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Accumulation of intermediate density lipoprotein in the plasma of cholesterol-fed hypothyroid rats
Accumulation
of Intermediate Density Lipoprotein in the Plasma of Cholesterol-Fed Hypothyroid Rats Toshio Murase, Nobuhiro Yamada, and Hidemasa Uchimura
The present study aimed to characterize the high cholesterol diet-induced hyperlipoproteinemia developed in thyroidectomized hypothyroid rats, in such animals the activities of hepatic triglyceride lipase having been shown to be decreased. Feeding a diet containing 1% cholesterol to normal control rats resulted in hypercholesterolemia characterized by increases of VLDL, IDL and LDL. The feeding of cholesterol to hypothyroid rats produced a marked accentuation of hypercholesterolemia characterized by a large increase in IDL with a lesser increase in LDL. The IDL contained an increased amount of apo E. The VLDL of cholesterol-fed hypothyroid rats contained &VLDL. These findings indicate a marked accumulation of remnant lipoproteins in the plasma of cholesterol-fed hypothyroid rats. Putting this and our previous findings together, we suggest that such an accumulation may be caused by impaired removal by the liver of remnant lipoproteins due to the decreased activities of hepatic triglyceride lipase.
W
E HAVE SHOWN
previously that thyroidec-
tomized hypothyroid rats have low activities of
postheparin
plasma
hepatic
triglyceride
lipase
(H-
TGL).’ There is at present debate concerning the physiologic function of this enzyme: this could play a role in the catabolism of intermediate density lipoprotein* or high density 1ipoprotein.3.4 Probably hypothyroid rats are an appropriate mode1 for assessing the function of H-TGL. It has been reported that rats fed a high cholesterol diet develop hypercholesterolemia with marked elevations in the composition of lipoprotein classes.536Such a dietary maneuver could exaggerate the abnormalities of lipoprotein metabolism due to low H-TGL, which might be presumably present in hypothyroid rats. In the present study, we attempted to characterize the plasma lipoproteins of hypothyroid rats fed a high cholesterol diet. Our special interest was directed to the changes of intermediate density lipoprotein (IDL) and high density lipoprotein (HDL) compositions. MATERIALS AND METHODS
Animals and Diet Male Wistar rats, weighing 150-170 g were allocated to three groups. Rats made hypothyroid by thyroidectomy were fed a standard laboratory chow for 3 wk and then placed on a high cholesterol diet containing 1% cholesterol plus 0.5% cholic acid for 2 wk. The high cholesterol diet controls were kept on the same dietary regime as that of the thyroidectomized rats. The normal diet controls were fed a standard laboratory chow as long as 5 wk. At the end of the period, the rats were anesthetized with sodium pentobarbital, 50 mg/kg of body weight, and blood was taken into tubes containing EDTA. 1 mg/ml of blood. This was performed between 10 A.M. and From The Third Department of Internal Medicine, Faculty of Medicine, University of Tokyo, Hongo, Tokyo, Japan. Received for publication August 24, 1982. Address reprini requests to Toshio Murase, The Third Department of Internal Medicine. Faculty of Medicine, University of Tokyo, Hongo, Tokyo, Japan. 0 1983 by Grune & Stratton, Inc. 0026-0495/83/3202-0010$01.00/0
146
12 P.M. without prior fasting. The respective body weights at sacrifice were shown in Table 1.
Analytical Method Cholesterol,’ triglyceride’ and phospholipids’ concentrations were measured enzymatically. Protein was determined by the method of Lowry et al.,” using bovine serum albumin as a standard. Plasma lipoproteins and lipoprotein fractions were analyzed by an agarose gel electrophoresis (Pol-E Film, Diagnostic Division, Pfizer Inc., NY). Plasma thyroxine was measured by radioimmunoassay Kit (Supplied by Eiken lmmunochemical Laboratory, Tokyo). Ultracentrifugal separation of plasma lipoproteins were carried out using a type 40-3 rotor (Beckman Instruments Inc., Palo Alto, CA).” The density was adjusted with KBr and KBr solutions of known density. Four fractions of very low density lipoprotein (VLDL, d c 1.006 g/ml) intermediate density lipoprotein (IDL, I .006-l .019), low density lipoprotein (LDL, I ,019-l ,063) and high density lipoprotein (HDL, I .063-1.210) were isolated by ultracentrifugation at 40,000 rpm for 16, 18, 18 and 40 hr. respectively. For the analysis of apoproteins, individual lipoprotein fractions of each group of rats were pooled. Each lipoprotein fraction was dialyzed and delipidated by solvent extraction with chloroform: methanol (2:1 vol/vol). The apoproteins thus obtained were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis by a modification of Weber and Osborn.” Apoproteins of VLDL (50 pg), IDL (20 rg). LDL (20 fig) and HDL (50 Fg) were applied to the gels.
Measurement of Postheparin Plasma Lipase Activities A separate set of rats on the same regimen was set up and used to measure H-TGL activity. Blood samples were withdrawn from the subclavian venous plexus 3 min after i.v. administration of heparin (Novo Industries, Denmark) at 200 U/100 g of body weight. The plasma was separated in a refrigerated centrifuge. Two lipases, i.e., H-TGL and lipoprotein lipase (LPL), in postheparin plasma were measured separately by an immunochemical method described previously.’ RESULTS
Plasma Thyroxine and Lipid Concentrations
The presence of hypothyroidism in the thyroidectomized rats was confirmed by the findings of diminished plasma thyroxine concentrations (Table 1).
