Atherosclerosis,
0
349
22 (1975) 349-368
Elsevier Scientific Publishing Company, Amsterdam
- Printed in The Netherlands
EFFECTS OF PALM-KERNEL OIL AND SUNFLOWER-SEED OIL ON SERUM LIPIDS AND ATHEROGENESIS IN ALLOXAN-DIABETIC R.4BBITS
J. KLOEZE Unilever
AND
Research,
A. M. M. ABDELLATIF Vlaardingen
(The Netherlands)
(Received October 28th, 1974) (Revised received February 26th, 1975) (Accepted February 28th, 1975)
SUMMARY
of metabolically normal (controls) and alloxan-diabetic adult female rabbits were fed semi-synthetic diets containing 40 Cal% palm-kernel oil (PKO) or sunflower-seed oil (SSO) for 54 weeks. In contrast to control rabbits fed PKO-diet, the alloxan-diabetic rabbits on this diet, developed no or only a negligible degree of atherosclerosis, although the serum levels of all lipid classes had increased in the diabetic rabbits above that of the controls during almost the whole experimental period. The diabetic rabbits and the controls fed SSO-diet were both free from any significant atherosclerotic involvement in spite of the fact that the SSO-diet appeared unable to suppress the very high levels of the various serum lipid classes induced by Groups
the diabetic
state. On both diets, the diabetic
rabbits
showed
a significantly
higher
cholesteryl linoleate/oleate ratio than the controls, which was caused by an increase in the cholesteryl linoleate level in the diabetics. No serious aorta atherosclerosis was found in rabbits with a cholesteryl linoleate/oleate ratio higher than 0.6, although no correlation was found between the atherosclerosis indices and these ratios at values lower than 0.6. Rabbits with cholesteryl linoleate/oleate ratios below 0.6 seemed to run a greater risk of developing atherosclerosis. It is suggested that insulin might be required for atherogenesis in addition to hyperlipemia and hypercholesterolemia.
Key words:
Alloxan - Atherosclerosis - Cholesteryl _ Glucose - Ketone bodies - Serum lipids
ester
- Diabetes
mellitus
-
INTRODUCTION
Epidemiological
investigations
and
clinical
observations
indicate
a close
350
J. KLOEZE, A. M. M. ABDELLATIF
relationship between diabetes mellitus and atherosclerosis. Atherosclerotic complications become manifest earlier, are more frequent, and are severer in diabetics than in the general population. In patients with no diabetic history, impaired carbohydrate tolerance is frequently found after the incidence of cardiovascular disease though this phenomenon often disappears with recovery. In contrast with the observations in diabetic man, various authorsr-5 have shown that alloxan-diabetic rabbits have a remarkable resistance against the genesis of cholesterol-induced atherosclerosis. This “protecting effect” of alloxan disappears on treatment of the diabetes with insulins. This suggests that the drug affects atherogenesis via its diabetogenic action. Indeed, Cook4 has shown that the “protecting effect” of alloxan was exclusively due to its effect on the pancreas. This is further demonstrated by the finding that cholesterol feeding
does induce
comparable
degrees
of atherosclerosis
in rabbits
treated
with subdiabetic doses of alloxan6, in rabbits recovered from an initially alloxandiabetic state1 and in control rabbits. One possible criticism with respect to the unexpected finding that alloxan-diabetes in rabbits prevents atherogenesis may be that cholesterol-induced atherosclerosis in rabbits is not comparable with the arterial disease in man. In the present experiment, atherosclerosis was induced by feeding fat-rich diets - without added cholesterol - containing high percentages of saturated and shortchain fatty acids. This type of diet induces arterial lesions in rabbits, which are very similar to those found in man’. Moreover, alloxan-diabetic and control rabbits were
TABLE 1 COMPOSITION
OF DIETS
Components
gllOO0 kcal
PKO or SSO Maize flour Casein Minerals& Water-soluble vitamin mixb Fat-soluble vitamin mixC Sawdust*
43.0 104.7 62.0 18.5 1.13 1.0 60.0
a Provided per 1000 kcal: Grams: sodium chloride 2.56; calcium phosphate 8.53; potassium chloride 4.27; magnesium carbonate 0.85; sodium citrate 0.43; magnesium oxide 1.50. Milligrams: ferrous sulphate 2 aq. 197.06; manganese sulphate 4 aq. 102.45; potassium aluminium sulphate 3 aq. 31.35; zinc sulphate 3 aq. 6.72; copper sulphate 5 aq. 6.72; potassium bromide 5.60; sodium fluoride 2.24; nickel sulphate 5 aq. 2.24; borax 1.12; cobalt sulphate 1.12; sodium molybdate 1.12; potassium iodide 1.12; arsenic trioxide 0.06. b Provided per 1000 kcal: Milligrams: choline chloride (50%) 594.0; myoinositol 293.3; aminobenzoic acid 146.6; nicotinic acid 44.6; thiamine 17.6; pantothenic acid 16.5; riboflavin 6.6; vitamin Bs 6.6; folic acid 2.5; vitamin K 1.2; biotin 0.5. c Provided per 1000 kcal: I.U. : vitamin A 4000; vitamin Da 400. Milligrams: vitamin E (a-tocopheryl acetate) 15. Powdered sugar added to 1.0 g. * Sterilised.
EFFECTS OF PALM-KERNEL
TABLE
OIL AND SUNFLOWER-SEED
OIL ON SERUM LIPIDS
351
2
FATTY
ACID
SPECTRA
Range
in parenthesis.
