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Pre- and post-natal development of lipoprotein lipase and hepatic triglyceride hydrolase activity in rat tissues
549
Atherosclerosis,
26 (1977)
0 Elsevier/North-Holland
549-561 Scientific Publishers, Ltd.
PRE- AND POST-NATAL DEVELOPMENT OF LIPOPROTEIN LIPASE AND HEPATIC TRIGLYCERIDE HYDROLASE ACTIVITY IN RAT TISSUES TOVA CHAJEK, OLGA STEIN and YECHEZKIEL
STEIN
Lipid Research Laboratory, Department of Medicine B, Hadassah University Hospital, and Department of Experimental Medicine and Cancer Research, Hebrew University-Hadassah Medical School, Jerusalem (Israel)
(By invitation, received November, 1976)
Summary The ontogenic development of lipoprotein lipase and liver triglyceride hydrolase was studied in the rat. The enzyme activity measured in extrahepatic tissues fulfilled the criteria of lipoprotein lipase from the onset of measurable activity, i.e. it was inhibited by protamine and 1 M NaCl and showed requirement for serum and heparin for optimal activity. In the liver, measurable amounts of triglyceride hydrolase, active at pH 8.6 were detected 6 days prior to birth. However, till the fourth postnatal day about 50% of this activity was inhibited by NaCl and its sensitivity towards protamine was also higher than that of the enzyme in adult liver. Three patterns of development of enzymic activity were observed in extrahepatic tissues. In the lung, the lipoprotein activity reached the adult values one day prior to birth, while in the kidney only 30% of adult activity were found at birth. A linear increase of enzyme activity was observed in the heart; only 25% of adult activity were detected at birth and 100% were reached only 20 days after birth. The increase in lipoprotein lipase activity in the heart was accompanied by morphological differentiation of cardiocytes and by a progressive development of the capillary bed, which might be related to the pattern of development of enzyme activity in this organ. Adipose tissue lipoprotein lipase activity in inguinal fat fell from values 15 times higher than adult values between the 4th and 40th postnatal days. The enzyme activity in epididymal fat increased steeply between day 10 and 40, at which time it exceeded the adult values very considerably. These findings indicate that the regulation of the development of lipoprotein lipase activity in extrahepatic tissues is governed by local factors, which can differ This paper is dedicated to Professor F.G. Schettler on the occasion of his sixtieth birthday. This investigation was supported in part by a grant from the United States--Israel Binational Foundation, and by a grant of the Israeli Ministry of Health to Y. Stein (Established Investigator of the Ministry of Health).
550
even in the same type of tissue, inguinal and epididymal fat. Key words:
Brain - Diaphragm Inguinal fat - Kidney
as exemplified
by the difference
- Electron microscopy -Liver - Lung -Muscle
-
Epididymal
fat -
between
Heart
-
Introduction Lipoprotein lipase and hepatic triglyceride hydrolase are the key enzymes which regulate the vascular component of chylomicron and very low density lipoprotein catabolism. During the past few years these two enzyme activities have been characterized and the enzymes have been isolated from different sources and purified [l--8]. The site of action of lipoprotein lipase has been localized to the endothelial cell surface [9]. The transport from the site of its synthesis to the lumenal surface of the endothelial cell proper has been shown to depend on the integrity of the microtubular system [ 10-131. Diet and hormones were shown to affect lipoprotein lipase activity differently, depending on the anatomical site, i.e., adipose tissue or muscle [ 14-201. Age related differences in lipoprotein lipase activity have been described in adipose tissue [21-231 and later studies have dealt with the relation between the cell size and enzyme activity [ 241. Presently we have undertaken a systematic study of ontogenic development of lipoprotein lipase in several tissues of the rat in order to gain more information as to other factors which might regulate the enzymic activity. Methods Animals
Male and female rats, 90-120 days old of the Hebrew University strain kept in constant temperature rooms and fed the pelleted Am-Rod 931 diet (25) were used. Mating pairs were caged separately for 12 h, from 20.00 to 8.00 and thereafter the females in which a vaginal plug was detected, were isolated and the gestation period was calculated starting from that date. The day of birth was considered as age zero, and fetal age was expressed in negative numbers from that date. Under these breeding conditions the error in the estimation of total age was less than 12 h. The newborn stayed with their mother during the first postnatal month. Thereafter they were separated according to sex and caged in groups of six; these animals were fed the pelleted Am-Rod 931 diet. Preparation hydrolase
of tissues for the assay of lipoprotein
lipase and hepatic
The activity of lipoprotein lipase was assayed in middle head of quadriceps muscle, brain, inguinal fat, trial fat, kidney and spleen. All adult tissues were guinated under light ether anesthesia. To obtain fetal
heart, lung, epididymal taken from tissues, the
triglyceride
diaphragm, fat, paramerats exsanuterus, con-
551
taining the embryos was removed from anaesthetized rats and placed on ice for 5 min, and the immobilized. fetuses were decapitated. The animals and respective organs were weighed and these data served as an additional criterion of fetal age (Table 1). Dried defatted tissue preparations were made by homogenization of lOO500 mg of fresh tissue in 50 ml of ice-cold acetone using. a Polytron (Kinematica GmbH, Luzern, Switzerland) homogenizer with pt lo-11 probe at maximum speed for .I min at 0°C. The homogenates were centrifuged at 15,000 rpm for 20 min at 4°C and the supernatant was discarded and the residue was re-extracted three times with 50 ml ice-cold acetone and twice with 50 ml of cold diethyl ether. The defatted preparations were dried at 0” under nitrogen and stored at -20°C in vacua. Under these conditions no measurable loss of lipolytic activity was detected for up to 7 days but assays were routinely performed within 24 h. This preparation was designated acetone powder. Determination of lipoprotein lipase activity For the assay of enzyme activity aliquots of the acetone powder were homogenized in ice cold 0.025 M NH,-HCl buffer, pH 8.1, to give 4-10 mg acetone powder/ml. Lipoprotein lipase activity was determined according to Schotz et al. [26], using triolein in the form of an emulsion as substrate. To prepare the emulsion the following ingredients were suspended in 15 ml of 0.2 M Tris-HCl buffer, pH 8.6, containing 0.15 M NaCl: 300 nmol of glycerol Tri[l-‘4C]oleate (spec. act. 54.9 Ci/mol, Amersham, Searly Corp., Arlington Heights, Ill.); 22.6 pmol carrier triolein (Sigma Chemical Co., St. Louis, MO.), 14.4 mg bovine serum albumin (fatty acid poor, Miles Lab., Kankakee, Ill.) and 0.36 ml of 1 : 1000 solution of Triton X-100 (B.D.H. Chemicals, Poole, U.K.). The suspension was subjected to ultmsonic irradiation in a Braun-Sonic 300 instrument (Braun, Melsungen, Germany) using a probe of 10 mm in diameter, at maximal scale for 4 min at 4°C. 4.8 ml of fasting human serum were added TABLE 1 ORGAN