Metabolism, Vol. 32, No. 2 (February), 1983
147
PLASMA LIPOPROTEINS IN HYPOTHYROID RATS
Table 1. Plasma Thyroxine and Lipid Concentrations Plasma Llptds Body Weight
Plasma Thyroxrxe
(9)
@g/dll
Condmon
Tnglyceride
Cholesterol
(mg/dll
imgldll
Phosphoirprds lmg/dil
Normal diet controls (n = 5)
409
c 13
5.7 * 0.3
162 t 23
70 + 5
40 r 5
462
+ 8t
6.0 + 0.5
226 t 12*
149 f 12t
78 + 8t
0.4 * 0.1tt
208 + 12
460 + 55ti
189 + 23tt
High cholesterol diet controls (n = 6) Cholesterol-fed hypothyroid rats bJ = 7)
239 ?z 5ti
Each value represents the mean + SE. *Significantly different at p < 0.05 from corresponding values of the normal diet controls. tsignificantly different at least at p < 0.01 from corresponding valuesof the normal diet controls. SSignificantly different at least at p < 0.01 from corresponding values of the high cholesterol diet controls.
Plasma concentrations of both cholesterol and phospholipids increased two-fold in the high cholesterol diet controls, while those of triglyceride increased only slightly (Table I). Concentrations of cholesterol and phospholipids in the plasma of cholesterol-fed hypothyroid rats were 3- and 2.4-fold higher than those of the high cholesterol diet controls. These two groups of rats had similar levels of plasma triglyceride.
VLDL (Fig. 1). The fractions of the high cholesterol diet controls did not contain such a band. These findings were very similar to those reported earlier.13 The LDL fraction of the high cholesterol diet controls showed an increase in the staining of P-band and that of the cholesterol-fed hypothyroid rats showed much heavier staining of it. The LDL fraction of these two groups of cholesterol-fed rats also contained an a-migrating band, which may referred to as HDL,, a particle described by Mahley and Holcombe.6 The HDL fraction of the cholesterol-fed hypothyroid rats was reduced in its staining of an a-lipoprotein band.
Lipoprotein Electrophoresis On agarose electrohporesis, marked alterations were found in plasma and individual lipoprotein fractions of the high cholesterol diet controls and the cholesterolfed hypothyroid rats. The plasma of the high cholesterol diet controls revealed a broad-p pattern. The plasma of cholesterol-fed hypothyroid rats also showed a broad-/3 pattern, however, the band had much stronger staining with slower mobility than that of the high cholesterol diet controls. The VLDL and IDL fractions of cholesterol-fed hypothyroid rats contained a P-migrating lipoprotein probably referred to as fl-
Lipid and Protein in Plasma Lipoprotein Fractions The high cholesterol diet controls had higher concentrations of both cholesterol and phospholipids in VLDL than those of the normal diet controls (Table 2). VLDL triglyceride concentrations were decreased in the cholesterol-fed hypothyroid rats compared to those in the high cholesterol diet controls, while VLDL cholesterol, phopholipids and protein of the cholester-
&:‘.*
C Fig. 1. Agarose electrophoresis of plasma lipoprotein fractions from the normal diet controls (CL high cholesterol diet controls (C,,J and cholesterol-fed hypothyroid rats (TX,,,).
CHC
TX. HC
C
CHIC
TX.HC
(
C
CW
TX.HC
C
Cnc
TX.tic
148
MURASE, YAMADA,
ol-fed hypothyroid rats were similar to those of the high cholesterol diet controls. The greatest change was found in the IDL fraction. Concentrations of triglyceride, cholesterol and phospholipids in IDL increased in the high cholesterol diet controls. These increases were much more so in the hypothyroid rats fed a high cholesterol diet. IDL protein, which did not increase in the high cholesterol diet controls, was greatly elevated in the cholesterolfed hypothyroid rats (Fig. 2). Both lipids and protein in LDL increased in the cholesterol-fed hypothyroid rats, however, these increases were to a lesser extent than those of IDL. HDL cholesterol, phospholipids and protein of the high cholesterol diet controls were lower than those of the normal diet controls. However, the cholesterol-fed hypothyroid rats had similar levels of all lipids and protein in HDL to those of the high cholesterol diet controls. SDS Gel Electrophoresis The SDS polyacrylamide gel electrophoretic patterns of each lipoprotein fraction obtained from three groups of rats are shown in Fig. 3. The most striking difference between the three groups can be seen in the IDL gels. IDL gel of the high cholesterol diet controls shows a stronger apo E band than that of the normal
Table 2. Chemical Composition Nwmal
Diet Controls (n = 51
of Plasma Lipoprotein
Fractions
High Cholesterol DfietControls (n = 6)
Cholesterol-Fed HypothyroidRats (n= 7)
122.8
VLDL 93.6
t 15.0
? 11.5
71.1
* 7.96
6.4 + 0.8
55.8
? 5.4t
90.5
i- 17.2t
PL
15.0 + 2.6
35.0
* 4.2t
46.3
2 8.8C
Protein
31.5
* 8.2
49.8
+ 5.2
40.3
? 3.4
24.7
k 3.1 t$
TG Chol
LDL + 3.4
36.8
+ 2.2’
46.3
Chol
7.4 + 1.2
23.6
* 2.2t
72.4
+ 6.3ts
PL
6.2 f 0.8
14.1 i
1.7t
37.9
+ 3.1 t§
zk 1.5t
36.6
+ 2.6tg
TG
Protein
13.2 + 1.4
20.6
r*ir*i
n/
TRlGLYCERlDE
AND UCHIMIJRA
r*i
CHOLESTEROL
FwSPHOLlPlDS
PROTEIN
Fig. 2. Lipid and protein concentrations in the plasma IDL fraction of the normal diet controls (Cl, high cholesterol diet controls (C,,) end cholesterol-fed hypothyroid rats (TX,,,). The bars indicate + SE.
