Effects of adrenaline on the turnover of lipoprotein lipase in rat adipose tissue

Biochimica Elsevier

et Biophysics

399

Acta 877 (1986) 399-405

BBA 52135

Effects of adrenaline on the turnover of lipoprotein lipase in rat adipose tissue Kathryn

L. Ball, Brian K. Speake and Donald

Department

of Biochemistry,

University of Leeds. Leeds LS2 9JT (U. K.)

(Received

Key words:

Lipoprotein

S. Robinson

December

lipase; Adrenaline;

16th, 1985)

Enzyme turnover;

(Rat adipose)

The mechanisms by which adrenaline brings about a reduction in the lipoprotein lipase activity of adipose tissue in vitro were investigated. The incorporation of [3H]leucine into lipoprotein lipase was measured during l-h pulse incubations of rat epididymal fat bodies that had been preincubated for 4 h in the presence of glucose, insulin and dexamethasone. When adrenaline was added to the incubation medium at the start of the pulse, the incorporation of 13H]leucine was markedly reduced, suggesting that the rate of the enzyme’s synthesis had decreased. On the other hand, the degradation of lipoprotein lipase, as measured by the loss of 3H-labelled enzyme protein during pulse-chase incubations of the epididymal fat bodies, was found to be significantly increased by the addition of adrenaline to the incubation medium at the start of the chase period. It is concluded that adrenaline is able both to inhibit the synthesis of lipoprotein lipase and to stimulate its degradation. Introduction The uptake of triacylglycerol fatty acids from the circulation by adipose tissue is dependent on the activity of lipoprotein lipase (EC 3.1.1.34) in the tissue. This enzyme is synthesized in the adipocytes and is then secreted to be eventually bound at the luminal surfaces of the capillary endothelial cells, where it catalyses the hydrolysis of triacylglycerols present in the plasma chylomicrons and very-low-density lipoproteins [l]. In the rat, the activity of lipoprotein lipase in adipose tissue varies markedly with changes in the physiological state of the animal and, on present evidence, regulation of its activity appears likely to be a complex matter involving the concerted action of several hormones [l-3]. Thus, it has been shown that insulin and glucocorticoids increase the activity of the enzyme, both in intact animals and during tissue incubations in vitro, whilst adrenaline antagonizes these effects. Recent studies have indicated that the effects of insulin and of glucocorticoids on the activity of 0005-2760/86/$03.50

0 1986 Elsevier Science Publishers

adipose tissue lipoprotein lipase are achieved through a stimulation of its synthesis [4-61. The present work was carried out specifically to investigate the mechanism of adrenaline’s action. The results suggest that it brings about both a decrease in the rate of synthesis of the enzyme and a concomitant increase in its rate of degradation. Materials and Methods Materials. r_-[4,5-H]Leucine (specific radioactivity 130-190 Ci/ mmol) was obtained from Amersham International, Amersham, Bucks., U.K. Bovine serum albumin (fraction V), adrenaline bitartrate and dexamethasone were obtained from Sigma Chemical Co., Kingston-upon-Thames, Surrey, U.K. Other materials were as previously specified [5] or were supplied by BDH Chemicals, Poole, Dorset, U.K. Animals. Specific pathogen-free male rats of the Wistar strain and with body weights ranging from 170 to 190 g were used (A. Tuck and Son, Rayleigh, Essex, U.K.). They were maintained on Oxoid

B.V. (Biomedical

Division)

