Activity of lipoprotein lipase in thyroidectomized rats

Biochimica et Biophysics Acta, 750 (1983) 533-538 Elsevier Biomedical

533

Press

BBA 51342

ACTIVITY OF LIPOPROTEIN NINA

SKOTT’OVA

LIPASE IN THYROIDECTOMIZED

*, LARS WALLINDER

and GUNILLA

RATS

BENGTSSON

Department of Chemistry, Section of Physiological Chemistry, Unioersiiy of Ume& S- 901 87 Umed (Sweden) (Received June 7th, 1982) (Revised manuscript received November

29th, 1982)

Key words: Lipoprotein lipase; Thyroidectomy; Heparin; Lipase turnover; (Liver)

Total plasma postheparin lipolytic activity as well as lipoprotein lipase activity in plasma was higher after heparin injection in thyroidectomized rats than in controls. In contrast, the activity of liver lipase was lower in thyroidectomized rats. Adipose tissue from thyroidectomized rats contained more lipoprotein lipase activity than adipose tissue from controls as measured both in extracts of tissue homogenates and medium from in vitro incubations of tissue .pieces. There were no differences between control and hy~thyroid rats in the disappearance of intravenously injected ‘251-labeled lipoprotein lipase, but when a low dose of heparin was injected before the labeled enzyme, the disappearance of ‘251-labeled lipoprotein lipase was more retarded in thyroidectomized rats. The elimination of heparin itself was slightly retarded by thyroidectomy.

Introduction

Heparin injection releases at least two triacylglycerol lipase activities into the blood stream [ 1,2], lipoprotein lipase and liver lipase, and markedly retards the disappearance of these enzymes from the blood [3], as well as the disappearance of intravenously injected ‘2fI-labeled bovine milk lipoprotein lipase [4]. Lipoprotein lipase has a well-defined role in the metabolism of triacylglycerol-rich lipoproteins [5,6], but the physiological function of liver lipase has not yet been established. There are several reports that hypothyroidism affects plasma lipoprotein concentrations [7-lo] and the activity of enzymes involved in plasma lipoprotein metabolism f I l- 171. In previous studies Skottova and PalkoviE [ lO,lS] found an increased plasma postheparin lipolytic activity in * TO whom

correspondence should be addressed at (present address): Institute of Chemistry, Medical Faculty, 3, Dr. S. Allende, 775 15 Olomouc, Czechoslovakia.

OOOS-2760/83/0000-0000/$03.00

0 1983 Elsevier Science Publishers

thyroidectomized rats. The aim of the present work was to analyze the cause of this. Therefore, the following studies were carried out: (a) lipoprotein lipase and liver lipase activities in plasma were followed after heparin injection, (b) lipoprotein lipase activity in adipose tissue extracts and in medium from in vitro incubations of adipose tissue was measured and (c) the disappearance from blood of ‘251-labeled lipoprotein lipase and of 3SS-labeled heparin was followed. Materials and Methods

Female Sprague-Dawley rats (AB Anticimex, Stockholm), weighing 170-230 g and fed a standard pellet diet (AB EWOS, Sijdertalje, Sweden) were surgically thyroidectomized. They were used for experiments on days 20-25 after the operation. Sham-operated rats were used as controls. The hypothyroid status was evident from the lack of body growth [ 19,201, the food intake of thyroidectomized rats was lower than in the controls (Table I).

534 TABLE I THE BODY WEIGHT OF EXPERIMENTS

OF CONTROL AND THYROIDECTOMIZED

RATS AT THE START AND

I DAY BEFORE

ENDING

The food intake (g/day) was calculated from the intake during the last 5 days before each experiment. I. plasma postheparin lipolytic activity, lipoprotein lipase and liver lipase activities; II, lipoprotein lipase activity of retroperitoneal adipose tissue; 111, experiments on disappearance of ‘251-labeled lipoprotein lipase. Values are mean * S.E., n = 4-6. Significance of differences from control groups: * P < 0.05; ** P < 0.02; ***p < 0.01. Group

Body weight (g) 1st day 1 day before the experiment Food intake (g/day)

