6515 + 0.025)

Soluble

aI-- < 0.01. bO.O1

46.95 f 2.3 (4.545 * 0.2)

TOtal

Group 1I.GroupVcomparedwit.h

68.8 ?r2.9 a (0.7126 f 0.025)

34.8 t 2.07 (0.5178 f 0.02)

NUClI?W fraction

77.9 f 5.1 (0.8091 * 0.05)

181.3 f 7.9 (10.99 f 0.23)

71.6 fr4.3 (0.7311 i-0.015)

241.5 f 11.1 a 107.1 t 4.9 a (14.66 + 0.3) (1.028 f 0.02)

155.4 _+10.3 (11.06 2 0.30)

201.1 f 10.5 a 119.2 f 6.6 a (11.98 f 0.25) (1.204 * 0.03)

160.1 f 6.8 a (10.18 f 0.24)

66.61 f 4.29 (6.46? 0.25)

TOti

23.75 + 1.4 (1.667 f 0.42)

Soluble

568.1 f 20.5 (5.579 f 0.12)

639 f 24.5 (5.898 f 0.16)

622.3 + 41.9 (6.208 f 0.32)

681.1 f 32.4 (6.11 + 0.12)

87 f 4.1 (3.593 + 0.21)

204.3 + 7.5a (8.357 f 0.2)

69.75 C 4.7 (3.641 f 0.09)

119.6 f 6.1 a (4.796 + 0.1)

567.7 f 23.8 a 86.67 + 4.4 a (5.506 f 0.14) (3.669 f 0.08)

378.9 f 21.2 (3.941 f 0.19)

Activityin lysosomes

/3-Glucuronidase activity mgp-nitrophenollhlg protein

valuesfrom the individualliverof 6 rats.Group IIis compared with Group I. Group IIIandIVcomparedwith

Cathepsinactivitymgtyrlhlgprotein

the

I

Group

Average of Group VI.

ENZYMEACTIVITYINDIFFERENTSUBCELLULARFRACTIONSOFLIVERINRATSOFVARIOUSGROUPS

TABLE2

288

At 15 min there was no significant difference in the release of hepatic and aortic enzyme. At 30 min, the release of hepatic and aortic enzyme was significantly greater in Group II rats (high fat-cholesterol diet + 100 IU of vitamin A) than in those of Group I (normal diet + 100 IU vitamin A). Rats of Group III (high fat-cholesterol diet + 4 IU of vitamin A) showed a further increase in enzyme release when compared to Group II, while the rats of Group IV (high fat-cholesterol diet + 2,000 IU of vitamin A) showed no significant alteration. In the hypervitaminotic Group V, release of hepatic enzyme was greater than in the pairfed control Group VI, while a significant decrease was observed here in the aortic enzyme. Similar results were observed after 45 min. At 60 min, 90% of total enzyme activity was released in Group II rats, while Group I rats showed only a 71--75% release. Thus, hepatic lysosomal stability -- as judged by enzyme release - decreased in rats fed the high fat- cholesterol diet: both vitamin A deficiency and hypervitaminosis further decreased this stability. In the case of the aorta, decreased stability was observed in rats fed the high fat-cholesterol diet. Vitamin A deficiency decreased this stability still further, but hypervitaminosis increased it. This difference between liver and aorta may be due to the much lower concentration of vitamin A in the aorta than in the liver, the high concentration in the latter tissue promoting lysosomal lysis. In this connection, Peters and De Duve [21] reported a progressive increase in the latency of aortic lysosomes in atheromatous rabbits; this increased stability seemed to be a direct consequence of the cholesterol load. This lack of agreement between the results now obtained and those reported by De Duve et al. may possibly be a species difference. (4) /3-Glucuronidase activity in the serum The results (Table 3) indicate no significant alteration in enzyme activity in the serum in any group. This, in the face of the increased release of lysosomal enzymes reported above, may be due to the presence in serum of inhibitors of this enzyme activity. It is relevant that lecithin and many sulphated glycosaminoglycans (gg) inhibit this enzyme [Sudhakaran and Kurup, unpublished observation] . Group I Group II Group III Euu”, E Group QI

60

Time (min)

90

Fig. 1. Rate of release of @-glucuronidase from hepatic lysosomes. Activity expressed as percentage of the total lysosomal activity.

289

60 Fig. 2. Rate of release of P-ghxcuronidase total lysosomal activity. TABLE 3 P-GLUCURONIDASE

ACTIVITY

from

aortic

IN THE SERUM

90 Ttme (min) lysosomes. Activity

OF RATS

OF VARIOUS

Average of the values from 6 rats. Group II has been compared Group II and Group V with Group VI. Not significant in aII cases. Group

P_Glucuronidase

I II III IV V VI

52.79 53.9 55.1 54.3 55.1 52.9

+ * f + f k

activity

expressed

of the

GROUPS

with Group

of serum mg p-nitrophenol/h/ml

as percentage

I. Group

III and IV with

serum

1.7 1.9 2.5 1.9 1.8 1.6

Thus the increased release of lysosomal enzymes, many of which degrade gg, in rats fed the high fat-cholesterol diet, can result in increased degradation and consequently decreased concentration of gg. Decreased concentration of gg in the atheromatous aorta has been observed in this species to be associated with increased lipid accumulation [ 451. Vitamin A deficiency may aggravate atherosclerosis and this might be due to the further decrease in lysosomal stability it brings about in the aorta of animals fed an atherogenic diet. Hypervitaminosis A has now been found to increase the stability of aortic lysosomes, resulting in decreased release of lysosomal enzymes with a probable consequent increase in the local concentration of gg. References Dim&. J.T., Studies on Fell, H.B. and Dingle, (1963) 403. Dingle, J.T. and Lucy, (1962) 611. Dingle, J.T. and Lucy. (1961) 497.

the mode of action of excess of vitamin A, Biochem. J., 79 (1961) 509. J.T., Studies on the mode of action of excess of vitamin A, Biochem.

J.. 87

J.A..

Studies

on the mode

of action

of excess

of vitamin

A. Biochem.

J.. 84

J.A..

Studies

on the mode

of action

of excess

of vitamin

A, Biochem.

J., 79

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to vitamin A, J. Cell. Biol., 27 (1955) 448. A on acid phosphatase content of guinea pig

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