OF DIETARY
Fatty acid
PKO (n = 3)a
6:0 8:O lo:o 12:o 14:o 16:0 16:l 18:O 18:l 18:2 18:3 20:o 2O:l 22:o
0.2 3.4 3.6 49.6 14.8 8.0 trace 2.7 14.6 2.2
& n = number
FATS DETERMINED
pO.3) (2.0-4.2) (3.5-3.8) (46.4-55.0) (13.0-16.0) (7.5-8.3) (2.5-3.2) (14.c15.3) (2.0-2.4)
BY GAS-LIQUID
CHROMATOGRAPHY
sso (n = 10)”
trace trace 6.7 (6.2-7.2) trace 4.1 (3.64.7) 24.8 (23.2-26.9) 62.5 (59.2-64.6) trace 0.3 (0.2-0.5) 0.3 (0.24.4) 0.7 (04.9)
of batches.
fed a diet rich in polyunsaturated fatty acids (PUFA) in order to examine whether such a diet decreases the high blood lipid levels occurring in diabetic rabbits, as it does in normal rabbits and normal man. Palm-kernel oil (PKO) was chosen as the atherogenic fat and sunflower-seed oil (SSO) as the one rich in PUFA. Both dietary fats were admixed
to a semi-synthetic
diet in a concentration
of 40 Cal%.
METHODS
Animals and diets One hundred
and six adult female Dutch rabbits
(l-lr/s
year old) were delivered
by the breeder in 2 batches with an interval of about 6 weeks. The rabbits were housed individually and given food and drinking water ad libitum. One to two weeks after delivery, the animals were gradually accustomed to eat semi-synthetic diets (Tables 1 and 2) for a pre-experimental period of about 3 weeks. The 2 batches of rabbits were treated separately, but in the end the data obtained were combined. The experiment lasted 54 weeks for both batches in order to allow the development of a reasonable degree of atherosclerosis. Alloxan treatment Fifty rabbits were accustomed to the PKO-diet and the remainder to the SSO-diet. From both dietary groups, two control groups were composed by selection of 12 rabbits (6 from each batch) with a similar mean initial body weight of about
352
J.
2200 g. The remaining
animals
KLOEZE,
of both groups were treated
A. M. M. ABDELLATIF
with alloxan
after fasting
for one night. The rabbits of batch I received 100 mg alloxan monohydrate (British Drug House, Poole, England) per kg body weight. The dose of alloxan was reduced for the rabbits
of batch II (90 mg/kg), because of the high mortality
in batch I. The incidence glucose
levels.
of diabetes
Rabbits
was controlled
were considered
blood glucose levels exceeded
9 mmol/l
by periodic
severely
diabetic
that had appeared
determination when
from 2 days after alloxan
their injection
of blood non-fasting up to the
end of the experiment (or up to death). The majority of the surviving rabbits responded in this way to the alloxan injection. A minority did not respond to alloxan or they recovered from an initial hyperglycemia. These animals were classified into groups referred to as “treated”. Thus 6 groups were obtained : PKOcontrol (n = 12) SSOcontrol (n = 12) PKO treated (n = 8) SSO treated (n = 2) PKO diabetic The mean data in the statistical
(n = 11)
SSO diabetic
(n =
9)
of the treated groups are presented, but these were not included analysis because of the low and unbalanced number of animals in
these groups. Determinations Body weight, mean food intake over 4 day periods, blood glucose, total serum cholesterol*, non-esterified cholesterols, free fatty acids10 (FFA), lipid phosphorus11 and total glycerol (Boehringer test set) were determined periodically in non-fasting blood or serum. At the end of the 54 weeks’ feeding period, the fatty acid composition of the serum cholesteryl esters was analysed by gas-liquid chromatography. Serum ketone bodies were determined semi-quantitatively by the Ames Acetest (Laboratoires Ames,
Epernon,
France).
The lipemia
of the serum
was estimated
visually.
Lipo-
proteins were analysed by agarose-gel electrophoresisra before and three times during the experimental period. Before this analysis the animals had fasted overnight. Serum insulin was assayed radio-immunologically (insulin Immunoassay Kit, The Radiochemical Centre, Amersham, England). Threefold determinations were carried out after an overnight fast and 30 min after a glucose load of 1 g/kg body weight administered by stomach tube (human insulin served as standard). In the same blood samples, glucose was determined as well. Periodically, urine was collected for the semiquantitative determinations of glucose and ketone bodies with Ames Labstix. Pathological procedure Except for those animals that died early in the experiment, all the other animals that died during the experiment were autopsied. Liver, kidney, spleen and pancreas as well as other organs showing macroscopic deviations were examined microscopically. The microscopic sections were stained with Masson’s trichrome and Harris hematoxylin-azophloxinrs. Additionally, the pancreas sections were stained by the Gomori
3
MONTHS)
AND
OF THE ANIMALS
AUTOPSIED
AT THE END
OF THE EXPERIMENT
Animals
PKO
SSO control
diabetic
treated
0.50 0 0
1.50 0.25 0.75
0.50
2.0
0.15
0.80
0 0 0 0.50 0.50 0.50 0.25
0.50 0.50 2.0 2.0 2.0 2.50 3.0 3.50 4.0 4.0 2.40
0 0.25 0.50 0.50 0.75 0.75
0.50 0.50 0.50 1.0 1.50 2.0 2.50
0 0.50 0.50 0.50 0.50 0.50 0.50 1.50 1.50
0.25 0.25 0.25
0.75
0.45
1.20
0.65
0.25
0.75
control
diabetic
Late mortalities
2.0
Average Autopsied
Average
treated
0.50
The AI’s observed in late mortality cases are in agreement with the findings at the end of the experiment. Histopathology. Two extra pancreatic changes appeared to be pathognomic for the diabetic state: the vacuolar changes (fatty infiltration) of the liver and the Armanni-Ebstein lesions in the kidney (Table 7). None of them, however, is specific of diabetes. The fatty deposition of the liver is indicative of excessive fat utilization by the diabetic animal. The Armanni-Ebstein lesion often occurred in human diabetics before the introduction of insulin therapy, nowadays being observed only in poorly treated diabetics 16. The lesion (clear cytoplasm) is caused by the deposition of glycogen intracellularly in the proximal convoluted tubulus and the straight portion of the Henle loopl’. DISCUSSION
