Effect of phospholipids on cholesterol-induced modifications in mouse brain

59

Atherosclerosis, 26 (1977) 59-66 @ Elsevier/North-Holland Biomedical

Press

EFFECT OF PHOSPHOLIPIDS ON CHOLESTEROL-INDUCED MODIFICATIONS IN MOUSE BRAIN

G. TOFFANO, FIDIA

A. LEON,

Research

(Received (Revised,

Laboratories,

26th April, received

(Accepted

D. BENVEGNC Abano

and F. CERRITO

Terme

*

(Italy)

1976)

23rd July,

4th August,

1976)

1976)

Summary Mice on an atherogenic diet for 40 days show a decrease in brain content of catecholamines, cyclic AMP and in dopamine degradation, and modification of the glycolytic pathway. The metabolic changes are paralleled by changes in behaviour, i.e. decrease in spontaneous motor activity and in conditioning avoidance response. The decrease in dopamine degradation and in behaviour parameters is partly due to the propylthiouracil present in the diet. Endovenous treatment with sonicated dispersions of bovine brain phospholipids induces a modification in the parameters of behaviour and metabolism. The possibility is discussed that some of the defects arising during the atherogenie diet are related with the establishment of a hypoxic state.

Key words:

Atherosclerosis -

Cerebral

-

glycolytic

Behaviour pathway

-

Bovine -

Cyclic

bmin

phospholipids

-

Catecholamines

AMP

Introduction Cholesterol-supplemented diet induces several biochemical changes in animal brain [ 1,2], i.e., phospholipid synthesis, phospholipid-fatty acids and activity of membrane bound enzymes, e.g., (Na+, K’) ATPase. All the above parameters were modified by administration of bovine brain phospholipids. Exploratory activity and cerebral catecholamines are also decreased in rats fed an atherogenic diet [ 31. * Institute

of Pharmacology.

Universita

Cattolica

Sacro

Chore.

Roma

(Italy).

60

Several questions arise. First, whether the presence of propylthiouracil, in the atherogenic diet, contributes to the alterations of behaviour and metabolic parameters. Second, whether catecholamine-dependent metabolic pathways, such as production of CAMP and rate of glycogenolysis, are modified. Third, whether the atherogenic diet produces changes which can be attributed to a hypoxic state. The present investigation aims to provide an answer to these ,questions. We will show that propylthiouracil affects behaviour parameters and dopamine degradation. Atherogenic diet induces a decrease in brain catecholamines which is accompanied by a decreased production of CAMP and a modification of the glycogenolysis rate. The increase of lactate/pyruvate ratio suggests a hypoxic state of the brain of animals kept under atherogenic diet [ 41. Finally we will show that treatment with bovine brain phospholipids modifies significantly the pattern due to the atherogenic diet and slightly that due to the administration of propylthiouracil. Material and Methods Treatment Male albino mice CD-1 (Charles River), weighing 25-30 g, were used throughout, and were divided into 5 groups. Group I: received normal laboratory diet and were considered as a control group ; Group II: received normal diet additioned with 0.3% propylthiouracil; Group III: received normal diet additioned with 0.3% propylthiouracil. From the 20th day, animals were treated intravenously with sonicated dispersion of bovine brain phospholipids (BC-PL), 100 mg/kg; Group IV: received atherogenic diet according to Gresham and Howard [ 51 and Thomas and Hartroft [6] in addition to normal diet. Atherogenic components were 3% cholesterol, 0.3% propylthiouracil, 2% sodium cholate and 40% arachis oil. The fatty acid composition of atherogenic diet consisted of 11.6% palmitic acid, 3.5% stearic acid, 54.4% oleic acid, 28.6% linoleic acid and trace amounts of arachic and linoleic acids (as % of total fatty acids). Group V: received atherogenic diet. From the 20th day, animals were treated intravenously with sonicated dispersion of bovine brain phospholipids (BC-PL), 100 mg/kg. Each group was divided into groups of 20 mice which were housed together in separated cages, fed food and water ad lib. and weighed weekly. Twenty days after the beginning of the diet, groups III and V received daily 100 mg/kg of BC-PL intravenously. Phospholipid vesicles were obtained by sonication, at 0°C in 0.05 M Tris-HCl pH 7.5, in an MSE apparatus for 8 min [ 71. After sonication, phospholipid composition and peroxide content remained unvaried. Intravenous injection of liposomes, in 0.1-0.2 ml buffer, was done in the tail vein within a few hours from their preparation; the control group received only buffer. The composition of employed BC-PL has already been reported [1,8]. It is characterized by a !arge amount of polyunsaturated fatty acids, such as and by lysophosphoCzoE4and CX:~, by both acidic and neutral phospholipids, lipids. Diet and phospholipid treatment were continued until the .4Oth day,

