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THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1385 by The American Society of Biological Chemists, Inc.

Vol . 260, No. 7, Issue of April 10, pp. 4424-4431,1385 Printed in U. S. A.

Maturation and Secretion of Lipoprotein Lipase in Cultured Adipose Cells I. INTRACELLULAR ACTIVATION OF THE ENZYME*

(Received for publication, May 16, 1984)

Christian VannierS, Ez-Zoubir Amril, Jacqueline Etienneg, Raymond NegrelS, and Gerard AilhaudS From the $Centre de Biochimie, Centre National de la Recherche Scientifique LP7300, Uniuersite de Nice, 06034 Nice, Cedex, France and the SLaboratoire de Biochimie, Facult4 de Medecine Saint-Antoine, 75012 Paris, France

The intracellular pathway and the activation of lipoprotein lipase have been examined in differentiated Ob17 cells. These adipose cells were previously shown to secrete lipoprotein lipase during exposure to heparin. Treatment of the cells with cycloheximide and heparin leads to enzyme depletion, as shown by activity measurement and immunofluorescence microscopy. The repletion phase has been studied in the presence of monensin or carbonyl cyanide m-chlorophenylhydrazone, ionophores known to affect the intracellular transport of membrane and secretory proteins. Monen- sin-treated cells synthesize fully active lipoprotein lipase. Under these conditions the antigen accumulates in the Golgi apparatus and the heparin-stimulated enzyme release is extensively reduced. Carbonyl cyanide m-chlorophenylhydrazone-treated cells do not contain any enzyme activity but show detectable antigen which accumulates in the endoplasmic reticulum. Competi- tion for binding to immobilized anti-lipoprotein lipase antibodies of mature and endoplasmic reticulum-sequestered antigens is observed. Carbonyl cyanide m- chlorophenylhydrazone removal is rapidly followed by a transient burst of enzyme activity and a redistribu- tion of the antigen in the different subcellular com- partments. Therefore, the results show that the activation of lipoprotein lipase is an intracellular event taking place after the enzyme exits from the endoplasmic reticulum and before it reaches the trans-Golgi cisternae.

Lipoprotein lipase is synthesized and secreted by differentiated cells of mesodermal origin (1). In adipose tissue, as well as in other tissues, the enzyme is transported by some un- known mechanism to the luminal surface of endothelial cells where its lipolytic activity is then expressed in the presence of apo-C2-containing lipoproteins (2-4). Available data indicate clearly that active enzyme molecules are already present in adipocytes before exportation (5-7). An unanswered ques- tion is the ability of lipoprotein lipase to increase its catalytic activity on its way to, at or when released from, the cell surface of adipocytes. Indirect evidence reported in the last decade has suggested the occurrence of an intracellular activation of lipoprotein lipase in adipocytes (8-10). This conclu-

* This work was supported by the Centre National de la Recherche Scientifique (LP 7300) and the Institut National de la Sante et de la Recherche Midicale (CRE 837011 and CRL 827-002). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduer- tisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

sion relies on a protein synthesis-independent activation observed in cycloheximide-treated fat bodies from rat (8). The presence of glucose and of certain sugars able to be converted to glucose 6-phosphate was required, suggesting the necessity of at least one glycosylation step for such activation (10). More recently Spooner et al. (11) have studied the regulation of lipoprotein lipase activity and its release in differentiated 3T3-Ll cells. Fructose, mannose, and glucosamine could sub- stitute for glucose in supporting, within a few minutes, an increase of lipoprotein lipase activity in insulin-pretreated, glucose-deprived cells. Among several possibilities, these observations could be in favor of hexoses being required as structural components for synthesis of the enzyme itself or glycoproteins essential for its activity. These observations would also favor, in adipose cells, the existence of a precursor form of lipoprotein lipase. The experiments reported below were carried out with preadipocyte Ob17 cells (12). Previous studies have shown that the lipoprotein lipase content is enhanced 20-50-fold during adipose conversion of Ob17 cells and that the enzyme content was modulated by insulin and triiodothyronine within physiological concentrations, both hormones which are specifically recognized by surface and nuclear receptors, respectively (13-16). The approach used below to study the maturation process of lipoprotein lipase was to provoke drug-induced perturbation of the intracellular transport of the enzyme, within different subcellular com- partments known to be involved in the process of protein secretion (17). Our results show for the first time that an inactive precursor form of lipoprotein lipase can be detected in endoplasmic reticulum and that its activation likely takes place before the enzyme reaches the trans-cisternae of the Golgi apparatus.

