97

Biochimica

et Biophysics Acta, 380 (1975) 97-105 @ Elsevier Scientific Publishing Company, Amsterdam

- Printed

in The Netherlands

BBA 56528

RELEASE OF LIPOPROTEIN LIPASE FROM FAT CELLS IN VITRO

DAVID

M. KORNHAUSER

and MARTHA

Laboratory of Cellular Metabolism, National Health, Bethesda, Md. 20014 (U.S.A.)

(Received

August

VAUGHAN Heart and Lung

Institute,

National

Institutes

of

8th, 1974)

Summary Release of lipoprotein lipase from rat fat cells incubated at 20°C in medium with albumin, but without glucose proceeded at a constant rate for 30 min. The initial rate of release was increased when serum was present in the medium. Maximal stimulation (loo-300%) was produced with 3.8% serum. The maximal increment in release caused by serum was always greater than that produced by heparin and when both were added release was greater than it was with either one alone. The active component(s) of serum, nondialyzable and stable for 30 min at 56”C, was present in sera from humans and rats in the fed or fasted state. Glucose plus insulin (but neither alone) enhanced the rate of lipase release in the presence of serum but not in its absence. The half-life of the lipase in basal medium at 20°C was 90 min. Heparin decreased this to about 50 min and serum markedly prolonged it whether or not heparin was present. Lipoprotein lipase activity in cells and fractions thereof was assayed in extracts of acetone powders. After centrifugation of fat cell homogenates at 600 X g for 15 min, only 50-60% of the activity was recovered in the supernatant. After centrifugation at 100 000 X g for 60 min, the supernatant contained about 10% of the total activity and the sediment 40%. In some experiments, most of the rest was recovered in the floating fat fraction. Total lipoprotein lipase activity of cells plus medium increased steadily during incubation of fat cells for 1 h at 30°C. The major increment occurred in the cells and activity in the medium was always less than 15% of the total. Our observations are consistent with the view that activation may be an important determinant of fat cell lipoprotein lipase activity as well as an integral part of the release process. Introduction [l-9]

Lipoprotein lipase is released or secreted from fat cells incubated in vitro and the rate of this process is enhanced when heparin is present in the

98

incubation medium [1,4,9]. Relatively little is kno,wn, however, about the mechanisms underlying control of intracellular lipoprotein lipase activity and release of the enzyme from fat cells. It has been suggested that activation of lipoprotein lipase occurs as a part of the release process [7,9] . We report below observations that are consistent with this view. In addition, data concerning the subcellular distribution of lipoprotein lipase are presented and some characteristics of the recently reported [8] effect of serum on lipoprotein lipase release are described. Although there are similarities between our findings and those of other workers [l-9] there are also certain differences, the latter related perhaps in large part to numerous differences in experimental conditions, e.g. nutritional state of animals from which fat cells were obtained, incubation medium and temperature, methods for assay of lipoprotein lipase activity. Methods Epididymal fat pads from Sprague-Dawley rats (150-200 g) which had been fasted for 48 h and then permitted access to food overnight were used for preparation of fat cells [lo] . The minced tissue was incubated at 37 “C for 60 min with collagenase (Worthington Biochemical Corp.), 1.6 mg/ml. For preparation, washing and incubation of fat cells Krebs-Ringer medium without phosphate, with 15 mM Tris buffer, and serum albumin(Fraction V from bovine serum, Armour Pharmaceutical Co.), 20 mg/ml, (final pH 7.4) was used. Samples of the washed cell suspension (2.5 ml containing 125-325 mg of cells) were transferred to polyethylene vials and incubated at 30°C with additions as indicated. For studies of medium lipase activity, l-ml samples of cells plus medium were removed from incubation vials and centrifuged for 1 min in a Beckman 152 Microfuge. The infranatant medium was collected and stored (400

No additions Heparin, 10 pg/ml Serum, 3.8% Heparin plus serum

varied from about 100 to 300% of the basal rate with different preparation of fat cells. Sera obtained from one person on different days and tested on the same preparation of fat cells, however, produced similar increments in lipase release (Table II). While these experiments were in progress Stewart and Schotz [8] reported that serum from fed rats increased lipase release from fat cells. As shown in Table II, rat serum and human serum were equally effective and sera from fed and fasted donors did not differ significantly in activity. Serum heated at 56°C for 30 min or dialyzed against normal saline was as active as it was before such treatment. Serum from which lipids had been extracted [16] was also effective. Stewart and Schotz [8] found that the active component(s) of rat serum was destroyed by incubation with papain or trypsin and was associated with material of molecular weight > 100 000. The serum factor(s) is evidently not included in Cohn Fraction V from bovine serum, present at a concentration of 20 mg/ml in all media in our experiments. In exploratory studies Fractions II and.111 were without effect but Fraction IV, 40-400 I.cg/ml, enhanced release in a concentration-dependent fashion. It has not been established, however, that the activity of Fraction IV is qualitatively like that of whole serum in all respects. Whether one of the apolipoproteins is responsible for the serum effect remains to be determined. Sera from patients TABLE EFFECT

II OF SERUM

ON RELEASE

OF LIPOPROTEIN

LIPASE

FROM

FAT CELLS

Cells were incubated at 30°C with or without serum (2%) as indicated and samples of medium were obtained from each vial after 0 and 30 min for assay of lipoprotein lipase activity and calculation of the amount released in 30 min. AU samples of human serum were obtained from the same donor, on dates as indicated. Lipase released is expressed relative to that of cells incubated in medium alone = 100. Expt No.