The high acute mortality caused by the alloxan treatment of rabbits as well as the death rate in the course of the experiment are responsible for the unbalance in the number of animals per group. A small reduction in the dose of alloxan administered to half of the rabbits did not result in a decreased mortality rate but it decreased the incidence of permanent diabetes. In vivo, radioimmunoassayable insulin could still be detected in the diabetic
362
J. KLOEZE, A. M. M. ABDELLATIF
TABLE 7 FREQUENCY AUTOPSIED
Organ
Pancreas
Kidney
OF HISTOPATHOLOGICAL
CHANGES
DIFFERING
FROM
BASE-LINE
PATHOLOGY
IN THE
ANIMALS
AT THE END OF THE EXPERIMENT
Change
PKO
sso
control
diabetic
treated
control
diabetic
treated
(n = IO)
(n = 6)
(n == 7)
(n = 9)
(n = 2)
(n = I)
0
1
0
0
2
0
0
1
1
diminished islet volume hydropic degeneration of /I-cells islet mainly composed of a-cells casts Kimmelstiel-Wilson change vacuolar nephropathy (Armanni-Ebstein lesions)
0
0
0
0
0 0
2
1
0
(slight) Liver Spleen Lungs
vacuolation (fatty infiltration)
3
6
1
1
1
0
congested/excessive deposition
0
3
1
1
0
0
1 1
0 0
0 0
0 0
0 0
0 0
pigment
atherosclerotic pulmonary arteries cholesterol granulomata
rabbits fasted overnight, though the mean levels were somewhat lower than those in the controls. However, the blood glucose levels were about 4 times higher in the diabetic rabbits than in the controls. Consequently, the stimulus for insulin secretion should have been many times greater in the diabetics than in the control rabbits. This failure to respond adequately to a glucose stimulus was also illustrated after an intragastric glucose load. Comparable results have been reported for juvenile onset human diabeticsrs. The alloxan-diabetic rabbits generally showed high blood lipid levels. Especially, the total glycerol fraction - reflecting triglyceride levels - was extremely increased. This increase paralleled an increase in pre-,&lipoproteins indicating that the triglycerides are of endogenous origin. Although the variability is extensive, the mobilization of triglycerides seems to be a prompt consequence of insulin deficiency. However, in some rabbits, the blood lipids remained at low levels during the whole experimental period, in spite of an existing insulin deficiency and very high blood glucose levels. In the non-diabetic controls an SSO-diet - rich in linoleic acid - caused significantly lower serum lipid levels than a PKO-diet. However, in the diabetics, the SSO-diet failed to prevent the increase of the serum lipid levels. In individual cases, even the highest total glycerol and total cholesterol concentrations were observed in this group.
EFFECTS OF PALM-KERNEL
Consequently,
the serum
OIL AND SUNFLOWER-SEED
lipid lowering
363
OIL ON SERUM LIPIDS
effect of linoleic
acid seems to require
the
presence of intact pancreatic /I-cells and normal insulin production. This assumption is supported by preliminary results of a later investigation showing that a linoleicacid rich diet lowers serum lipid levels in alloxan-diabetic rabbits treated with exogenous insulin even when the treatment is not optimal. The earlier findingIthat alloxan diabetes prevents atherogenesis in rabbits has been confirmed by the present experiment. Induction of atherosclerotic lesions similar to that found in man7 was reached by feeding a diet without added cholesterol. This indicates that alloxan cholesterol or by dietary
diabetes prevents atherogenesis whether induced fat without cholesterol addition. The controls
by dietary fed PKO
showed considerable aorta atherosclerosis but the alloxan-diabetic rabbits developed no or only a mild atherosclerosis. This happened in spite of comparable or even higher serum cholesterol and other serum lipid levels in both the diabetics fed SSO as well as in those fed PKO. As could be expected,
the controls
fed SSO showed low serum
lipid levels and a negligible degree of atherosclerosis. The results suggest that, besides hypercholesterolemia and hyperlipemia, an additional unknown factor(s) is required for induction of atherosclerosis. At the moment, we can only speculate about the nature of this factor. Duff and Payne2 assumed that the simultaneous increase of the serum phospholipidlevelinproportion to the increase in the serum cholesterol level was the causal factor in the prevention of atherogenesis in alloxandiabetic rabbits. Moreover, Moore and Williams 1s stated that severe atherosclerosis is found only in those rabbits ratio smaller than 1.
showing
a serum phospholipid/free
cholesterol
molar
In our material, the serum phospholipid/free cholesterol molar ratio was never lower than 1, but nevertheless, we found cases of severe atherosclerosis in the control PKO-group. The degree of atherosclerosis did not correlate with either the individual phospholipid/total cholesterol or the individual phospholipid/free cholesterol molar ratios averaged over the whole experimental period. The discrepancy between our results and those of Duff and Payne2 may be due to the fact that we added no cholessterol to the diets. Consequently, the serum cholesterol in our experiment was of endogenous origin. The serum free cholesterol level increased relatively more in the alloxan-diabetic rabbits than the esterified cholesterol level, which is in accordance with the results of Payne and DutFJ. But, nevertheless, the mean esterified cholesterol levels were not lower in the diabetics than in the controls fed PKO. Thus, the differences in aorta atherosclerosis indices between the control and the diabetic rabbits cannot be explained by differences in serum cholesteryl ester concentrations. Analysis of the fatty acid composition of serum cholesteryl esters showed that the relative as well as the absolute amount of cholesteryl linoleate is higher in serum of the diabetic rabbits than in that of the non-diabetic rabbits, irrespective of the dietary fat. The cholesteryl oleate concentration was only proportionally higher in the serum of the control rabbits than in that of the diabetic rabbits. No differences were found in the molar serum concentrations of cholesteryl oleate in these groups. These findings do not agree with those of Schrade et aLzl in human diabetics.