Experiment I

II

III

control thyroidectomized

263.00 f 1.92 260.00 k 1.89

256.50 * 2.49 256.50 k 5.85

177.83 k 9.21 177.67 k 9.03

control thyroidectomized control thyroidectomized

271.25 f 2.69 250.50+3.80 *** 12.43 * 0.52 10.40 + 0.42 *

261.75k4.77 237.75k4.91 ** 12.93 k 0.30 10.03+0.40 ***

225.67 & 5.02 195.33 + 7.20 ** 12.67kO.59 9.85 kO.32 ***

For determination of the postheparin plasma lipoprotein lipase and liver lipase activities the rats were starved for 16 h and then anesthetized with diethyl ether. 25 I.U. of heparin (Kabi, AB, Sweden) per 100 g of body weight was injected in an exposed jugular vein. Blood was collected 3, 5, 10, 30 and 60 min later from the opposite jugular vein into ice-cold tubes containing 0.25 M sodium citrate (50 ~1 per 1 ml of blood). The plasma was obtained after centrifugation for 5 min at 4°C in a Beckman Microfuge and was immediately used for the measurement of lipase activities by the method previously described [2 11. The triacylglycerol/gum arabic emulsion used as substrate was prepared as follows: 25 mg of olive oil were mixed with a trace amount of [ ‘H]oleic acid-labeled triolein (2 . 10’ cpm) in heptane and the solvent was evaporated under nitrogen. 1 ml of 10% gum arabic, 1.25 ml 1 M Tris-HCl buffer, pH 8.5, and 2 ml of distilled water were added and the mixture was sonicated chilled in ice-water for 8 min with a Branson Sonifier. To this emulsion, 0.45 ml human serum, 1.5 ml 1 M NaCl and 3.8 ml distilled water were added. This mixture was incubated for 60 min at 37°C. Then, 2.5 ml 10% (w/v) bovine serum albumin (fraction V, Sigma, MO, U.S.A.), pH adjusted to 8, was added. 150 ~1 of this mixture were used for each determination of total lipase activity in postheparin plasma (postheparin lipolytic activ-

ity), in adipose tissue extracts or in media from incubations of pieces of adipose tissue. For determination of liver lipase activity the same basic incubation mixture was used but the serum was omitted and the final concentration of NaCl was increased from 0.1 to 1 M. All incubations were carried out in a total volume of 200 ~1 for 60 min at 37°C. The free fatty acids released were extracted and determined as described [22]. The lipase activities are expressed as pmol of free fatty acids released per min. A rabbit antiserum was prepared against liver lipase purified by heparinSepharose chromatography of heparin perfusates from rat livers [23]. This antiserum inhibited all lipase activity measured at 1 M NaCl, which thus represents total liver lipase activity. The antiserum inhibited 55% of the plasma postheparin lipolytic activity measured in the assay with serum and 0.1 M NaCl, demonstrating that this assay detected both liver lipase and lipoprotein lipase. The inhibitable activity of liver lipase in this assay was the same as the activity detected at 1 M NaCl. Therefore, in most of the present experiments the total lipoprotein lipase activity could be calculated by subtracting the liver lipase activity (assay at 1 M NaCl) from the total postheparin plasma activity (assay at 0.1 M NaCl in the presence of serum). For studies of release of lipoprotein lipase from

535

addipose tissue in vitro, rats were anesthetized with diethyl ether, exsanguinated through the abdominal aorta and retroperitoneal adipose tissue was removed and rinsed in cold 0.9% saline. Suitable pieces (about 100 mg) were cut and distributed into incubation flasks containing 1 ml of bicarbonate-buffered medium, pH 7.4 [24], gassed with O/CO, (95 : 5). The medium contained 4% human serum (v/v) and 5% bovine serum albumin (w/v). The tissue samples were incubated for 30 min at 37’C. The amounts of lipoprotein lipase activity in the media were then determined as described above. The activity was fully inhibited with 1 M NaCl and 4-fold stimulated by human serum. Homogenates of adipose tissue were prepared in ice-cold 2.1 M glycine buffer (pH 8.3) containing 100 I.U. of heparin per ml (100 mg tissue/ml). The homogenates were centrifuged for 5 min at 4°C in a Beckman Microfuge and lipoprotein lipase activity was measured in the supernatants as described above. The activity was expressed as pmol free fatty acid/g wet weight per h of incubation or as mmol free fatty acid/g protein per h of incubation. Proteins were measured according to the method of Lowry et al. 1251. The lipase activity was inhibited by 1 M NaCl and was stimulated by serum. For studies of the disappearance of ‘251-labeled lipoprotein lipase from the blood, lipoprotein lipase purified from bovine milk [26] and then labeled with ‘25I by the lactoperoxidase method [4] was used. 200 ~1 of the labeled enzyme solution (about 0.1 mg per ml in 2 M NaCl/S mM sodium Verona1 buffer, pH 7.4) were mixed with 1 ml of 20% bovine serum albumin in 0.05 M Tris-HCl buffer, pH 8.5, on ice. 0.3 ml of this solution (about lo6 cpm) was injected into a jugular vein of a diethyl ether-anesthetized rat. Blood samples of 0.1-0.2 ml were taken from the opposite jugular vein (about 10 samples during 15 min). The samples were put.into weighed tubes. The radioactivity of the blood samples was measured in a 1275 Minigamma, LKB Wallac, Sweden. For calculation of lipoprotein lipase the total amount of “‘I-labeled remaining in the blood, it was assumed that the blood volume was 5.5% of the rats’ body weight [41. “S-labeled