diet controls. This is much more so in the hypothyroid rats fed a high cholesterol diet. The cholesterol-fed hypothyroid rats showed an increase in apo A- 1 of IDL The apo E in HDL of two groups of rats fed a high cholesterol diet was reduced, such a finding noted by Mahley and Holcombe.6 Between two groups of cholesterol-fed rats, no difference of the apoprotein pattern of HDL was found. Postheparin Plasma H-TGL and LPL Activities Postheparin plasma H-TGL activity in the cholesterol-fed hypothyroid rats was 0.777 rf: 0.050 pmole FFA/ml/min (mean + SE, n = 8), significantly lower than that in the high cholesterol diet controls (1.212 + 0.133, n = 6, p < 0.02) and that in the normal diet controls (1.076 t 0.048, n = 7, p -C 0.01). The cholesterol-fed hypothyroid rats had no different LPL activity from that of the high cholesterol diet controls (0.640 k 0.041 versus 0.627 + 0.064 pmole FFA/ ml/min). The cholesterol-fed hypothyroid rats had slightly, but not significantly, higher LPL activity than the normal diet controls (0.537 k 0.033, 0.05 < p < 0.1).
HDL TG
19.2 + 2.7
23.7
k 1.3
27.2
+ 2.5
Chol
56.5
49.0
* 5.3
39.0
* 4.9’
PL
17.7 + 1.5
14.7 * 2.2
10.6 i
Protein
96.2
97.2
74.6
+ 3.1 k 5.4
r 10.1
1.2t
f 6.5,
Each value represents the mean k SE, mg/dl. Abbreviations: TG, triglyceride; Chol, cholesterol: PL, phospholipids. *Significantly different at p < 0.05 from corresponding values of the normal diet controls. tsignificantly different at least at p < 0.01
from the corresponding
values of the normal diet controls. SSignificantly different at p < 0.05 from corresponding values of the high cholesterol diet controls. $Significantly different at least at p < 0.01 values of the high cholesterol diet controls.
from the corresponding
DISCUSSION
The present study demonstrates a marked accumulation of IDL in the plasma of thyroidectomized hypothyroid rats fed a high cholesterol diet. We speculate that such a change reflects the impaired removal of remnant lipoproteins by the liver, and that this may be caused by the decreased activities of H-TGL. In our previous study, thyroidectomized hypothyroid rats fed a standard laboratory chow had normal levels of both plasma cholesterol and triglyceride, despite a marked decrease in H-TGL activities in postheparin plasma.’ Since hypothyroid rats become
PLASMA LIPOPROTEINS IN HYPOTHYROID RATS
149
VLDL c
CHC Tntc
IDL C
Cttc T&m:
c
0%
TX. HC
-8
-A-IV -E Fig. 3. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of plasma lipoprotein fractions from the normal diet controls (0, high cholesterol diet controls (C,,) and cholesterol-fed hypothyroid rats IT,,,, I.