61

after which the animals were sacrified or submitted animal was used for one determination and randomly

to behavioural chosen.

tests. Each

Behavioural tests (1) Spontaneous motor activity test. The procedure was that described by Dews [9]. Square actometric cages were divided by two plexiglass into four equal parts. The movements were counted by a photoelectrical cell situated at the base of the cages and registered on a Battaglia-Rangoni counter. (2) Conditioning avoidance response test. The apparatus consisted of a series of double compartment chambers with a communication hole (shuttle-box). Electrical current alternately ran across the floor of each compartment composed of a grating of stainless stell bars. A bell, which rang 5 set prior to the arrival of the current on the grating, was employed as a conditioning stimulus. Each cycle had a duration of 30 set and each experimental trial consisted of 100 cycles. A photoelectrical cell connected to an electromechanical counter, counted the animal passages occurring during the ringing of the bell, i.e., before reception of the electrical discharge. Such passage (avoidances) were subsequently plotted. Before the diet-treatment, 20 mice per group underwent one session consisting of 5 trials. Those capable of 85-90s avoidance were considered and kept in separated cages throughout the experiment. Ca techolamine and homovanillic acid Three pooled brains of decapitated mice were homogenized with 5 volumes of 0.4 N perchloric acid containing 0.1% Na&Os. Dopamine (DA) and norepinephrine (NE) were assayed in 2 ml of perchloric acid extract according to Chang [lo], while homovanillic acid (HVA) was assayed in 3 ml of the same neutralized perchloric acid extract according to Korf et al. [ 111. Glycogen, glucose, lactate and pyruvate Animals were killed by decapitation so as to allow the head to fall The brain hemispheres were removed, immediately into liquid nitrogen. powdered under liquid nitrogen together with frozen 0.5 ml 0.3 N perchloric acid and allowed to warm to 0-4°C. Glycogen, glucose, lactate and pyruvate were estimated on perchloric acid extract of each brain according to Lowry et al. [12]. Cyclic AMP The liquid nitrogen frozen brain was powdered together with 1 ml 3% trichloroacetic acid. Cyclic AMP was separated according to Krishna et al. [13] and assayed according to Kuo and Greengard [ 141. Results Table 1 shows that propylthiouracil (group II) reduces growth. Inclusion of propylthiouracil in the atherogenic diet suppresses growth and induces mortality. BC-PL do not affect either growth or mortality. Figure 1 shows that the spontaneous motor activity and the conditioning avoidance response, as measured after 40 days, were decreased by addition of

62

TABLE

1

EFFECT

OF

WEIGHT

AND

PROPYLTHIOURACIL, ON

ATHEROGENIC

MORTALITY,

Groups

40

Number

DAYS

AFTER

of

DIET

AND

INITIATION

Body

weight

OF

BC-PL

TREATMENT

ON

(9)

Number

animals/group Final

A wt

Control

120

28.33

+ 0.02

36.40

f 0.04

+8.07

120

Propylthiouracil

120

28.12

f 0.04

32.07

+ 0.04

+3.95

120

28.75

f 0.02

33.10

f 0.03

+4.35

(100

+ BC-PL

80

80

mg/kg)

Atherogenic

diet

Atherogenic

diet

(100

of

survivals Initial

Propylthiouracil

BODY

TREATMENT

160 + BC-PL

80

28.12

* 0.03

24.86

? 0.05

-3.26

142

28.43

+ 0.03

24.58

+ 0.05

-3.85

73

mgFg)