AND BODY WEIGHT DURING PRE- AND POST-NATAL
DEVELOPMENT
OF THE RAT
Values of body weight are means f SE of 6-8 determinations. Organ weights are means of 6-8 tions for each time interval. For fetal tissues no distinction of the sex of the donor was made. Age (days) -6 -5 -3 -1 0 +4 +10 +20 +30
Body weight (9) 0.47 + 0.80 f 2.08 ? 4.67 ? 5.56 + 9.36 ? 13.60 r 30.30+ 56.4Oi
0.03 0.01 0.04 0.3 0.5 0.1 0.3 1.2 1.9
Liver (g)
Brain (g)
Heart (mg)
0.03 0.05 0.14 0.25 0.29 0.29 0.40 1.25 2.99
0.05 0.06 0.13 0.18 0.23 0.40 0.14 1.28 1.35
2.4 5.3 10.9 20.0 25.7 46.3 71.4 154.2 247.0
7.8 9.1
1.59 1.58
545.4 626.3
determina-
Lung (mg)
11.7 42.7 71.0 88.4 157.1 242.6 311.0 438.4
Adult
female male
185.0 237.0
+ 7.4 + 9.4
929.3 1011.0
552
to 15 ml of the substrate emulsion which was used within 30 min of preparation. The reaction was terminated by addition of 4 ml of isopropanol/l.5 M HzS04 (40 : 1, v/v) according to Schotz et al. [26] and the free fatty acids were adsorbed on Amberlite IRA 400 (B.D.H. Chemicals, Poole, U.K.) as described by Kelley [27]. After the resin was washed four times with 5 ml of hexane, 1 ml of Soluene 100 (Packard Inst. Co., Downers Grove, Ill.) and 15 ml of scintillation fluid (4 g 2,5-diphenyloxazole and 100 mg 1,4-bis-[2-(4-methyl-5phenyloxazolyl)]-benzene in 1000 ml toluene) were added and the samples were counted in a Tricarb liquid /?-scintillation spectrometer 3380 equipped with an absolute activity analyzer, model 544. The radioactivity present in the hexane phase in samples incubated without the enzyme was substracted from all experimental values. The assay system consisted of 0.2 ml of acetone powder homogenate, 0.2 ml of 0.15 M NaCl containing heparin 5 u/ml (Evans Medical Corp., Liverpool, U.K.) and 0.6 ml of the emulsified substrate. Incubations were carried out in duplicate for 90 min at 27” C in a shaking incubator. The reaction was linear up to 120 min of incubation and the activity was expressed as nmoles of free fatty acid released per mg acetone powder during 1 h of incubation. To measure the effect of protamine and NaCl on the lipolytic activity, 0.2 ml of defatted tissue homogenate were preincubated for 10 min at 27°C with 0.2 ml of 0.15 M NaCl containing 6 mg/ml of protamine sulfate (salmine, Sigma Chemical Co., St. Louis, MO.) and heparin 5 u/ml or with 0.2 ml of 2 A4 NaCl containing heparin 5 u/ml. Lipoprotein lipase assays were performed also in the absence of serum or heparin in the reaction mixture. Preparation
of tissues for microscopy
Fixation was carried out with 2% osmium tetroxide in acetate veronal buffer. Following dehydration in graded ethanols the tissues were embedded in Epon [ 281. 0.5 /J sections were prepared and stained with toluidine blue. Results Lipolytic enzyme activity was detected in most tissues about 6 days prior to birth. The activation by heparin and serum and the inhibition by protamine and 1 M NaCl of enzymic activity were determined during pre- and post-natal development on organs obtained at day -3, -1, 0, +4, +lO and +30. Since the requirement of serum and heparin for optimal activity and the degree of inhibition by 1 M NaCl were quite similar at each age group examined, the data for each organ were pooled and are presented in Table 2 under a common heading of “fetal” tissues. Adult tissues were assayed at the same time as the fetal tissues and again the values obtained for each organ in different experiments were pooled. The data summarized in Table 2 show that the enzymic activity measured during pre- and post-natal development was reduced to about 50% in the absence of heparin. Inhibition by 1 M NaCl and protamine was almost complete in all instances and did not vary from the sensitivity of the enzyme in adult tissues. The degree of serum dependence did not vary significantly from that of the corresponding adult tissue. These findings provide evidence that the enzymic activity determined throughout the pre- and post-natal period fulfilled the criteria of lipoprotein lipase.
553 TABLE 2 COMPARISON OF COFACTOR FETAL AND ADULT TISSUES
REQUIREMENT
AND INHIBITION
OF LIPOPROTEIN
LIPASE IN
The data represent percent of control activity, measured in the complete system. Values are means k SE of 3-5 experiments for fetal tissues and of 22 experiments for adult tissues. For definition of “fetal” organs see Results. Organ
Lipoprotein lipase activity Percent activity in absence of heparin
Percent inhibition by 1 M NaCl
Protamine
Lung
“Fetal” Adult
55.3 f 2.9 51.8 k 3.7
91.5 + 2.9 97.6 f 0.8
89.9 t 2.0 91.1 + 1.9
Muscle
“Fetal” Adult
53.3 f 3.4 52.1 + 4.5
97.5 * 1.0 96.7 + 1.7
89.9 * 1.9 91.6 + 3.3
Diaphragm
Adult
48.2
98.3
93.0
Heart
“Fetal” Adult
55.9 f 4.6 59.5 f 2.9
95.3 + 1.3 92.8 f. 1.3
90.0 + 2.3 91.3 * 2.1
Kidney
“Fetal” Adult
63.7 f 6.5 60.5 ? 3.7
90.3 * 2.0 90.1 + 2.8
83.0 .? 5.7 86.5 + 6.2
Brain
“Fetal” Adult
55.4 f 5.0 57.5 + 3.8
95.7 + 2.0 90.9 + 7.2
88.2 + 1.4 92.3 + 5.0
InguinaI fat
“Fetal” Adult
68.8 f 6.6 54.8 * 3.7
98.2 f 1.0 99.0 f 1.0
93.2 + 1.8 83.4 2 2.3
Epididymal fat
Adult
48.4
97.8
95.1
The change in enzyme activity as a function of age fell into distinct patterns which varied in different organs. The lipoprotein lipase activity of lung (Fig. 1, lower panel) increased between day -6 and -1 and then showed small fluctuations around the adult value. A similar behavior was seen also in the spleen; in this organ the earliest time interval measured was +4. The enzyme activity in the kidney at birth was 30% of adult value and increased gradually till +20 days. A very prominent change in lipoprotein lipase activity was observed in the heart (Fig. 1, upper panel), which showed a linear increase till day +20 at which time it reached the adult values. At birth the activity of lipoprotein lipase in the heart was about 25% of adult value, at which time it was already much higher than that of the kidney and spleen (Fig. 1). An attempt was made to correlate this steep increase in enzyme activity with some morphological changes in the myocardium. Fig. 2a-d shows representative sections of the heart taken between day -3 and +20. It can be seen that the cells are filled with myofilaments and have prominent mitochondria at all time intervals examined. There is a progressive fall in the size of the nuclei and an increase in the diameter of the myocytes. At -3 the cells are separated by intercellular spaces and between birth and +20 days there is a progressive increase in capillaries which develop in the intercellular spaces. A second pattern in the change of enzyme activity was seen in the brain and skeletal muscle, even though the absolute values in the brain were much lower
I’ .’
~--.Jyn9 “I
~...-i____‘._________..~~,” , I
630
Kidney
1
10
20 Oars
30
AOULT
Fig. 1. Lipoprotein lipase activity in fetal and post-natal period of lung, kidney and spleen (lower panel) and of heart as a function of age. Values are means f SE of 3-5 determinations in fetal tissues and 22 determinations in adult tissues.