EXPERIMENTAL PROCEDURES

Materials-Dulbecco's modified Eagle's medium was purchased from Seromed, Munchen, Germany (catalog No. T-041). Fetal calf serum was a product of Flow Laboratories. Glycerol tri[9,10-3H]oleate and ~-[4,5-~H]leucine were obtained from Amersham Corp. Rhoda- mine-conjugated rabbit anti-goat IgG was a product of Cappel Labo- ratories, Cochranville, PA. Crystalline insulin, triiodothyronine, cycloheximide, heparin, and Triton X-114 were products of Sigma. Other compounds were obtained as follows: triolein, Bast of Copen- hagen; sodium dodecyl sulfate, Merck; monensin and CCCP,' Calbi- ochem-Behring.

Cell Culture-Ob17 cells were plated at 2 X lo3 cells/cm* in 60- or 100-mm diameter dishes in the presence of Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 200 units of penicillin.ml", 50 pg of streptomycin 'ml", 33 pM biotin, and 17 p~ pantothenate. After confluence (5 days after seeding) cells were

The abbreviation used is: CCCP, carbonyl cyanide m-cblorophen- ylhydrazone.

4424

Intracellular Activation of Lipoprotein Lipase 4425

maintained in the same medium in the presence of 17 nM insulin and 1.5 nM triiodothyronine, in order to obtain optimal adipose conversion (16). This final medium, defined as differentiation medium, was changed every 2 days. Under such conditions, lipoprotein lipase reached its maximal level 11-13 days after confluence, as previously shown (9, 17). The experiments reported below were performed between days 7 and ll postconfluence, 18 h after the last medium change. Before use, cells in such medium were first exposed to 3 pg. ml" of heparin and/or to 15 p~ cycloheximide in an atmosphere of air/COz (955). All subsequent operations were carried out in fresh differentiation medium. In some instances, cells were grown on glass coverslips (used for immunofluorescence staining) or on plastic (Thermanox; Flow Laboratories) coverslips (used for secretion rates determined by the continuous flow technique, see below). Cell viabil- ity was routinely checked by erythrosin B exclusion and was found to be 98-100% under all conditions. Stock solutions of 10 mM CCCP and 1 mM monensin were prepared in absolute ethanol and stored up to 4 weeks at -80 "C before use.

PHILeucine Incorporation into Cell Proteins-The rate of protein synthesis was determined on cells grown in 60-mm diameter dishes, in the presence of 2.5 ml of differentiation medium containing 25 pCi of ~-[4,5-~H]leucine and 80 PM leucine. Under these conditions the rate of leucine incorporation was linear up to 12 h. At the end of the labeling period, cells were systematically washed a t 0 "C, first with isotope-free medium and next with a solution of 10 mM Tris-C1 buffer, pH 7.4, 0.15 M NaCl. CCCP- or monensin-treated cells were washed with such media containing either 10 p M CCCP or 1 p M monensin. After cell solubilization in 10 mM Tris-HC1 buffer, pH 7.4, 0.15 M NaC1, and 0.1% sodium dodecyl sulfate, the radioactivity was determined in the trichloroacetic acid-precipitable material.

Kinetics of Lipoprotein Lipase Secretion-Lipoprotein lipase secreted in the differentiation medium, in the presence of 3 pg. ml" of heparin, shows at 37 "C a loss of activity with a half-life of 20 rnin.' As a consequence, the determination of enzyme activities by taking aliquots from the culture medium leads to a significant underestimation which increases with time following heparin addition. In order to circumvent this difficulty, accurate determinations of the secretion rate were made possible by the use of a continuous flow technique.' Briefly, cells grown on two rectangular (6 cm') plastic coverslips were disposed, facing each other, in a plastic parallelepipedic chamber (0.4 ml). The differentiation medium containing 3 pg. ml" of heparin was perfused a t a flow rate of 70 pl . rnin". The effluent was immediately and continuously mixed with a solution of 5 mM sodium barbital buffer, pH 7.4, 1 M glycerol maintained a t -6 "C. Fractions were collected every 5 min a t 4 "C. Each fraction was stored in liquid nitrogen before assay. Since no loss of enzyme activity takes place in the mixture of 4 "C before deep freezing and subsequently during storage at -180 "C, the only loss was due to the inactivation occurring before mixing; this inactivation leads to consistent underestimation of activity values by less than 15% (not shown). An additional advantage of this technique is that the cells are exposed to a medium which is replenished continuously. Lipoprotein lipase activity of each fraction was determined, and the first derivative of the secretion curve was then integrated.