0.3% propylthiouracil. Atherogenic diet further depressed both activities in mice. Phospholipid treatment, initiated 20 days after the diet, improved the behaviour pattern in both groups. When phospholipid treatment was initiated, propylthiouracil had not yet induced behaviour modifications while atherogenie diet had decreased the spontaneous motor activity by 15% and the conditioning avoidance response by 35%. It has been suggested that behaviour performances might be influenced by brain catecholamines [15,16] ; atherogenic diet affected parallely NE and DA content, as reported in Table 2. After 20 days, only DA and its HVA catabolite were decreased, while after 40 days NE was also affected. Propylthiouracil alone did not produce any significant variation in catecholamine content at the 20th and 40th day, while it strongly decreased HVA. Phospholipid treatment produced increase of catecholamines and HVA in the atherogenic diettreated group, while in the propylthiouracil-treated group only slight modifications were observed. It is well known that catecholamines produce increase of adenylate cyclase

% avoidance

Control

Atther;genic dietPropylthiour .

ltrol erogenic

05

Fig. tests test.

25

1. Effect in mice

65

45

of

40

days

60

Propylthiour

40

Atherogenic

dwt

8 5 Time(h)

propylthiouracil,

after

BC~PL diet .

atherogenic

of treatment.

Values

diet

and

BC-PL

are mean

f SE.

treatment

(100

Sixteen

mice

mg/kg, per group

i.v.)

on behavioural

were

used

in each

63 TABLE

2

NOR.EPINEPHRINE AND 40th DAY

(NE),

DDPAMlNE

(DA)

CND

HOMOVANILLIC

ACID

Group

DA

NE * (fig/g) = Control

0.59

f 0.01

Propylthiouracil

0.55

Propylthiouracil

Atherogenic

+

@g/g) a 1.00

+ 0.02

(10)

(10)

Atherogenic

IN MICE BRAINS

AT 20th

40 days

20 days

BC-PI, (100

(HVA)

OF TREATMENT

+ 0.02

HVA

NE

DA

(@g/g) a

(@g/g) a

@g/g)

0.30

0.66

0.97

f 0.02

0.94

? 0.03

0.30

t 0.03

f 0.01

0.66

(9)

(9)

(9)

(8)

_

_

_

0.68

mg/kg)

f 0.02

0.96

0.66

diet +

f 0.03

0.88

t 0.03

c

0.22

f 0.01

0.59

c

(10)

(10)

(10)

(8)

_

_

_

0.67

BC-PL (100 mg/kg)

0.90

t 0.03 * 0.01

C P < 0.01;

b P > 0.02;

Statistical significances versus atherogcnic diet: e P < 0.02:

h

0.80

+ 0.02

0.07

c

0.90

+ 0.01

d

? 0.01

d

(8) 0.10

f 0.03

(7) f 0.02

d

0.07

i 0.008

d

(8)

(8) f 0.03

f 0.01

(8)

(7)

(6)

.a Mean f standard error (II). Statistical significances versus control:

0.27

f 0.03

(8)

(7)

diet

tug/g) =

(8)

(8)

(10)

HVA a

k 0.02

f

0.14

+ 0.02

d.f

(6)

(6)

d P c 0.001.

f p < 0.01.

activity and induce an accumulation of CAMP in animal brain. Table 3 reports the effect of atherogenic diet and phospholipid treatment on cerebral CAMP. After 20 days, CAMP was strongly decreased and this decrease persisted until the end of treatment. BC-PL treatment completely restored the CAMP content to normal values. The role of intracellular CAMP as a brain regulatory factor, because of its involvement in the activation of phosphorylase and acceleration ‘of glycogenolysis, has already been pointed out [17,18]. Tables 4 and 5 show that at both the 20th and 40th day, the propylthiouracil group showed only a slight decrease of lactate and pyruvate in respect to the control group, and that the BC-PL treatment did not affect these variations. The atherogenic diet caused after 20 days a marked decrease of brain glycogen, glucose, lactate and pyruvate and also of blood glucose (3.5 + 0.25 pm/ml compared to 5.6 f 0.43