(Fig. 3, lower panel). In both the brain and muscle the prenatal enzyme activity was higher than that in the respective adult tissues and peaked at day +4, when the activity was 4-fold that of the adult muscle. The higher absolute activity in the diaphragm than in the thigh muscle decreased as well to adult values between day +4 and day +30 (Fig. 3). The measurement of lipoprotein lipase activity in adipose tissue was confined to the post-natal period, and while the inguinal fat was well developed at day +4, the epididymal fat was not readily identified prior to day +lO. As seen in Fig. 4, a progressive decrease to adult values was seen in the inguinal fat, the peak activity being 15-fold that of the adult. No difference was observed in the inguinal fat of the two sexes. Lipoprotein lipase in the epididymal and parametrial fat showed a sharp increase with a peak at 40 days and a decline to adult values at 60 days. The enzymic activity in the epididymal fat was consistently higher than in the parametrial fat (Fig. 4). Hepatic triglyceride lipase activity was measurable at -6 days and the level of activity reached the adult levels at day -3 (Table 3). Thereafter, a slight increase was observed betweec day -1 and +4. However, unlike the extrahepatic activity, the liver enzyme examined at various fetal ages showed a changing cofactor requirement and sensitivity to 1 M NaCl and protamine
Fig. 2a-d. Sections of rat myocardium increase in cell size and a progressive spaces. X1350.
at day -3, 0.10 and 20. There is a decrease in the nuclerlr size, an increase in the number of capillaries (arrows) in the intercellular
(Table 3). Between day -6 and +4 the activity in the absence of heparin ranged between 70-80% of control value, while at later time intervals it was similar to the adult, i.e. 85% of control value. The enzymic activity did not require serum for optimal activity, and in that respect was similar to that of the adult. The most prominent difference was in the inhibition by 1 M NaCl, which was about 50% at the early pre-natal period and was still 40% at day +4. Only at
1 ii /’‘.\‘1.\
---.-._ I\.\
i
‘k
i
‘\
‘.\.,Diaphragm
I “‘\f-.- _._,_+
i
\ .A
Thigh muscle -1
I
‘f-.,
/
--I
/’
-6 -3 0
f-S
‘\
‘f---__,
Brain -_ ---.
I
I
I
I
k
10
20
30
I ADULT
Fig. 3. Lipoprotein lipase activity in fetal and post-natal period of brain (lower panel) and skeletal muscle (upper panel) as a function of age. Values are means + SE of 3-5 determinations in fetal tissues and 22 determinations in adult tissue.
!
5
2
s 120 r tr \
g 1f
It
poraovarian\ fat
‘L_.p
30 0t
0
10
20
30
LO
50
60
AOULT
Days Fig. 4. Post-natal lipoprotein lipase activity of rat adipose tissue as a function of age. Values ? SE of 3-5 determinations in fetal tissues and 15-24 determinations in adult tissue.
are means
TABLE
3
COMPARISON
OF
HYDROLASE Control
Age
activity
(days)
COFACTOR
REQUIREMENT
IN DEVELOPING was determined
Exp.
No.
Fatty
AND
INHIBITION
OF
HEPATIC
TRIGLYCERIDE
LIVER in the complete acid
nmoles/mg powder/h)
released
system
described
Enzyme
activity
in Methods. as percent
Values
are means
+ SE.
of control
acetone In absence
of
In presence
Heparin
Serum
1 M N&l
of Protamine
-6
3
12.0
f 1.6
70.3
103.1
? 5
43.3
? 4.8
-
-5
4
10.3
i- 1.3
79.2
104.6
+
57.6
+ 5.0
43.3
-3
4
15.1
+ 1.4
78.0
+ 2.7
52.2
+ 3.0
48.5
f 2.6
-1
5
23.0
+ 1.2
80.1
+ 4.4
97.7
f 6.0
64.5
* 3.8
54.0
f 5.9
0
f 2.6
3
22.2
f 3.0
79.3
+ 1.1
114.0
+ 1.7
59.3
+ 4.7
54.8
f 5.4
+4
3
18.9
+ 0.7
78.4
f 4.2
108.3
+ 8.3
63.3
+ 5.3
60.5
+ 6.1
+10
4
16.5
+ 1.6
88.7
f 5.0
119.3
+ 5.7
97.3
f 1.5
60.1
f 5.1
+20
5
16.6
+ 1.4
84.3
i
5.9
112.8
f 6.9
104.4
f 9.4
71.1
* 4.9
4
13.5
+ 1.7
85.3
+ 6.0
113.3
f 8.6
119.3
?- 6.3
70.8
t 7.6
22
15.4
f 0.7
85.1
+ 2.3
112.2
i- 2.7
108.0
f 4.8
67.8
+ 3.5
+30 Adult
day +20 was there also the slight enhancement of enzyme activity in the presence of 1 M NaCl encountered in the adult. Likewise a somewhat greater inhibition of enzyme activity by protamine occurred between day -5 and +4. Discussion Lipoprotein lipase, the key enzyme in the degradation of very low density lipoprotein and chylomicron triglyceride on the endothelial cell surface responds differently to the same stimulus in adipose tissue and in muscle. Thus fasting results in a fall in lipoprotein lipase in adipose tissue and an increase in the enzymic activity of heart and skeletal muscle [29]. Moreover, while enzyme activity in adipose tissue was shown to have a positive correlation with plasma insulin concentration [l&20,30], a negative correlation was found for lipoprotein lipase activity in the heart and skeletal muscle [13,20]. Yet, another difference in the behavior of this enzyme in the two types of tissues, was shown presently in the pattern of pre- and post-natal development. The lipoprotein lipase activity of the heart at birth attained a level of only 25% of the adult and the activity expressed per mg of acetone powder was similar to that of skeletal muscle and lung. The further post-natal increase in enzyme activity, which was measured in the fed state, is most probably related to the post-natal development of the heart, as the final differentiation of this organ in the rat occurs only after birth [31,32]. The main changes during development of the heart consist in the alignment of cells, differentiation and orientation of myofibrils, maturation and redistribution of mitochondria, and these are completed at the age of about 24 days [31]. The development of the transverse system, which is concomitant with an increase in the sarcolemmal cell surfaces occurs between 14 and 31 days of age [ 311. In contradistinction to skeletal muscle, cell division and differentiation are concurrent in cardiac muscle, as synthesis of DNA, of myofibrils and of connective tissue ground
substance occurs at the same time [33]. Thus it seems that the pattern of development of lipoprotein lipase is not related to an inability of the younger cardiocytes to synthesize the enzyme protein, as was suggested by findings in cell culture [34]. Another aspect of the structural development of the heart is the formation of a well-developed capillary bed, which progresses between birth and 3 weeks of age s[321. In view of the fact that lipoprotein lipase acts at the capillary cell surface, to which it has to be transported, one might envisage the possibility that the differentiation of the sarcolemmal membrane and the increase in the number of capillaries play a key role in the post-natal increase of cardiac lipoprotein lipase. The relatively high dietary intake of lipids during the suckling period could also contribute to this increase. In the adult rat, fed for 3 days a 50 Cal% fat diet there was a 3-fold increase in the lipoprotein lipase of the heart [35]. It seems of interest to point out that enzymes active in the oxidation of fatty acids, such as palmitoylcarnitine transferase show an analogous pattern of post-natal development in the heart [ 361. No explanation can be suggested so far for the diagonally opposite response of lipoprotein lipase in skeletal muscle (presumably red) and diaphragm in which a decrease to adult values was observed during the post-natal period between 4 and 30 days. The relatively high activity of lipoprotein lipase in the brain between birth and 10 days of age occurred at a time during which there is a striking increase in cholesterol content as well as a high rate of weight gain [ 371. Since at the same time 3-hydroxy-3-methylglutaryl-CoA reductase activity was found to increase &fold [38] over adult values, it seems that local cholesterol synthesis was responsible for this increase. As free cholesterol is an integral part of cellular membranes it seems plausible that the high lipoprotein lipase activity might contribute towards the supply of fatty acids needed for increased membrane phospholipid synthesis, during the perinatal period. Lipoprotein lipase activity in adipose tissue differed markedly according to the location, and so did the developmental pattern. Higher activity of lipoprotein lipase in omental than in subcutaneous tissue was described in the human [ 391. Lipoprotein lipase activity of peritoneal and inguinal adipose tissue was compared in the duck, and no difference was seen in tissues from adult birds [22]. In that study a fall in enzyme activity was reported between 2 and 8 weeks of age and throughout that period the activity in the peritoneal fat was c.onsistently higher than in the inguinal fat. The gradual decline in enzyme activity was considered to be related to the specific growth rates of fat depots in different regions, the inguinal fat being the earliest and the peritoneal the latest in development [22]. In the rat lipoprotein lipase activity in epididymal fat of 4-5-week-old rats was found to be 8 times higher than that of adult rats [21], while in another study the highest enzymic activity was found on day 2 and fell gradually till day 19 [23]. In the female rat a fall in adipose tissue lipoprotein lipase was encountered during lactation and was related to prolactin secretion [40]. In the present study it became evident that regulation of enzyme activity in adipose tissue is dependent on local factors as well. The earlier development of the inguinal fat and the later appearance of epididymal tissue could be related to the onset of lipoprotein lipase activity. If the adipocyte should be considered as the source of the enzyme, one could