Enzyme Preparation-Cell-free homogenates were prepared as follows. Cells, rinsed twice with ice-cold Buffer A, pH 7.4 (5 mM sodium barbital, 280 mM mannitol, 1.8 mM CaCl', 0.5 mM MgClz, and 1 M glycerol), scraped with a rubber policeman, and suspended in the same buffer a t a ratio of 0.05 ml/cm' of cell monolayer, were homog- enized (Potter-Elvehjem homogenizer, 25 strokes). The protein concentration was between 4 and 8 mg.ml-'. These homogenates could be used for enzyme assays immediately or stored a t -20 "C without inactivation. Since lipoprotein lipase is a secretory enzyme sequestered in closed-membrane vesicles: treatment of the homogenate with detergent was required prior to enzyme assays in order to unmask all latent activity. Two protocols were used to achieve complete solubilization. Protocol 1 (used for enzyme assays): 2 h at 4 "C after adjusting Buffer A to 0.15 M NaCl, 10 mM Tris-HC1, pH 7.4, and 0.2% Triton X-114. As described by Bordier (18), the detergent was removed by heat treatment of the solubilized homogenate for 10 min a t 30 "C followed by sedimentation (10 min, 12,000 X g). Under such conditions the recovery of lipoprotein lipase was 98-100% whereas >95% of the initial amount of detergent was removed. The supernatant was used for enzyme assays within l h without loss of activity. Protocol 2 (used for immunoprecipitation and immunoinhibition

C. Vannier et aL, manuscript in preparation.

experiments): 10 min a t 20 "C followed by 3 h at 4 "C after adjusting Buffer A to 0.05% Triton X-114 and 3 mM sodium dodecyl sulfate. In this case, no heat treatment and sedimentation are required. When experiments were carried out with cells grown and maintained in culture dishes instead of coverslips, the heparin-releasable enzyme was stabilized in the culture medium by adjusting the concentrations to 2.5 mM sodium barbital, pH 7.4, 140 mM mannitol, and 0.5 M glycerol. This preparation could be used a t once for assays or stored a t -20 "C up to 5 days until use.

Enzyme Assays-Lipoprotein lipase activity was determined as previously described (13) as the serum-dependent hydrolysis of glycerol tri[9,10-3H]oleate (10 Ci/mol). Highly concentrated cell homogenates had to be prepared (0.04-0.1 mg of protein/assay) since the concentrations of detergents in the assays were severely controlled in order to avoid inhibition of enzyme activity. Therefore, the final concentration of sodium dodecyl sulfate had to be kept a t S0.6 mM when present alone or a t CO.1 mM when present in combination with Triton X-114, the concentration of which could not exceed 0.005%. Specific activities were expressed in milliunits.mg" of protein. All assays were performed on a t least duplicate aliquots of the same preparation obtained from a minimum of two pooled culture dishes. Variability between assays was not more than 5%. Control experiments showed that variability between mean values from three separate dishes never exceeded 15%.

Zmmunotitration-An anti-lipoprotein lipase antiserum was raised in a goat against the rat adipose tissue enzyme purified to homoge- neity by preparative isoelectrofocusing (19). In the present work the anti-lipoprotein lipase antibodies were from the IgG fraction prepared by ammonium sulfate precipitation and DEAE-cellulose ion exchange chromatography. Purified IgG were kept a t -20 "C in Ca2+- and M e - free phosphate-buffered saline, pH 7.4, (Pi/NaC1) until use. As described previously (14) such antibodies could be used for the detection by immunofluorescence staining of lipoprotein lipase in differentiat- ing and in differentiated Ob17 cells. It should be pointed out that increasing concentrations of these antibodies are able to inhibit, in a parallel manner, the heparin-releasable activity determined in the presence or in the absence of apo-Cz (not shown). Therefore, the inhibitory action of these antibodies seems to involve the recognition between the enzyme and the substrate interface and does not seem to interfere with the recognition site(s) for apo-Cz. We took advantage of these observations in order to detect a population of enzymatically inactive antigens. In both protocols used for immunotitration experiments, the reference curves were obtained with homogenates of differentiated cells never exposed to any drug (control cells).