TABLE

3

EFFECT

OF

BC-PL

ON

CEREBRAL

Group

CAMP

CAMP

COIltXOl

IN MICE

(Pm/g)

diet

1453

1237

t 78

diet

t 41

b

b P <

-f standard 0.001.

error

-

(II).

f 62

832

k 53 b

(10) 1142 (10)

a Mean

days

(5)

929

+ BC-PL

ATHEROGENIC

a 40

(10) Atherogenic

WITH

20 days

(5) Atherogenic

FED

k 59

DIET

OF

4

diet

+

significances

Statistical

versus

versus

(n).

DAYS

enxx

significances

? standard

mg/kg)

+

mg/kg)

40

Statistical

a Mean

(100

Atherogenic

BC-PL

diet

(100

Atherogenic

BC-PL

PropyIthiouraciI

Propylthiouracil

Control

Group

(n).

versus

(14)

2.26

(14)

2.66

(10)

2.69

b

0.01:

d

0.001;

diet:

b P <

atherogenic

c * 0.04

* 0.02

t 0.03

f 0.03

c P < 0.001. P < 0.001.

(10)

0.77

f 0.06

2.00

(10)

(10)

0.50

2.05

(10)

(lo)

(10)

? 0.05

0.74

2.64

? 0.10

(8)

0.77

2.73

(8)

(8)

? 0.12

f 0.11

2.65

a + 0.02

(/m/g) 0.76

Glucose

a

@m/g)

c

d

c

AND

+ 0.03

+ 0.03

DIET

(8)

a

DIET

f 0.03

Glycogen

C

(14)

0.56

(14)

0.71

(lo)

0.76

c P < 0.001.

ATHEROGENIC

TREATMENT

control:

OF

b P <

* 0.08

f 0.04

f 0.12

Glucose @m/g)

ATHEROGENIC

@m/g)

a

AND

Glycogen

control:

PROPYLTHIOURACIL,

OF

AFTER

EFFECT

5

errm

significance

BRAINS

TABLE

diet

+ standard

Statistica!

a Mean

Atherogenic

PROPYLTHIOURACIL

TREATMENT

OF

Propylthiowacil

Control

Group

DAYS

EFFECT

TABLE

BC-PL

ON

f 0.05

f 0.06

(10)

2.00

(10)

1.50

(10)

2.20

(8)

2.36

(8)

2.70

@m/g)

a

k 0.10

+ 0.09

+ 0.06

f 0.07

f 0.09

Lactate

TREATMENT

(14)

1.89

(14)

2.42

(1 O)

f 0.07

c

c,d

c

c

b

c

ON

(10)

0.127

(10)

0.073

(10)

0.158

(8)

0.162

(8)

0.187

* 0.005

r 0.002

+ 0.001

+ 0.003

+ 0.004

a

c

AND

c-d

c

c

c

GLUCOSE,

f 0.005

r 0.005

Pyruvate (!&n/g)

a ? 0.007

GLYCOGEN.

(14)

0.096

(14)

0.126

(10)

0.146

(/m/g)

2.80

Pyruvate

LACTATE

Lactate =

GLUCOSE,

t/m/g)

GLYCOGEN,

r 0.07

? 0.09

f. 0.11

c

(10)

2.59

* 0.17

c.d

(10)

15.74

(10)

20.54 (10)

4.10

13.92 (10) ‘- 0.12

+ 0.13 (10)

3.56

(8)

14.56

3.54 (8)

(8)

14.43 (8) ? 0.12

r 0.10

* 0.60

? 0.53

? 0.19

r 0.09

IN

o.l@

? 0.12

Lactatel

f

f 0.09

pyruvate 3.48

d

C

AFTER

+ 0.11

Glycogenl

PYRUVATE

(14)

19.68

(14)

19.20

(10)

19.18

glucose

LACTATE

(14)

4.04

(14)

3.75

(10)