hypophagic and plasma lipids are largely influenced by diet, diminished food intake as well as low cholesterol intake might have been responsible for the normal levels of plasma lipids in such animals. We anticipated that a cholesterol feeding could provoke the metabolic abnormalities presumably present in hypothyroid rats with low H-TGL activity and conducted the current study. Feeding a high cholesterol diet to normal control rats produced hypercholesterolemia characterized by increases of IDL, VLDL and LDL, the findings being consistent with the observations of others.5.h The present study also demonstrates that hypothyroid rats fed the same cholesterol-rich diet showed a marked accentuation of hypercholesterolemia characterized by a large increase in IDL. This IDL contained an increased amount of apo E. These observations are similar to those found in other studies with cholesterolfed propylthiouracil treated rats.” Such findings indicate an accumulation of remnant lipoproteins in the plasma. The finding that a significant amount of apo A- 1 was present in the cholesterol-fed hypothyroid rats indicates that the IDL includes cholomicron remnants. The appearance of a P-VLDL also suggests the presence of remnant lipoproteins.‘4 However, since rat livers have been shown to secrete such lipoproteins,‘5.‘6 an alternative is that the secretion might increase in the cholesterol-fed animals. The concentration of LDL was also elevated in the cholesterol-fed hypothyroid rats. This LDL contained less triglyceride than cholesterol compared with the LDL of high cholesterol diet controls. It has been suggested that the appropriate density range for IDL in rats is d = 1.006-l .030.” If so, then the LDL fraction in the current study may contain some IDL contamination. In addition, this LDL fraction may contain the HDL, particles,(’
although in the current study we have not characterized fully such lipoproteins, Plasma triglyceride concentrations were high in these three groups of rats, because our experiments were done on ad libitum fed rats. Plasma triglyceride concentrations of the high cholesterol diet controls were higher than those of the normal diet controls. We speculate that a high cholesterol diet may stimulate appetite and increase food intake, resulting in an elevation of plasma triglyceride. This is consistent with a large body weight gain in cholesterol-fed rats. In cholesterol-fed hypothyroid rats, VLDL-triglyceride was significantly lower than that in the high cholesterol diet controls. This may be a consequence of diminished food intake in hypothyroid animals. A marked accumulation of IDL in the plasma of cholesterol-fed hypothyroid rats could be attributed to a defective removal of the liver of remnant lipoproteins. Such a possibility is supported by the recent observations made by Redgrave and Snibson,‘” and FlorCn and Nilson,” both of them found that in hypercholesterolemic hypothyroid rats the clearance of injected chylomicron remnants was delayed. The mechanism by which the removal of remnant lipoproteins is impaired in hypothyroid rats is of particular interest. Recently, we have shown that hypothyroid rats have low activities of H-TGL in postheparin plasma.’ In the current study, we have confirmed that the hypothyroid rats fed a high cholesterol diet have low H-TGL activities as well. As far as concerning the physiological role of H-TGL, studies made by Kuusi et al.3 and Jansen et a1.,4 in which antibody to H-TGL was infused into fasting rats, have suggested that this enzyme could play a role in the metabolism of HDL. If this was the case, then hypothyroid rats with low
150
MURASE, YAMADA,
H-TGL activity should have elevated concentrations of plasma HDL. However, as our present study as well as other previous reportI shows, the concentration of HDL did not increase in such animals. We have had different observations on the ad libitum fed rats that treatment with the antibody prepared against H-TGL caused a significant increase of IDL in plasma.* Consequently, we speculated that H-TGL mediate the uptake of remnant lipoproteins by the liver. The findings of the present study strengthen this view. Thus, we suggest that a marked accumulation of IDL in the plasma of cholesterol-fed hypothyroid rats may be caused by impaired removal by the liver of remnant lipoproteins due to decreased activities of H-TGL. The question then arises how this enzyme participates in this process. Hydrolysis by H-TGL of triglyceride might be essential for remnant to be taken up by the liver. However, recent studies suggest that remnant uptake by the liver proceeds with concomitant uptake
AND UCHIMURA
of both triglyceride and cholesterol rather than with a selective removal of remnant triglyceride.20~2’ It is possible that H-TGL acts at the step when remnant lipoproteins bind to the receptor site in the liver, but this awaits further studies. ACKNOWLEDGMENT The authors the manuscript
wish to thank Dr. K. Kosaka for his critical and valuable comments.
review of
ADDENDUM After submitting this manuscript, we have finished setting up the radioimmunoassay method for measuring rat plasma apo E. This method is essentially the same as that for human plasma ape E described elsewhere (Horm Metab Res, in press). With the use of this method, we have measured plasma apo E concentrations in cholesterol-fed rats. Plasma stored frozen at -8OOC was used. Plasma apo E concentrations of cholesterol-fed hypothyroid rats were 51.2 + 4.2 mg/dl, significantly higher than those of the high cholesterol diet controls (22.1 + 1.6,~ < 0.001).