Serum source

Lipase released (% of control)

1.

Rat. fasted Rat, fed Human, fasted, 9125 Human, fasted, 10124 Human, fasted, 10127 Human, fed, 10127

249 205 222 186 196 207

102 TABLE III EFFECT OF GLUCOSE,

INSULIN AND SERUM ON RELEASE

OF LIPOPROTEIN

LIPASE

Experiments were carried out as described in Table II. Lipase activity released is expressed relative to that released by cells incubated for 30 min in basal medium = 100. Additions

Serum 3.8%

Lipase released (Relative amount)

0 +

100 233 91 262 81 221 107 322

NOlIt?

Glucose, 1.5 mg/ml Insulin, 1 munitlml Glucose plus insulin -

with a variety of diagnoses and plasma from patients with Types I-V hyperlipoproteinemia (courtesy of Dr Robert I. Levy) all enhanced release of lipoprotein lipase. In three experiments like that shown in Table III, lipase release in the presence of serum was significantly (p < 0.025) augmented by the addition of glucose plus insulin. Unlike earlier workers (71, however, we observed no consistent effects of glucose or insulin alone with or without serum in the medium. The effect of heparin, 10 (ug/ml, on release was always less than that of 3.8% serum and was more variable. In 14 experiments, the amount of lipase released in the presence of heparin, 10 pg/ml, ranged from 95 to 200% of that released from the same preparation of cells in basal medium; the mean increment (? S.E.) produced by heparin was 37 f 7.3%. A maximal effect of heparin was observed with concentrations of 4-10 pg/ml*. These observations are similar to those reported by others [ 1,4,9]. In medium containing a maximally effective concentration of serum the addition of heparin produced a further increment in lipoprotein lipase release (Table IV) from which we infer that these agents may act in different ways to enhance lipase release.

Distribution

of lipoprotein

lipase in homogenates

of fat cells

After centrifugation of fat cell homogenates at 100 000 X g for 60 min, about 40% of the lipoprotein lipase activity was recovered in the sediment fraction and approx. 10% in the supernatant. In two of the four experiments reported in Table V, much of the remainder of the activity was recovered in the fat fraction. Lipoprotein lipase activity in the whole homogenate was stable for 60 min at 4°C and when acetone powders were prepared from the contents of tubes remixed after centrifugation the activity recovered was essentially equal to that of the original whole homogenate (Table V). For reasons that are not clear, the

* Equivalent amounts of heparin added to the assay system had no effect on the activity of the enzyme released into medium without heparin (with or without serum).

103 TABLE

IV

EFFECT

OF HEPARIN

AND SERUM

ON RELEASE

OF LIPOPROTEIN

Experiments were carried out and data are reported as described relative to that of cells incubated in medium alone = 100. Additions

Lipase released (% of control)

Serum, 3.8% Serum, 7.4% Heparin. 0.4 pg/ml Heparin. 1 pg/ml Heparin. 4 Wml Heparin. 10 pg/ml Heparin, 20 fig/ml Heparin, 10 &&/ml plus serum. 3.8%

226 230 132 142 145 176 169 308

LIPASE

in Table II. Lipase released is expressed

activity of cells plus medium added to acetone without homogenization was consistently slightly lower than that of the paired homogenate. When homogenates were centrifuged at 600 X g for 15 min, only 50-60% of the total activity was recovered in the supernatant fraction (Table V). The observed distribution of lipoprotein lipase activity after centrifugal fractionation of fat cell homogenates could reflect subcellular localization of the enzyme or may be simply an artifact resulting from the methodology. In any case, it is apparent that unless total fat cell lipase activity is measured significant changes could be overlooked. It should be noted that these studies were carried out with homogenates prepared from cells immediately after removal of collagenase. The distribution may be different in cells in which lipoprotein lipase activity has increased after incubation (see below).