Zmmunoinhibition-Experiments were carried out in a final volume of 0.2 ml. A fixed amount of lipoprotein lipase units from control cell homogenate was incubated in the presence of increasing amounts of soluble anti-lipoprotein lipase IgG. The incubation medium at pH 7.4 contained 2.5 mM sodium barbital, 5 mM Tris-C1, 0.5 M glycerol, and 10 FM phenylmethylsulfonyl fluoride. Competition experiments were performed by using a mixture containing 0.075 mg of protein from control cell homogenate (specific activity, 18 milliunits. mg") and either 0.095 or 0.19 mg of protein from CCCP-treated cells. Since these treated cells happened to contain some residual lipoprotein lipase activity (0.73 milliunits. mg"), two reference curves had to be drawn. In the first one, 0.075 mg of protein from the homogenate of control cells was used. In the second one, 0.083 mg of protein from the same homogenate was used, thus giving before any antibody addition a number of enzyme units identical to that present in the competition experiment. Under the three conditions used, the total protein concentration was kept constant by adding a solubilized homogenate from undifferentiated Ob17 cells (in which no lipoprotein lipase could be detected either by immunofluorescence staining or by enzyme assay (14)). In each case, the residual lipoprotein lipase activity was assayed after a 20-h incubation a t 4 "C.

Immunoprecipitation-Immobilization of anti-lipoprotein lipase antibodies was carried out by covalently coupling 2.8 mg of IgG/ml of packed beads (Affi-Gel 10 beads, Bio-Rad). Immunoprecipitation was performed in a final volume of 0.45 ml by incubating a fixed amount of lipoprotein lipase units (50 pg of cellular proteins from homogenate of control cells at a specific activity of 28.4 milliunits. mg") with increasing amounts of immobilized anti-lipoprotein lipase IgG. In the reference and competition curves (see Fig. 7), 220 pg of protein from solubilized homogenates of either undifferentiated cells or CCCP-treated cells were added, respectively.

After 20 h of end-over-end rotation a t 4 "C, the slurries were sedimented (4 min, 12,000 X g). The supernatants were used for

4426 Intracellular Actiuation of Lipoprotein Lipase

enzyme assays. No loss of enzyme activity was observed when immobilized antibodies were not added.

Fluorescence Microscopy-Cells were grown on glass coverslips and used 9 days after confluence. The localization of the different antigenic forms of lipoprotein lipase (see "Results") was examined with the goat anti-lipoprotein lipase IgG fraction described above. Proc- essing of the cells for immunofluorescence staining followed the procedure described by Louvard et al. (20) with slight modifications. All operations were carried out at 20 "C. Cells were rapidly rinsed in phosphate-buffered saline, pH 7.4,O.g mM CaCl2, and 0.5 mM MgCI,, fixed with 3% formaldehyde in the same buffer for 40 min, and washed twice with PJNaCl. Remaining aldehyde groups were quenched with 50 mM NH,Cl in Pi/NaC1 for 30 min. After permea- bilization of cell membranes with 0.05% saponin in Pi/NaC1 for 15 min, cells were washed three times in Pi/NaCI containing 0.2% gelatin (used for all subsequent steps) and incubated for 30 min in the presence of 0.06 mg.ml" of the anti-lipoprotein lipase IgG fraction. Subsequently, the cells were washed four times as above and incubated with rabbit anti-goat IgG labeled with rhodamine isothiocyan- ate (1:40 dilution, 30 rnin). After three additional washes, cells were mounted in 0.5 M glycine saline buffer, pH 8.6, containing 90% glycerol. They were viewed and photographed on Kodak Tri-X film using epifluorescence on a Zeiss photomicroscope I11 equipped with a filter for rhodamine and with X 40 and X 63 lenses.