REFERENCES 1. Murase T, Uchimura H: A selective decline of postheparin plasma hepatic triglyceride lipase in hypothyroid rats. Metabolism 29:797-801, 1980 2. Murase T, Itakura H: Accumulation of intermediate denstiy lipoprotein in plasma after intravenous administration of hepatic triglyceride lipase antibody in rats. Atherosclerosis 39:293-300, 1981 3. Kuusi T, Kinnunen PKJ, Nikkila EA: Hepatic endotherial lipase antiserum influences rat plasma low and high density lipoproteins in vivo. FEBS Letters 104:384-388, 1979 4. Jansen H, van To1 A, Hillsmann WC: On the metabolic function of heparin-releasable liver lipase. Biochem Biophys Res Commun 92:53-59, 1980 5. Lasser NL, Roheim PS, Edelstein D, et al: Serum lipoproteins of normal and cholesterol-fed rats. J Lipid Res 14:1-S, 1973 6. Mahley RW, Holcombe KS: Alterations of the plasma lipoproteins and apoproteins following cholesterol feeding in the rat. J Lipid Res: 18:31&324, 1977 7. Allain CC, Poon LS, Chan CSG, Richmond W, et al: Enzymatic determination of total serum cholesterol. Clin Chem 20:470475.1974 8. Bucolo G, David H: Quantitative determination of serum triglycerides by the use of enzymes. Clin Chem 19:476-482, 1973 9. Takayama M, Ito S, Nagasaki T, et al: A new enzymatic method for determination of serum cholinecontaining phospholipids. Clin Chim Acta 79:93-98, 1977 10. Lowry OH, Rosebrough NJ, Farr AL, et al: Protein measurement with the Folin phenol reagent. J Biol Chem 193:265-275, 1951 Il. Have1 RJ, Eder AH, Bragton JH: The distribution and chemical composition of ultracentrifugally separated lipoproteins in human serum. J Clin Invest 34: 1345-l 353, 1955
12. Weber K, Osborn M: The reliability determined by dodecyl sulfate-polyacrylamide Biol Chem 244:4406-4412,1969
of molecular weight gel electrophoresis. J
13. DeLamatre JG, Roheim PS: Effect of cholesterol feeding on apoB and apoE concentrations and distributions in euthyroid and hypothyroid rats. J Lipid Res 22:297-306, 1981 14. Ross AC, Zilversmit DB: Chylomicron remnant cholesteryl esters as the major constituent of very low density lipoproteins in plasma of cholesterol-fed rabbits. J Lipid Res 18: 169-l 8 1, 1977 15. Noel S-P, Wong L, Dolphin PJ, et al: Secretion of cholestrolrich lipoproteins by perfused livers of hypercholesterolemic rats. J Clin Invest 64:674-683, 1979 16. Kris-Etherton PM, Cooper AD: Studies on the etiology of the hypoerlipemia in rats fed an atherogenic diet. J Lipid Res 21:435442, 1980 17. Dory L, Roheim PS: Rat plasma lipoproteins and apolipoproteins in experimental hypothyroidism. J Lipid Res 22:287-296, 1981 18. Redgrave TG, Snibson DA: Clearance of chylomicron triacylglycerol and cholesterol ester from the plasma of streptozotocininduced diabetic and hypercholesterolemic hypothyroid rats. Metabolism 26:493-503, 1977 19. Floren C-H, Nilsson A: Effects of hypothyroidism and cholesterol feeding on the clearance of chylomicron remnants in vivo and by rat hepatocyte monolayers. Europ J Clin Invest 1I:1 l-18, 1981 20. Cooper AD: The metabolism of chylomicron isolated perfused rat liver. Biochim Biophy Acta 1977
remnants by 488:46&474,
21. Sherrill BC, Dietschy JM: Characterization of the sinusoidal tranport process responsible for uptake of chylomicrons by the liver. J Biol Chem 253:1859-1867, 1978
of Intermediate Density Lipoprotein in the Plasma of Cholesterol-Fed Hypothyroid Rats Toshio Murase, Nobuhiro Yamada, and Hidemasa Uchimura
The present study aimed to characterize the high cholesterol diet-induced hyperlipoproteinemia developed in thyroidectomized hypothyroid rats, in such animals the activities of hepatic triglyceride lipase having been shown to be decreased. Feeding a diet containing 1% cholesterol to normal control rats resulted in hypercholesterolemia characterized by increases of VLDL, IDL and LDL. The feeding of cholesterol to hypothyroid rats produced a marked accentuation of hypercholesterolemia characterized by a large increase in IDL with a lesser increase in LDL. The IDL contained an increased amount of apo E. The VLDL of cholesterol-fed hypothyroid rats contained &VLDL. These findings indicate a marked accumulation of remnant lipoproteins in the plasma of cholesterol-fed hypothyroid rats. Putting this and our previous findings together, we suggest that such an accumulation may be caused by impaired removal by the liver of remnant lipoproteins due to the decreased activities of hepatic triglyceride lipase.
W
E HAVE SHOWN
previously that thyroidec-
tomized hypothyroid rats have low activities of
postheparin
plasma
hepatic
triglyceride
lipase
(H-
TGL).’ There is at present debate concerning the physiologic function of this enzyme: this could play a role in the catabolism of intermediate density lipoprotein* or high density 1ipoprotein.3.4 Probably hypothyroid rats are an appropriate mode1 for assessing the function of H-TGL. It has been reported that rats fed a high cholesterol diet develop hypercholesterolemia with marked elevations in the composition of lipoprotein classes.536Such a dietary maneuver could exaggerate the abnormalities of lipoprotein metabolism due to low H-TGL, which might be presumably present in hypothyroid rats. In the present study, we attempted to characterize the plasma lipoproteins of hypothyroid rats fed a high cholesterol diet. Our special interest was directed to the changes of intermediate density lipoprotein (IDL) and high density lipoprotein (HDL) compositions. MATERIALS AND METHODS
Animals and Diet Male Wistar rats, weighing 150-170 g were allocated to three groups. Rats made hypothyroid by thyroidectomy were fed a standard laboratory chow for 3 wk and then placed on a high cholesterol diet containing 1% cholesterol plus 0.5% cholic acid for 2 wk. The high cholesterol diet controls were kept on the same dietary regime as that of the thyroidectomized rats. The normal diet controls were fed a standard laboratory chow as long as 5 wk. At the end of the period, the rats were anesthetized with sodium pentobarbital, 50 mg/kg of body weight, and blood was taken into tubes containing EDTA. 1 mg/ml of blood. This was performed between 10 A.M. and From The Third Department of Internal Medicine, Faculty of Medicine, University of Tokyo, Hongo, Tokyo, Japan. Received for publication August 24, 1982. Address reprini requests to Toshio Murase, The Third Department of Internal Medicine. Faculty of Medicine, University of Tokyo, Hongo, Tokyo, Japan. 0 1983 by Grune & Stratton, Inc. 0026-0495/83/3202-0010$01.00/0