364
J. KLOEZE,
A. M. M. ABDELLATlt
They found that the fatty acid composition of the cholesteryl esters in well-controlled juvenile and elderly diabetics with normal serum levels of these esters was similar to that in the controls. If the serum cholesteryl ester fraction was high, particularly in diabetic coma, the proportions of the monounsaturated fatty acids in the juvenile diabetics and the proportions of the saturated fatty acids in the elderly diabetics were significantly higher than in the controls. As a consequence, the proportions of the diand poly-unsaturated
fatty acids were in both cases significantly
lower than in the
controls. In contrast to our rabbits, these patients were either treated with insulin or were able to produce it. It is conceivable that the presence or absence of insulin could be responsible for the fatty acid composition of the cholesteryl esters. Besides by a possible effect of insulin,
the fatty acid composition
of the serum cholesteryl
esters
is determined by the fatty acid composition of the diet. This is important as the significance of the cholesteryl ester composition lies in a suggested relationship with atherosclerosis. Various authors22-24 have found elevated concentrations of serum cholesteryl oleate in CHD-patients, whereas others 25-35 have shown that cholesteryl oleate accumulates in early atherosclerotic lesions. This has resulted in a certain suspicion against cholesteryl oleate as being the sclerogenic factor. However, the present results
show that although
an elevated
cholesteryl
oleate level in the normal
and the
alloxan-treated rabbits both fed PKO is associated with a high degree of aorta atherosclerosis, a similar molar level of cholesteryl oleate causes only a negligible degree of atherosclerosis in the alloxan-diabetic rabbits fed PKO. Consequently, a high cholesteryl oleate level in the circulating blood seems not to be the only prerequisite for the development of atherosclerotic lesions. On the other hand, dietary fats with a high linoleic acid content are known to prevent atherogenesis and atherosclerotic complications. Thus, the high level of cholesteryl linoleate might have protected the diabetic rabbits fed PKO against the sclerogenic effects of cholesteryl oleate. If this assumption is correct, a comparison of the two groups of normal rabbits fed PKO or SSO shows that the “antisclerotic” effect of cholesteryl linoleate is limited. The two groups show a rather similar level of cholesteryl linoleate (Table 5). However, cholesteryl oleate levels lie far apart as do the AI’s (Table 6). Consequently, the cholesteryl linoleate/cholesteryl oleate (18 :2/18 : 1) ratio seems a better criterion than the molar concentrations of cholesteryl oleate or cholesteryl linoleate. The present results seem to suggest that a 18 :2/l 8 : 1 ratio above 0.6 can prevent atherogenesis in rabbits almost completely. However, below the ratio of 0.6, no proportionality between the AI’s and the 18:2/18:1 ratios was found. This may indicate that rabbits run a higher risk of developing serious aorta atheroslerosis at 18 :2/18:1 ratios below 0.6, but an additional factor seems to be required for the manifestation of the lesions. The period during which a low 18:2/18:1 ratio has existed in the individual rabbits could be this factor, but we have no information about that as we analysed the serum cholesteryl esters in the terminal phase only. On the other hand, deficiency of insulin might have prevented atherogenesis in the diabetic rabbits, as various authorsashave stated that hyperinsulinemia may promote it.
EFFECTS OF PALM-KERNEL OIL AND SUNFLOWER-SEED OIL ON SERUM LIPIDS
365
Indeed,
and
a common
factor in the alloxan-diabetic
those in previous investigations atherogenesis is understandable body,
including
that the hormone
the synthesis favours
rabbits
in the present experiment
is insulin deficiency. Theoretically, a role of insulin in as this hormone promotes anabolic processes in the of glycogen,
deposition
protein
and storage
and lipid.
of materials
Moreover, including
it implies lipids, prob-
ably not only in the organs specialized for that but also in the vessel walls. In the case of insulin deficiency, the hormonal equilibrium is shifted and catabolic processes prevail. Under these conditions, no fatty materials might be deposited in the arterial walls, because just now breakdown and mobilization of stored and even structural tissue materials prevails everywhere in the body. If correct, this assumption is of far-reaching importance. Even the unsolved cause of the more frequent occurrence of cardiovascular controlled availability anabolic
disease
equilibrium
in western
between
of excess food in western state. Moreover,
society
anabolism
might and
countries,
in many individuals
be connected
catabolism. the population
with the insulin-
Because
of the easy
as a whole is in an
the basal concentrations
of insulin
in
the circulation and/or the insulin response to a physiological stimulus measured in the peripheral blood is often higher than would be desirable. These situations are common in the case of lack of physical activity, obesity or mild adult onset diabetes mellitus whereas it has long been known that under these circumstances the risk for cardiovascular diseases is highly increased. But also in the circulating blood of the insulin-dependent diabetic patient, who is treated with exogenous insulin, the concentration of it is during the major part of 24 h much higher than would be desirable physiologically, because of both the unphysiological route of administration and the unphysiological dosing of the hormone. ACKNOWLEDGEMENTS
Thanks are due to Mrs W. Arentz-Eskes der Zwan for their technical assistance.
and to Messrs.
M. Boers and M. Van
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EFFECTS OF PALM-KERNEL
35
36 37 38 39 40
OIL AND SUNFLOWER-SEED
OIL ON SERUM LIPIDS
367
Part 4 (Uptake and composition of cholesteryl ester in morphologically defined regions of atherosclerotic lesions), Atherosclerosis, 15 (1972) 273. DAY, A. J., BELL, F. P., MOORE, S. AND FRIEDMAN,R., Lipid composition and metabolism of thrombo-atherosclerotic lesions produced by continued endothelial damage in normal rabbits, Circulation, 48 (1973) 157. TZAGOURNIS, M., SEIDENSTICKER, J. F. AND HAMWI, G. J., Serum insulin, carbohydrate and lipid abnormalities in patients with premature coronary heart disease, Ann. Intern. Med., 67 (1967) 42. STOUT,R. W., Insulin-stimulated lipogenesis in arterial tissue in relation to diabetes and atheroma, Lancer, 2 (1968) 702. STOUT,R. W. AND VALLANCEOWEN, J., Insulin and atheroma, Lancet, 1 (1969) 1078. STOUT,R. W., Insulin and atherosclerosis, Lancet, 2 (1969) 372. STOUT, R. W., Development of vascular lesions in insulin treated animals fed a normal diet, Brit. Med. J., 3 (1970) 685.