heparin

(11.3

mCi

per

g, Radio-

chemical Centre, Amersham, U.K.) was injected intravenously into rats, and blood was collected at intervals during 30 min. The amount of injected heparin corresponded to a dose of 5 I.U. per 100 g body weight. The blood was centrifuged and the radioactivity of plasma was measured in a Packard Scintillation Spectrometer. The statistical significance of the data was analyzed by Student’s f-test.

Results As Table II shows, the plasma postheparin lipolytic activity was higher in thyroidectomized rats than in controls during the period studied after heparin injection (3-60 min). Lipoprotein lipase activity displayed a similar pattern - increased enzyme activity in thyroidectomized rats (Fig. 1). On the other hand, the liver lipase activity was lower in the thyroidectomized rats, except at 60 min (Fig. 1). From Fig. 1 it is evident that the disappearance of both lipases from the circulation was retarded in the thyroidectomized rats. Three possible causes for the higher lipoprotein lipase activity in postheparin plasma from thyroidectomized rats were investigated: 1, that thyroidectomized rats had a higher tissue lipoprotein lipase activity and/or that lipoprotein lipase was released in higher amounts from their tissues; 2, that the clearance of lipoprotein lipase from the blood might be impaired in thyroidectomized rats; 3,

TABLE

11

PLASMA POSTHEPARIN LIPOLYTIC INJECTION OF HEPARIN TO THYROIDECTOMIZED RATS

ACTIVITY CONTROL

AFTER AND

Values are mean *SE., n = 4. Significance of differences control groups: *P < 0.05; l * P < 0.001. Time after heparin (min)

3 5 IO 30 60

from

Plasma postheparin lipolytic activity (pmol free fatty acid/ml plasma per min) Control

Thyroidectomized

2.91 k 0.09 2.74+0.21 2.80+0.19 I .76 * 0.05 0.89 + 0.08

4.36 + 0.43 4.61 kO.31 * 3.88 k 0.28 3.85+0.11 ** 2.95 f 0.06

536

4.0

3.0

3.0

.5 E -E 2.0 : E a.

1.0

i Oh t

r

I

I

I

10

30

60min

h< sparin

1

I

20min

60min

-

S)hepL

heparini

i”ll-LPL5

lo

”

Fig. 1. Activity of lipoprotein lipase (left) and liver lipase (centre) in the plasma of control (0) and thyroidectomized injection of 25 LU. of heparin per 100 g of body weight. Each point is mean f SE. of four rats. Fig. 2. Disappearance of ‘251-labeIed lipoprotein Iipase ( t2sI-LPL) in control (open symbols) and thyroidectomized rats when heparin (5 (circles) or 25 (squares) I.U. per 100 g of body weight) was injected 1 min before the lipoprotein no heparin.

that the clearance of heparin might differ between thyroidectomized and control rats. Lipoprotein lipase activity in extracts of retroperitoneal adipose tissue was 2-2.5-fold higher in thyroidectomized rats than in controls (Table III).

TABLE

(0) rats after

(filled symbols) lipase. Triangles.

This was evident both when the activities were expressed per g wet tissue and per g protein. Pieces of adipose tissue from thyroidectomized rats released more lipoprotein lipase activity to the medium when incubated in the presence of serum and albumin than did corresponding pieces from control rats (Table IV).

III

LIPOPROTEIN LIPASE ACTIVITY IN RETROPERITONEAL ADIPOSE TISSUE OF CONTROL AND THYROIDECTOMIZED RATS. Values are mean f S.E., n = 4. Significance of differences the control group: * P < 0.05; ** P c:0.001. Group

Control Thyroidectomized

Lipoprotein

from

TABLE

IV

LIPOPROTEIN LIPASE ACTIVITY SECRETED IN VITRO FROM PIECES OF RETROPERITONEAL ADIPOSE TISSUE Values are mean i S.E., n = 9- 12. Significance from the control group: * P 4 0.05.

hpase activity

of differences

pm01 free fatty acid/h per g wet weight

mm01 free fatty acid/h per g protein

Group

Lipoprotein lipase activity in medium after 30 min incubation (pmol free fatty acid/g wet weight per h)

36.63 rt 0.65 70.21 ~fr10.87 *

2.17+0.05 5.33*0.39

Control Thyroidectomized

4.31 kO.70 12.39rt: 1.79 *

**

537

t “S-heparin

Fig. 3. Disappearance of 3’S-labeled heparin from the circulation in control (0) and thyroidectomized (0) rats.

