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Age-related changes in blood and liver lipids of male wistar rats
Arch. Gerontol. Geriatr., 16 (1993) 249-262 © 1993 Elsevier Science Publishers B.V. All fights reserved. 0167-4943/93/$06.00
249
A G G 00506
Age-related changes in blood and liver lipids of male Wistar rats A l f r e d o C a n t a f o r a a, R o b e r t a Masella a, Elena Pignatelli a a n d R o b e r t o Verna b alstituto Superiore di. Sanit~, Dept. of Metabolism and Pathological Biochemistry, Rome and bCattedra di Patologia Clinica, Universitit degfi Studi, L 'Aquila, Italy (Received 9 January 1993; revised version received 30 April 1993; accepted 3 May 1993)
Summary The results of this study indicate that the age-dependent plasma cholesterol increase observed in male Wistar rats is correlated with changes in both the distribution of high-density lipoprotein fractions and the storage of hepatic cholesterol. Specifically, the lipoprotein distribution showed a significant increase in the proportion of HDL l and a symmetrical decrease in both the HDL 2 and HDL 3 fractions during the 3 month to 18 month age period. There were no significant changes in the very-low density and lowdensity lipoprotein fractions. The chemical composition of lipoproteins showed many age-related variations, especially in the proportion of cholesteryl ester and in the distribution of HDL subfractions. A study of fatty acyl composition of the major lipid classes showed that, within cholesteryl ester found in liver, there was an increase in the proportion of saturated fatty acids. Polyunsaturated fatty acids increased in the cholesteryl esters found in high-density lipoproteins of older rats. These observations suggest that the age-dependent accumulation of body cholesterol occurs by a reduced catabolism of HDL I fraction, and modifications in plasma and liver lipids. Rat; Liver; Lipoprotein; Erythrocyte; Lipids; Cholesterol; Aging
Introduction The age-related increase in human serum cholesterol concentration is thought to be correlated with the higher incidence of coronary and atherosclerotic pathologies observed in aged populations (Kannel, 1988). This justifies the interest in establishing animal models for the study of age-related cholesterol changes. Rats are commonly used in these types of experimental studies and show an age-related increase in both serum and liver cholesterol levels (Choi et al., 1987; Choi et al., Correspondence to: Dr. Alfredo Cantafora, Istituto Superiore di SanitY, Viale Regina Elena, 299, 00161 Roma, Italy.
250 1987). However, they have not been frequently used in such studies on age-related cholesterol increase, because of uncertainty about the causes for these increases. The rise of cholesterol levels in rats has been attributed to changes in the activity of either cholesteryl ester hydrolase (Tanaka et al., 1987) or HMG-CoA reductase (Shefer et al., 1972). Modifications in the rate of either cholesterol absorption (Yamamoto and Yamamura, 1971; Hollander and Morgan, 1978) or turnover (Story and Kritchevsky, 1974; Stange and Dietchy, 1984) and bile steroid secretion (Yamamoto and Yamamura, 1971; Hruza and Wachtlova, 1969) have also been implicated in elevated cholesterol levels. A possible involvement of altered lipoprotein metabolism has not been considered in this context, and the same 'normal distribution' of plasma cholesterol carriers has only occasionally been reported, in spite of the frequent use of rats in metabolic studies (Chapman, 1986; Malhotra and Kritchevsky, 1978). In previous studies we have demonstrated that changes in the distribution of highdensity lipoprotein (HDL) classes were involved in the cholesterol increase (Giganti et al., 1991; Cantafora et al., 1992). Furthermore, we showed that the HDL 1 fraction, whose concentration is strongly age-related, induced in the perfused rat liver a rapid increase in hepatic cholesterol concentration and biliary lipid output (Rivabene et al., 1992). Here, we show that alterations in lipid composition in both liver and blood compartments are strictly associated with the age-related changes in lipoprotein profile. This suggests that Wistar rats are an interesting model for pharmacological and dietary studies on the role of liver in age-related cholesterol increase. Materials and Methods
Materials The chemicals used were analytical grade reagents and solvents produced by C. Erba (Milan, Italy). Enzymatic kits for the assay of triglycerides, free and total cholesterol were purchased from Boehringer Mannheim Italia (Milan, Italy).
Animals and plasma preparation Male Wistar rats of different ages were purchased from Charles River Italia (Calco-Como, Italia). The animals were subjected to light cycling and were allowed free access to water and food (Standard rodent diet by Mucedola s.r.l., Settimo Milanese, Italy) for at least 1 week before beginning the study. The animals were submitted to 1 h fasting before being killed by decapitation under ether anesthesia. The blood was collected in plastic tubes containing 3 mg/ml EDTA as anticoagulant. Plasma was immediately separated from erythrocytes by low-speed centrifugation at 4°C. The erythrocytes were washed three times with normal saline. The red blood cell membranes were isolated by centrifugation after hemolysis of erythrocyte with water. The liver was excised from the opened abdomen. An aliquot was homogenised and used for preparing the lipid extract.
Lipoprotein fractionation Plasma lipoproteins were separated by discontinuous ultracentrifugation according to Chapman et al. (1981). Briefly, 3 ml of plasma brought to a density of 1.21
251 g/ml with NaBr was layered over 2 ml of a solution at a density of 1.24 g/ml. Next, 2 ml, 2.5 ml and 3 ml of solutions at densities of 1.21, 1.063 and 1.006 were layered, in that order. The tubes were transferred into a SW 41 Ti rotor and were centrifuged for 20 h at 40 000 rev/min and 22°C with a L-70 Beckman ultracentrifuge. Then, the gradients were fractionated with an apparatus produced by Hoefer Scientific Instruments (San Francisco, CA) by upward displacement with a solution of NaBr at 1.34 g/ml density pumped into the bottom of the tube at the flow rate of 0.2 ml/min. The gradient passed through the flow cell of an UV detector set at 280 nm (Monitor UV-M, Pharmacia, Sweden) before being collected in pre-weighed tubes using a fraction collector. Aliquots of 100/~1 taken from each tube were used for the determination of protein, free and total cholesterol and lipid phosphorous concentrations. The lipoprotein fractions were collected by pooling the contents of the tubes according to the above mentioned analyses. The pooled fractions were analyzed for their concentrations of triglyceride, free and total cholesterol, total protein, apolipoprotein, and lipid phosphorous. Some lipoprotein preparations used for electron microscopy examination were extensively dialysed overnight at 4°C against a solution containing 0.14 M NaC1 and 1.0 mM sodium azide. Lipids from plasma, erythrocyte and liver samples were extracted with chloroform/methanol (2:1, by vol) according to Folch et al. (1957).
Analytical procedures The protein content of plasma and lipoproteins was determined by Bradford's method (Bradford, 1976) using bovine serum albumin as a calibration standard. Free and total cholesterol and triacylglycerols were determined according to the instructions given in enzymatic commercial kits. The class distribution of phospholipids and the fatty acid composition of the major lipid classes in lipoprotein and liver extracts was determined by thin-layer chromatography and gas-liquid chromatography, as previously described (Alvaro et al., 1986). The distribution of apolipoproteins was determined by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) with the Phast-System (Pharmacia, Uppsala, Sweden) as previously described (Di Biase et al., 1989). A drop of the dialysed fraction was observed by transmission EM after staining with sodium phosphotungstate (Forte and Nordhausen, 1986). Observations were made at 50 000 x magnification with a Zeiss EM 10C electron microscope operating at 60 kV. The size of particles was determined on micrographs at 200 000 x magnification in a field of 500-1000 particles. Analytical data were statistically evaluated by two-tailed Student's t-test. Data were considered significant from levels of P < 0.05. Results
The age-related changes in lipoprotein class distribution observed in male Wistar rats included a slight, but not significant, increase in the proportions of both VLDL and LDL, and a significant increase in the proportion of HDLI (Table I). These increases corresponded to decreases in the proportions of both HDL 2 and HDL 3
b~
TABLE 1 Percent distribution of lipoprotein classes and plasma total lipoprotein concentration in plasma samples of male Wistar rats of different ages The total amount was calculated by the sum of lipid and protein components of each lipoprotein fraction isolated by density gradient ultracentrifugation. The data represent means 4- S.D. Age (month)
n
VLDL (%)
3 8 10 13 18
5 5 4 3 3
9.5 9.5 9.8 10.2 10.4
~ ± • 44-
LDL (%) 0.5 0.6 0.6 1.0 0.7
4.4 4.7 4.9 4.8 6.4
4± 44±
HDLI (%) 0.9 1.5 0.8 0.7 1.0
19.4 20.9 25.1 32.9 33.4
+ ± ± 4+
HDL 2 (%) 1.4 1.6 3.5 1.3"* 1.0"**
46.0 46.8 39.9 36.6 34.6
44± 4+
Significance in comparison with 3-month-old values: *P < 0.05, **P < 0.01, ***P < 0.001.