0
349
22 (1975) 349-368
Elsevier Scientific Publishing Company, Amsterdam
- Printed in The Netherlands
EFFECTS OF PALM-KERNEL OIL AND SUNFLOWER-SEED OIL ON SERUM LIPIDS AND ATHEROGENESIS IN ALLOXAN-DIABETIC R.4BBITS
J. KLOEZE Unilever
AND
Research,
A. M. M. ABDELLATIF Vlaardingen
(The Netherlands)
(Received October 28th, 1974) (Revised received February 26th, 1975) (Accepted February 28th, 1975)
SUMMARY
of metabolically normal (controls) and alloxan-diabetic adult female rabbits were fed semi-synthetic diets containing 40 Cal% palm-kernel oil (PKO) or sunflower-seed oil (SSO) for 54 weeks. In contrast to control rabbits fed PKO-diet, the alloxan-diabetic rabbits on this diet, developed no or only a negligible degree of atherosclerosis, although the serum levels of all lipid classes had increased in the diabetic rabbits above that of the controls during almost the whole experimental period. The diabetic rabbits and the controls fed SSO-diet were both free from any significant atherosclerotic involvement in spite of the fact that the SSO-diet appeared unable to suppress the very high levels of the various serum lipid classes induced by Groups
the diabetic
state. On both diets, the diabetic
rabbits
showed
a significantly
higher
cholesteryl linoleate/oleate ratio than the controls, which was caused by an increase in the cholesteryl linoleate level in the diabetics. No serious aorta atherosclerosis was found in rabbits with a cholesteryl linoleate/oleate ratio higher than 0.6, although no correlation was found between the atherosclerosis indices and these ratios at values lower than 0.6. Rabbits with cholesteryl linoleate/oleate ratios below 0.6 seemed to run a greater risk of developing atherosclerosis. It is suggested that insulin might be required for atherogenesis in addition to hyperlipemia and hypercholesterolemia.
Key words:
Alloxan - Atherosclerosis - Cholesteryl _ Glucose - Ketone bodies - Serum lipids
ester
- Diabetes
mellitus
-
INTRODUCTION
Epidemiological
investigations
and
clinical
observations
indicate
a close
350
J. KLOEZE, A. M. M. ABDELLATIF
relationship between diabetes mellitus and atherosclerosis. Atherosclerotic complications become manifest earlier, are more frequent, and are severer in diabetics than in the general population. In patients with no diabetic history, impaired carbohydrate tolerance is frequently found after the incidence of cardiovascular disease though this phenomenon often disappears with recovery. In contrast with the observations in diabetic man, various authorsr-5 have shown that alloxan-diabetic rabbits have a remarkable resistance against the genesis of cholesterol-induced atherosclerosis. This “protecting effect” of alloxan disappears on treatment of the diabetes with insulins. This suggests that the drug affects atherogenesis via its diabetogenic action. Indeed, Cook4 has shown that the “protecting effect” of alloxan was exclusively due to its effect on the pancreas. This is further demonstrated by the finding that cholesterol feeding
does induce
comparable
degrees
of atherosclerosis
in rabbits
treated
with subdiabetic doses of alloxan6, in rabbits recovered from an initially alloxandiabetic state1 and in control rabbits. One possible criticism with respect to the unexpected finding that alloxan-diabetes in rabbits prevents atherogenesis may be that cholesterol-induced atherosclerosis in rabbits is not comparable with the arterial disease in man. In the present experiment, atherosclerosis was induced by feeding fat-rich diets - without added cholesterol - containing high percentages of saturated and shortchain fatty acids. This type of diet induces arterial lesions in rabbits, which are very similar to those found in man’. Moreover, alloxan-diabetic and control rabbits were
TABLE 1 COMPOSITION
OF DIETS
Components
gllOO0 kcal
PKO or SSO Maize flour Casein Minerals& Water-soluble vitamin mixb Fat-soluble vitamin mixC Sawdust*
43.0 104.7 62.0 18.5 1.13 1.0 60.0
a Provided per 1000 kcal: Grams: sodium chloride 2.56; calcium phosphate 8.53; potassium chloride 4.27; magnesium carbonate 0.85; sodium citrate 0.43; magnesium oxide 1.50. Milligrams: ferrous sulphate 2 aq. 197.06; manganese sulphate 4 aq. 102.45; potassium aluminium sulphate 3 aq. 31.35; zinc sulphate 3 aq. 6.72; copper sulphate 5 aq. 6.72; potassium bromide 5.60; sodium fluoride 2.24; nickel sulphate 5 aq. 2.24; borax 1.12; cobalt sulphate 1.12; sodium molybdate 1.12; potassium iodide 1.12; arsenic trioxide 0.06. b Provided per 1000 kcal: Milligrams: choline chloride (50%) 594.0; myoinositol 293.3; aminobenzoic acid 146.6; nicotinic acid 44.6; thiamine 17.6; pantothenic acid 16.5; riboflavin 6.6; vitamin Bs 6.6; folic acid 2.5; vitamin K 1.2; biotin 0.5. c Provided per 1000 kcal: I.U. : vitamin A 4000; vitamin Da 400. Milligrams: vitamin E (a-tocopheryl acetate) 15. Powdered sugar added to 1.0 g. * Sterilised.