RESULTS

Depletion of Intracellular and Heparin-releasable Lipopro- tein Lipase in Cycloheximide-treated Cells and Repletion after Drug Removal-As shown in Fig. 1A (inset), the blockade of protein synthesis in Ob17 cells occurs within 2 min after cycloheximide addition. When heparin is absent from the incubation medium, the small increase observed in the intracellular lipoprotein lipase activity is rapidly followed by a dramatic decrease and no activity remains after 2 h. In the absence of heparin, whether or not the cells are treated with cycloheximide, the rate of spontaneous release of the enzyme, if any, is below detection ( e50 microunits.h") (Fig. IC). Thus, this loss in activity should correspond to a degradation of enzyme molecules and t o a true decrease in enzyme content. In agreement with this hypothesis, Ob17 cells treated for 2 h with cycloheximide are devoid of lipoprotein lipase, as detected by immunofluorescence staining (Fig. 4E) , in contrast to control cells (Fig. 4A). When heparin is present in the incubation medium of cycloheximide-treated cells, no t ran- sient increase in intracellular enzyme activity takes place, but the subsequent decrease persists (Fig. 1A). Regarding the heparin-releasable enzyme, cells not treated with cycloheximide are able to secrete lipoprotein lipase for extended periods of time and show a biphasic curve. It is of interest that, during secretion, the intracellular lipoprotein lipase activity remains unchanged at any time (not shown). Thus, these control cells are able to replenish constantly their steady-state enzyme content. In contrast, cycloheximide-treated cells, at first able to secrete high amounts of lipoprotein lipase (Fig. le), show a secretion which levels off within 1 h (Fig. 1C). Interestingly, the total amount of releasable activity is the same as the amount of intracellular activity initially present at the time of cycloheximide addition. Therefore, such treated cells do secrete in toto the intracellular enzyme initially present, im- plying a very limited intracellular degradation in heparin- and cycloheximide-treated cells.

Repletion of lipoprotein lipase has been examined by drug removal following a prior depletion of the cells treated with cycloheximide and heparin. On one hand, intracellular activity is restored by 35-40% within 30 min and complete recovery occurs after 6 h. The ra te of recovery remains unchanged in the presence of heparin in the incubation medium (Fig. 1B). This repletion is clearly illustrated by immunofluorescence staining (Fig. 4F). On the other hand, the heparin-releasable

MINUTES

1. Kinetics of disaouearance and reamearance of lipoprotein lipase activity &-Ob17 cells treatedwith cycloheximide and heparin. Experiments were carried out with 9-day-old postconfluent cells in 60-mm diameter dishes. Cell homogenates from duplicate dishes were pooled, and the lipoprotein lipase activity (LPL) was measured after detergent solubilization (as described under "Ex- perimental Procedures"). A , disappearance of cellular activity as a function of time during exposure from time zero to 15 p~ cycloheximide, with (0) or without (0) 3 pg. ml" of heparin in the differentiation medium. Inset, [3H]leucine incorporation in cells incubated in the presence (0) or in the absence (A) of 15 WM cycloheximide from time zero. Incorporated radioactivity is plotted as a percentage by taking as 100% the value determined in untreated cells after 30 min incubation (1.75 X lo5 dpm.mg" of cell protein). B, reappearance of cellular activity as a function of time. After prior exposure for 2 h to 15 p~ cycloheximide and 3 pg.rn1-I of heparin, cells were washed extensively with four 3-ml portions of differentiation medium for a period not exceeding 9 min, then maintained in this medium with (0) or without (0) 3 pg.ml" of heparin. Lipoprotein lipase activity was determined in cell homogenates at the indicated times. Thus, lipoprotein lipase-depleted cells at time zero are identical to cells at time 120 min in A . The specific activities in untreated control cells, taken as 10076, were 11.1 and 10.7 milliunits.mg" in A and B, respectively. C, lipoprotein lipase secretion in the absence (V, V) or in the presence (0, 0) of 3 pg.ml" of heparin by cells exposed (0, V) or not exposed (0, V) from time zero to 15 pM cycloheximide. The curves shown are calculated integrals obtained by the continuous flow technique (see "Experimental Procedures"). D, recovery of releasable lipoprotein lipase from cells first depleted of enzyme activity as described in B, in the absence (V) or in the presence (0) of 3 pg. ml" of heparin. In C and D, the specific activity and the total amount of enzyme activity in untreated cells were 16 milliunits. mg" and 26 milliunits, respectively. The curues shown in A , E , C, and D are representative of 10, 6, 4, and 2 separate experiments, respectively.