HDL 3 (%) 1.2 0.8 3.6 1.2"* 1.8"*
20.7 18.1 20.3 15.5 15.2
4444±
Conc. (mg/dl) 1.8 2.9 3.0 1.0" 0.8*
435 434 439 453 504
+ ~ ± ± 4-
23 24 22 34 25**
T A B L E II A g e - r e l a t e d c h a n g e s in the d i s t r i b u t i o n o f the m a j o r c o m p o n e n t s o f l i p o p r o t e i n f r a c t i o n s i s o l a t e d b y e l u t i o n o f u l t r a c e n t r i f u g a t i o n d e n s i t y g r a d i e n t s F C H , free c h o l e s t e r o l ; C E , c h o l e s t e r y l ester; T G , triglyceride; P L , p h o s p h o l i p i d s . T h e d a t a r e p r e s e n t m e a n s 4- S . D . T h e n u m b e r o f s a m p l e s p e r a g e is the s a m e as r e p o r t e d in T a b l e I. Lipopro. fraction
Age (month)
FCH (%)
CE (%)
TG (%)
PL (%)
VLDL
3 8 10 13 18
4.3 4.2 4.9 4.4 5.9
± 444. 4-
0.6 0.4 0.6 1.3 3.6
3.6 3.8 4.5 12.1 14.0
4. 44± 4-
l.l 1.4 1.2 5.3** 5.8**
67.7 65.0 62.1 57.2 48.8
444± 4-
LDL
3 8 10 13 18
9.7 10.4 9.3 10.1 11.4
44444-
1.1 2.2 0.8 2.6 1.7
18.0 18.2 22.4 21.0 21.3
4. 4. 444-
1.7 1.5 1.9"* 2.9 2.1"
24.5 23.7 23.2 23.8 21.4
HDL l
3 8 10 13 18
11.2 10.6 10.0 10.5 9.0
444. 44-
1.4 1.6 1.3 1.2 1.4
23.8 23.9 23.8 25.4 25.7
4444. 4-
2.1 2.8 1.6 4.3 4.1
HDL 2
3 8 10 13 18
3.7 5.8 5.3 6.8 5.4
44444-
0.8 0.4 0.4 1.5 1.2
26.3 23.8 20.4 22.5 21.5
4444. 4-
HDL 3
3 8 10 13 18
0.7 1.2 1.7 1.6 1.2
i 4+ 44-
0.4 0.4 0.9 0.9 0.7
1.5 4.6 6.2 3.9 3.3
± 4± + 4-
2.9 3.9 3.1"* 3.1"* 2.1"**
Protein (%)
15.2 17.3 18.2 16.5 19.1
44444-
2.4 2.6 2.9 1.8 2.7
9.2 9.7 10.3 9.8 12.2
44444-
2.0 2.3 1.2 1.3 2.6
4- 3.3 4. 4.0 -4- 2.4 4- 2.3 4- 1.4
27.7 26.9 24.5 25.5 25.5
444. 44-
2.4 2.6 5.2 2.6 2.3
20.1 20.8 20.6 19.6 20.6
44444-
2.9 3.0 3.0 2.6 2.4
5.9 5.9 6.7 4.4 5.7
44444-
1.1 0.9 3.2 0.8 1.3
32.2 32.9 32.5 30.8 30.0
4. 4. 4. 44.
2.3 2.7 0.9 1.9 3.6
26.9 27.0 27.0 28.9 29.6
4. 44+ 4-
2.5 2.9 2.6 3.9 4.7
1.8 0.9* 0.8*** 4.1 3.7*
1.0 1.8 2.0 2.0 2.1
44. 4± 4-
0.5 0.8 0.9 0.8 0.7*
34.5 34.5 35.7 33.4 34.2
4. 44. 44-
2.2 1.2 i.! 2.4 2.6
34.5 34.1 36.8 35.3 36.8
4. 4. + 44-
1.8 1.7 1.2 3.0 4.4
0.6 !.8"* 2.4** 0.9** 0.8*
3.9 3.2 3.8 3.6 3.5
4± 4± 4-
0.3 0.1 0.4 0.7 0.5
43.1 43.5 43.3 46.3 47.3
4- 3.1 -4- 7.0 q- 3.5 + 3.8 ± 3.1
50.9 47.5 44.8 44.6 44.7
+ + ± + 4-
2.9 4.9 3.5 3.4* 3.2*
Significance in c o m p a r i s o n w i t h 3 - m o n t h - o l d values: * P < 0.05, * * P < 0.01, ***P < 0.001.
TABLE Ill Age-related changes in plasma (mg/dl) and liver (mg/g) concentrations of free cholesterol (FCH), cholesteryl ester (CE) and total cholesterol (TCH) in male Wistar rats The data represent means + S.D. Age
n
(month) 3 8 10 13 18
Plasma FCH
5 5 4 3 5
21 22 25 33 35
+ ± ± + +
Liver CE
3.0 3.2 4.2 5.4** 6.2**
92 141 152 158 162
TC H 44± ± +
10.5 13.2"** 13.0"** 13.5"** 12.4"**
76 105 116 127 132
+ 44+ ±
FCH 9.2 11.8"* 11.6"* 15.6"* 15.8"**
Significance in comparison with 3-month-old values: **P < 0.01, ***P < 0.001.
1.0 1.0 0.9 0.9 0.8
± ± 444-
CE 0.14 0.16 0.18 0.18 0.21
0.7 0.9 1.1 1.2 1.4
TCH ± ± ± ± ±
0.17 0.14 0.14"* 0.18"* 0.18"*
1.3 1.5 1.5 1.6 1.7
± 4± ± 4-
0.28 0.26 0.25 0.27 0.29
255
fractions. The concentration of plasma total lipoproteins showed an age-related increase. However, this increase was significant only in the oldest animals (Table I). The variations in the lipoprotein class distribution were accompanied by changes in the chemical composition of each lipoprotein fraction that had been isolated by elution from an ultracentrifugation gradient (Table II). Cholesteryl ester (CE) appeared to be the lipid class showing most clear age-related variations. Specifically, CE proportion showed a decreasing trend in the HDL2 class and increased in the other lipoprotein classes. A detailed study showed that, whereas the esterified cholesterol concentrations were significantly increased in liver and plasma, the free cholesterol concentration significantly increased only in the plasma (Table III). It is interesting to note that the higher levels in circulating free cholesterol did not correlate with a higher proportion of free cholesterol in erythrocyte membrane. In fact, the molar ratios of choles-
40
C 0
g 3O
0 I
~c
~
HDLI
HDL2
20
0
g 1 0 ¢J I
o
10
~
LDL VLDL
HDLJ
;o Age (months) Fig. 1. Age-related changes in total cholesterol concentration of individual lipoprotein classes isolated from plasma of male Wistar rats. Each point in the curves represents the mean of 3-5 different determinations.
256
terol to phospholipids in erythrocyte membrane were 0.77 4- 0.025, 0.76 ~- 0.030 and 0.78 4- 0.045 in 3-, 10- and 18-month-old rats, respectively. If the contribution of individual lipoprotein fractions to the total plasma cholesterol concentration is considered (Fig. 1), it is immediately evident that the cholesterol increase in plasma of the older animals is related to an increase in HDL1 that is not completely compensated for by a decrease in HDL 2. LDL and VLDL fractions appeared to play a minor role to this effect. The important role of the HDL subfractions in cholesterol intercellular trafficking of rats (Chapman, 1986) led us to study the fatty acid composition of both CE and phosphatidylcholine (PC) classes in the three HDL subfractions isolated. The fatty
T A B L E IV Age-related changes in the fatty acid d i s t r i b u t i o n in cholesteryl esters isolated from high-density lipoprotein subfractions of male W i s t a r rats The data represent m e a n s ± S.D. The n u m b e r of samples per age is given in parentheses.
Fatty
acid
Age
(n)
3 (5) Fraction HDLt 16:0 16:1 18:0 18:1 18:2 18:3 20:3 20:4
40.6 8.6 3.7 14.9 23.9 tr tr 8.3
Fraction HDL e 16:0 16:1 18:0 18:1 18:2 18:3 20:3 20:4
34.5 5.3 5.7 15.1 26.7 tr tr 12.7
8 (5)
+ + 4± 4-
2.2 4.0 1.2 4.2 2.9
10-13 (5)
18 (3)
32.3 6.0 2.3 17.3 16.5 0.4 0.6 24.6
± 4444+ 44-
2.1"** 1.1 1.1 2.1 2.5** 0.2 0.2 1.5"**
28.7 4.5 3.3 8.7 17.0 0.4 0.6 36.8
44± 44± 44-
2.7*** 1.5 0.9 2.5* 1.8'* 0.2 0.2 3.1'**
26.1 4.3 2.6 8.0 21.7 0.5 0.7 36.1
4444+ 444-
4- 3.9
20.0 4.3 1.4 8.3 21.6 0.3 0.5 43.6
444444± ±
1.5"** 0.9 0.4** 1.7"* 2.6 0.1 0.1 4.2***
17.3 3,8 1.9 7,0 22.4 0.4 0.7 46.0
4± 444± + 4-
1.3"** 0.8 0.4** 1.2"* 1.8 0.1 0.1 2.8***
16.7 3.8 1.6 7.3 23.4 0.6 0.9 45.7
± 1.1"** -4- 0.6 4- 0.5** 4- 1.0"* 4- 1.1 + 0.1 4- 0.1 4- 3.2***
Fraction HDL 3 16:0 49.1 + 4.1 16:l 9.2 ± 3.4 18:0 10.3 4- 1.5 18:l l l . l 4- l.O 18:2 10.8 4- 2.3 18:3 0.2 4- O.l 20:3 0.3 ± O. 1 20:4 9.0 4- 4.1
45.0 7.2 8.2 8.5 12.6 0.2 0.3 18.0
44444± 4±
2.6 1.4 1.4 1.1** 2.7 O.1 O.l 4.4**
41.5 5.8 6.1 8.2 15.5 0.2 0.3 22.4
± 4+ 444± 4-
2.3** 1.2 1.2"* 1.6"* 2.4* O.1 O.l 4.3***
31.1 4.8 3.7 7.1 21.2 0.3 0.4 31.4
± 44+ ± 4+ +
4- 5.1
44444-
5.7 1.5 2.5 4.0 4.6
2.3*** 1.2 1.8 1.9' 1.2 0.2 0.2 3.0***
3.6*** 1.4 1.4"** 1.8"* 2.4*** O.l 0.2 3.8***
Significance in c o m p a r i s o n with 3-month-old values: *P < 0.05, **P < 0.01, *P < 0.001.