400

pasteurized diet 41B (Herbert Styles, Bewdley, Worcs., U.K.) and were starved for 24 h before the experiments were begun. Incubation of epididymal fat-bodies. In each experiment the required numbers of epididymal fatbodies were collected as described by Ashby et al. [7]. Groups of four fat-bodies were then incubated in flasks containing 10 ml of medium for varying periods of time at 37°C. The medium was KrebsHenseleit bicarbonate buffer (pH 7.3-7.5) [8] gassed continuously with O,/CO, (19: 1) and supplemented with amino acids [5], glucose (10 mM), insulin (0.002 I.U./ml), dexamethasone (400 nM) and bovine serum albumin (2%, w/v) that had been previously dialysed against water for 24 h. Adrenaline (10 PM) and [3H]leucine (10 pCi/ ml) were present where indicated in the text. At the end of each incubation, each group of fat-bodies was removed from its incubation medium, washed for 15 s in water at room temperature and homogenised in 10 ml of casein solution (2%, w/v) that had been adjusted to pH 7.2. The homogenates were delipidated by extraction with acetone and diethyl ether [5], and the delipidated tissue residues were dried under vacuum and stored at - 20°C for up to 48 h. The incubation media were stored under the same conditions. Because an increase in the activity of the tissue mobilizing lipase was anticipated during the incubations in the presence of adrenaline, albumin was included in all the incubation media to act as an acceptor of the released free fatty acids. Concentrations of free fatty acid in the tissue and in the medium were measured in four preliminary experiments as previously described [14], groups of four fat-bodies being used for each determination. The mean (AS.D.) initial tissue free fatty acid concentration was 2.82 + 0.32 pmol/fat-body and, as expected, this fell to 2.05 -t 0.05 pmol/ fat-body after incubation in the presence of glucose, insulin and dexamethasone for 5 h. When the fat-bodies were then incubated in fresh medium in the absence or presence of adrenaline (i.e. under the conditions of the experiment described in Table II), the free fatty acid concentrations at the end of a further 2 h were respectively 1.55 + 0.20 and 2.35 f 0.45 pmol/ fat-body. Media concentrations at the end of the 2-h incubations were 1.85 + 0.53 and 3.00 k 0.45 pmol/fat-body equivalent

in the absence and presence of adrenaline, respectively. Thus, despite increased activity of the mobilizing lipase in the presence of adrenaline, the tissue free fatty acid concentration remains throughout below that in the fat-bodies immediately after their removal from the rats. Incorporation of [3H]Ieucine into lipoprotein lipase. We have previously described a method for measuring the incorporation of radioactivity into lipoprotein lipase during incubations of rat epididymal fat-bodies in the presence of [3H]leucine in vitro [5]. The procedure involves the quantitative isolation of the enzyme from extracts of the delipidated tissue by affinity chromatography on heparin-Sepharose, followed by SDS-polyacrylamide gel electrophoresis. A single band of 3Hlabelled protein corresponding to lipoprotein lipase is obtained and the incorporation of [3H]leucine into the enzyme is calculated from the radioactivity (expressed as cpm/fat-body) detected in the relevant gel slices. The foregoing procedure was employed in the present study to measure the extent of [3H]leucine incorporation into lipoprotein lipase during l-h pulse incubations of epididymal fat-bodies and also during pulse-chase incubations in which the chase-period was up to 2 h. Some of the labelled lipoprotein lipase that is synthesized during such incubations is released into the medium and, as a proportion of the total, the amount can become significant after 2-3 h [9]. For this reason, in all such experiments the extracts of the delipidated tissue were combined with the corresponding incubation media immediately prior to the chromatography on heparin-Sepharose. Preliminary experiments showed that the incorporation of [3H]leucine measured in this way was the same as the sum of the incorporations into the tissue and medium enzyme when these were determined separately. Whereas duplicate determinations of [ 3H]leutine incorporation into lipoprotein lipase within an experiment were always in good agreement with each other, incorporations measured under equivalent incubation conditions but at different times sometimes showed considerable differences (see Table II). These last could well be related to the substantial variations in fat-body lipoprotein lipase activity from one consignment of animals to

401

vents the rise in lipoprotein lipase activity that occurs when epididymal fat-bodies from starved rats are incubated in the presence of glucose, insulin and dexamethasone at 37°C [12]. The results in Fig. 1 confirm this finding and show further that, when adrenaline is added 5 h after the start of such incubations, the elevated enzyme activity that has been achieved by this time falls rapidly towards the starting value.

another that have been noted previously [7] and that may perhaps be accounted for by seasonal factors. Such factors, as well as the presence of albumin in the incubation media, may contribute to the somewhat lower incorporations in the present study than we have previously reported for equivalent incubations in the absence of adrenaline [5,9]. Incorporation of [-‘H]leucine into total adipose tissue protein. The incorporation of [ 3H]leucine into total fat-body protein was determined during the pulse and the pulse-chase incubations of the epididymal fat-bodies by the method of Mans and Novelli [lo]. The values in Tables I and II include the radioactivity due to 3H-labelled protein that appeared in the incubation medium, which was always measured separately from that in the delipidated tissue extracts. The amounts of such labelled medium protein were never greater than 10% of those found in the tissue. Measurement of tissue lipoprotein lipase activity. This was determined in extracts of the delipidated fat-bodies as previously described [ll]. No significant enzyme activity could be detected in the media under any of the incubation conditions. One unit of activity is defined as the release of 1 pmol of free fatty acid/h.