146
12 P.M. without prior fasting. The respective body weights at sacrifice were shown in Table 1.
Analytical Method Cholesterol,’ triglyceride’ and phospholipids’ concentrations were measured enzymatically. Protein was determined by the method of Lowry et al.,” using bovine serum albumin as a standard. Plasma lipoproteins and lipoprotein fractions were analyzed by an agarose gel electrophoresis (Pol-E Film, Diagnostic Division, Pfizer Inc., NY). Plasma thyroxine was measured by radioimmunoassay Kit (Supplied by Eiken lmmunochemical Laboratory, Tokyo). Ultracentrifugal separation of plasma lipoproteins were carried out using a type 40-3 rotor (Beckman Instruments Inc., Palo Alto, CA).” The density was adjusted with KBr and KBr solutions of known density. Four fractions of very low density lipoprotein (VLDL, d c 1.006 g/ml) intermediate density lipoprotein (IDL, I .006-l .019), low density lipoprotein (LDL, I ,019-l ,063) and high density lipoprotein (HDL, I .063-1.210) were isolated by ultracentrifugation at 40,000 rpm for 16, 18, 18 and 40 hr. respectively. For the analysis of apoproteins, individual lipoprotein fractions of each group of rats were pooled. Each lipoprotein fraction was dialyzed and delipidated by solvent extraction with chloroform: methanol (2:1 vol/vol). The apoproteins thus obtained were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis by a modification of Weber and Osborn.” Apoproteins of VLDL (50 pg), IDL (20 rg). LDL (20 fig) and HDL (50 Fg) were applied to the gels.
Measurement of Postheparin Plasma Lipase Activities A separate set of rats on the same regimen was set up and used to measure H-TGL activity. Blood samples were withdrawn from the subclavian venous plexus 3 min after i.v. administration of heparin (Novo Industries, Denmark) at 200 U/100 g of body weight. The plasma was separated in a refrigerated centrifuge. Two lipases, i.e., H-TGL and lipoprotein lipase (LPL), in postheparin plasma were measured separately by an immunochemical method described previously.’ RESULTS
Plasma Thyroxine and Lipid Concentrations
The presence of hypothyroidism in the thyroidectomized rats was confirmed by the findings of diminished plasma thyroxine concentrations (Table 1).
Metabolism, Vol. 32, No. 2 (February), 1983
147
PLASMA LIPOPROTEINS IN HYPOTHYROID RATS
Table 1. Plasma Thyroxine and Lipid Concentrations Plasma Llptds Body Weight
Plasma Thyroxrxe
(9)
@g/dll
Condmon
Tnglyceride
Cholesterol
(mg/dll
imgldll
Phosphoirprds lmg/dil
Normal diet controls (n = 5)
409
c 13
5.7 * 0.3
162 t 23
70 + 5
40 r 5
462
+ 8t
6.0 + 0.5
226 t 12*
149 f 12t
78 + 8t
0.4 * 0.1tt
208 + 12
460 + 55ti
189 + 23tt
High cholesterol diet controls (n = 6) Cholesterol-fed hypothyroid rats bJ = 7)
239 ?z 5ti
Each value represents the mean + SE. *Significantly different at p < 0.05 from corresponding values of the normal diet controls. tsignificantly different at least at p < 0.01 from corresponding valuesof the normal diet controls. SSignificantly different at least at p < 0.01 from corresponding values of the high cholesterol diet controls.
Plasma concentrations of both cholesterol and phospholipids increased two-fold in the high cholesterol diet controls, while those of triglyceride increased only slightly (Table I). Concentrations of cholesterol and phospholipids in the plasma of cholesterol-fed hypothyroid rats were 3- and 2.4-fold higher than those of the high cholesterol diet controls. These two groups of rats had similar levels of plasma triglyceride.
VLDL (Fig. 1). The fractions of the high cholesterol diet controls did not contain such a band. These findings were very similar to those reported earlier.13 The LDL fraction of the high cholesterol diet controls showed an increase in the staining of P-band and that of the cholesterol-fed hypothyroid rats showed much heavier staining of it. The LDL fraction of these two groups of cholesterol-fed rats also contained an a-migrating band, which may referred to as HDL,, a particle described by Mahley and Holcombe.6 The HDL fraction of the cholesterol-fed hypothyroid rats was reduced in its staining of an a-lipoprotein band.
Lipoprotein Electrophoresis On agarose electrohporesis, marked alterations were found in plasma and individual lipoprotein fractions of the high cholesterol diet controls and the cholesterolfed hypothyroid rats. The plasma of the high cholesterol diet controls revealed a broad-p pattern. The plasma of cholesterol-fed hypothyroid rats also showed a broad-/3 pattern, however, the band had much stronger staining with slower mobility than that of the high cholesterol diet controls. The VLDL and IDL fractions of cholesterol-fed hypothyroid rats contained a P-migrating lipoprotein probably referred to as fl-
Lipid and Protein in Plasma Lipoprotein Fractions The high cholesterol diet controls had higher concentrations of both cholesterol and phospholipids in VLDL than those of the normal diet controls (Table 2). VLDL triglyceride concentrations were decreased in the cholesterol-fed hypothyroid rats compared to those in the high cholesterol diet controls, while VLDL cholesterol, phopholipids and protein of the cholester-
&:‘.*
C Fig. 1. Agarose electrophoresis of plasma lipoprotein fractions from the normal diet controls (CL high cholesterol diet controls (C,,J and cholesterol-fed hypothyroid rats (TX,,,).