257
acid proportions in cholesteryl ester showed an age-related decrease in saturated and monounsaturated fatty acid and an increase in the polyunsaturated fatty acid (Table IV). The fatty acid composition of PC class showed reciprocal variations to those described for CE (Table V). These age-related changes in the fatty acid composition of CE and PC in HDL fractions were different from those observed in the same lipid classes isolated from liver lipids (Tables VI and VII, for CE and PC, respectively). Both CE and PC classes isolated from liver extracts showed an age-related increase
TABLE V
Age-related changes in the fatty acid distribution in phosphatidylcholine isolated from high-density lipoprotein fractions of male Wistar rats The data represent means .4. S.D. The number of samples per age is the same as in Table I. Fatty acid
Age (n) 3 (5)
8 (5)
10-13 (5)
18 (3)
50.3 1.5 23.2 8.0 13.4
Fraction H D L I 16:0 16:1 18:0 18:1 18:2 18:3 20:3 20:4 20:5
32.9 ± 3.3 ± 20.6 ± 10.6 ± 20.8 .4. 0.4.40.4 ± 9.3 ± 1.7 .4.
2.9 1.0 1.8 0.8 1.9 0.2 0.2 1.8 0.8
38.3 .4. 2.1 + 25.5 ± 8.5 ± 16.8 ± 0.2± 0.4 ± 7.5 ± 0.8 ±
1.9"* 0.6 2.4** 2.3 2.2* 0.1 0.2 1.6 0.5
41.0 1.4 28.3 7.3 14.I 0.2
.4. 2.6** ± 0.4** ± 2.3*** ± 2.2* ± 3.5** ±:0.1
37.6 1.5 22.6 10.8 16.3 0.2 0.3 10.1 0.6
.4. ± ± ± ± ± ± ± ±
3.2 0.4 2.7 3.5 4.7 0.1 0.1 2.2 0.1
41.5 2.2 27.0 8.4 12.6 0.1 0.2 7.0 1.0
.4. ± .4. ± ± .4. ± ± a-
2.6 0.6 3.6 2.3 2.5 0.0 0.1 2.5 0.9
40.2 1.8 30.1 6.7 12.6 0.1 tr 7.3 1.2
± ± ± ± ± ±
41.2 2.7 19.9 13.5 10.8 0.3 0.3 10.3 1.0
a- 2.8 ± 1.5 ± 3.2 .4. 3.3 ± 2.9 .4. 0.1 ± 0.1 ± 2.7 ± 0.9
44.8 2.1 22.2 8.8 10.9 0.1 0.2 10.0 0.9
a4± a± a± .4. ±
4.5 0.9 2.2 1.2" 1.3 0.1 0.1 1.0 0.1
44.5 1.9 25.3 8.8 8.7 0.1 0.2 9.6 0.9
+ ± .4. .4. .4. ± ± .4. ±
tr 7.1 ± 1.4 0.7 ± 0.3*
± 2.2*** .4. 0.4** .4. 2.3 .4. 2.0* .4. 2.4***
tr tr 3.4 ± 1.3"** 0.3 ± 0.1"*
Fraction H D L 2 16:0 16:1 18:0 18:1 18:2 18:3 20:3 20:4 20:5
1.9 0.4 2.3** 1.8 2.8 0.1
± 2.2 ± 0.6
46.5 2.1 24.0 7.5 14.7
± ± ± ± ±
2.2*** 0.9 2.3 1.7 4.6
tr 0.1 ± 0.1 3.9 ± 0.9*** 1.2 .4. 0.8
Fraction H D L 3 16:0 16:1 18:0 18:1 18:2 18:3 20:3 20:4 20:5
2.9 0.5 35* 1.2" 2.1 0.1 0.1 3.1 0.1
47.7 1.1 28.7 5.7 9.5 0.1 0.2 6.1 0.9
± ± ± ± ± ± ± ± ±
2.0*** 0.6 2.3** 2.3** 1.5 0.1 0.0 1.7" 0.0
Significance in comparison with 3-month-old values: * P < 0.05, **P < 0.01, ***P < 0.001.
258 TABLE VI Age-related changes in the fatty acid composition in cholesteryl ester isolated from liver of male Wistar rats The data represent means ~ S.D. The number of samples per age is given in parentheses. Fatty acid
Age (n) 3 (5)
16:0 16:1 18:0 18:1 18:2 18:3 20:3 20:4
53.3 4.8 9.5 18.4 8.6 0.7 0.5 4.2
8 (4) ± + + ± ± ± ± ±
6.2 0.7 1.4 2.2 2.6 0.2 0.3 1.1
69.3 3.9 9.0 8.1 5.8 0.6 0.2 3.1
13-20 (4) ± ± ± ± ± ± + ±
4.0** 1.3 1.3 3.0*** 1.2 0.2 0.1 0.8
69.7 2.8 9.0 9.6 5.3 0.7 0.3 2.5
± + ± ± ± ± ± ±
2.2** 1.0 0.9 1.8"** 1.0' 0.3 0.2 0.8*
Significance in comparison with 3-month-old values: *P < 0.05, **P < 0.01, ***P < 0.001.
in the proportion of saturated fatty acids and a decrease in the polyunsaturated fatty acids. Electron microscopy of the lipoprotein fractions isolated by gradient ultracentrifugation showed that the larger diameter of HDL1 particles in comparison with other HDL fractions (14.0 4- 5.5, 8.8 4- 3.6, 4.4 + 2.1 nm for HDLI, HDL2 and
TABLE VII Age-related changes in the fatty acid composition in phosphatidylcholine isolated from liver of male Wistar rats The data represent means ± S.D. The number of samples per age is given in parentheses. Fatty acid
Age (n) 3 (5)
16:0 16:1 18:0 18:1 18:2 18:3 20:3 20:4 20:5 22:4 22:5 22:6
26.4 1.8 23.2 10.7 14.8 0.8 1.7 16.4 0.6 0.3 0.7 2.6
8 (4) ± + ± ± ± ± ± ± ± ± ± ±
2.1 0.9 2.3 1.1 1.6 0.1 0.5 2.5 0.4 0.1 0.4 1.1
27.6 1.8 27.0 9.0 12.2 0.7 0.8 15.8 0.8 0.3 0.8 3.2
13-20 (4) ± ± ± ± ± ± ± ± ± ± ± ±
1.6 0.5 3.6 1.6 1.7 0.2 0.4 2.0 0.5 0.1 0.2 0.6
34.8 2.7 23.6 14.2 10.7 0.6 0.4 11.6 0.2 0.1 0.2 1.9
± + ± ± ± + ± ± ± ± ± ±
2.0*** 1.2 1.1 1.2"* 2.0* 0.1 0.2** 3.1" 0.1 0.1 0.1" 0.4
Significance in comparison with 3-month-old values: *P < 0.05, **P < 0.01, ***P < 0.001.
