Vol. 166, No. 3, 1990 February 14, 1990

INHIBITION

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1118-1125

BY SERUM COMPONENTS

LIPOPROTEIN

OF THE EXPRESSION

OF

LIPASE GENE UPON STIMULATION BY GROWTH HORMONE

Anne PRADINE!+FIGUERES, Sylvie BARCELLINI-COUGET, Christian DANI, Christian BAUDOIN, and G&ard AlLHAUD*

Centre

Received

de Biochimie du CNRS, UPR-7300, Faculte des Sciences, Universite de Nice-Sophia Antipolis, Part Valrose, 06034 Nice cedex, France

December

20,

1989

Growth hormone regulates in a positive way the expression of the lipoprotein lipase gene at a transcriptional level in preadipocyte Ob1771 cells. Inhibition by serum components of this expression was investigated upon stimulation by growth hormone. Low-molecular weight, lipid-soluble components (a serum lipid extract, corticosterolds and oleic acid) and high-molecular weight, hydrophilic components (TGF-6 and those present in delipidated serum) were inhibitory. Inhibition of the expression of LPL mRNAs and that of LPL activity were parallel. It is concluded that the regulation of the expression of LPL gene occurs likely at a transcriptional level and that a balance between multiple effecters present in serum are active in an opposite manner. m 1990 Academic Press, Inc. Numerous studies performed on intact fat pads as well as on isolated adipocytes have documented the role of insulin, catecholamines and glucocortico’ids in the control of lipoprotein lipase (LPL) synthesis and secretion (1). We have shown in previous studies that, in preadipocyte cells, the emergence of LPL is coupled to growth arrest (2) whereas growth hormone (GH) (3) was only able to increase moderatly (24 hours),

out,as

of LPL activity

GH at 0.1% BS, remains unaffected

stimulation

is shown at 8% BS. It should be

overcome and a - 2-fold increase in the in agreement with previous data (3).

also be pointed

expression

inhibition

in part,

to a decrease

concentration

01 12 0.1 %bovlneserum

2

4

of serum

6

in the before

8

FiPure 1: Inhibitory effect of bovine serum on the expression of LPL activity upon stimulation by growth hormone Cells were maintained in the incubation medium (see”Materials and Methods”) in the absence (A) or in the presence (B) of varying concentrations of BS for 15 hours before performing the experiments. At time zero, in the presence of the incubation medium supplemented with varying concentrations of BS as indicated, cells were exposed (0.0) or not (0.m) to 4 nM GH during 7 hours. Cellular LPL activities were assayed: each value represents the mean of pooled triplicate 35-mm dishes. 1120

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exposure to GH. As shown in Figure lB, this does not appear to be the case as cells pre-exposed for 15 hours to concentrations of serum from 0.1 to 8% BS, before adding or not GH for the next 7 hours, showed very similar values of LPL activity, in the absence of GH at any serum concentration, and responded in a very similar way to GH addition. In order to identify some putative factor(s) of serum able to inhibit the GH-stimulated expression of LPL, various sera were used at a concentration of 8% (v/v) under conditions described in Figure 1A : fetal bovine serum, extensively dialysed bovine serum and heat-treated (56”C, 30 min) bovine serum gave identical results to those obtained with bovine serum. Thus either low molecular weight factor(s) were not required or, even it were so, one could exclude that in addition other non-dialysable and/or heat-stable factor(s) of serum could well be required. LPL and pOb24 mFWAs are known to behave as early markers of adipose cell differentiation. Among the serum factors which play a regulatory role, TGF-6 was shown to decrease LPL and pOb24 gene expression (10,ll) but the regulation of LPL gene expression and that of LPL activity by corticosterolds and fatty acids, which are known to be present in bovine serum (12,13), were not thoroughly investigated. Data of Table 1 indicate that addition of 1% lipid extract (equivalent to 10% BS) led to a complete suppression of the GH-promoted increase in the expression of LPL

inhibition

of the expression

Table 1 of LPI activity LPL activity

culture

(mU/mg

conditions

Incubation

extract

Delipidated

serum

components

of cell protein1

Acknticn

medium

BS 10% (v/v) Lipid

by various

1% (v/v) BS 10% (v/v)

Oleic acid 30 m Dexamethasone

1 nM

Corticosterone

10 nM

Bovine serum albumin Fatty acid -free bovine serum albumin TGFI3 80 pM

0.3%

(w/v)

O.~%(W/V)

None

OH

1.8

10.9

1.7

1.4

1.9

1.8

1.7

2.5

1.8

4.4

1.9

1.5

1.6

3

1.7

5.7

2.1 2.2

11.8 3

Cells were maintained In the incubation medium serum) supplemented or not as indicated. Determination performed from pooled triplicate 3.Grnm dishes.