activity only becomes detectable after 35 min and then secretion proceeds linearly (Fig. ID). This lag period observed between the appearance of intracellular activity and that of heparin-releasable activity should correspond to the minimal time needed for enzyme synthesis, accumulation, and exportation induced by heparin. Therefore, under these defined conditions where complete depletion can be followed by a repletion phase during which no enzyme can be secreted in the absence of heparin (Fig. I D ) , it was possible to delineate the subcellular level at which the activation of lipoprotein lipase, if any, could occur.


Intracellular and Heparin-releasable Activities in Monensin- treated Cells-In order to see whether such enzyme activation could be an early or a late event during transport from the endoplasmic reticulum to the cell surface, Ob17 cells were depleted of lipoprotein lipase by prior treatment with cycloheximide and heparin. The replation phase was followed in the absence or presence of monensin. This drug has been previously shown in different cellular models so far described to block the transport process of secreted and membrane proteins at the level of cisternae of the Golgi apparatus (21- 23); it has also been shown to impede the transport of proteins from medial to trans-Golgi cisternae and therefore the com- pletion of oligosaccharide maturation (24, 25). The curves of Fig. 2A indicate that the lipoprotein lipase activity is not only detectable but even higher in monensin-treated cells than in untreated cells. Since no spontaneous release of lipoprotein lipase can be observed either in control or monensin-treated cells (Fig. 2B), this result suggests that accumulation of the enzyme in the Golgi apparatus occurs when cells are exposed to monensin, under conditions where protein synthesis remains unchanged (not shown). In agreement with this hypothesis, histograms of Fig. 2B show a marked decrease in the secretion of lipoprotein lipase in monensin-treated cells exposed to heparin; these results would favor the hypothesis of a reduction in the exit of lipoprotein lipase from the postcisternal Golgi compartment en route to the cell surface. If the enzyme degradation taking place in cycloheximide- treated cells (Fig. lA) happened to be a post-Golgi event, such reduction could lead, at least in part, to a lower intracellular degradation of lipoprotein lipase in monensin-treated cells. The results of Fig. 3 are in agreement with this prediction. At early times during the repletion phase, the enzyme degrada-

1234 St YMJTES

FIG. 2. Effect of monensin on the recovery of lipoprotein lipase activity in lipoprotein lipase-depleted cells. Nine days after confluence lipoprotein lipase-depleted cells, first obtained as described in Fig. IB, were maintained from time zero in differentiation medium supplemented or not supplemented with l ~ L M monensin. A , kinetics of recovery of lipoprotein lipase activity in the absence (0) or in the presence (0) of the drug. Activities are plotted as a percentage of the specific activity (35.2 milliunits. mg") determined in control cells before treatment for enzyme depletion. B, cellular (white bars) and secreted lipoprotein lipase (hatched bars) activities were determined after a repletion phase performed in the absence (bars 2-4) or in the presence (bars 5-8) of 1 p~ monensin. The releasable activity was determined after 90 min of the repletion phase followed by exposure (bars 3 and 7) or no exposure (bars 4 and 8) to 3 pg.ml" of heparin for 30 min. Extracellular media from a t least triplicate dishes were pooled and aliquots were withdrawn for enzyme assays. The specific activity of cellular lipoprotein lipase was determined before heparin treatment (bars I and 5 ) and after heparin treatment (bars 2 and 6) . Specific activities are expressed in milliunits. mg" of cellular proteins. The data reported are representative of three separate experiments in both A and B.

MINUTES

FIG. 3. Changes in lipoprotein lipase activity after cycloheximide addition during the repletion phase. This experiment is identical to that described in the legend to Fig. 2 A , except that at times indicated by arrows, 15 p~ cycloheximide was added to recover- ing cells maintained (0) in the presence of 1 p~ monensin or with no monensin (0). The activity values reported were determined in cells exposed (-----) or not exposed (--) to cycloheximide. Activities are plotted as a percentage of the specific activity (22.5 milliunits. mg") determined in control cells before treatment for enzyme depletion. The curves are representative of two separate experiments.

tion is very slow in cells treated with the ionophore. At later times this degradation does occur but remains significantly slower than that observed in control cells; this slow but significant degradation is likely related to incomplete blockade by the drug of enzyme transport (Fig. 2B), as already reported for different proteins in other cellular systems (25, 26).