EFFECTS OF PALM-KERNEL
TABLE
OIL AND SUNFLOWER-SEED
OIL ON SERUM LIPIDS
351
2
FATTY
ACID
SPECTRA
Range
in parenthesis.
OF DIETARY
Fatty acid
PKO (n = 3)a
6:0 8:O lo:o 12:o 14:o 16:0 16:l 18:O 18:l 18:2 18:3 20:o 2O:l 22:o
0.2 3.4 3.6 49.6 14.8 8.0 trace 2.7 14.6 2.2
& n = number
FATS DETERMINED
pO.3) (2.0-4.2) (3.5-3.8) (46.4-55.0) (13.0-16.0) (7.5-8.3) (2.5-3.2) (14.c15.3) (2.0-2.4)
BY GAS-LIQUID
CHROMATOGRAPHY
sso (n = 10)”
trace trace 6.7 (6.2-7.2) trace 4.1 (3.64.7) 24.8 (23.2-26.9) 62.5 (59.2-64.6) trace 0.3 (0.2-0.5) 0.3 (0.24.4) 0.7 (04.9)
of batches.
fed a diet rich in polyunsaturated fatty acids (PUFA) in order to examine whether such a diet decreases the high blood lipid levels occurring in diabetic rabbits, as it does in normal rabbits and normal man. Palm-kernel oil (PKO) was chosen as the atherogenic fat and sunflower-seed oil (SSO) as the one rich in PUFA. Both dietary fats were admixed
to a semi-synthetic
diet in a concentration
of 40 Cal%.
METHODS
Animals and diets One hundred
and six adult female Dutch rabbits
(l-lr/s
year old) were delivered
by the breeder in 2 batches with an interval of about 6 weeks. The rabbits were housed individually and given food and drinking water ad libitum. One to two weeks after delivery, the animals were gradually accustomed to eat semi-synthetic diets (Tables 1 and 2) for a pre-experimental period of about 3 weeks. The 2 batches of rabbits were treated separately, but in the end the data obtained were combined. The experiment lasted 54 weeks for both batches in order to allow the development of a reasonable degree of atherosclerosis. Alloxan treatment Fifty rabbits were accustomed to the PKO-diet and the remainder to the SSO-diet. From both dietary groups, two control groups were composed by selection of 12 rabbits (6 from each batch) with a similar mean initial body weight of about
352
J.
2200 g. The remaining
animals
KLOEZE,
of both groups were treated
A. M. M. ABDELLATIF
with alloxan
after fasting
for one night. The rabbits of batch I received 100 mg alloxan monohydrate (British Drug House, Poole, England) per kg body weight. The dose of alloxan was reduced for the rabbits
of batch II (90 mg/kg), because of the high mortality
in batch I. The incidence glucose
levels.
of diabetes
Rabbits
was controlled
were considered
blood glucose levels exceeded
9 mmol/l
by periodic
severely
diabetic
that had appeared
determination when
from 2 days after alloxan
their injection
of blood non-fasting up to the
end of the experiment (or up to death). The majority of the surviving rabbits responded in this way to the alloxan injection. A minority did not respond to alloxan or they recovered from an initial hyperglycemia. These animals were classified into groups referred to as “treated”. Thus 6 groups were obtained : PKOcontrol (n = 12) SSOcontrol (n = 12) PKO treated (n = 8) SSO treated (n = 2) PKO diabetic The mean data in the statistical
(n = 11)
SSO diabetic
(n =
9)
of the treated groups are presented, but these were not included analysis because of the low and unbalanced number of animals in
these groups. Determinations Body weight, mean food intake over 4 day periods, blood glucose, total serum cholesterol*, non-esterified cholesterols, free fatty acids10 (FFA), lipid phosphorus11 and total glycerol (Boehringer test set) were determined periodically in non-fasting blood or serum. At the end of the 54 weeks’ feeding period, the fatty acid composition of the serum cholesteryl esters was analysed by gas-liquid chromatography. Serum ketone bodies were determined semi-quantitatively by the Ames Acetest (Laboratoires Ames,
Epernon,
France).
The lipemia
of the serum
was estimated
visually.
Lipo-
proteins were analysed by agarose-gel electrophoresisra before and three times during the experimental period. Before this analysis the animals had fasted overnight. Serum insulin was assayed radio-immunologically (insulin Immunoassay Kit, The Radiochemical Centre, Amersham, England). Threefold determinations were carried out after an overnight fast and 30 min after a glucose load of 1 g/kg body weight administered by stomach tube (human insulin served as standard). In the same blood samples, glucose was determined as well. Periodically, urine was collected for the semiquantitative determinations of glucose and ketone bodies with Ames Labstix. Pathological procedure Except for those animals that died early in the experiment, all the other animals that died during the experiment were autopsied. Liver, kidney, spleen and pancreas as well as other organs showing macroscopic deviations were examined microscopically. The microscopic sections were stained with Masson’s trichrome and Harris hematoxylin-azophloxinrs. Additionally, the pancreas sections were stained by the Gomori
3
MONTHS)
AND
OF THE ANIMALS
AUTOPSIED
AT THE END
OF THE EXPERIMENT
Animals
PKO
SSO control
diabetic
treated
0.50 0 0
1.50 0.25 0.75
0.50
2.0
0.15
0.80
0 0 0 0.50 0.50 0.50 0.25
0.50 0.50 2.0 2.0 2.0 2.50 3.0 3.50 4.0 4.0 2.40
0 0.25 0.50 0.50 0.75 0.75
0.50 0.50 0.50 1.0 1.50 2.0 2.50
0 0.50 0.50 0.50 0.50 0.50 0.50 1.50 1.50
0.25 0.25 0.25
0.75
0.45
1.20
0.65
0.25
0.75
control
diabetic
Late mortalities
2.0
Average Autopsied
Average
treated
0.50
The AI’s observed in late mortality cases are in agreement with the findings at the end of the experiment. Histopathology. Two extra pancreatic changes appeared to be pathognomic for the diabetic state: the vacuolar changes (fatty infiltration) of the liver and the Armanni-Ebstein lesions in the kidney (Table 7). None of them, however, is specific of diabetes. The fatty deposition of the liver is indicative of excessive fat utilization by the diabetic animal. The Armanni-Ebstein lesion often occurred in human diabetics before the introduction of insulin therapy, nowadays being observed only in poorly treated diabetics 16. The lesion (clear cytoplasm) is caused by the deposition of glycogen intracellularly in the proximal convoluted tubulus and the straight portion of the Henle loopl’. DISCUSSION