3.54

LactateI

B-RAINS

pyruvate

AND

MICE

Glycogenl

c

IN

glucose

PYRUVATE

MICE

20

:

65

pm/ml in the control animals). The changes in the glycolytic intermediates to the decrease in glycogen. The glycogen/glucose ratio were not proportional increased above the control group while the lactate/pyruvate ratio remained normal. After 40 days the cerebral lactate/pyruvate ratio increased. Phospholipid treatment led to a decreased glycogen/glucose ratio and to a decreased lactate/pyruvate ratio in the atherogenic group. Discussion Propylthiouracil reduces in mice growth and causes decrease of both the spontaneous motor activity and the conditioning avoidance response. In this case NE and DA are not affected, while HVA is strongly reduced. This suggests a correlation between impairment of catecholamine utilization and behaviour modifications. It has been reported that hypothyroid animals have a decreased motor response and an increased adrenergic activity consequent to a defective sensitivity of the adrenergic receptors [ 19,201. Atherogenic diet decreased behaviour activities and brain catecholamines. The decrease of CAMP, although an activation of phosphodiesterase activity cannot be excluded, is probably consequent to the decrease of both catecholamine availability and adenylate cyclase activity. The latter may be related with a change of acyl groups of membrane phospholipids [ 1,2]. The decrease of CAMP parallels the effect of the atherogenic diet on the glycolytic pathway, i.e., increase of glycogen/glucose ratio. After 40 days the atherogenic diet increases the lactate/pyruvate ratio. This ratio is in equilibrium with the cytosol NADH/NAD’ ratio. Thus an increase in the lactate/pyruvate ratio, can be interpreted as an indication of hypoxic damage [ 4,121. This observation is in accord with the view that at least some of the damages due to the atherogenic diet are due to the presence of an hypoxic state in the brain. The view is further supported by the fact that hypoxia causes, similarly to the atherogenic diet, modifications of the conditioned avoidance response [15], locomotor activity [ 161, and a decreased catecholamine synthesis [ 21,221. Analogous modifications in glycogenolysis were found in the aortic arch of rabbits fed an atherogenic diet (unpublished data). Sonicated dispersions of BC-PL modify a number of physiological and biochemical parameters induced by the atherogenic diet. The modification of the cerebral parameters is independent of an effect of BC-PL on mortality and body weight. The mechanism by which BC-PL exert their effect is not yet understood. In normal mice BC-PL increase the movements of brain catecholamines and stimulate the adenylate cyclase activity [ 231, and increase the brain glucose content [24]. These observations indicate an effect of phospholipids on the central aminergic activity. Whatever the mechanism through which the effect is achieved, a modification of the activity of the catecholaminergic system would explain most of the present observations. It would explain the improvement in behaviour, the rise in CAMP and, furthermore, the decrease in glycogen/glucose ratio on the basis of an activation of glycogen phosphorylase. On the other hand, the modifications induced by BC-PL on the lactate/pymvate ratio are more likely to be related with an

improved oxygenation ergic system [ 251.