Effect of adrenaline on the incorporation of [3H]leucine into lipoprotein lipase We have recently reported that the rate of [ 3H]leucine incorporation into lipoprotein lipase is markedly enhanced during incubations of fat-bodies from starved rats at 37°C in the presence of glucose, insulin and dexamethasone and we have interpreted this as indicative of an increased rate of synthesis of the enzyme under such conditions [6]. In order to determine the effect of adrenaline on this enhanced rate, we have carried out experiments in which fat-bodies have been first incubated for 4 h at 37OC in the presence of glucose, insulin and dexamethasone. [ 3H]leucine has then been added to the incubation medium and its incorporation into the lipoprotein lipase of the fat-bodies has been determined during a further hour’s incubation, either in the presence or in the absence of adrenaline. The results in Table I show that incorporation of [3H]leucine into the enzyme is markedly decreased during the incubation in the presence of adrenaline, whereas incorporation into the total

Results Effect of adrenaline on lipoprotein lipase activity during incubations of epididymal fat-bodies We have shown previously that adrenaline preTABLE

I

EFFECT

OF ADRENALINE

ON THE INCORPORATION

OF [3H]LEUCINE

INTO

LIPOPROTEIN

LIPASE

Epididymal fat-bodies were incubated at 37“C for 5 h in the presence of glucose (10 mM), insulin (0.002 I.U./ml) and dexamethasone (400 nM). [3H]Leucine (10 pCi/ml) was present during the final 1 h of the incubation. Adrenaline (10 PM) was added to some of the incubation media at the same time as the [‘Hlleucine. At the end of each incubation, the incorporation of [ 3Hlleucine into the total lipoprotein lipase and the total protein of the incubation system was measured. The data from two separate experiments are shown. In each experiment, the determinations were in duplicate and the results are expressed as the mean of these f half the range. Experiment

Incubation

conditions

Incorporation of sH into lipoprotein lipase (cpm/fat-body)

1

adrenaline adrenaline

absent present

95*7 25f2

2.41 * 0.18 1.s5*0.03

2

adrenaline adrenaline

absent present

107+2 29*4

2.82 + 0.10 2.53 kO.44

Incorporation of 3H into total fat-body protein (10s cpm/fat-body)

TABLE

II

EFFECT OF ADRENALINE OF LIPOPROTEIN LIPASE

j

o1

2

4

6 Incubation

8 time

IO

I2

Ih)

Fig. 1. Effect of adrenaline on lipoprotein lipase activity. Groups of epididymal fat-bodies were incubated at 37°C in media containing glucose (10 mM) insulin (0.002 I.U./ml) and dexamethasone (400 nM). Adrenaline (10 PM) was absent throughout (0) or present from the beginning of the incubation (A), or added after 5 h (0). At the times shown, the fat-bodies were removed from the incubation media and delipidated. Lipoprotein lipase was then measured in extracts of the delipidated preparations. The results are typical of three experiments.

fat-body protein is only modestly affected. It appears, therefore, that the decrease in the activity of lipoprotein lipase brought about by adrenaline in the experiment of Fig. 1 may be due, at least in part, to a rapid and specific inhibition of lipoprotein lipase synthesis in the tissue. The possibility that the findings could be explained by a change in the pool size of the labelled leucine available for protein synthesis would appear to be ruled out by the relatively small effect of the hormone on the incorporation into total fat-body protein. Effect of adrenaline on the degradation of lipoprotein lipase Previous work has shown that lipoprotein lipase that is newly synthesized during the incubation of epididymal fat-bodies from starved rats in the presence of glucose and insulin is rapidly degraded again [9]. The results of pulse-chase experiments carried out during the present investigation show that there is a similar rapid rate of degradation when the rate of synthesis of the enzyme is