The high acute mortality caused by the alloxan treatment of rabbits as well as the death rate in the course of the experiment are responsible for the unbalance in the number of animals per group. A small reduction in the dose of alloxan administered to half of the rabbits did not result in a decreased mortality rate but it decreased the incidence of permanent diabetes. In vivo, radioimmunoassayable insulin could still be detected in the diabetic
362
J. KLOEZE, A. M. M. ABDELLATIF
TABLE 7 FREQUENCY AUTOPSIED
Organ
Pancreas
Kidney
OF HISTOPATHOLOGICAL
CHANGES
DIFFERING
FROM
BASE-LINE
PATHOLOGY
IN THE
ANIMALS
AT THE END OF THE EXPERIMENT
Change
PKO
sso
control
diabetic
treated
control
diabetic
treated
(n = IO)
(n = 6)
(n == 7)
(n = 9)
(n = 2)
(n = I)
0
1
0
0
2
0
0
1
1
diminished islet volume hydropic degeneration of /I-cells islet mainly composed of a-cells casts Kimmelstiel-Wilson change vacuolar nephropathy (Armanni-Ebstein lesions)
0
0
0
0
0 0
2
1
0
(slight) Liver Spleen Lungs
vacuolation (fatty infiltration)
3
6
1
1
1
0
congested/excessive deposition
0
3
1
1
0
0
1 1
0 0
0 0
0 0
0 0
0 0
pigment
atherosclerotic pulmonary arteries cholesterol granulomata
rabbits fasted overnight, though the mean levels were somewhat lower than those in the controls. However, the blood glucose levels were about 4 times higher in the diabetic rabbits than in the controls. Consequently, the stimulus for insulin secretion should have been many times greater in the diabetics than in the control rabbits. This failure to respond adequately to a glucose stimulus was also illustrated after an intragastric glucose load. Comparable results have been reported for juvenile onset human diabeticsrs. The alloxan-diabetic rabbits generally showed high blood lipid levels. Especially, the total glycerol fraction - reflecting triglyceride levels - was extremely increased. This increase paralleled an increase in pre-,&lipoproteins indicating that the triglycerides are of endogenous origin. Although the variability is extensive, the mobilization of triglycerides seems to be a prompt consequence of insulin deficiency. However, in some rabbits, the blood lipids remained at low levels during the whole experimental period, in spite of an existing insulin deficiency and very high blood glucose levels. In the non-diabetic controls an SSO-diet - rich in linoleic acid - caused significantly lower serum lipid levels than a PKO-diet. However, in the diabetics, the SSO-diet failed to prevent the increase of the serum lipid levels. In individual cases, even the highest total glycerol and total cholesterol concentrations were observed in this group.
EFFECTS OF PALM-KERNEL
Consequently,
the serum
OIL AND SUNFLOWER-SEED
lipid lowering
363
OIL ON SERUM LIPIDS
effect of linoleic
acid seems to require
the
presence of intact pancreatic /I-cells and normal insulin production. This assumption is supported by preliminary results of a later investigation showing that a linoleicacid rich diet lowers serum lipid levels in alloxan-diabetic rabbits treated with exogenous insulin even when the treatment is not optimal. The earlier findingIthat alloxan diabetes prevents atherogenesis in rabbits has been confirmed by the present experiment. Induction of atherosclerotic lesions similar to that found in man7 was reached by feeding a diet without added cholesterol. This indicates that alloxan cholesterol or by dietary
diabetes prevents atherogenesis whether induced fat without cholesterol addition. The controls
by dietary fed PKO
showed considerable aorta atherosclerosis but the alloxan-diabetic rabbits developed no or only a mild atherosclerosis. This happened in spite of comparable or even higher serum cholesterol and other serum lipid levels in both the diabetics fed SSO as well as in those fed PKO. As could be expected,
the controls
fed SSO showed low serum
lipid levels and a negligible degree of atherosclerosis. The results suggest that, besides hypercholesterolemia and hyperlipemia, an additional unknown factor(s) is required for induction of atherosclerosis. At the moment, we can only speculate about the nature of this factor. Duff and Payne2 assumed that the simultaneous increase of the serum phospholipidlevelinproportion to the increase in the serum cholesterol level was the causal factor in the prevention of atherogenesis in alloxandiabetic rabbits. Moreover, Moore and Williams 1s stated that severe atherosclerosis is found only in those rabbits ratio smaller than 1.
showing
a serum phospholipid/free
cholesterol
molar
In our material, the serum phospholipid/free cholesterol molar ratio was never lower than 1, but nevertheless, we found cases of severe atherosclerosis in the control PKO-group. The degree of atherosclerosis did not correlate with either the individual phospholipid/total cholesterol or the individual phospholipid/free cholesterol molar ratios averaged over the whole experimental period. The discrepancy between our results and those of Duff and Payne2 may be due to the fact that we added no cholessterol to the diets. Consequently, the serum cholesterol in our experiment was of endogenous origin. The serum free cholesterol level increased relatively more in the alloxan-diabetic rabbits than the esterified cholesterol level, which is in accordance with the results of Payne and DutFJ. But, nevertheless, the mean esterified cholesterol levels were not lower in the diabetics than in the controls fed PKO. Thus, the differences in aorta atherosclerosis indices between the control and the diabetic rabbits cannot be explained by differences in serum cholesteryl ester concentrations. Analysis of the fatty acid composition of serum cholesteryl esters showed that the relative as well as the absolute amount of cholesteryl linoleate is higher in serum of the diabetic rabbits than in that of the non-diabetic rabbits, irrespective of the dietary fat. The cholesteryl oleate concentration was only proportionally higher in the serum of the control rabbits than in that of the diabetic rabbits. No differences were found in the molar serum concentrations of cholesteryl oleate in these groups. These findings do not agree with those of Schrade et aLzl in human diabetics.