1121

(containing of LPL

0.1% activities

bovine were

Vol. 166, No. 3, 1990

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Cant

Cont

Dex

AND BIOPHYSICAL

XiF-D

D,..~

Lip

l&-J3

RESEARCH COMMUNICATIONS

&x

‘KS4

D,

l-fp

Lip

“T-B LiP

I

1

+GH

Fi@ure 2: Inhibitory effect of dexamethasone. lipid extract and TGF-8 on the ttxpe~son of LFL activity upon stimulation by m hormone Cells were exposed to the incubation medium in the presence or in the absence of 4 r&l GH (Cant). When supplemented with GH, 0.1 nM dexamethasone (Dex), 8 pM TGF-j3 or 0.3OA lipid extract (Lip) or a combination of the various effecters were added or not as indicated. After 8 hours, LPL activities were determined on triplicate dishes (errors bars correspond to f s.d.1.

activity. Both corticosterone and dexamethasone as well as oleic acid were clearly inhibitory: corticosterone and oleic acid were active within a physiological range of concentrations: howewer, 10% delipidated bovine serum were also able to bring the same phenomenon, implying some hydrophilic components as inhibitors. To identify further the component(s) present in delipidated bovine serum, the effects of BSA and delipidated-BSA were compared when present at 0.3% (w/v), i.e. at a concentration similar to that obtained with 10% BS (v/v). The data of Table 1 show that delipidated BSA had no effect whereas BSA had only a partial inhibitory effect. This suggests that lipophilic components present in both BS and BSA were partially responsible for the observed effects but that additional factors present in delipidated-BS but not in delipidatedBSA should be involved. As shown in Table 1, TGF-j3 at a low concentration was very effective in inhibiting the GH-promoted increase of LPL activity. To determine wether the various inhibitors were acting or not synergistically, sub-maximal concentrations of the various effecters were assayed separately and in combination. The results of Figure 2 show that their effects on the expression of LPL activity were additive but not synergistic. Since the regulation of LPL activity has been reported by various authors to occur from a transcriptional to a post-translational levels (4.14-18). LPL activity and LPL mRNA content were determined. The results of Figure 3A show that, in the presence of GH. the inhibitory effect of BS, BSA or delipidated-BSA on the expression of LPL activity was 1122

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dBsA

F&ure 3:Comparative effect of bovine serum and serum albumin on the GE&promoted increase of LPL mRNAs level and LPL activity:

A : Cells were exposed in the presence or in the absence of 4 nM GH to the incubation medium supplemented or not (CONT) with loo/b BS (BS), 0.3% (w/v) bovine serum albumin (BSAI or 0.3O/6 (w/v1 fatty acid-free bovine serum albumin (dBSA) during 8 hours. Total FWAs were isolated from four pooled lOO-mm dishes, electrophoresed and probed for LPL mRNAs (3.3 and 3.7 kb species) (hatched bars). Cellular LPL activity was determined from pooled duplicate dishes (stippled bars). The results are expressed as fold increase above the values obtained for cells not exposed to GH. B : Northern-blot analysis of LPL mRNAs and GAPDH mRNA of cells incubated as above (same series of cells) for 8 hours. The arrows indicate the 3.3 and 3.7 kb species of LPL mFWA.

parallel to that observed for the expression of LPL mFWAs. The two species of 3.3 and 3.7 kb decreased in parallel ( Figure 3B). It is likely that the components involved in the regulation of the expression of LPL activity are active at a transcriptional level and not at a posttranscriptional, translational and/or a post-translational level, as GH has been shown to regulate the expression of LPL gene primarily at a transcriptional level, whereas LPL mRNAs. LPL protein and LPL activity increased in parallel (4). The results of Figure 4A are in agreement with this hypothesis as, after stimulation of the expression of LPL activity, either GH removal or BS addition led to an identical time-dependent decrease of LPL activity. Since the decrease of the LPL mRNA content (and LPL activity) after GH removal was parallel to that observed after blockade of gene transcription by actinomycin D (t1/2 - 2.5 hours) (4). it is clear that the degradation rate of LPL mRNAs is independent of the presence of GH; it is tempting to postulate that the various factors present in bovine serum were active at a transcriptional level. The results of Figure 4B show that, after stimulation by GH. both dexamethasone and the lipid extract led to inhibition of the expression of LPL activity similar to that observed with bovine serum, despite the continous presence of GH. It has been excluded that a disappearance of cell surface GH receptors in cells exposed to high serum concentrations was responsible for the inhibition of the expression of LPL activity stimulated by GH since GH was shown to bind to intact Ob 1771 cells under these conditions (Kd 1123

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4 6 hours

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No BS addition

Dex

Lip

Fiare 4: Inhibition of LPL activity following bovine serum, dexamethasone or lipid extract addition in cells pre-exposed to growth hormone A ; At day 4 post-confluence, Ob1771 cells were exposed to the incubation medium supplemented with 4 nM (W, 0) or not supplemented (0) with 4 nM GH. Twenty-four hours later, either GH was removed (0) or 10% BS (a) were added. At the times indicated, duplicate dishes were pooled for the determination of LPL activity. The results are expressed as percent of the values obtained at time zero for cells pre-exposed to GH . B ; cells from a different series were exposed to the incubation medium containing (black and hatched bars) or not (open bars) 4 nM GH. Twenty four hours later (defining time zero). cells were exposed or not to 10% BS (BS), 1 nM dexamethasone (Dex) or 1% lipid extract (Lip) as indicated (hatched bars). After 8 hours, duplicate 35-mm dishes were pooled for the determination of LPL activity.