559
envisage the possibility that the onset of enzyme synthesis precedes the transition between pre-adipocytes and adipocytes. In contradistinction to the other organs, the levels of hepatic triglyceride hydrolase activity were similar to those in the adult, with a transient increase during the immediate perinatal period. The pronounced sensitivity to 1 M NaCl, which was prominent during the early development might have suggested the presence of two enzyme species, differing in their response to NaCl. Since in the fetal liver hematopoietic cells comprise about 50% of the organ’s volume [ 411, the possibility could be considered that the NaCl inhibited lipase originates in these cells. However, while the fractional volume contributed by the hematopoietic cells drops sharply between day -3 and 0 [41], during that time there is no change in the lability of the enzyme towards NaCl (Table 3). More recently it has been shown that the canine liver secretes a triglyceride hydrolase which is inhibited by 1 M NaCl and by protamine and requires apolipoprotein C-I for optimal activity [ 421. The presently described enzyme activity in fetal liver was inhibited by 1 M NaCl to a greater extent than by protamine but was not activated by the addition of serum, however, activation by C-I was not determined. The ontogeny of peripheral lipoprotein lipase and hepatic triglyceride hydrolase differs completely from that of pancreatic lipase [43]. In the rat pancreas the activity of lipase A followed a sigmoidal curve with relatively low activity between day -10 and -6 and a very steep rise between day -6 and -2, during which time the enzyme activity increased by 3 orders of magnitude, to almost adult values [43]. The rapid increase in pancreatic lipase (as well as other pancreatic enzymes) occurs during the period of cytodifferentiation in which the acinar cells become filled with rough endoplasmic reticulum and zymogen granules [44]. An attempt was made during this study to seek ultrastructural changes which could correspond to the development of lipoprotein lipase. Lipoprotein lipase is transported from the site of its synthesis to the site of its action on the endothelial cell plasma membrane and this process is inhibited by colchicine [ 10-121. However the ultrastructural pathway of the transport has not been defined so far, and no analogs of zymogenic granules have been shown to contain the enzyme activity. It was only in the heart that the increase of lipoprotein lipase coincided with a structural change in the form of extensive capillarization, which might have been instrumental in the regulation of enzymic activity. Acknowledgement The excellent edged.
technical
help of Miss R. *Ben-Moshe is gratefully
acknowl-
References 1 Greten, H.. Walter, B. and Brown, W.V.. Purification of a human post-heparin plasma triglyceride lipase,FEBS Letters. 27 (1972) 306-310. 2 Egelrud, T. and Olivecrona, T. The purification of a lipoprotein lipase from bovine skim milk, J. Biol. Chem.. 247 (1972) 6212-6217. 3 Fielding, P.E., Shore. V.G. and Fielding, C.J., Lipoprotein lipase - Properties of the enzyme isolated from post-heparin plasma, Biochemistry. 13 (1974) 4318-4232.
560 4 Greten, H. and Walters, B., Purification of rat adipose tissue lipoprotein lipase, FEBS Letters, 35 (1973) 36-40. 5 Bensadoun. A., Enholm. C., Steinberg, D. and Brown, W.V., Purification and characterization of lipoprotein lipase from pig adipose tissue, J. Biol. Chem.. 249 (1974) 2220-2227. 6 Enholm. C., Bensadoun, A. and,Brown, W.V., Separation and partial purification of two types of triglyceride lipase from swine postheparin plasma, J. Clin. Invest. (Abstract). 52 (1973) 26a. 7 Enholm. C., Shaw, W.. Greten, H. and Brown, W.V., J. Biol. Chem., In press. 8 Krauss, R.M., Levy, R.I. and Fredrickson, D.S.. Selective measurement of two lipase activities in postheparin plasma from normal subjects and patients with hyperlipoproteinemia. J. Clin. Invest., 54 (1974) 1107-1124. 9 Blanchette-Mackie. E.J. and Scow, R.O., Sites of lipoprotein lipase activity in adipose tissue perfused with chylomicrons - Electron microscope cytochemical study, J. Cell Biol., 51 (1971) l-25. 10 Chajek, T.. Stein, 0. and Stein, Y., Colchicine-induced inhibition of plasma lipoprotein lipase release in the intact rat, Biochim. Biophys. Acta. 380 (1975) 12’7-131. 11 Chajek, T., Stein, 0. and Stein, Y., Interference with the transport of hew&n-releasable lipoprotein lipase in the perfused rat heart by colchicine and vinblastine. Biochim. Biophys. Acta, 388 (1975) 260-267. 12 Borensztajn, J., Rone, M.S. and Sandros, T.. Effects of colchicine and cycloheximide on the functional and non-functional lipoprotein lipase fractions of rat heart, Biochim. Biophys. Acta, 398 (1975) 394-400. 13 Cryer, A.. McDonald, A., Williams, H.R. and Robinson, D.S., Colchicine inhibition of the heparinstimulated release of clearing-factor llpase from isolated fat-cells, Biochem. J., 152 (1975) 717-720. 14 Borensztajn. J. and Robinson, D.S. The effect of fasting on the utilization of chylomicron triglyceride fatty acids in relation to clearing factor lipase (lipoprotein lipase) releasable by heparin in the perfused rat heart, J. Lipid Res., 11 (1970) 111-117. 15 Robinson, D.S.. The effect of changes in nutritional state on the lipolytic activity of rat adipose tissue, J. Lipid Res., 1 (1960) 332-338. 16 Wing. D.R., Salaman, M.R. and Robinson, D.S., Clearing-factor lipase in adipose tissue - Factors influencing the increase in enzyme activity produced on incubation of tissue from starved rats in vitro, Biochem. J., 99 (1966) 648-656. 1,7 Kessler, J.I., Effect of diabetes and insulin on the activity of myocardial and adipose tissue lipoprotein lipase of rats, J. Clin. Invest., 42 (1963) 362-367. 18 Cryer, A., Riley. S.E., Williams, E.R. and Robinson, D.S., Effect of nutritional status on rat adipose tissue, muscle and postheparin plasma clearing factor lipase activities - Their relationship to triglyceride fatty acid uptake by fat-cells and to plasma insulin concentrations. Clin. Sci. Mol. Med., 50 (1976) 213-221. 19 Borensztajn, J.. Otway, S. and Robinson. D.S., Effect of fasting on the clearing factor lipase (lipoprotein lipase) activity of fresh and defatted preparations of rat heart muscle, J. Lipid Res., 11 (1970) 102-110. 20 Borensztajn, S., Samols. D.R. and Rubinstein, A.H.. Effects of insulin on lipoprotein lipase activity in the rat heart and adipose tissue. Amer. J. Physiol.. 223 (1972) 1271-1275. 21 Chlouverakis. C.. Adipose tissue lipoprotein lipase activity in rats of two different ages, Nature (Land.). 196 (1962) 1103. 22 23
24 25
26 27 28 29
30
Evans. A.J., Lipoprotein lipase activity in adipose tissues of the domestic duck - The effect of age, sex, and nutritional state, Int. J. Biochem., 3 (1972) 199-206. Hahn, P. and Drahota. Z., The activities of citrate cleavage enzyme, acetyl-CoA synthetase and lipoProtein lipase in white and brown adipose tissue and the liver of the rat during development, Experientia (Base]). 22 (1966) 706-707. Nestel. P.J., Austin, W. and Foxman, C., Lipoprotein lipase content and triglyceride fatty acid uptake in adipose tissue of rats of differing body weights, J. Lipid Res., 10 (1969) 383-387. Rachmilewitz, D.. Stein, 0.. Roheim, P.S. and Stein. Y., Metabolism of iodinated high density lipoproteins in the rat, Part 2 (Autoradiographic localization in the liver), Biochim. Biophys. Acta. 270 (1972) 414-425. Schotz. M.C., Garfinkel, A.S., Huebotter. R.J. and Stewart, J.E., A rapid assay for lipoprotein lipase. J. Lipid Res., 11 (1970) 68-69. Keller, T.F., Rapid assay of labeled free fatty acids in mixtures of labeled lipids, J. Lipid Res., 9 (1968) 799-800. Luft, J.H., Fine structure of capillary and endocapillary layers as revealed by ruthenium red, Fed. Proc.. 25 (1966) 1773. Robinson, D.S., Assimilation, distribution and storage. In: M. Florkin and E.H. Stotz (Eds.), Comprehensive Biochemistry, Vol. 18 (Lipid Metabolism). Elsevier. Amsterdam. 1970, Ch. 1, pp. 51116. Pykalisto.