CHC
TX. HC
C
CHIC
TX.HC
(
C
CW
TX.HC
C
Cnc
TX.tic
148
MURASE, YAMADA,
ol-fed hypothyroid rats were similar to those of the high cholesterol diet controls. The greatest change was found in the IDL fraction. Concentrations of triglyceride, cholesterol and phospholipids in IDL increased in the high cholesterol diet controls. These increases were much more so in the hypothyroid rats fed a high cholesterol diet. IDL protein, which did not increase in the high cholesterol diet controls, was greatly elevated in the cholesterolfed hypothyroid rats (Fig. 2). Both lipids and protein in LDL increased in the cholesterol-fed hypothyroid rats, however, these increases were to a lesser extent than those of IDL. HDL cholesterol, phospholipids and protein of the high cholesterol diet controls were lower than those of the normal diet controls. However, the cholesterol-fed hypothyroid rats had similar levels of all lipids and protein in HDL to those of the high cholesterol diet controls. SDS Gel Electrophoresis The SDS polyacrylamide gel electrophoretic patterns of each lipoprotein fraction obtained from three groups of rats are shown in Fig. 3. The most striking difference between the three groups can be seen in the IDL gels. IDL gel of the high cholesterol diet controls shows a stronger apo E band than that of the normal
Table 2. Chemical Composition Nwmal
Diet Controls (n = 51
of Plasma Lipoprotein
Fractions
High Cholesterol DfietControls (n = 6)
Cholesterol-Fed HypothyroidRats (n= 7)
122.8
VLDL 93.6
t 15.0
? 11.5
71.1
* 7.96
6.4 + 0.8
55.8
? 5.4t
90.5
i- 17.2t
PL
15.0 + 2.6
35.0
* 4.2t
46.3
2 8.8C
Protein
31.5
* 8.2
49.8
+ 5.2
40.3
? 3.4
24.7
k 3.1 t$
TG Chol
LDL + 3.4
36.8
+ 2.2’
46.3
Chol
7.4 + 1.2
23.6
* 2.2t
72.4
+ 6.3ts
PL
6.2 f 0.8
14.1 i
1.7t
37.9
+ 3.1 t§
zk 1.5t
36.6
+ 2.6tg
TG
Protein
13.2 + 1.4
20.6
r*ir*i
n/
TRlGLYCERlDE
AND UCHIMIJRA
r*i
CHOLESTEROL
FwSPHOLlPlDS
PROTEIN
Fig. 2. Lipid and protein concentrations in the plasma IDL fraction of the normal diet controls (Cl, high cholesterol diet controls (C,,) end cholesterol-fed hypothyroid rats (TX,,,). The bars indicate + SE.
diet controls. This is much more so in the hypothyroid rats fed a high cholesterol diet. The cholesterol-fed hypothyroid rats showed an increase in apo A- 1 of IDL The apo E in HDL of two groups of rats fed a high cholesterol diet was reduced, such a finding noted by Mahley and Holcombe.6 Between two groups of cholesterol-fed rats, no difference of the apoprotein pattern of HDL was found. Postheparin Plasma H-TGL and LPL Activities Postheparin plasma H-TGL activity in the cholesterol-fed hypothyroid rats was 0.777 rf: 0.050 pmole FFA/ml/min (mean + SE, n = 8), significantly lower than that in the high cholesterol diet controls (1.212 + 0.133, n = 6, p < 0.02) and that in the normal diet controls (1.076 t 0.048, n = 7, p -C 0.01). The cholesterol-fed hypothyroid rats had no different LPL activity from that of the high cholesterol diet controls (0.640 k 0.041 versus 0.627 + 0.064 pmole FFA/ ml/min). The cholesterol-fed hypothyroid rats had slightly, but not significantly, higher LPL activity than the normal diet controls (0.537 k 0.033, 0.05 < p < 0.1).
HDL TG
19.2 + 2.7
23.7
k 1.3
27.2
+ 2.5
Chol
56.5
49.0
* 5.3
39.0
* 4.9’
PL
17.7 + 1.5
14.7 * 2.2
10.6 i
Protein
96.2
97.2
74.6
+ 3.1 k 5.4
r 10.1
1.2t
f 6.5,
Each value represents the mean k SE, mg/dl. Abbreviations: TG, triglyceride; Chol, cholesterol: PL, phospholipids. *Significantly different at p < 0.05 from corresponding values of the normal diet controls. tsignificantly different at least at p < 0.01
from the corresponding
values of the normal diet controls. SSignificantly different at p < 0.05 from corresponding values of the high cholesterol diet controls. $Significantly different at least at p < 0.01 values of the high cholesterol diet controls.
from the corresponding
DISCUSSION
The present study demonstrates a marked accumulation of IDL in the plasma of thyroidectomized hypothyroid rats fed a high cholesterol diet. We speculate that such a change reflects the impaired removal of remnant lipoproteins by the liver, and that this may be caused by the decreased activities of H-TGL. In our previous study, thyroidectomized hypothyroid rats fed a standard laboratory chow had normal levels of both plasma cholesterol and triglyceride, despite a marked decrease in H-TGL activities in postheparin plasma.’ Since hypothyroid rats become
PLASMA LIPOPROTEINS IN HYPOTHYROID RATS
149
VLDL c
CHC Tntc
IDL C
Cttc T&m:
c
0%
TX. HC
-8
-A-IV -E Fig. 3. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of plasma lipoprotein fractions from the normal diet controls (0, high cholesterol diet controls (C,,) and cholesterol-fed hypothyroid rats IT,,,, I.