The existence of a monensin-imposed block at the level of Golgi cisternae is strengthened by immunofluorescence staining of newly synthesized lipoprotein lipase. The localization of the antigen is clearly restricted to this well-recognized subcellular organelle (Fig. 4G), in contrast to control cells which show a punctate fluorescence pattern in addition to that observed within the Golgi region (Fig. 4A). Altogether, these experiments indicate that active enzyme molecules are present in medial Golgi cisternae (24) and that the enzyme degradation is a post-Golgi event. Therefore, the activation of lipoprotein lipase, if any, should be an event occurring at the very latest in medial Golgi cisternae or even earlier. Investigations reported below show that such activation does not take place in the endoplasmic reticulum, but rather takes place at a later step.

Intracellular Lipoprotein Lipase Activity in CCCP-treated Cells-CCCP causes the accumulation of secretory proteins in the endoplasmic reticulum, by blocking the budding process involved in the formation of vesicles that carry these proteins from the transitional elements of the endoplasmic reticulum to the Golgi complex (25,27-29). This blockade can be rapidly reversed by drug removal (30). In our experiments, prior exposure of Ob17 cells to cycloheximide and heparin led to complete enzyme depletion. The removal of both agents was followed within less than 5 s by the addition of CCCP. Under these repletion conditions, the rate of protein synthesis is significant, being approximately 15% of that of control cells after 1 h (Fig. 5), whereas the lipoprotein lipase activity remains nil at all times. This lack of activity is not due to the

4428 Intracellular Activation of Lipoprotein Lipase

FIG. 4. Immunofluorescence of lipoprotein lipase in Ob17 cells exposed to different drugs. Seven-day postconfluent cells were processed under all conditions as described under "Experimental Procedures." A, control differentiated cells. Note the labeling of almost all of the subcellular structures except the nuclei (see B). The presence of a discrete punctate fluorescence pattern is visible, in addition to a more intense and unipolar fluorescence concentrated in the vicinity of nuclei. B, same cells as in A, phase contrast. C, control differentiated cells exposed to the nonimmune IgG fraction. D, same cells as in C, phase contrast. E, differentiated cells after treatment for 2 h with 15 ~ L M cycloheximide and 3 pg. ml" of heparin. No labeling can be observed. F, differentiated cells after enzyme depletion as in E and a repletion phase of 45 min after cycloheximide and heparin removal. The labeling pattern is reminiscent of that observed in control cells (A) . G and H, differentiated (G) and undifferentiated ( H ) cells from the same coverslip, after enzyme depletion as in E and a repletion phase of 90 min in the presence of 1 p~ monensin. Note in differentiated cells the restricted labeling at one pole of the perinuclear region where the reticular network of the Golgi complex appears. Z and J , differentiated (Z) and undifferentiated (J) cells after enzyme depletion as in E and a repletion phase of 45 min in the presence of 10 p~ CCCP. The diffused labeling is observed all around the nucleus and the pattern is very different from that of control cells (A) and of monensin- treated cells (G). K, differentiated cells after enzyme depletion as in E followed by a first repletion.phase of 45 min in the presence of 10 p~ CCCP and a second phase of 20 min after CCCP removal. Note the restoration of a labeling pattern similar to that of control cells ( A ) or of repleted cells (F). Typical patterns of remaining uncommitted cells, i.e. insusceptible to adipose conversion, are shown in H and J. No lipoprotein lipase can be detected in these cells, as previously reported (9). Bar equals 20 pm.