- 1 nM, 42,000 sites/ cell) (19). for GH is not very likely as the expression of c-fos gene (20) and determined in the presence of 8% respectively. in fair agreement with

Moreover a change in receptor affinity concentration of GH required for the IGF-I gene (8) to half-maximal level, BS, were found to be 1nM and 0.5 nM, the Kd value of the GH receptor.

To conclude, the expression of the LPL gene appears to be under the control of various effecters interplaying in a rather subtle way as GH acts as a positive effector whereas the serum factors identified in the present study act as negative effecters. Therefore, when studying the regulation of LPL in vivo, caution should be taken when extrapolating from in vitro data, as various factors acting in opposite manner may change simultaneously in vivo. ACKNOWLEDGMENTS

Thanks are due to Drs M.C. Schotz and T. Kirchesgessner (Los Angeles, USA) for providing us with a LPL cDNA probe, to Dr P. Fort (Montpellier, France) for the kind kigft of pR GAPDH-13 probe and to Dr A. Skottner-Lundin (Kabi Vitrum. Stockholm, Sveden) for the generous gift of recombinant human GH. This work was supported by the “Centre National de la Recherche Scientifique” (UPR 7300). 1124

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REFERENCES

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11, 12. 13. 14. 15. 16. 17. 18. 19. 20.

Eckel R.H. (1987) Adipose tissue Ifpoprotefn Ifpase. In: Borensztajn J. (ed.) Lipoprotein Ifpase. Everner Publishers, Inc, Chicago. pp 79-132 Amrf E.. C. Danf. A. Doglfo, P. Grfmaldf, and G. Aflhaud. (1986) Biochem.Bfophys.Res.Commun. 137. 903-910 DogIio A, C. Dani, P. GrfmaIdi. and G. Aflhaud. (1986) Bi0chem.J. 238, 123-129 Pradines-Figu&res, A.. Barcellinf-Couget, S.. Danf. C.. Vannier. C., and Ailhaud. G. (1989) submitted for publication. Amrf E.. C. Danf, A. DogIfo, J. Etfenne, P. Grfmaldi. and G. Aflhaud. (1986) Bi0chem.J. 238, 115122. Bordier C. (1981) J.Bfol.Chem. 256, 1604-1607. Vannfer C., E. Amri, J. Etfenne, R Negrel, and G. Aflhaud. (1985) J.Biol.Chem. 260, 4424-4431 DogIio A., C. Danf, G. Fredrikson, P. GrfmaIdi. and G. Aflhaud. (1987) Embo.J. 6. 401 l-4016 Cham, B.E., and Knowles, B.R. (1976) J.Lfpfd Res. 17, 176-181 Dani C., E. Amri, B. Bertrand, S. Enerback, G. Bjursell, P. Grfmaldf, and G. AfIhaud. (1989) JCeILBiochem., in press. Dani, C , Doglio, A., Amri, 2.. Bardon, S.. Fort, P., Bertrand, B., Grimaldi, P., and Ailhaud. G. (1989) J.Biol.Chem. 264, 10119-10125 Hauner. H., Entenmann. G., Wabitsch, M., Gaillard. D., Aflhaud, G., Negrel. R., and Pfeiffer, E.F. (1989) J.Clin.Invest. 84, 1663-1670 Gaillard, D.. Negrel, R.. Lagarde, M., and Aflhaud, G. (1989) Bi0chem.J. 257, 389-397 Vannier C., S. Deslex, A. Pradines-Figueres, and G. Aflhaud. (1989) J.Biol.Chem. 264, 13 199- 13205 Pradines-Figutres, A., Vannier, C!.. and Aflhaud, G. (1989) submitted for publication. Semenkovfch, C.F., Wims, M., Noe. L., Etienne. J., Chan, L. (1989) J.Biol.Chem. 264, 9030-9038 Ong, J.M., Kfrchgessner, T.G., Schotz, M.C., and Kern, P.A. (1988) J.Biol.Chem. 263, 12933-12938 Ong, J.M., and Kern, P.A. (1989) J.CIin.Invest. 84, 305-311 Doglio A. , E. Amrf, C. Dani, P. GrimaIdi, S. Deslex, R. Negrel, and G. Aflhaud. (1987) In: Isaksson 0 (ed) Growth hormone. Basic and clinical aspects, Elsevfer Science Publishers, Amsterdam, p 299-305 Doglio, A., Dani, C., Grimaldi, P., and Ailhaud G. (1989) Proc.NatI .Acad.Sci.USA 86, 1148- 1152

1125

Inhibition by serum components of the expression of lipoprotein lipase gene upon stimulation by growth hormone.

Growth hormone regulates in a positive way the expression of the lipoprotein lipase gene at a transcriptional level in preadipocyte Ob1771 cells. Inhi...
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