O.J..
Smith.
P.H. and
Brunzell.
J.D.,
Determinants
of human
adipose
tissue lipoprotein
561
31 32
33 34 35
36
37 38 39 40
41
42 43 44
lipase - Effect of diabetes and obesity on basal- and diet-induced activity, J. Clin. Invest., 56 (1975) 1108-1117. Untersuchungen am Herzmuskel der Schiebler, T.H. and Wolff, H.H., Electronenmikroskopische Ratte wiihrend der Entwicklung. Z. Zellforsch., 69 (1966) 22-40. Toth, A. and Schiebler, T.H., Ueber die Entwicklung der Arbeits- und Erregungsleitungsmuskulatur des Herzens van Ratte und Meerschweinchen - Histologische, histochemische und electrophysiologische Untersuchungen, 2. Zellforsch., 76 (1967) 543-567. Weinstein, R.B. and Hay. E.D., Deoxyribonucleic acid synthesis and mitosis in differentiated cardiac muscle cells of chick embryos, J. Cell Biol.. 47 (1970) 310-316. Pinson, A.. Frelin, Ch. and Padieu, P.. The lipoprotein lipase activity in cultured beating heart cells of the post-natal rat. Biochimie. 55 (1973) 1261-1264. Jansen. H.. Hulsmann, W.C.. Van Zuylen-Van Wiggen. A., Struijk, C.B. and Houtsmuller. U.M.T., Influence of rapeseed oil feeding on the lipase activities of rat heart, Biochem. Biophys. Res. Commun., 64 (1975) 747-751. Warshaw. J.B.. Cellular energy metabolism during fetal development, Part 4 (Fatty acid activation, acyl transfer and fatty acid oxidation during development of the chick and rat), Develop. Biol., 28 (1972) 537-544. Cuzner, M.L. and Davison, A.N., The lipid composition of rat brain my&n and subcellular fractions during development, Biochem. J.. 106 (1968) 29-34. Sudjic, M.M. and Booth, R., Activity of 3-hydroxy-3-methylglutaryl-coenzyme A reductase in brains of adult and 7-day-old rats, Biochem. J.. 154 (1976) 559-560. Nestel, P.J. and Havel, R.J., Lipoprotein lipase in human adipose tissue, Proc. Sot. Exptl. Biol. Med., 109 (1962) 985-987. Scow, R.O.. Blanchette-Mackie, E.J.. Mendelson, C.R., Hamosh, M. and Zinder, O., Incorporation of dietary fatty acids into milk triglyceride - Mechanism and regulation, In: Milk and Lactation (Modern Problems in Paediatry, Vol. 15). Karger. Bawl, 1975. pp. 31-45. Knox, W.E., Patterns of composition and tissue differentiation in a single set of analyses of rat tissues, In: Enzyme Patterns in Fetal, Adult and Neoplastic Rat Tissues, Karger, Basel, 1972. Chapter IX. pp. 185-221. Ganesan, G., Ganesan. D. and Bradford, R.H., Characterization of a triglyceride hydrolase secreted by canine liver maintained in vitro, Proc. Sot. Exp. Biol. Med.. 151 (1976) 390-394. Bradshaw, W.S. and Rutter. W.J.. Multiple pancreatic lipases - Tissue distribution and pattern of accumulation during embryological development, Biochemistry, 11 (1972) 1517-1528. Pictet, R.L., Clark, W.R.. Williams. R.H. and Rutter, W.J., An ultra~tructural analysis of the developing embryonic pancreas. Development. Biol.. 29 (1972) 436-467.
Atherosclerosis,
26 (1977)
0 Elsevier/North-Holland
549-561 Scientific Publishers, Ltd.
PRE- AND POST-NATAL DEVELOPMENT OF LIPOPROTEIN LIPASE AND HEPATIC TRIGLYCERIDE HYDROLASE ACTIVITY IN RAT TISSUES TOVA CHAJEK, OLGA STEIN and YECHEZKIEL
STEIN
Lipid Research Laboratory, Department of Medicine B, Hadassah University Hospital, and Department of Experimental Medicine and Cancer Research, Hebrew University-Hadassah Medical School, Jerusalem (Israel)
(By invitation, received November, 1976)
Summary The ontogenic development of lipoprotein lipase and liver triglyceride hydrolase was studied in the rat. The enzyme activity measured in extrahepatic tissues fulfilled the criteria of lipoprotein lipase from the onset of measurable activity, i.e. it was inhibited by protamine and 1 M NaCl and showed requirement for serum and heparin for optimal activity. In the liver, measurable amounts of triglyceride hydrolase, active at pH 8.6 were detected 6 days prior to birth. However, till the fourth postnatal day about 50% of this activity was inhibited by NaCl and its sensitivity towards protamine was also higher than that of the enzyme in adult liver. Three patterns of development of enzymic activity were observed in extrahepatic tissues. In the lung, the lipoprotein activity reached the adult values one day prior to birth, while in the kidney only 30% of adult activity were found at birth. A linear increase of enzyme activity was observed in the heart; only 25% of adult activity were detected at birth and 100% were reached only 20 days after birth. The increase in lipoprotein lipase activity in the heart was accompanied by morphological differentiation of cardiocytes and by a progressive development of the capillary bed, which might be related to the pattern of development of enzyme activity in this organ. Adipose tissue lipoprotein lipase activity in inguinal fat fell from values 15 times higher than adult values between the 4th and 40th postnatal days. The enzyme activity in epididymal fat increased steeply between day 10 and 40, at which time it exceeded the adult values very considerably. These findings indicate that the regulation of the development of lipoprotein lipase activity in extrahepatic tissues is governed by local factors, which can differ This paper is dedicated to Professor F.G. Schettler on the occasion of his sixtieth birthday. This investigation was supported in part by a grant from the United States--Israel Binational Foundation, and by a grant of the Israeli Ministry of Health to Y. Stein (Established Investigator of the Ministry of Health).