259 HDL 3 fractions in 3-month-old animals) was not affected by the age of the animal (Results not shown). The agreement with previous data on the size and composition of HDLI particles (Oschry and Eisenberg, 1982) suggested that this fraction was properly isolated. The histology examination of liver and kidney sections fixed in paraffin and stained with H&E did not show any relevant morphological alteration in older animals as compared with younger animals. In particular, we failed to observe any relevant glomerular lesions in the older animals (results not shown). Discussion
The results of this study confirm that male Wistar rats show a significant agerelated increase in total cholesterol in both plasma and liver compartments (+74% and +31%, respectively). Cholesteryl ester also exhibits age-related increase, but shows the greatest increase in the liver compartment (+76% in plasma and + 133% in liver). In spite of the well known differences in lipoprotein metabolism between humans and murine species (Chapman, 1986), there are some analogies with findings in humans (Kreisberg and Kasim, 1987). In the case of man, serum cholesterol accumulation is due to the increase in both VLDL and LDL proportions (Kreisberg and Kasim, 1987). In turn, the increase in these lipoprotein fractions is attributed to a reduced hepatic LDL-receptor expression (i.e., down-regulation of apolipoprotein (apo) B receptor) rather than to an increased synthesis of apo B containing lipoproteins (Ericsson et al., 1991). In the case of Wistar rats, we observed that the serum cholesterol increase was mostly due to a higher pla:sma concentration of HDL l, rich in both apo E and cholesterol (Giganti et al., 1991; Cantafora et al., 1992). We were not able to determine if this increase in HDL l concentration was due to increased secretion or to reduced turnover of apo-E containing lipoproteins. However, it is evident that both the HDL~ increase in rats and the LDL increase in man coincide with the age-related accumulation of cholesterol in the liver (Dupont et al., 1985; Miller, 1987). Our findings on this point are in good agreement with previous reports (Kalen et al., 1989; Stahlberg et al. 1991) who showed a similar increase in both the total and esterified cholesterol content of rat liver. The apparent role of liver in this age-related process is supported by the observation of greater accumulation of cholesteryl ester in liver than in circulating lipoproteins. This probably occurs because of increased acyl CoA:cholesterol acyl transferase (ACAT) activity (Stahlberg et al., 1991), which leads to the increased incorporation of palmitic acid into liver CE of the older animals. Such correlations between ACAT activity and the formation of molecular species of CE preferentially containing saturated fatty acids have been made previously (Goodman et al., 1964; Hashimoto et al., 1984). Stahlberg et al. (1991) have reported an age-related decrease in HMG-CoA reductase activity and an increase in ACAT activity in the liver. Such changes are likely to have led to the stability of free cholesterol levels that we found in livers of aged animals. Reduced (or constant) levels of FCH in reticulo-endothelial cells were indicated by the lack of significant age-related variations in the cholesterol to phospholipids molar ratio in erythrocyte membranes. The increase in both plasma CE concentration ana ~ts degree of unsaturation
260 seems to be related to the age-related increase in lecithin:cholesterol acyi transferase (LCAT) activity. This is suggested by the fact that HDL 2 can be converted to the larger HDL l by a LCAT-mediated process (Eisenberg, 1984) and that LCAT shows a higher substrate specificity for polyunsaturated fatty acids at the sn-2 position of phosphatidylcholine (Subbaiah et al., 1988). It is noteworthy that the age-related increase in LCAT also has been observed in humans (Miller et al., 1988). The distribution of lipoprotein components (Table II) showed that HDL l has a proportion of free cholesterol much higher than that observed in other HDL fractions. This finding may be explained as follows. The ratio of free cholesterol to phospholipids (FCH/PL, both surface components in lipoprotein particles) is in HDL1 very close to that of fl-lipoproteins and different from that of a-lipoproteins (0.32 4. 0.02, 0.28 4- 0.02, 0.38 ± 0.03, 0.15 + 0.03, and 0.03 4. 0.01 HDL~, VLDL, LDL, HDL 2 and HDL 3, respectively). This suggests that the metabolic transformation of the smaller HDLs involves the transfer not only of apo E (Eisenberg, 1984), but also of surface lipids, from lipolysed 3-1ipoproteins to HDL I. Furthermore, it is known that remodeling of HDL includes the interaction of HDL 2 and HDL 3 with peripheral cells that results in an enrichment in apo E, free cholesterol and phospholipids (Johnson et al., 1991). It has been suggested recently that alterations in circulating lipoproteins similar to those described by us in aged Wistar rats also occurred in rats of Milan Normotensive Strain as a consequence of a spontaneous chronic progressive nephrotic syndrome (Tarugi et al., 1991). We cannot exclude that some slight alteration in the kidney may play a role in the age-related lipid changes observed by us. However, the lack of the relevant glomerular alterations, commonly observed in 2-3-year-old laboratory rats (Gray, 1977), makes it unlikely that this plays a major role in our older rats for this parameter. In conclusion, it appears that the age-related changes in the lipoprotein profile include alterations in both the plasma and liver compartments. The mechanisms by which this occurs are not completely clear. A possibility is that in older animals the reduced uptake of cholesterol by peripheral tissues (a result of the decline in the growth-rate of the animal) is not completely compensated by either a reduction in cholesterol hepatic synthesis or an increase in cholesterol incorporation into bile steroid secretion. This is suggested by the fact that both the 7a-cholesterol hydroxylase activity (Stahlberg et al., 1991; Hruza and Wachtlova, 1969) and the bile acid excretion (Yamamoto and Yamamura, 1971) are reduced rather than increased by the aging process. The incorporation of excessive free cholesterol into both cellular and circulating CE by the increase in both ACAT and LCAT activities is intended to expand the slowly exchangeable pool of cholesterol and to protect cells from major damages. In fact, free cholesterol may move from plasma to cells by diffusion, without receptor regulation. Furthermore, its accumulation in cell membranes is known to affect the activity of enzyme systems, especially those located in endoplasmic reticulum of liver (Innis, 1986; Brenner, 1990).
Acknowledgements The expert technical assistance of Mrs. Tiziana Marinelli is gratefully acknowl-
261
edged. This investigation was partially supported by the Progetto Finalizzato Invecchiarnento of the National Research Council of Italy (Grants No. 93.00470.PF40 and No. 92.00477.PF40. Publ. Code: INV. 93/293). References Alvaro, D., Angelico, M., Cantafora, A., Di Bias¢, A., Gaeta, G.B., Ginanni Corradini, S., Tripodi, M.F., Attili, A.F. and Utili, R. (1986): Influence of tauroursodeoxycholic and taurodeoxycholic acids on hepatic metabolism and biliary secretion of phosphatidylcholine in the isolated rat liver. Biochim. Biophys. Acta, 878, 216-224. Bradford, M. (1976): A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of dye-binding. Anal. Biochem., 72, 248-254. Brenner, R.R. (1990): Role of cholesterol in the microsomal membrane. Lipids, 25, 581-585. Cantafora, A., Masella, R., Pignatelli, E. and Verna, R. (1992): Influence of age on the lipoprotein profile of male Wistar rats. Arch. Gerontol. Geriatr., Suppl. 3, 93-100. Chapman, M.J. (1986): Comparative analysis of mammalian plasma lipoproteins. Methods Enzymol., 128, 70-143. Chapman, M.J., Goldstein, S., Lagrange, D. and Lapland, P.M. (1981): A density gradient ultracentrifugal procedure for the isolation of the major lipoprotein classes from human serum. J. Lipid Res., 22, 339-358. Choi, Y.S., Ide, T. and Sugano, M. (1987): Age-related changes in the regulation of cholesterol metabolism rats. Exp. Crerontol., 22, 339-349. Choi, Y.S., Tomari, K., Sugano, M. and lde, T. (1987): Effects of short-term cholestyramine feeding on cholesterol metabolism in differently aged rats. Mech. Ageing Dev., 41, 149-159. Di Biase, A., Bravo, E., Puoti, C., Patane, F., Blotta, I. and Cantafora, A. (1989): Changes of lipoprotein composition associated with liver cirrhosis. Ital. J. Gastroenterol., 21,204-209. Dupont, J., Ewens-Luby, S. and Mathias, M.M. (1985): Cholesterol metabolism in relation to aging and dietary fats in rats and humans. Lipids, 26, 349-352. Eisenberg, S, (1984) High density lipoprotein metabolism. J. Lipid Res., 25, 1017-1058. Ericsson, S., Eriksson, M., Vitols, S., Einarsson, K., Berglund, L. and Angelin, B. (1991): Influence of age on the metabolism of plasma low density lipoproteins in healthy males. J. Clin. Invest., 87, 591-596. Folch, J., Lees, M. and Sloane-Stanley, G.H. (1957): A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem., 226, 497-509. Forte, T.M. and Nordhausen, R.W. (1986): Electron microscopy of negatively stained lipoproteins. Methods Enzymol., 128, 442-457. Giganti, M.G., Pignatelli, E., Modesti, D., Masella, R., Cantafora, A., and Verna, R. (1991): On the mechanism of aging. Age-related changes of transmembrane Na and K fluxes: their relationship with membrane and circulating lipids. In: Biotechnology of Cell Regulation, pp. 457-470. Editors: R. Verna and Y. Nishizuka. Raven Press, N.Y. Goodman, D.S., Deykin, D. and Shiratori, T. (1964): The formation of cholesteryl ester with rat liver enzyme. J. Biol. Chem., 239, 1335-1345. Gray, J.E. (1977): Chronic progressive nephrosis in the albino rat. CRC Crit. Rev. Toxicol., 5, 115-144. Hashimoto, S., Dayton, S., Alfin-Slater, R.B., Bui, P.T., Baker, N. and Wilson, L. (1984) Characteristics of the cholesterol esterifying activity in normal and atherosclerotic rabbit aortas. Circ. Res., 34, 176-183. Hollander, D. and Morgan, D. (1979): Increase in cholesterol intestinal absorption with aging in the rat. Exp. Gerontol., 14, 201-204. Hruza, Z. and Wachtlova, M. (1969): Decrease of cholesterol turnover in old rats. Exp. Gerontol., 4, 245-250. Innis, S.M. (1986): The activity of 3-hydroxy-3-methylglutaryl CoA reductase and acyl-CoA:cholesterol acyltransferase in hepatic microsomes from male, female and pregnant rats. The effect of cholestyramine treatment and the relationship of enzyme activity to microsomal lipid composition. Biochim. Biophys. Acta, 875, 353-361.