of the brain tissue rather than with changes of the amin-

References 1 Toffano, G.. Gonzato. P., Aporti. F. and Castellani, A., Variations of brain enzymatic activities in experimental atherosclerosis, Atherosclerosis, 20 (1974) 427. 2 Toffano. G.. Gonzato, P. and Benvegnti, D., In: G. Porcellati (Ed.), Function and Metabolism of Phospholipids in CNS and PNS. Plenum Press, New York, 1976, p. 293. 3 PiIecki, R., Samochowiec, L. and Szyszka, K., The influence of atherogenic diet and “essential” phospholipids upon the contents of noradrenaline and dopamine in the brains of rats and their exploratory activity, Atherosclerosis, 22 (1975) 401. 4 Bachelard. H.S., Lewis. L.D., Ponten, K. and Siesjii, B.K., Mechanism activating glycogenolysis in the brain in arterial hypoxia, J. Neurochem., 22 (1975) 561. 5 Gresham, G.A. and Howard, A.N., The independent production of atherosclerosis and thrombosis in rat, Brit. J. Exp. Path.. 61 (1960) 395 6 Thomas, W.A. and Hartrof, W.S.. Myocardial infarction in rats fed diets containing high fat cholesterol, thiouracil, and sodium cholate, Circulation, 19 (1959) 65. 7 Papahadjopoulus. D. and Kimelberg, H.K., In: S.G. Dawison (Ed.), Progress in Surface Science, Pergamon Press, Oxford, 1973, p. 162. 8 Bruni. A., Leon, A. and Boarato, E., In: G. Porcellati (Ed.). Function and Metabolism of Phospholipids in CNS and PNS, Plenum Press, New York, 1976, p. 271. 9 Dews, P.B., The measurement of the influence of drugs on voluntary activity in mice, Brit. J. Pharmacol., 8 (1953) 46. 10 Chang, C.C., A sensitive method for spectrophotofluorometric assay of catecholamines. Int. J. Neuropharmacol.. 3 (1964) 643. 11 Korf, J.. Ottema, S. and Van der Veen. I., Fluorimetric determination of homovanillic acid in biological materials after isolation on Sephadex G-10. Analyt. Biochem., 40 (1971) 187. 12 Lowry, O.H., Passonneau, J.V., Hasselberger, F.X. and Schultz, D.W., Effect of ischemia on known substrates and cofactors of the glycolytic pathway in brain, J. Biol. Chem., 239 (1964) 18. 13 Krishna, G., Weiss, B. and Brodie. B.B.. A simple sensitive method for the assay of adenyl cyclase, J. Pharmacol. Exp. Ther.. 163 (1968) 379. 14 Kuo, J.F. and Greengard, P., Cycloc nucleotide-dependent protein kinases, Part 8 (An assay method for the measurement of adenosine 3’,5’-monophosphate in various tissues and a study of agents influencing its level in adipose cells), J. Biol. Chem., 245 (1970) 4067. 15 Brown. R., Davis, J.N. and Carlsson, A., Dopa reversal of hypoxia-induced disruption of the conditioned avoidance response. J. Pharm. Pharmacol.. 25 (1973) 412. 16 Brown, R. and Engel, I., Evidence for catecholamine involvement in the suppression of locomotor activity due to hypoxia, J. Pharm. Pharmacol., 25 (1973) 815. 17 Edwards, G.. Nahorski, S.R. and Rogers, K.J.. In viva changes of cerebral cyclic adenosine 3’,5’monophosphate induced by biogenic amines - Association with phosphorylase activation, J. Neurothem.. 22 (1974) 565. 18 Nahorski. S.R. and Rogers, K.J.. The incorporation of glucose into brain glycogen and the activities of cerebral glycogen phosphorylase and synthetase - Some affects of amphetamine. J. Neurochem., 23 (1974) 579. 19 Emlen. W., Segal, D.S. and Mandell. A.J., Thyroid state - Effects on pre- and postsynaptic central noradrenergic mechanisms, Science, 175 (1972) 79. 20 Parker, L.N.. The turnover of norepinephrine in the brain stems of dysthyroid rats, J. Neurochem., 19 (1972) 1611. 21 Davis, I.N. and Carlsson, A., The effect of hypoxia on monoamine synthesis, levels and metabolism in rat brain, J. Neurochem.. 21 (1973) 783. 22 Brown, R.M., Carlsson, A., Ljunggren, B., Siesja, B.K. and Snider, S.R.. Effect of ischemia on monoamine metabolism in the brain, Acta Physiol. &and., 90 (1974) 789. 23 Leon, A. and Toffano, G., in: G. Porcellati (Ed.). Function and Metabolism of Phospholipids in CNS and PNS, Plenum Press, New York, 1976. 24 Bmni, A., Leon, A.. Toffano, G. and Boarato, E., Pharmacological effects of phosphatidylserine liposomes, Nature (Land.). 260 (No. 5549) (1976) 331. 25 Williamson, D.H., Lund, P. and Krebs. H.A., The redox state of free nicotinamide-adenine dinucleotide in the cytoplasm and mitochondria of rat liver, Biochem. J., 103 (1967) 514.