550
even in the same type of tissue, inguinal and epididymal fat. Key words:
Brain - Diaphragm Inguinal fat - Kidney
as exemplified
by the difference
- Electron microscopy -Liver - Lung -Muscle
-
Epididymal
fat -
between
Heart
-
Introduction Lipoprotein lipase and hepatic triglyceride hydrolase are the key enzymes which regulate the vascular component of chylomicron and very low density lipoprotein catabolism. During the past few years these two enzyme activities have been characterized and the enzymes have been isolated from different sources and purified [l--8]. The site of action of lipoprotein lipase has been localized to the endothelial cell surface [9]. The transport from the site of its synthesis to the lumenal surface of the endothelial cell proper has been shown to depend on the integrity of the microtubular system [ 10-131. Diet and hormones were shown to affect lipoprotein lipase activity differently, depending on the anatomical site, i.e., adipose tissue or muscle [ 14-201. Age related differences in lipoprotein lipase activity have been described in adipose tissue [21-231 and later studies have dealt with the relation between the cell size and enzyme activity [ 241. Presently we have undertaken a systematic study of ontogenic development of lipoprotein lipase in several tissues of the rat in order to gain more information as to other factors which might regulate the enzymic activity. Methods Animals
Male and female rats, 90-120 days old of the Hebrew University strain kept in constant temperature rooms and fed the pelleted Am-Rod 931 diet (25) were used. Mating pairs were caged separately for 12 h, from 20.00 to 8.00 and thereafter the females in which a vaginal plug was detected, were isolated and the gestation period was calculated starting from that date. The day of birth was considered as age zero, and fetal age was expressed in negative numbers from that date. Under these breeding conditions the error in the estimation of total age was less than 12 h. The newborn stayed with their mother during the first postnatal month. Thereafter they were separated according to sex and caged in groups of six; these animals were fed the pelleted Am-Rod 931 diet. Preparation hydrolase
of tissues for the assay of lipoprotein
lipase and hepatic
The activity of lipoprotein lipase was assayed in middle head of quadriceps muscle, brain, inguinal fat, trial fat, kidney and spleen. All adult tissues were guinated under light ether anesthesia. To obtain fetal
heart, lung, epididymal taken from tissues, the
triglyceride
diaphragm, fat, paramerats exsanuterus, con-
551
taining the embryos was removed from anaesthetized rats and placed on ice for 5 min, and the immobilized. fetuses were decapitated. The animals and respective organs were weighed and these data served as an additional criterion of fetal age (Table 1). Dried defatted tissue preparations were made by homogenization of lOO500 mg of fresh tissue in 50 ml of ice-cold acetone using. a Polytron (Kinematica GmbH, Luzern, Switzerland) homogenizer with pt lo-11 probe at maximum speed for .I min at 0°C. The homogenates were centrifuged at 15,000 rpm for 20 min at 4°C and the supernatant was discarded and the residue was re-extracted three times with 50 ml ice-cold acetone and twice with 50 ml of cold diethyl ether. The defatted preparations were dried at 0” under nitrogen and stored at -20°C in vacua. Under these conditions no measurable loss of lipolytic activity was detected for up to 7 days but assays were routinely performed within 24 h. This preparation was designated acetone powder. Determination of lipoprotein lipase activity For the assay of enzyme activity aliquots of the acetone powder were homogenized in ice cold 0.025 M NH,-HCl buffer, pH 8.1, to give 4-10 mg acetone powder/ml. Lipoprotein lipase activity was determined according to Schotz et al. [26], using triolein in the form of an emulsion as substrate. To prepare the emulsion the following ingredients were suspended in 15 ml of 0.2 M Tris-HCl buffer, pH 8.6, containing 0.15 M NaCl: 300 nmol of glycerol Tri[l-‘4C]oleate (spec. act. 54.9 Ci/mol, Amersham, Searly Corp., Arlington Heights, Ill.); 22.6 pmol carrier triolein (Sigma Chemical Co., St. Louis, MO.), 14.4 mg bovine serum albumin (fatty acid poor, Miles Lab., Kankakee, Ill.) and 0.36 ml of 1 : 1000 solution of Triton X-100 (B.D.H. Chemicals, Poole, U.K.). The suspension was subjected to ultmsonic irradiation in a Braun-Sonic 300 instrument (Braun, Melsungen, Germany) using a probe of 10 mm in diameter, at maximal scale for 4 min at 4°C. 4.8 ml of fasting human serum were added TABLE 1 ORGAN
AND BODY WEIGHT DURING PRE- AND POST-NATAL
DEVELOPMENT
OF THE RAT
Values of body weight are means f SE of 6-8 determinations. Organ weights are means of 6-8 tions for each time interval. For fetal tissues no distinction of the sex of the donor was made. Age (days) -6 -5 -3 -1 0 +4 +10 +20 +30
Body weight (9) 0.47 + 0.80 f 2.08 ? 4.67 ? 5.56 + 9.36 ? 13.60 r 30.30+ 56.4Oi
0.03 0.01 0.04 0.3 0.5 0.1 0.3 1.2 1.9
Liver (g)
Brain (g)
Heart (mg)
0.03 0.05 0.14 0.25 0.29 0.29 0.40 1.25 2.99
0.05 0.06 0.13 0.18 0.23 0.40 0.14 1.28 1.35
2.4 5.3 10.9 20.0 25.7 46.3 71.4 154.2 247.0
7.8 9.1
1.59 1.58
545.4 626.3
determina-
Lung (mg)
11.7 42.7 71.0 88.4 157.1 242.6 311.0 438.4
Adult
female male
185.0 237.0
+ 7.4 + 9.4
929.3 1011.0
552
to 15 ml of the substrate emulsion which was used within 30 min of preparation. The reaction was terminated by addition of 4 ml of isopropanol/l.5 M HzS04 (40 : 1, v/v) according to Schotz et al. [26] and the free fatty acids were adsorbed on Amberlite IRA 400 (B.D.H. Chemicals, Poole, U.K.) as described by Kelley [27]. After the resin was washed four times with 5 ml of hexane, 1 ml of Soluene 100 (Packard Inst. Co., Downers Grove, Ill.) and 15 ml of scintillation fluid (4 g 2,5-diphenyloxazole and 100 mg 1,4-bis-[2-(4-methyl-5phenyloxazolyl)]-benzene in 1000 ml toluene) were added and the samples were counted in a Tricarb liquid /?-scintillation spectrometer 3380 equipped with an absolute activity analyzer, model 544. The radioactivity present in the hexane phase in samples incubated without the enzyme was substracted from all experimental values. The assay system consisted of 0.2 ml of acetone powder homogenate, 0.2 ml of 0.15 M NaCl containing heparin 5 u/ml (Evans Medical Corp., Liverpool, U.K.) and 0.6 ml of the emulsified substrate. Incubations were carried out in duplicate for 90 min at 27” C in a shaking incubator. The reaction was linear up to 120 min of incubation and the activity was expressed as nmoles of free fatty acid released per mg acetone powder during 1 h of incubation. To measure the effect of protamine and NaCl on the lipolytic activity, 0.2 ml of defatted tissue homogenate were preincubated for 10 min at 27°C with 0.2 ml of 0.15 M NaCl containing 6 mg/ml of protamine sulfate (salmine, Sigma Chemical Co., St. Louis, MO.) and heparin 5 u/ml or with 0.2 ml of 2 A4 NaCl containing heparin 5 u/ml. Lipoprotein lipase assays were performed also in the absence of serum or heparin in the reaction mixture. Preparation
of tissues for microscopy
Fixation was carried out with 2% osmium tetroxide in acetate veronal buffer. Following dehydration in graded ethanols the tissues were embedded in Epon [ 281. 0.5 /J sections were prepared and stained with toluidine blue. Results Lipolytic enzyme activity was detected in most tissues about 6 days prior to birth. The activation by heparin and serum and the inhibition by protamine and 1 M NaCl of enzymic activity were determined during pre- and post-natal development on organs obtained at day -3, -1, 0, +4, +lO and +30. Since the requirement of serum and heparin for optimal activity and the degree of inhibition by 1 M NaCl were quite similar at each age group examined, the data for each organ were pooled and are presented in Table 2 under a common heading of “fetal” tissues. Adult tissues were assayed at the same time as the fetal tissues and again the values obtained for each organ in different experiments were pooled. The data summarized in Table 2 show that the enzymic activity measured during pre- and post-natal development was reduced to about 50% in the absence of heparin. Inhibition by 1 M NaCl and protamine was almost complete in all instances and did not vary from the sensitivity of the enzyme in adult tissues. The degree of serum dependence did not vary significantly from that of the corresponding adult tissue. These findings provide evidence that the enzymic activity determined throughout the pre- and post-natal period fulfilled the criteria of lipoprotein lipase.