262 Johnson, W.W., Mahlberg, F.H., Rothblat, G.H. and Phillips, M.C. (1991): Cholesterol transport between cells and high-density lipoproteins. Biochim. Biopbys. Acta, 1085, 273-289. Kalen, A., Appelkvist, E.L and Dallner, G. (1989): Age-related changes in the lipid compositions of rat and humans tissues. Lipids, 24, 579-584. Kannel, W.D. (1988): Nutrition and the occurrence and prevention of cardiovascular disease in the elderly. Nutr. Rev., 46, 68-78. Kreisberg, R.A. and Kasim, S. (1987): Cholesterol metabolism and aging. Am. J. Med., 82 (Suppl. 1B), 54-60. Malhotra, S. and Kritchevsky, D. (1978): The distribution and lipid composition of ultracentrifugally separated lipoproteins of young and old rat plasma. Mech. Ageing Dev., 8, 445-452. Miller, N.E. (1987): On the associations of body cholesterol pool size with age, HDL cholesterol and plasma total cholesterol concentration in humans. Atherosclerosis, 67, 163-172. Miller, N.E., Nanjee, M.N. and Albers, J.J. (1988): Plasma cholesterol, ageing and lecithin:cholesterol acyltransferase. Atherosclerosis, 70, 275-276. Oschry, Y. and Eisenberg, S. (1982): Rat plasma lipoproteins: reevaluation of lipoprotein system in an animal devoid of cholesterol ester transfer activity. J. Lipid Res., 23, 1099-1106. Rivabene, R., Cantafora, A., Yah, C.C., Castellano, F., Bruscalupi, G. and Bravo, E. (1992): Effect of HDL I infusion on biliary secretion in perfused rat liver. Biosci. Rep., 12, 425-432. Shefer, S., Hauser, S., Lapar, V. and Mosbach, E.H. (1972): HMG CoA reductase of intestinal mucosa and liver of the rat. J. Lipid Res., 13, 402-412. Stahlberg, D., Angelin, B. and Einarsson, K. (1991): Age related changes in the metabolism of cholesterol in rat liver microsomes. Lipids, 26, 349-352. Stange, E.F. and Dietchy, J.M. (1984): Age-related decreases in tissue sterol acquisition are mediated by changes in cholesterol synthesis and not low density lipoprotein uptake in the rat. J. Lipid Res., 25, 703-713. Story, J.A. and Kritchevsky, D. (1974): Cholesterol oxidation by rat liver preparations: effect of age. Experientia, 30, 242-243. Subbaiah, P.V. and Monshizadegan H. (1988) Substrate specificity of human plasma lecithin-cholesterol acyltransferase towards molecular species of phosphatidylcholine in native plasma. Biochim. Biophys. Acta, 963, 445-455. Tanaka, M., Toshihiro, I. and Tabata, T. (1987): The activity and properties of a hepatic acid cholesteryl ester hydrolase obtained from rats of different age groups. Lipids, 22, 677-679. Tarugi, P., Nicolini, S., Albertazzi, L., Calandra, S., Salvati, P., Festi, C. and Patrono. C. (1991): Dyslipoproteinemia in an inbread rat strain with spontaneous chronic progressive nephrotic syndrome. J. Lipid Res., 32, 1675-1687. Yamamoto, M. and Yamamura, Y. (1971): Changes of cholesterol metabolism in the ageing rat. Atherosclerosis, 13, 365-374.
249
A G G 00506
Age-related changes in blood and liver lipids of male Wistar rats A l f r e d o C a n t a f o r a a, R o b e r t a Masella a, Elena Pignatelli a a n d R o b e r t o Verna b alstituto Superiore di. Sanit~, Dept. of Metabolism and Pathological Biochemistry, Rome and bCattedra di Patologia Clinica, Universitit degfi Studi, L 'Aquila, Italy (Received 9 January 1993; revised version received 30 April 1993; accepted 3 May 1993)
Summary The results of this study indicate that the age-dependent plasma cholesterol increase observed in male Wistar rats is correlated with changes in both the distribution of high-density lipoprotein fractions and the storage of hepatic cholesterol. Specifically, the lipoprotein distribution showed a significant increase in the proportion of HDL l and a symmetrical decrease in both the HDL 2 and HDL 3 fractions during the 3 month to 18 month age period. There were no significant changes in the very-low density and lowdensity lipoprotein fractions. The chemical composition of lipoproteins showed many age-related variations, especially in the proportion of cholesteryl ester and in the distribution of HDL subfractions. A study of fatty acyl composition of the major lipid classes showed that, within cholesteryl ester found in liver, there was an increase in the proportion of saturated fatty acids. Polyunsaturated fatty acids increased in the cholesteryl esters found in high-density lipoproteins of older rats. These observations suggest that the age-dependent accumulation of body cholesterol occurs by a reduced catabolism of HDL I fraction, and modifications in plasma and liver lipids. Rat; Liver; Lipoprotein; Erythrocyte; Lipids; Cholesterol; Aging
Introduction The age-related increase in human serum cholesterol concentration is thought to be correlated with the higher incidence of coronary and atherosclerotic pathologies observed in aged populations (Kannel, 1988). This justifies the interest in establishing animal models for the study of age-related cholesterol changes. Rats are commonly used in these types of experimental studies and show an age-related increase in both serum and liver cholesterol levels (Choi et al., 1987; Choi et al., Correspondence to: Dr. Alfredo Cantafora, Istituto Superiore di SanitY, Viale Regina Elena, 299, 00161 Roma, Italy.
250 1987). However, they have not been frequently used in such studies on age-related cholesterol increase, because of uncertainty about the causes for these increases. The rise of cholesterol levels in rats has been attributed to changes in the activity of either cholesteryl ester hydrolase (Tanaka et al., 1987) or HMG-CoA reductase (Shefer et al., 1972). Modifications in the rate of either cholesterol absorption (Yamamoto and Yamamura, 1971; Hollander and Morgan, 1978) or turnover (Story and Kritchevsky, 1974; Stange and Dietchy, 1984) and bile steroid secretion (Yamamoto and Yamamura, 1971; Hruza and Wachtlova, 1969) have also been implicated in elevated cholesterol levels. A possible involvement of altered lipoprotein metabolism has not been considered in this context, and the same 'normal distribution' of plasma cholesterol carriers has only occasionally been reported, in spite of the frequent use of rats in metabolic studies (Chapman, 1986; Malhotra and Kritchevsky, 1978). In previous studies we have demonstrated that changes in the distribution of highdensity lipoprotein (HDL) classes were involved in the cholesterol increase (Giganti et al., 1991; Cantafora et al., 1992). Furthermore, we showed that the HDL 1 fraction, whose concentration is strongly age-related, induced in the perfused rat liver a rapid increase in hepatic cholesterol concentration and biliary lipid output (Rivabene et al., 1992). Here, we show that alterations in lipid composition in both liver and blood compartments are strictly associated with the age-related changes in lipoprotein profile. This suggests that Wistar rats are an interesting model for pharmacological and dietary studies on the role of liver in age-related cholesterol increase. Materials and Methods
Materials The chemicals used were analytical grade reagents and solvents produced by C. Erba (Milan, Italy). Enzymatic kits for the assay of triglycerides, free and total cholesterol were purchased from Boehringer Mannheim Italia (Milan, Italy).
Animals and plasma preparation Male Wistar rats of different ages were purchased from Charles River Italia (Calco-Como, Italia). The animals were subjected to light cycling and were allowed free access to water and food (Standard rodent diet by Mucedola s.r.l., Settimo Milanese, Italy) for at least 1 week before beginning the study. The animals were submitted to 1 h fasting before being killed by decapitation under ether anesthesia. The blood was collected in plastic tubes containing 3 mg/ml EDTA as anticoagulant. Plasma was immediately separated from erythrocytes by low-speed centrifugation at 4°C. The erythrocytes were washed three times with normal saline. The red blood cell membranes were isolated by centrifugation after hemolysis of erythrocyte with water. The liver was excised from the opened abdomen. An aliquot was homogenised and used for preparing the lipid extract.