hypophagic and plasma lipids are largely influenced by diet, diminished food intake as well as low cholesterol intake might have been responsible for the normal levels of plasma lipids in such animals. We anticipated that a cholesterol feeding could provoke the metabolic abnormalities presumably present in hypothyroid rats with low H-TGL activity and conducted the current study. Feeding a high cholesterol diet to normal control rats produced hypercholesterolemia characterized by increases of IDL, VLDL and LDL, the findings being consistent with the observations of others.5.h The present study also demonstrates that hypothyroid rats fed the same cholesterol-rich diet showed a marked accentuation of hypercholesterolemia characterized by a large increase in IDL. This IDL contained an increased amount of apo E. These observations are similar to those found in other studies with cholesterolfed propylthiouracil treated rats.” Such findings indicate an accumulation of remnant lipoproteins in the plasma. The finding that a significant amount of apo A- 1 was present in the cholesterol-fed hypothyroid rats indicates that the IDL includes cholomicron remnants. The appearance of a P-VLDL also suggests the presence of remnant lipoproteins.‘4 However, since rat livers have been shown to secrete such lipoproteins,‘5.‘6 an alternative is that the secretion might increase in the cholesterol-fed animals. The concentration of LDL was also elevated in the cholesterol-fed hypothyroid rats. This LDL contained less triglyceride than cholesterol compared with the LDL of high cholesterol diet controls. It has been suggested that the appropriate density range for IDL in rats is d = 1.006-l .030.” If so, then the LDL fraction in the current study may contain some IDL contamination. In addition, this LDL fraction may contain the HDL, particles,(’
although in the current study we have not characterized fully such lipoproteins, Plasma triglyceride concentrations were high in these three groups of rats, because our experiments were done on ad libitum fed rats. Plasma triglyceride concentrations of the high cholesterol diet controls were higher than those of the normal diet controls. We speculate that a high cholesterol diet may stimulate appetite and increase food intake, resulting in an elevation of plasma triglyceride. This is consistent with a large body weight gain in cholesterol-fed rats. In cholesterol-fed hypothyroid rats, VLDL-triglyceride was significantly lower than that in the high cholesterol diet controls. This may be a consequence of diminished food intake in hypothyroid animals. A marked accumulation of IDL in the plasma of cholesterol-fed hypothyroid rats could be attributed to a defective removal of the liver of remnant lipoproteins. Such a possibility is supported by the recent observations made by Redgrave and Snibson,‘” and FlorCn and Nilson,” both of them found that in hypercholesterolemic hypothyroid rats the clearance of injected chylomicron remnants was delayed. The mechanism by which the removal of remnant lipoproteins is impaired in hypothyroid rats is of particular interest. Recently, we have shown that hypothyroid rats have low activities of H-TGL in postheparin plasma.’ In the current study, we have confirmed that the hypothyroid rats fed a high cholesterol diet have low H-TGL activities as well. As far as concerning the physiological role of H-TGL, studies made by Kuusi et al.3 and Jansen et a1.,4 in which antibody to H-TGL was infused into fasting rats, have suggested that this enzyme could play a role in the metabolism of HDL. If this was the case, then hypothyroid rats with low
150
MURASE, YAMADA,
H-TGL activity should have elevated concentrations of plasma HDL. However, as our present study as well as other previous reportI shows, the concentration of HDL did not increase in such animals. We have had different observations on the ad libitum fed rats that treatment with the antibody prepared against H-TGL caused a significant increase of IDL in plasma.* Consequently, we speculated that H-TGL mediate the uptake of remnant lipoproteins by the liver. The findings of the present study strengthen this view. Thus, we suggest that a marked accumulation of IDL in the plasma of cholesterol-fed hypothyroid rats may be caused by impaired removal by the liver of remnant lipoproteins due to decreased activities of H-TGL. The question then arises how this enzyme participates in this process. Hydrolysis by H-TGL of triglyceride might be essential for remnant to be taken up by the liver. However, recent studies suggest that remnant uptake by the liver proceeds with concomitant uptake
AND UCHIMURA
of both triglyceride and cholesterol rather than with a selective removal of remnant triglyceride.20~2’ It is possible that H-TGL acts at the step when remnant lipoproteins bind to the receptor site in the liver, but this awaits further studies. ACKNOWLEDGMENT The authors the manuscript
wish to thank Dr. K. Kosaka for his critical and valuable comments.
review of
ADDENDUM After submitting this manuscript, we have finished setting up the radioimmunoassay method for measuring rat plasma apo E. This method is essentially the same as that for human plasma ape E described elsewhere (Horm Metab Res, in press). With the use of this method, we have measured plasma apo E concentrations in cholesterol-fed rats. Plasma stored frozen at -8OOC was used. Plasma apo E concentrations of cholesterol-fed hypothyroid rats were 51.2 + 4.2 mg/dl, significantly higher than those of the high cholesterol diet controls (22.1 + 1.6,~ < 0.001).
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