553 TABLE 2 COMPARISON OF COFACTOR FETAL AND ADULT TISSUES
REQUIREMENT
AND INHIBITION
OF LIPOPROTEIN
LIPASE IN
The data represent percent of control activity, measured in the complete system. Values are means k SE of 3-5 experiments for fetal tissues and of 22 experiments for adult tissues. For definition of “fetal” organs see Results. Organ
Lipoprotein lipase activity Percent activity in absence of heparin
Percent inhibition by 1 M NaCl
Protamine
Lung
“Fetal” Adult
55.3 f 2.9 51.8 k 3.7
91.5 + 2.9 97.6 f 0.8
89.9 t 2.0 91.1 + 1.9
Muscle
“Fetal” Adult
53.3 f 3.4 52.1 + 4.5
97.5 * 1.0 96.7 + 1.7
89.9 * 1.9 91.6 + 3.3
Diaphragm
Adult
48.2
98.3
93.0
Heart
“Fetal” Adult
55.9 f 4.6 59.5 f 2.9
95.3 + 1.3 92.8 f. 1.3
90.0 + 2.3 91.3 * 2.1
Kidney
“Fetal” Adult
63.7 f 6.5 60.5 ? 3.7
90.3 * 2.0 90.1 + 2.8
83.0 .? 5.7 86.5 + 6.2
Brain
“Fetal” Adult
55.4 f 5.0 57.5 + 3.8
95.7 + 2.0 90.9 + 7.2
88.2 + 1.4 92.3 + 5.0
InguinaI fat
“Fetal” Adult
68.8 f 6.6 54.8 * 3.7
98.2 f 1.0 99.0 f 1.0
93.2 + 1.8 83.4 2 2.3
Epididymal fat
Adult
48.4
97.8
95.1
The change in enzyme activity as a function of age fell into distinct patterns which varied in different organs. The lipoprotein lipase activity of lung (Fig. 1, lower panel) increased between day -6 and -1 and then showed small fluctuations around the adult value. A similar behavior was seen also in the spleen; in this organ the earliest time interval measured was +4. The enzyme activity in the kidney at birth was 30% of adult value and increased gradually till +20 days. A very prominent change in lipoprotein lipase activity was observed in the heart (Fig. 1, upper panel), which showed a linear increase till day +20 at which time it reached the adult values. At birth the activity of lipoprotein lipase in the heart was about 25% of adult value, at which time it was already much higher than that of the kidney and spleen (Fig. 1). An attempt was made to correlate this steep increase in enzyme activity with some morphological changes in the myocardium. Fig. 2a-d shows representative sections of the heart taken between day -3 and +20. It can be seen that the cells are filled with myofilaments and have prominent mitochondria at all time intervals examined. There is a progressive fall in the size of the nuclei and an increase in the diameter of the myocytes. At -3 the cells are separated by intercellular spaces and between birth and +20 days there is a progressive increase in capillaries which develop in the intercellular spaces. A second pattern in the change of enzyme activity was seen in the brain and skeletal muscle, even though the absolute values in the brain were much lower
I’ .’
~--.Jyn9 “I
~...-i____‘._________..~~,” , I
630
Kidney
1
10
20 Oars
30
AOULT
Fig. 1. Lipoprotein lipase activity in fetal and post-natal period of lung, kidney and spleen (lower panel) and of heart as a function of age. Values are means f SE of 3-5 determinations in fetal tissues and 22 determinations in adult tissues.
(Fig. 3, lower panel). In both the brain and muscle the prenatal enzyme activity was higher than that in the respective adult tissues and peaked at day +4, when the activity was 4-fold that of the adult muscle. The higher absolute activity in the diaphragm than in the thigh muscle decreased as well to adult values between day +4 and day +30 (Fig. 3). The measurement of lipoprotein lipase activity in adipose tissue was confined to the post-natal period, and while the inguinal fat was well developed at day +4, the epididymal fat was not readily identified prior to day +lO. As seen in Fig. 4, a progressive decrease to adult values was seen in the inguinal fat, the peak activity being 15-fold that of the adult. No difference was observed in the inguinal fat of the two sexes. Lipoprotein lipase in the epididymal and parametrial fat showed a sharp increase with a peak at 40 days and a decline to adult values at 60 days. The enzymic activity in the epididymal fat was consistently higher than in the parametrial fat (Fig. 4). Hepatic triglyceride lipase activity was measurable at -6 days and the level of activity reached the adult levels at day -3 (Table 3). Thereafter, a slight increase was observed betweec day -1 and +4. However, unlike the extrahepatic activity, the liver enzyme examined at various fetal ages showed a changing cofactor requirement and sensitivity to 1 M NaCl and protamine
Fig. 2a-d. Sections of rat myocardium increase in cell size and a progressive spaces. X1350.
at day -3, 0.10 and 20. There is a decrease in the nuclerlr size, an increase in the number of capillaries (arrows) in the intercellular
(Table 3). Between day -6 and +4 the activity in the absence of heparin ranged between 70-80% of control value, while at later time intervals it was similar to the adult, i.e. 85% of control value. The enzymic activity did not require serum for optimal activity, and in that respect was similar to that of the adult. The most prominent difference was in the inhibition by 1 M NaCl, which was about 50% at the early pre-natal period and was still 40% at day +4. Only at
1 ii /’‘.\‘1.\
---.-._ I\.\
i
‘k
i
‘\
‘.\.,Diaphragm
I “‘\f-.- _._,_+
i
\ .A
Thigh muscle -1
I
‘f-.,
/
--I
/’
-6 -3 0
f-S
‘\
‘f---__,
Brain -_ ---.
I
I
I
I
k
10
20
30
I ADULT
Fig. 3. Lipoprotein lipase activity in fetal and post-natal period of brain (lower panel) and skeletal muscle (upper panel) as a function of age. Values are means + SE of 3-5 determinations in fetal tissues and 22 determinations in adult tissue.
!
5
2
s 120 r tr \
g 1f
It
poraovarian\ fat
‘L_.p
30 0t
0
10
20
30
LO
50
60
AOULT
Days Fig. 4. Post-natal lipoprotein lipase activity of rat adipose tissue as a function of age. Values ? SE of 3-5 determinations in fetal tissues and 15-24 determinations in adult tissue.
are means
TABLE
3
COMPARISON
OF
HYDROLASE Control
Age
activity
(days)
COFACTOR
REQUIREMENT
IN DEVELOPING was determined
Exp.
No.