Lipoprotein fractionation Plasma lipoproteins were separated by discontinuous ultracentrifugation according to Chapman et al. (1981). Briefly, 3 ml of plasma brought to a density of 1.21
251 g/ml with NaBr was layered over 2 ml of a solution at a density of 1.24 g/ml. Next, 2 ml, 2.5 ml and 3 ml of solutions at densities of 1.21, 1.063 and 1.006 were layered, in that order. The tubes were transferred into a SW 41 Ti rotor and were centrifuged for 20 h at 40 000 rev/min and 22°C with a L-70 Beckman ultracentrifuge. Then, the gradients were fractionated with an apparatus produced by Hoefer Scientific Instruments (San Francisco, CA) by upward displacement with a solution of NaBr at 1.34 g/ml density pumped into the bottom of the tube at the flow rate of 0.2 ml/min. The gradient passed through the flow cell of an UV detector set at 280 nm (Monitor UV-M, Pharmacia, Sweden) before being collected in pre-weighed tubes using a fraction collector. Aliquots of 100/~1 taken from each tube were used for the determination of protein, free and total cholesterol and lipid phosphorous concentrations. The lipoprotein fractions were collected by pooling the contents of the tubes according to the above mentioned analyses. The pooled fractions were analyzed for their concentrations of triglyceride, free and total cholesterol, total protein, apolipoprotein, and lipid phosphorous. Some lipoprotein preparations used for electron microscopy examination were extensively dialysed overnight at 4°C against a solution containing 0.14 M NaC1 and 1.0 mM sodium azide. Lipids from plasma, erythrocyte and liver samples were extracted with chloroform/methanol (2:1, by vol) according to Folch et al. (1957).
Analytical procedures The protein content of plasma and lipoproteins was determined by Bradford's method (Bradford, 1976) using bovine serum albumin as a calibration standard. Free and total cholesterol and triacylglycerols were determined according to the instructions given in enzymatic commercial kits. The class distribution of phospholipids and the fatty acid composition of the major lipid classes in lipoprotein and liver extracts was determined by thin-layer chromatography and gas-liquid chromatography, as previously described (Alvaro et al., 1986). The distribution of apolipoproteins was determined by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) with the Phast-System (Pharmacia, Uppsala, Sweden) as previously described (Di Biase et al., 1989). A drop of the dialysed fraction was observed by transmission EM after staining with sodium phosphotungstate (Forte and Nordhausen, 1986). Observations were made at 50 000 x magnification with a Zeiss EM 10C electron microscope operating at 60 kV. The size of particles was determined on micrographs at 200 000 x magnification in a field of 500-1000 particles. Analytical data were statistically evaluated by two-tailed Student's t-test. Data were considered significant from levels of P < 0.05. Results
The age-related changes in lipoprotein class distribution observed in male Wistar rats included a slight, but not significant, increase in the proportions of both VLDL and LDL, and a significant increase in the proportion of HDLI (Table I). These increases corresponded to decreases in the proportions of both HDL 2 and HDL 3
b~
TABLE 1 Percent distribution of lipoprotein classes and plasma total lipoprotein concentration in plasma samples of male Wistar rats of different ages The total amount was calculated by the sum of lipid and protein components of each lipoprotein fraction isolated by density gradient ultracentrifugation. The data represent means 4- S.D. Age (month)
n
VLDL (%)
3 8 10 13 18
5 5 4 3 3
9.5 9.5 9.8 10.2 10.4
~ ± • 44-
LDL (%) 0.5 0.6 0.6 1.0 0.7
4.4 4.7 4.9 4.8 6.4
4± 44±
HDLI (%) 0.9 1.5 0.8 0.7 1.0
19.4 20.9 25.1 32.9 33.4
+ ± ± 4+
HDL 2 (%) 1.4 1.6 3.5 1.3"* 1.0"**
46.0 46.8 39.9 36.6 34.6
44± 4+
Significance in comparison with 3-month-old values: *P < 0.05, **P < 0.01, ***P < 0.001.
HDL 3 (%) 1.2 0.8 3.6 1.2"* 1.8"*
20.7 18.1 20.3 15.5 15.2
4444±
Conc. (mg/dl) 1.8 2.9 3.0 1.0" 0.8*
435 434 439 453 504
+ ~ ± ± 4-
23 24 22 34 25**
T A B L E II A g e - r e l a t e d c h a n g e s in the d i s t r i b u t i o n o f the m a j o r c o m p o n e n t s o f l i p o p r o t e i n f r a c t i o n s i s o l a t e d b y e l u t i o n o f u l t r a c e n t r i f u g a t i o n d e n s i t y g r a d i e n t s F C H , free c h o l e s t e r o l ; C E , c h o l e s t e r y l ester; T G , triglyceride; P L , p h o s p h o l i p i d s . T h e d a t a r e p r e s e n t m e a n s 4- S . D . T h e n u m b e r o f s a m p l e s p e r a g e is the s a m e as r e p o r t e d in T a b l e I. Lipopro. fraction
Age (month)
FCH (%)
CE (%)
TG (%)
PL (%)
VLDL
3 8 10 13 18
4.3 4.2 4.9 4.4 5.9
± 444. 4-
0.6 0.4 0.6 1.3 3.6
3.6 3.8 4.5 12.1 14.0
4. 44± 4-
l.l 1.4 1.2 5.3** 5.8**
67.7 65.0 62.1 57.2 48.8
444± 4-
LDL
3 8 10 13 18
9.7 10.4 9.3 10.1 11.4
44444-
1.1 2.2 0.8 2.6 1.7
18.0 18.2 22.4 21.0 21.3
4. 4. 444-
1.7 1.5 1.9"* 2.9 2.1"
24.5 23.7 23.2 23.8 21.4
HDL l
3 8 10 13 18
11.2 10.6 10.0 10.5 9.0
444. 44-
1.4 1.6 1.3 1.2 1.4
23.8 23.9 23.8 25.4 25.7
4444. 4-
2.1 2.8 1.6 4.3 4.1
HDL 2
3 8 10 13 18
3.7 5.8 5.3 6.8 5.4
44444-
0.8 0.4 0.4 1.5 1.2
26.3 23.8 20.4 22.5 21.5
4444. 4-
HDL 3
3 8 10 13 18
0.7 1.2 1.7 1.6 1.2
i 4+ 44-
0.4 0.4 0.9 0.9 0.7
1.5 4.6 6.2 3.9 3.3
± 4± + 4-
2.9 3.9 3.1"* 3.1"* 2.1"**
Protein (%)
15.2 17.3 18.2 16.5 19.1
44444-
2.4 2.6 2.9 1.8 2.7
9.2 9.7 10.3 9.8 12.2
44444-
2.0 2.3 1.2 1.3 2.6
4- 3.3 4. 4.0 -4- 2.4 4- 2.3 4- 1.4
27.7 26.9 24.5 25.5 25.5
444. 44-
2.4 2.6 5.2 2.6 2.3
20.1 20.8 20.6 19.6 20.6
44444-
2.9 3.0 3.0 2.6 2.4
5.9 5.9 6.7 4.4 5.7
44444-
1.1 0.9 3.2 0.8 1.3
32.2 32.9 32.5 30.8 30.0
4. 4. 4. 44.
2.3 2.7 0.9 1.9 3.6
26.9 27.0 27.0 28.9 29.6
4. 44+ 4-
2.5 2.9 2.6 3.9 4.7
1.8 0.9* 0.8*** 4.1 3.7*
1.0 1.8 2.0 2.0 2.1
44. 4± 4-
0.5 0.8 0.9 0.8 0.7*
34.5 34.5 35.7 33.4 34.2
4. 44. 44-
2.2 1.2 i.! 2.4 2.6
34.5 34.1 36.8 35.3 36.8
4. 4. + 44-
1.8 1.7 1.2 3.0 4.4
0.6 !.8"* 2.4** 0.9** 0.8*
3.9 3.2 3.8 3.6 3.5
4± 4± 4-
0.3 0.1 0.4 0.7 0.5
43.1 43.5 43.3 46.3 47.3
4- 3.1 -4- 7.0 q- 3.5 + 3.8 ± 3.1
50.9 47.5 44.8 44.6 44.7
+ + ± + 4-
2.9 4.9 3.5 3.4* 3.2*
Significance in c o m p a r i s o n w i t h 3 - m o n t h - o l d values: * P < 0.05, * * P < 0.01, ***P < 0.001.
TABLE Ill Age-related changes in plasma (mg/dl) and liver (mg/g) concentrations of free cholesterol (FCH), cholesteryl ester (CE) and total cholesterol (TCH) in male Wistar rats The data represent means + S.D. Age
n
(month) 3 8 10 13 18
Plasma FCH
5 5 4 3 5
21 22 25 33 35
+ ± ± + +
Liver CE
3.0 3.2 4.2 5.4** 6.2**
92 141 152 158 162
TC H 44± ± +
10.5 13.2"** 13.0"** 13.5"** 12.4"**
76 105 116 127 132
+ 44+ ±
FCH 9.2 11.8"* 11.6"* 15.6"* 15.8"**
Significance in comparison with 3-month-old values: **P < 0.01, ***P < 0.001.