TABLE

V

DISTRIBUTION

OF LIPOPROTEIN

LIPASE

ACTIVITY

IN HOMOGENATES

OF FAT CELLS

After collagenase treatment and washing, cells were homogenized in a glass tissue grinder. One portion of the homogenate was added to acetone immediately. Others were centrifuged as indicated and acetone powders prepared from the fractions or from the remixed contents of the centrifuge tube. Recovery of activity is expressed as a percentage of that in the acetone powder of the whole homogenate. Acetone powder of homogenate fraction

100000 X g X 60 min Sediment Supematant Fat Total remixed 6OOXgX 15min Supematant Cells plus medium*

Percentage

of activity

of whole homogenate

Expt 1

Expt 2

Expt 3

Expt 4

Expt 5

Mean f S.E.

44 5

34 10 5

38 7 35 100

34 7 3 79

41 19 23

38 f 2.0 10 + 2.5

92

95

88

52 91

* Samples of cells plus medium were removed just before homogenization of acetone powders as was done in the experiments in Table VI.

61 87

91 + 1.4

and used for preparation

104 TABLE

VI

LIPOPROTEIN

LIPASE

ACTIVITY

IN CELLS

AND MEDIUM

Cells were incubated in basal medium without additions and at the indicated times acetone powders were prepared from samples of cells plus medium and from separated medium. Cell activity equals the activity of cells plus medium minus the activity of the paired medium sample. For each experiment lipase activities were then expressed relative to the activity of the cells at zero time. Data are presented as the mean k S.E. of values from five experiments. Incubation time (min)

Cells

Medium

0 30 60

100 140 + 4 17025

7il 10 z!z1 23 t 5

Total lipoprotein -__-

Changes in fat cell lipoprotein

lipase

lipase during incubation

Total lipoprotein lipase activity (cells plus medium) increased steadily during incubation of fat cells at 30°C almost doubling in 60 min; it was essentially constant for this period at 4°C. As shown in Table VI, the major increment in lipoprotein lipase activity during incubation at 30°C occurred in the cells. The rate of accumulation of lipase activity in the medium was greater during the second 30 min than it was during the first. Even after 60 min, however, activity in the medium represented less than 15% of the total in cells plus medium. Cunningham and Robinson [2] reported that after incubation of fat cells from starved rats in suitable medium for 3.5 h total lipoprotein lipase activity in the system was increased and most of the increase was accounted for by enzyme in the medium. Stewart and Schotz [7] observed that lipase activity in cells from rats fed ad libitum and incubated at 23°C with glucose was essentially unchanged for 45 min while lipase accumulated in the medium and the total activity in the system increased almost 3-fold. Thus, after 45 min the medium contained more than twice as much activity as the cells. Their findings were similar when protein synthesis was inhibited with cycloheximide and they concluded that release of lipoprotein lipase was associated with activation of the enzyme. In addition, they have recently reported that heparin augments lipase activation as well as release [9]. In preliminary experiments, we have found that when lipase accum~atjon in the medium is enhanced by the addition of serum the cell activity is unchanged. Thus it seems that the effect of serum on rele;mee is also accompanied by increased activation of lipoprotein lipase. Probably because of the specific experimental conditions chosen which differ in many ways from those used by others we found accumulation of apparently newly activated lipase in the ceils rather than only in the medium as earlier reported [ 2,7,9 ] . These observations are clearly consistent with the view that activation of ceil associated lipase precedes release and may facilitate efforts to define and study these two processes.

105

References 1 Pokrajac, N., Lossow, W.J. and Chaikoff, I.L. (196’7) Biochim. Biophys. Acta 139.123-132 2 CunnIngham. V.J. and Robinson, D.S. (1969) Biochem. J. 112, 203-209 3 4 5 6 7 8 9 10 11 12 13 14 15 16

Stewart, J.E., Whelan, C.F. and Schotz, M.C. (1969) Biochem. Biophys. Res. Commun. 34, 376-381 Patten, R.L. and HoIIenberg. C.H. (1969) J. Lipid Res. 10, 374-382 Patten, R.L. (1970) J. Biol. Chem. 245. 5577-5584 Robinson, D.S. and Wing, D.R. (1970) Harm. Metab. Res. 2, Suppi. 2, 4146 Stewart, J.E. and Schotz, M.C. (1971) J. Biol. Chem. 246, 5749-5753 Stewart, J.E. and Schotz, M.C. (1973) Nat. New Biol. 244, 250-251 Stewart, J.E. and Schotz. M.C. (1974) J. Biol. Chem. 249,904-907 Rodbell, M. (1964) J. Biol. Chem. 239, 375-380 B&rage, P. and Vaughan, M. (1969) J. Lipid Res. 10.341-344 Carroll, K.K. (1961) J. Lipid Res. 2.135-141 Chen, R.F. (1967) J. Biol. Chem. 242,173-181 RodbeII, M. (1966) J. Biol. Chem. 241.3909-3917 Wing, D.R., Salaman, M.R. and Robinson, D.S. (1966) Biochem. J. 99. 648-655 WiIIiams. C.D. and Avigan, J. (1972) Biocbim. Biophys. Acta 260, 413-423