Fatty
AND
INHIBITION
OF
HEPATIC
TRIGLYCERIDE
LIVER in the complete acid
nmoles/mg powder/h)
released
system
described
Enzyme
activity
in Methods. as percent
Values
are means
+ SE.
of control
acetone In absence
of
In presence
Heparin
Serum
1 M N&l
of Protamine
-6
3
12.0
f 1.6
70.3
103.1
? 5
43.3
? 4.8
-
-5
4
10.3
i- 1.3
79.2
104.6
+
57.6
+ 5.0
43.3
-3
4
15.1
+ 1.4
78.0
+ 2.7
52.2
+ 3.0
48.5
f 2.6
-1
5
23.0
+ 1.2
80.1
+ 4.4
97.7
f 6.0
64.5
* 3.8
54.0
f 5.9
0
f 2.6
3
22.2
f 3.0
79.3
+ 1.1
114.0
+ 1.7
59.3
+ 4.7
54.8
f 5.4
+4
3
18.9
+ 0.7
78.4
f 4.2
108.3
+ 8.3
63.3
+ 5.3
60.5
+ 6.1
+10
4
16.5
+ 1.6
88.7
f 5.0
119.3
+ 5.7
97.3
f 1.5
60.1
f 5.1
+20
5
16.6
+ 1.4
84.3
i
5.9
112.8
f 6.9
104.4
f 9.4
71.1
* 4.9
4
13.5
+ 1.7
85.3
+ 6.0
113.3
f 8.6
119.3
?- 6.3
70.8
t 7.6
22
15.4
f 0.7
85.1
+ 2.3
112.2
i- 2.7
108.0
f 4.8
67.8
+ 3.5
+30 Adult
day +20 was there also the slight enhancement of enzyme activity in the presence of 1 M NaCl encountered in the adult. Likewise a somewhat greater inhibition of enzyme activity by protamine occurred between day -5 and +4. Discussion Lipoprotein lipase, the key enzyme in the degradation of very low density lipoprotein and chylomicron triglyceride on the endothelial cell surface responds differently to the same stimulus in adipose tissue and in muscle. Thus fasting results in a fall in lipoprotein lipase in adipose tissue and an increase in the enzymic activity of heart and skeletal muscle [29]. Moreover, while enzyme activity in adipose tissue was shown to have a positive correlation with plasma insulin concentration [l&20,30], a negative correlation was found for lipoprotein lipase activity in the heart and skeletal muscle [13,20]. Yet, another difference in the behavior of this enzyme in the two types of tissues, was shown presently in the pattern of pre- and post-natal development. The lipoprotein lipase activity of the heart at birth attained a level of only 25% of the adult and the activity expressed per mg of acetone powder was similar to that of skeletal muscle and lung. The further post-natal increase in enzyme activity, which was measured in the fed state, is most probably related to the post-natal development of the heart, as the final differentiation of this organ in the rat occurs only after birth [31,32]. The main changes during development of the heart consist in the alignment of cells, differentiation and orientation of myofibrils, maturation and redistribution of mitochondria, and these are completed at the age of about 24 days [31]. The development of the transverse system, which is concomitant with an increase in the sarcolemmal cell surfaces occurs between 14 and 31 days of age [ 311. In contradistinction to skeletal muscle, cell division and differentiation are concurrent in cardiac muscle, as synthesis of DNA, of myofibrils and of connective tissue ground
substance occurs at the same time [33]. Thus it seems that the pattern of development of lipoprotein lipase is not related to an inability of the younger cardiocytes to synthesize the enzyme protein, as was suggested by findings in cell culture [34]. Another aspect of the structural development of the heart is the formation of a well-developed capillary bed, which progresses between birth and 3 weeks of age s[321. In view of the fact that lipoprotein lipase acts at the capillary cell surface, to which it has to be transported, one might envisage the possibility that the differentiation of the sarcolemmal membrane and the increase in the number of capillaries play a key role in the post-natal increase of cardiac lipoprotein lipase. The relatively high dietary intake of lipids during the suckling period could also contribute to this increase. In the adult rat, fed for 3 days a 50 Cal% fat diet there was a 3-fold increase in the lipoprotein lipase of the heart [35]. It seems of interest to point out that enzymes active in the oxidation of fatty acids, such as palmitoylcarnitine transferase show an analogous pattern of post-natal development in the heart [ 361. No explanation can be suggested so far for the diagonally opposite response of lipoprotein lipase in skeletal muscle (presumably red) and diaphragm in which a decrease to adult values was observed during the post-natal period between 4 and 30 days. The relatively high activity of lipoprotein lipase in the brain between birth and 10 days of age occurred at a time during which there is a striking increase in cholesterol content as well as a high rate of weight gain [ 371. Since at the same time 3-hydroxy-3-methylglutaryl-CoA reductase activity was found to increase &fold [38] over adult values, it seems that local cholesterol synthesis was responsible for this increase. As free cholesterol is an integral part of cellular membranes it seems plausible that the high lipoprotein lipase activity might contribute towards the supply of fatty acids needed for increased membrane phospholipid synthesis, during the perinatal period. Lipoprotein lipase activity in adipose tissue differed markedly according to the location, and so did the developmental pattern. Higher activity of lipoprotein lipase in omental than in subcutaneous tissue was described in the human [ 391. Lipoprotein lipase activity of peritoneal and inguinal adipose tissue was compared in the duck, and no difference was seen in tissues from adult birds [22]. In that study a fall in enzyme activity was reported between 2 and 8 weeks of age and throughout that period the activity in the peritoneal fat was c.onsistently higher than in the inguinal fat. The gradual decline in enzyme activity was considered to be related to the specific growth rates of fat depots in different regions, the inguinal fat being the earliest and the peritoneal the latest in development [22]. In the rat lipoprotein lipase activity in epididymal fat of 4-5-week-old rats was found to be 8 times higher than that of adult rats [21], while in another study the highest enzymic activity was found on day 2 and fell gradually till day 19 [23]. In the female rat a fall in adipose tissue lipoprotein lipase was encountered during lactation and was related to prolactin secretion [40]. In the present study it became evident that regulation of enzyme activity in adipose tissue is dependent on local factors as well. The earlier development of the inguinal fat and the later appearance of epididymal tissue could be related to the onset of lipoprotein lipase activity. If the adipocyte should be considered as the source of the enzyme, one could
559
envisage the possibility that the onset of enzyme synthesis precedes the transition between pre-adipocytes and adipocytes. In contradistinction to the other organs, the levels of hepatic triglyceride hydrolase activity were similar to those in the adult, with a transient increase during the immediate perinatal period. The pronounced sensitivity to 1 M NaCl, which was prominent during the early development might have suggested the presence of two enzyme species, differing in their response to NaCl. Since in the fetal liver hematopoietic cells comprise about 50% of the organ’s volume [ 411, the possibility could be considered that the NaCl inhibited lipase originates in these cells. However, while the fractional volume contributed by the hematopoietic cells drops sharply between day -3 and 0 [41], during that time there is no change in the lability of the enzyme towards NaCl (Table 3). More recently it has been shown that the canine liver secretes a triglyceride hydrolase which is inhibited by 1 M NaCl and by protamine and requires apolipoprotein C-I for optimal activity [ 421. The presently described enzyme activity in fetal liver was inhibited by 1 M NaCl to a greater extent than by protamine but was not activated by the addition of serum, however, activation by C-I was not determined. The ontogeny of peripheral lipoprotein lipase and hepatic triglyceride hydrolase differs completely from that of pancreatic lipase [43]. In the rat pancreas the activity of lipase A followed a sigmoidal curve with relatively low activity between day -10 and -6 and a very steep rise between day -6 and -2, during which time the enzyme activity increased by 3 orders of magnitude, to almost adult values [43]. The rapid increase in pancreatic lipase (as well as other pancreatic enzymes) occurs during the period of cytodifferentiation in which the acinar cells become filled with rough endoplasmic reticulum and zymogen granules [44]. An attempt was made during this study to seek ultrastructural changes which could correspond to the development of lipoprotein lipase. Lipoprotein lipase is transported from the site of its synthesis to the site of its action on the endothelial cell plasma membrane and this process is inhibited by colchicine [ 10-121. However the ultrastructural pathway of the transport has not been defined so far, and no analogs of zymogenic granules have been shown to contain the enzyme activity. It was only in the heart that the increase of lipoprotein lipase coincided with a structural change in the form of extensive capillarization, which might have been instrumental in the regulation of enzymic activity. Acknowledgement The excellent edged.
technical
help of Miss R. *Ben-Moshe is gratefully
acknowl-
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