1.0 1.0 0.9 0.9 0.8
± ± 444-
CE 0.14 0.16 0.18 0.18 0.21
0.7 0.9 1.1 1.2 1.4
TCH ± ± ± ± ±
0.17 0.14 0.14"* 0.18"* 0.18"*
1.3 1.5 1.5 1.6 1.7
± 4± ± 4-
0.28 0.26 0.25 0.27 0.29
255
fractions. The concentration of plasma total lipoproteins showed an age-related increase. However, this increase was significant only in the oldest animals (Table I). The variations in the lipoprotein class distribution were accompanied by changes in the chemical composition of each lipoprotein fraction that had been isolated by elution from an ultracentrifugation gradient (Table II). Cholesteryl ester (CE) appeared to be the lipid class showing most clear age-related variations. Specifically, CE proportion showed a decreasing trend in the HDL2 class and increased in the other lipoprotein classes. A detailed study showed that, whereas the esterified cholesterol concentrations were significantly increased in liver and plasma, the free cholesterol concentration significantly increased only in the plasma (Table III). It is interesting to note that the higher levels in circulating free cholesterol did not correlate with a higher proportion of free cholesterol in erythrocyte membrane. In fact, the molar ratios of choles-
40
C 0
g 3O
0 I
~c
~
HDLI
HDL2
20
0
g 1 0 ¢J I
o
10
~
LDL VLDL
HDLJ
;o Age (months) Fig. 1. Age-related changes in total cholesterol concentration of individual lipoprotein classes isolated from plasma of male Wistar rats. Each point in the curves represents the mean of 3-5 different determinations.
256
terol to phospholipids in erythrocyte membrane were 0.77 4- 0.025, 0.76 ~- 0.030 and 0.78 4- 0.045 in 3-, 10- and 18-month-old rats, respectively. If the contribution of individual lipoprotein fractions to the total plasma cholesterol concentration is considered (Fig. 1), it is immediately evident that the cholesterol increase in plasma of the older animals is related to an increase in HDL1 that is not completely compensated for by a decrease in HDL 2. LDL and VLDL fractions appeared to play a minor role to this effect. The important role of the HDL subfractions in cholesterol intercellular trafficking of rats (Chapman, 1986) led us to study the fatty acid composition of both CE and phosphatidylcholine (PC) classes in the three HDL subfractions isolated. The fatty
T A B L E IV Age-related changes in the fatty acid d i s t r i b u t i o n in cholesteryl esters isolated from high-density lipoprotein subfractions of male W i s t a r rats The data represent m e a n s ± S.D. The n u m b e r of samples per age is given in parentheses.
Fatty
acid
Age
(n)
3 (5) Fraction HDLt 16:0 16:1 18:0 18:1 18:2 18:3 20:3 20:4
40.6 8.6 3.7 14.9 23.9 tr tr 8.3
Fraction HDL e 16:0 16:1 18:0 18:1 18:2 18:3 20:3 20:4
34.5 5.3 5.7 15.1 26.7 tr tr 12.7
8 (5)
+ + 4± 4-
2.2 4.0 1.2 4.2 2.9
10-13 (5)
18 (3)
32.3 6.0 2.3 17.3 16.5 0.4 0.6 24.6
± 4444+ 44-
2.1"** 1.1 1.1 2.1 2.5** 0.2 0.2 1.5"**
28.7 4.5 3.3 8.7 17.0 0.4 0.6 36.8
44± 44± 44-
2.7*** 1.5 0.9 2.5* 1.8'* 0.2 0.2 3.1'**
26.1 4.3 2.6 8.0 21.7 0.5 0.7 36.1
4444+ 444-
4- 3.9
20.0 4.3 1.4 8.3 21.6 0.3 0.5 43.6
444444± ±
1.5"** 0.9 0.4** 1.7"* 2.6 0.1 0.1 4.2***
17.3 3,8 1.9 7,0 22.4 0.4 0.7 46.0
4± 444± + 4-
1.3"** 0.8 0.4** 1.2"* 1.8 0.1 0.1 2.8***
16.7 3.8 1.6 7.3 23.4 0.6 0.9 45.7
± 1.1"** -4- 0.6 4- 0.5** 4- 1.0"* 4- 1.1 + 0.1 4- 0.1 4- 3.2***
Fraction HDL 3 16:0 49.1 + 4.1 16:l 9.2 ± 3.4 18:0 10.3 4- 1.5 18:l l l . l 4- l.O 18:2 10.8 4- 2.3 18:3 0.2 4- O.l 20:3 0.3 ± O. 1 20:4 9.0 4- 4.1
45.0 7.2 8.2 8.5 12.6 0.2 0.3 18.0
44444± 4±
2.6 1.4 1.4 1.1** 2.7 O.1 O.l 4.4**
41.5 5.8 6.1 8.2 15.5 0.2 0.3 22.4
± 4+ 444± 4-
2.3** 1.2 1.2"* 1.6"* 2.4* O.1 O.l 4.3***
31.1 4.8 3.7 7.1 21.2 0.3 0.4 31.4
± 44+ ± 4+ +
4- 5.1
44444-
5.7 1.5 2.5 4.0 4.6
2.3*** 1.2 1.8 1.9' 1.2 0.2 0.2 3.0***
3.6*** 1.4 1.4"** 1.8"* 2.4*** O.l 0.2 3.8***
Significance in c o m p a r i s o n with 3-month-old values: *P < 0.05, **P < 0.01, *P < 0.001.
257
acid proportions in cholesteryl ester showed an age-related decrease in saturated and monounsaturated fatty acid and an increase in the polyunsaturated fatty acid (Table IV). The fatty acid composition of PC class showed reciprocal variations to those described for CE (Table V). These age-related changes in the fatty acid composition of CE and PC in HDL fractions were different from those observed in the same lipid classes isolated from liver lipids (Tables VI and VII, for CE and PC, respectively). Both CE and PC classes isolated from liver extracts showed an age-related increase
TABLE V
Age-related changes in the fatty acid distribution in phosphatidylcholine isolated from high-density lipoprotein fractions of male Wistar rats The data represent means .4. S.D. The number of samples per age is the same as in Table I. Fatty acid
Age (n) 3 (5)
8 (5)
10-13 (5)
18 (3)
50.3 1.5 23.2 8.0 13.4
Fraction H D L I 16:0 16:1 18:0 18:1 18:2 18:3 20:3 20:4 20:5
32.9 ± 3.3 ± 20.6 ± 10.6 ± 20.8 .4. 0.4.40.4 ± 9.3 ± 1.7 .4.
2.9 1.0 1.8 0.8 1.9 0.2 0.2 1.8 0.8
38.3 .4. 2.1 + 25.5 ± 8.5 ± 16.8 ± 0.2± 0.4 ± 7.5 ± 0.8 ±
1.9"* 0.6 2.4** 2.3 2.2* 0.1 0.2 1.6 0.5
41.0 1.4 28.3 7.3 14.I 0.2
.4. 2.6** ± 0.4** ± 2.3*** ± 2.2* ± 3.5** ±:0.1
37.6 1.5 22.6 10.8 16.3 0.2 0.3 10.1 0.6
.4. ± ± ± ± ± ± ± ±
3.2 0.4 2.7 3.5 4.7 0.1 0.1 2.2 0.1
41.5 2.2 27.0 8.4 12.6 0.1 0.2 7.0 1.0
.4. ± .4. ± ± .4. ± ± a-
2.6 0.6 3.6 2.3 2.5 0.0 0.1 2.5 0.9
40.2 1.8 30.1 6.7 12.6 0.1 tr 7.3 1.2
± ± ± ± ± ±
41.2 2.7 19.9 13.5 10.8 0.3 0.3 10.3 1.0
a- 2.8 ± 1.5 ± 3.2 .4. 3.3 ± 2.9 .4. 0.1 ± 0.1 ± 2.7 ± 0.9
44.8 2.1 22.2 8.8 10.9 0.1 0.2 10.0 0.9
a4± a± a± .4. ±
4.5 0.9 2.2 1.2" 1.3 0.1 0.1 1.0 0.1
44.5 1.9 25.3 8.8 8.7 0.1 0.2 9.6 0.9
+ ± .4. .4. .4. ± ± .4. ±
tr 7.1 ± 1.4 0.7 ± 0.3*
± 2.2*** .4. 0.4** .4. 2.3 .4. 2.0* .4. 2.4***
tr tr 3.4 ± 1.3"** 0.3 ± 0.1"*
Fraction H D L 2 16:0 16:1 18:0 18:1 18:2 18:3 20:3 20:4 20:5
1.9 0.4 2.3** 1.8 2.8 0.1
± 2.2 ± 0.6
46.5 2.1 24.0 7.5 14.7
± ± ± ± ±
2.2*** 0.9 2.3 1.7 4.6
tr 0.1 ± 0.1 3.9 ± 0.9*** 1.2 .4. 0.8
Fraction H D L 3 16:0 16:1 18:0 18:1 18:2 18:3 20:3 20:4 20:5
2.9 0.5 35* 1.2" 2.1 0.1 0.1 3.1 0.1
47.7 1.1 28.7 5.7 9.5 0.1 0.2 6.1 0.9
± ± ± ± ± ± ± ± ±
2.0*** 0.6 2.3** 2.3** 1.5 0.1 0.0 1.7" 0.0
Significance in comparison with 3-month-old values: * P < 0.05, **P < 0.01, ***P < 0.001.