364
J. KLOEZE,
A. M. M. ABDELLATlt
They found that the fatty acid composition of the cholesteryl esters in well-controlled juvenile and elderly diabetics with normal serum levels of these esters was similar to that in the controls. If the serum cholesteryl ester fraction was high, particularly in diabetic coma, the proportions of the monounsaturated fatty acids in the juvenile diabetics and the proportions of the saturated fatty acids in the elderly diabetics were significantly higher than in the controls. As a consequence, the proportions of the diand poly-unsaturated
fatty acids were in both cases significantly
lower than in the
controls. In contrast to our rabbits, these patients were either treated with insulin or were able to produce it. It is conceivable that the presence or absence of insulin could be responsible for the fatty acid composition of the cholesteryl esters. Besides by a possible effect of insulin,
the fatty acid composition
of the serum cholesteryl
esters
is determined by the fatty acid composition of the diet. This is important as the significance of the cholesteryl ester composition lies in a suggested relationship with atherosclerosis. Various authors22-24 have found elevated concentrations of serum cholesteryl oleate in CHD-patients, whereas others 25-35 have shown that cholesteryl oleate accumulates in early atherosclerotic lesions. This has resulted in a certain suspicion against cholesteryl oleate as being the sclerogenic factor. However, the present results
show that although
an elevated
cholesteryl
oleate level in the normal
and the
alloxan-treated rabbits both fed PKO is associated with a high degree of aorta atherosclerosis, a similar molar level of cholesteryl oleate causes only a negligible degree of atherosclerosis in the alloxan-diabetic rabbits fed PKO. Consequently, a high cholesteryl oleate level in the circulating blood seems not to be the only prerequisite for the development of atherosclerotic lesions. On the other hand, dietary fats with a high linoleic acid content are known to prevent atherogenesis and atherosclerotic complications. Thus, the high level of cholesteryl linoleate might have protected the diabetic rabbits fed PKO against the sclerogenic effects of cholesteryl oleate. If this assumption is correct, a comparison of the two groups of normal rabbits fed PKO or SSO shows that the “antisclerotic” effect of cholesteryl linoleate is limited. The two groups show a rather similar level of cholesteryl linoleate (Table 5). However, cholesteryl oleate levels lie far apart as do the AI’s (Table 6). Consequently, the cholesteryl linoleate/cholesteryl oleate (18 :2/18 : 1) ratio seems a better criterion than the molar concentrations of cholesteryl oleate or cholesteryl linoleate. The present results seem to suggest that a 18 :2/l 8 : 1 ratio above 0.6 can prevent atherogenesis in rabbits almost completely. However, below the ratio of 0.6, no proportionality between the AI’s and the 18:2/18:1 ratios was found. This may indicate that rabbits run a higher risk of developing serious aorta atheroslerosis at 18 :2/18:1 ratios below 0.6, but an additional factor seems to be required for the manifestation of the lesions. The period during which a low 18:2/18:1 ratio has existed in the individual rabbits could be this factor, but we have no information about that as we analysed the serum cholesteryl esters in the terminal phase only. On the other hand, deficiency of insulin might have prevented atherogenesis in the diabetic rabbits, as various authorsashave stated that hyperinsulinemia may promote it.
EFFECTS OF PALM-KERNEL OIL AND SUNFLOWER-SEED OIL ON SERUM LIPIDS
365
Indeed,
and
a common
factor in the alloxan-diabetic
those in previous investigations atherogenesis is understandable body,
including
that the hormone
the synthesis favours
rabbits
in the present experiment
is insulin deficiency. Theoretically, a role of insulin in as this hormone promotes anabolic processes in the of glycogen,
deposition
protein
and storage
and lipid.
of materials
Moreover, including
it implies lipids, prob-
ably not only in the organs specialized for that but also in the vessel walls. In the case of insulin deficiency, the hormonal equilibrium is shifted and catabolic processes prevail. Under these conditions, no fatty materials might be deposited in the arterial walls, because just now breakdown and mobilization of stored and even structural tissue materials prevails everywhere in the body. If correct, this assumption is of far-reaching importance. Even the unsolved cause of the more frequent occurrence of cardiovascular controlled availability anabolic
disease
equilibrium
in western
between
of excess food in western state. Moreover,
society
anabolism
might and
countries,
in many individuals
be connected
catabolism. the population
with the insulin-
Because
of the easy
as a whole is in an
the basal concentrations
of insulin
in
the circulation and/or the insulin response to a physiological stimulus measured in the peripheral blood is often higher than would be desirable. These situations are common in the case of lack of physical activity, obesity or mild adult onset diabetes mellitus whereas it has long been known that under these circumstances the risk for cardiovascular diseases is highly increased. But also in the circulating blood of the insulin-dependent diabetic patient, who is treated with exogenous insulin, the concentration of it is during the major part of 24 h much higher than would be desirable physiologically, because of both the unphysiological route of administration and the unphysiological dosing of the hormone. ACKNOWLEDGEMENTS
Thanks are due to Mrs W. Arentz-Eskes der Zwan for their technical assistance.
and to Messrs.
M. Boers and M. Van
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EFFECTS OF PALM-KERNEL
35
36 37 38 39 40
OIL AND SUNFLOWER-SEED
OIL ON SERUM LIPIDS
367
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