The disappearance of intravenously injected lipop rotein lipase was the same in I ’ 5I-labeled both groups of rats (Fig. 2). Injection of heparin 1 min before the labeled enzyme retarded its disappearance. This effect was more pronounced in hypothyroid rats than in controls at the lower dose of heparin (Fig. 2). The curves of disappearance of 35S-labeled heparin indicate a biexponential decay (Fig. 3). Initially, the radioactivity disappeared with no difference between control and thyroidectomized rats with a half-life of about 5 min. Then the disappearance slowed down, with a half-life of about 14 min for control rats and 18 min for thyroidectomized ones. Discussion Skottova and PalkoviE [ 10,181 have recently reported that the plasma postheparin lipolytic activity is higher in thyroidectomized than in control rats. The present study demonstrates that the increase is due to an increased lipoprotein lipase activity. In hypothyroidism an increased lipoprotein lipase activity in several tissues has been described [ lo,1 1,13,15,16]. This was confirmed here, under our conditions, for adipose tissue. The increased plasma postheparin lipoprotein lipase activity in thyroidectomized rats may thus result from release of higher amounts of lipoprotein lipase from extrahepatic tissues into the blood stream. It

has been shown that food intake increases the lipoprotein lipase activity [6]. This intake is higher in normal than in hypothyroid animals ([7,27], Table I). From this point of view, the increased lipoprotein lipase activity in thyroidectomized rats in somewhat surprising. It seems that mechanisms leading to the accumulation of lipoprotein lipase activity in hypothyroid state are more complex, probably involving interactions with other hormones. On the other hand, the plasma liver lipase activity was decreased in the thyroidectomized rats. It has been shown previously that the lipase activity released by heparin from perfused livers is lower in hypothyroid rats than in controls [28]. The low liver lipase activities are also in accord with the results of Murase and Uchimura [ 171. These authors, however, did not find an increased lipoprotein lipase activity in postheparin plasma. This discrepancy may reflect differences in experimental conditions, perhaps mainly with respect to the diet. Our experiments were performed on fasted rats, whereas those of Murase and Uchimura were on nonfasted animals, and they measured the plasma lipoprotein lipase activity only shortly after heparin injection. In their experiments, the higher food intake might have increased the lipoprotein lipase activity in controls and could thus erase the differences in the postheparin lipoprotein lipase activity. However, our experiments showed an increase in the lipoprotein lipase activity in adipose tissue of thyroidectomized rats as well as in nonfasted animals [29]. Furthermore, we measured the postheparin lipoprotein lipase activity at different time intervals after heparin injection. We found that shortly after heparin administration the difference in lipoprotein lipase activity between control and thyroidectomized rats was not so great as later on, at 30- and 60-min intervals after heparin injection. These results led us to carry out studies of the lipoprotein lipase disapearance from blood. In our experiments, we injected labeled bovine milk lipoprotein lipase into both groups of rats, Previously, it was shown that the labeled enzyme is rapidly removed from the circulation and that heparin injected before the enzyme retards its disappearance [4]. In the present study we found no difference between controls and thyroidectomized rats in the disappearance rate. However, when a

538

amount of heparin was injected before the labeled lipoprotein lipase, the enzyme disappearance was more retarded in thyroidectomized rats. This may contribute to the elevated lipoprotein lipase activity in the blood of thyroidectomized rats at longer time intervals after heparin injection. These results suggest that in thyroidectomized rats the metabolism of lipoprotein lipase may be impaired, but the question why the more retarded disappearance of lipoprotein lipase was found only after heparin ad~nistration cannot be answered on the basis of the obtained data. The disappearance of heparin itself was also somewhat retarded in thyroidectomized rats. This may also contribute to the slower disappearance of lipoprotein lipase in thyroidectomized rats. In thyroid hormone deficiency an increased output of very low density lipoproteins from liver [9], a reduction of plasma very low density lipoprotein concentration and an increase in the amounts of lipoproteins of higher densities have been demonstrated [7,8, IO]. The high lipoprotein lipase activity may be, at least partially, responsible for the changed lipoprotein spectrum. The exact physiological role of liver lipase is not known, but the enzyme seems to be involved in the removal of cholesterol from the blood [30]. If this is correct, the low liver lipase activity would be one factor contributing to the high cholesterol levels which are usually seen in hypothyroidism. low

Acknowledgements This work was supported by grants from the Swedish Medical Research Council ( 13X-00727) and the Medical Faculty, University of Umea. We thank Ms. Marianne Borg for preparing the manuscript. References 1 Krauss, R.M., Windmueller.

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