ON THE

DEGRADATION

Epididymal fat-bodies were incubated at 37°C as described in the legend to Table I. 13H]Leucine (lOrCi/ml) was added after 4 h and the incubation was continued for a further 1 h. At the end of this period, the fat-bodies were removed and washed in an excess (20 ml per fat-body) of non-radioactive medium for 6 min at 37°C. They were then incubated for a further 2 h in fresh non-radioactive medium, with or without adrenaline (10 PM). Glucose (10 mM), insulin (0.002 I.U./ml) and dexamethasone (400 nM) were present in all the media. The incorporation of [3H]leucine into the total lipoprotein lipase and into the total protein of the incubation system was measured. The mean vlues (+S.D.) of the results from six separate experiments are shown. Incubation

details

Incorporation during l-h pulse Label remaining after 2-h chase (adrenaline absent) Label remaining after 2-h chase (adrenaline present) S degradation (adrenaline % degradation (adrenaline

‘H-labelled lipoprotein lipase (cpm/fat-body)

‘H-labelled total body-fat protein (lo5 cpm/ fat-body)

186k55

2.83*

0.61

91*35

2.46k

0.42

33510

2.56*

0.57

absent)

51+

5

12

*9

present)

82+

4

9

*13

enhanced by the additional presence of dexamethasone in the incubation medium (Table II). Table II also shows, however, that, when adrenaline is added at the beginning of the chase period, there is a substantial further increase in the rate of degradation above that observed in adrenaline’s absence. When analysed by the paired sample t-test this increase is highly significant (P < 0.001). The loss of label from the total fat-body protein during the chase period, though somewhat variable, is always comparatively small in the absence of adrenaline and is not increased in its presence. The time-course of lipoprotein lipase degradation in the presence and absence of adrenaline is shown in Fig. 2. The enhancement of the degradation in adrenaline’s presence occurs mainly within the first 30 min after the addition of hormone. Thereafter, degradation continues at a rate similar

403

I

!

0.5

I.0 Time

I h)

after

I

I

I.5

2.0

pulse

Fig. 2. Effect of adrenaline on the degradation of lipoprotein lipase, as measured during incubations of epididymal fat-bodies under pulse-chase conditions. The experiment was carried out as described in the legend to Table II, except that the chase incubations were for the times shown, with adrenaline absent (0). or present (0). The determinations at each time point were carried out in duplicate and the results are expressed as the mean of these f half the range. Similar results have been obtained in a second separate experiment.

to that observed in the absence of adrenaline. In the experiments shown in Fig. 2, radioactivity associated with total fat-body protein at 0.5 h was the same as that at 0 h, both in the presence and absence of adrenaline. This indicates that the washing procedure was very effective in removing free [3H]leucine from the system and seems to exclude any possibility that continuing effects of adrenaline on lipoprotein lipase synthesis could result in an apparent enhanced loss of label from the enzyme during the chase period which we have interpreted as an effect on degradation. Discussion Effect of adrenaline on lipoprotein lipase synthesis Catecholamines have long been recognised as potential regulators of lipoprotein lipase [l-3] and the rapid decline in the activity of the enzyme which has been found in the present study to follow the addition of adrenaline to appropriate fat-body incubation systems (Fig. 1) is clearly consistent with such a role. The effects of catecholamines on the activity of the enzyme that have been demonstrated in earlier investigations have been mimicked by dibutyryl cyclic AMP, and by other effecters which act by increasing the

intracellular concentration of cyclic AMP [13-181, and it has been concluded therefore that cyclic AMP is the probable mediator of the catecholamine effect. The mechanism of cyclic AMP’S action has, however, remained obscure and, in particular, no evidence of cyclic AMP-dependent phosphorylation of the enzyme has yet been obtained [19,20]. Indeed, such phosphorylation would appear to be precluded by the vesicular location of lipoprotein lipase within the adipocyte. The data presented in Table I now show that, under conditions that lead to an adrenaline-induced decline in lipoprotein lipase activity, there is also a dramatic reduction in the incorporation of [3H]leucine into the enzyme. This suggests that the fall in activity may be due, at least in part, to a reduction in the rate of lipoprotein lipase synthesis and, in this respect, therefore, the regulation by adrenaline may be related to other instances where cyclic AMP acts as the mediator of hormone action through effects on enzyme synthesis rates [20-291. In all such cases, the effects have been accompanied by changes in the levels of the respective mRNA species. This indicates an action of cyclic AMP at the level of gene transcription and indeed the gene coding for phosphoenolpyruvate carboxykinase has been shown to contain a cyclic AMP-responsive regulatory sequence [3W The rapidity of adrenaline’s effect on lipoprotein lipase synthesis might seem inconsistent with an action on gene transcription. However, it should be noted that changes in the levels of mRNA coding for tyrosine aminotransferase [21,22] and for phosphoenolpyruvate carboxykinase [23] occur within minutes of the administration of cyclic AMP. Effect of adrenaline on lipoprotein lipase degradation Experiments showing a rapid decline in the amounts both of immunologically detectable lipoprotein lipase in culture adipocytes after the addition of cycloheximide [31] and in labelled enzyme during pulse-chase incubations of epididyma1 fat-bodies [9] have indicated that lipoprotein lipase in adipose tissue is turning over rapidly. Since non-specific protein degradation in hepatocytes [32], and the degradation of certain specific