258 TABLE VI Age-related changes in the fatty acid composition in cholesteryl ester isolated from liver of male Wistar rats The data represent means ~ S.D. The number of samples per age is given in parentheses. Fatty acid
Age (n) 3 (5)
16:0 16:1 18:0 18:1 18:2 18:3 20:3 20:4
53.3 4.8 9.5 18.4 8.6 0.7 0.5 4.2
8 (4) ± + + ± ± ± ± ±
6.2 0.7 1.4 2.2 2.6 0.2 0.3 1.1
69.3 3.9 9.0 8.1 5.8 0.6 0.2 3.1
13-20 (4) ± ± ± ± ± ± + ±
4.0** 1.3 1.3 3.0*** 1.2 0.2 0.1 0.8
69.7 2.8 9.0 9.6 5.3 0.7 0.3 2.5
± + ± ± ± ± ± ±
2.2** 1.0 0.9 1.8"** 1.0' 0.3 0.2 0.8*
Significance in comparison with 3-month-old values: *P < 0.05, **P < 0.01, ***P < 0.001.
in the proportion of saturated fatty acids and a decrease in the polyunsaturated fatty acids. Electron microscopy of the lipoprotein fractions isolated by gradient ultracentrifugation showed that the larger diameter of HDL1 particles in comparison with other HDL fractions (14.0 4- 5.5, 8.8 4- 3.6, 4.4 + 2.1 nm for HDLI, HDL2 and
TABLE VII Age-related changes in the fatty acid composition in phosphatidylcholine isolated from liver of male Wistar rats The data represent means ± S.D. The number of samples per age is given in parentheses. Fatty acid
Age (n) 3 (5)
16:0 16:1 18:0 18:1 18:2 18:3 20:3 20:4 20:5 22:4 22:5 22:6
26.4 1.8 23.2 10.7 14.8 0.8 1.7 16.4 0.6 0.3 0.7 2.6
8 (4) ± + ± ± ± ± ± ± ± ± ± ±
2.1 0.9 2.3 1.1 1.6 0.1 0.5 2.5 0.4 0.1 0.4 1.1
27.6 1.8 27.0 9.0 12.2 0.7 0.8 15.8 0.8 0.3 0.8 3.2
13-20 (4) ± ± ± ± ± ± ± ± ± ± ± ±
1.6 0.5 3.6 1.6 1.7 0.2 0.4 2.0 0.5 0.1 0.2 0.6
34.8 2.7 23.6 14.2 10.7 0.6 0.4 11.6 0.2 0.1 0.2 1.9
± + ± ± ± + ± ± ± ± ± ±
2.0*** 1.2 1.1 1.2"* 2.0* 0.1 0.2** 3.1" 0.1 0.1 0.1" 0.4
Significance in comparison with 3-month-old values: *P < 0.05, **P < 0.01, ***P < 0.001.
259 HDL 3 fractions in 3-month-old animals) was not affected by the age of the animal (Results not shown). The agreement with previous data on the size and composition of HDLI particles (Oschry and Eisenberg, 1982) suggested that this fraction was properly isolated. The histology examination of liver and kidney sections fixed in paraffin and stained with H&E did not show any relevant morphological alteration in older animals as compared with younger animals. In particular, we failed to observe any relevant glomerular lesions in the older animals (results not shown). Discussion
The results of this study confirm that male Wistar rats show a significant agerelated increase in total cholesterol in both plasma and liver compartments (+74% and +31%, respectively). Cholesteryl ester also exhibits age-related increase, but shows the greatest increase in the liver compartment (+76% in plasma and + 133% in liver). In spite of the well known differences in lipoprotein metabolism between humans and murine species (Chapman, 1986), there are some analogies with findings in humans (Kreisberg and Kasim, 1987). In the case of man, serum cholesterol accumulation is due to the increase in both VLDL and LDL proportions (Kreisberg and Kasim, 1987). In turn, the increase in these lipoprotein fractions is attributed to a reduced hepatic LDL-receptor expression (i.e., down-regulation of apolipoprotein (apo) B receptor) rather than to an increased synthesis of apo B containing lipoproteins (Ericsson et al., 1991). In the case of Wistar rats, we observed that the serum cholesterol increase was mostly due to a higher pla:sma concentration of HDL l, rich in both apo E and cholesterol (Giganti et al., 1991; Cantafora et al., 1992). We were not able to determine if this increase in HDL l concentration was due to increased secretion or to reduced turnover of apo-E containing lipoproteins. However, it is evident that both the HDL~ increase in rats and the LDL increase in man coincide with the age-related accumulation of cholesterol in the liver (Dupont et al., 1985; Miller, 1987). Our findings on this point are in good agreement with previous reports (Kalen et al., 1989; Stahlberg et al. 1991) who showed a similar increase in both the total and esterified cholesterol content of rat liver. The apparent role of liver in this age-related process is supported by the observation of greater accumulation of cholesteryl ester in liver than in circulating lipoproteins. This probably occurs because of increased acyl CoA:cholesterol acyl transferase (ACAT) activity (Stahlberg et al., 1991), which leads to the increased incorporation of palmitic acid into liver CE of the older animals. Such correlations between ACAT activity and the formation of molecular species of CE preferentially containing saturated fatty acids have been made previously (Goodman et al., 1964; Hashimoto et al., 1984). Stahlberg et al. (1991) have reported an age-related decrease in HMG-CoA reductase activity and an increase in ACAT activity in the liver. Such changes are likely to have led to the stability of free cholesterol levels that we found in livers of aged animals. Reduced (or constant) levels of FCH in reticulo-endothelial cells were indicated by the lack of significant age-related variations in the cholesterol to phospholipids molar ratio in erythrocyte membranes. The increase in both plasma CE concentration ana ~ts degree of unsaturation
260 seems to be related to the age-related increase in lecithin:cholesterol acyi transferase (LCAT) activity. This is suggested by the fact that HDL 2 can be converted to the larger HDL l by a LCAT-mediated process (Eisenberg, 1984) and that LCAT shows a higher substrate specificity for polyunsaturated fatty acids at the sn-2 position of phosphatidylcholine (Subbaiah et al., 1988). It is noteworthy that the age-related increase in LCAT also has been observed in humans (Miller et al., 1988). The distribution of lipoprotein components (Table II) showed that HDL l has a proportion of free cholesterol much higher than that observed in other HDL fractions. This finding may be explained as follows. The ratio of free cholesterol to phospholipids (FCH/PL, both surface components in lipoprotein particles) is in HDL1 very close to that of fl-lipoproteins and different from that of a-lipoproteins (0.32 4. 0.02, 0.28 4- 0.02, 0.38 ± 0.03, 0.15 + 0.03, and 0.03 4. 0.01 HDL~, VLDL, LDL, HDL 2 and HDL 3, respectively). This suggests that the metabolic transformation of the smaller HDLs involves the transfer not only of apo E (Eisenberg, 1984), but also of surface lipids, from lipolysed 3-1ipoproteins to HDL I. Furthermore, it is known that remodeling of HDL includes the interaction of HDL 2 and HDL 3 with peripheral cells that results in an enrichment in apo E, free cholesterol and phospholipids (Johnson et al., 1991). It has been suggested recently that alterations in circulating lipoproteins similar to those described by us in aged Wistar rats also occurred in rats of Milan Normotensive Strain as a consequence of a spontaneous chronic progressive nephrotic syndrome (Tarugi et al., 1991). We cannot exclude that some slight alteration in the kidney may play a role in the age-related lipid changes observed by us. However, the lack of the relevant glomerular alterations, commonly observed in 2-3-year-old laboratory rats (Gray, 1977), makes it unlikely that this plays a major role in our older rats for this parameter. In conclusion, it appears that the age-related changes in the lipoprotein profile include alterations in both the plasma and liver compartments. The mechanisms by which this occurs are not completely clear. A possibility is that in older animals the reduced uptake of cholesterol by peripheral tissues (a result of the decline in the growth-rate of the animal) is not completely compensated by either a reduction in cholesterol hepatic synthesis or an increase in cholesterol incorporation into bile steroid secretion. This is suggested by the fact that both the 7a-cholesterol hydroxylase activity (Stahlberg et al., 1991; Hruza and Wachtlova, 1969) and the bile acid excretion (Yamamoto and Yamamura, 1971) are reduced rather than increased by the aging process. The incorporation of excessive free cholesterol into both cellular and circulating CE by the increase in both ACAT and LCAT activities is intended to expand the slowly exchangeable pool of cholesterol and to protect cells from major damages. In fact, free cholesterol may move from plasma to cells by diffusion, without receptor regulation. Furthermore, its accumulation in cell membranes is known to affect the activity of enzyme systems, especially those located in endoplasmic reticulum of liver (Innis, 1986; Brenner, 1990).
Acknowledgements The expert technical assistance of Mrs. Tiziana Marinelli is gratefully acknowl-
261
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