404

cytosolic [33] and secreted [34] proteins, is stimulated by cyclic AMP, it seemed appropriate to carry out experiments in the present study to see whether adrenaline also affected the degradation of lipoprotein lipase, especially in the light of previous data suggesting post-translational regulation of the enzyme by adrenaline [7]. The results shown in Table II and Fig. 2 indicate that the degradation of lipoprotein lipase during incubations of epididymal fat-bodies is indeed markedly increased in the presence of adrenaline. The site of degradation of lipoprotein lipase in adipose tissue is unknown but, since the enzyme is synthesised and secreted by the adipocyte, and thereafter transported to the luminal surface of the endothelial cells, a number of possibilities can be envisaged. For example, degradation of a proportion of the newly synthesised enzyme could take place prior to its secretion, as has been shown for several other secretory proteins [35]. On the other hand, degradation could occur following release from the adipocyte. Degradation at the endothelial cell surface, for instance, would have considerable regulatory potential, enabling rapid changes in activity at the functional site. Alternatively, the adipocyte could take up extracellular enzyme again and degrade it and there is already some evidence for such endocytotic uptake [36]. In the present pulse-chase experiments the most marked effect of adrenaline on the loss of 3Hlabelled enzyme occurred during the initial 30 min of the chase period. After this time, the amount of labelled enzyme decreased at the same rate as in the absence of the hormone (Fig .2). This could suggest that any regulation of degradation by adrenaline occurred shortly after the enzyme’s synthesis, but that degradation unaffected by adrenaline also took place at a later stage. The possibility that the decreased incorporation of [3H]leucine into lipoprotein lipase in the presence of adrenaline observed during the l-h pulse incubations (Table I) could partly reflect increased degradation ,rather than decreased synthesis, of the enzyme clearly needs to be considered. While the rate of degradation in the presence of adrenaline in the experiment shown in Fig. 2 (50% in 1 h) seems insufficient to account entirely for the decreased incorporation of label during a 1 h pulse, precise calculations are not possible because

of the uncertainty regarding the site of lipoprotein lipase degradation. For example, it is conceivable that, in the experiment shown in Fig. 2, a proportion of the labelled enzyme present at the end of the 1 h pulse had already traversed the site at which adrenaline-stimulated degradation occurs. Addition of the hormone at the start of the chase period would then have no effect on the degradation of this population of labelled enzyme molecules, leading to an underestimation of the effect of adrenaline. Further studies are therefore needed to ascertain the precise changes in the rates of synthesis and degradation of the enzyme brought about by adrenaline. Finally, it should be noted that our interpretation of the results of the present study is based on the general assumption that all the labelled lipoprotein lipase binds to columns of heparin-Sepharose and is subsequently eluted by high concentrations of NaCl (see Materials and Methods). Although our previous work has shown that at least 95% of the lipoprotein lipase activity applied to such columns is recovered in fractions eluted by 2 M NaCl [5], it is just conceivable that adrenaline could bring about inactivation of the enzyme by a process which, while not involving its turnover, did reduce its affinity for heparin. Such a process would be indistinguishable from an effect of adrenaline on the enzyme’s turnover using the methodology employed in this study. The incubation of epididymal fat-bodies with adrenaline does not by itself reduce the affinity of the active lipoprotein lipase in tissue extracts of the fat-bodies for heparin (K.L. Ball, B.K. Speake and D.S. Robinson, unpublished data). Nevertheless, the additional presence of inactive enzyme with a low affinity for heparin in such extracts cannot be entirely ruled out. Further work to exclude the possibility is, therefore, needed. Acknowledgements

We are grateful to the Medical Council for financial support.

Research

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405

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