MINUTES

FIG. 5. Effect of CCCP on the recovery of cellular lipoprotein lipase activity and on protein synthesis in lipoprotein lipase-depleted cells. Lipoprotein lipase depletion was performed on 9-day-old postconfluent cells as described in Fig. 1B. Cells were then treated ( - - - - - ) or not treated (-) with 10 p~ CCCP. Two sets of the same series of cells were used for the [3H]leucine incorporation into proteins (A) and for recovery of cellular lipoprotein lipase (0). The results are expressed as a percentage of the value determined in CCCP-untreated cells after 60 min of repletion phase. Variability in the determinations of lipoprotein lipase activity and of protein synthesis in drug-treated cells never exceeded 15% of the mean values reported. The specific activity of lipoprotein lipase after a 60-min exposure to CCCP was below 0.003 milliunit. mg" as compared to 6.45 milliunits. mg" in control cells. The curves are representative of three separate experiments.

absence of antigen, since lipoprotein lipase can be detected by immunofluorescence staining in differentiated CCCP- treated cells but not in undifferentiated cells (Fig. 41 compared to Fig. 45). The antigen seems to be localized in the endoplasmic reticulum of the perinuclear region. The fluorescence is weak but highly significant since no labeling can be observed in cycloheximide-treated cells (Fig. 4E). In any event, this fluorescence pattern is different from that of control (Fig. 4 4 ) and monensin-treated cells (Fig. 4G). These results would indicate that CCCP-treated cells do accumulate some enzymatically inactive form(s) of lipoprotein lipase in the endoplasmic reticulum and that this accumulation increases with time of exposure of the cells to CCCP. Since the effect of CCCP is very rapidly reversible (Fig. 4K), one would expect an increase of enzyme activity following CCCP removal. Moreover, this increase should be faster than that observed for cycloheximide-treated cells in which no enzyme and no antigen are present (Fig. 1 and Fig. 4E). Experiments reported in Fig. 6A show clearly that this is indeed the case. The existence of an accumulation of lipoprotein lipase in CCCP- treated cells is also strengthened by additional experiments in which cells remaining for 45 min in the presence of cycloheximide and CCCP were compared to CCCP-only-treated cells. In both cases, these treatments were followed by removal of all inhibitors. As shown in the histograms of Fig. 6A, the recovery of lipoprotein lipase activity after 15 min was approximately 2-fold higher in CCCP-only-treated cells. The differences observed in the rate of accumulation of lipoprotein lipase in CCCP-treated cells (Fig. 6A) cannot be attributed to differences in the rate of protein synthesis since control experiments reported in Fig. 6B indicate no differences in the incorporation of [3H]leucine into proteins after removal of

MINUTES

FIG. 6. Effect of the removal of cycloheximide and CCCP on the recovery of cellular lipoprotein lipase activity (A) and of protein synthesis (B) . Nine-day-old postconfluent cells were depleted of lipoprotein lipase and washed as described in the legend to Fig. 1B. A first set of lipoprotein lipase-depleted cells was maintained from time zero to 60 min in differentiation medium alone (0). A second set of cells was first maintained for 45 min in the same medium containing 10 ~ L M CCCP; then removal of CCCP (defining time zero) from CCCP-treated cells was followed by extensive washes as described in the legend to Fig. lB, and these cells were further maintained in differentiation medium alone for 60 min (0, CCCP- treated cells). Inset, the histogram bars give the specific activity values of cellular lipoprotein lipase expressed by taking as 100% (bar I ) the value obtained 15 min after cycloheximide removal from lipoprotein lipase-depleted cells (1.94 milliunits. mg"). Bar 2, value obtained 15 min after CCCP removal from cells first treated with CCCP as described above. Bar 3, value obtained 15 min after CCCP and cycloheximide removal from cells first treated for 45 min with 10 p M CCCP and 15 pM cycloheximide. All the data reported are representative of three separate experiments.

either drug at time zero (cycloheximide) or at time 45 min (CCCP). It is likely that, after CCCP removal from the cells, the transport can be reinitiated and that the sequestered inactive enzyme can become activated in a compartment posterior to the endoplasmic reticulum. Immunofluorescence staining of lipoprotein lipase in CCCP-treated cells after CCCP removal indicates that the antigen localization (Fig. 4K) is reminiscent of that observed both in control cells (Fig. 4A) and in cycloheximide-treated cells after cycloheximide removal (Fig. 4 0 .

Binding to Anti-lipoprotein Lipase Antibodies of Mature and Endoplasmic Reticulum-sequestered Lipoprotein Lipase Anti- gens-Using the IgG fraction of anti-lipoprotein lipase antibodies, we detected the presence of antigen in the endoplasmic reticulum of CCCP-treated cells (Fig. 4 0 . Surprisingly, however, such cells did not contain any significant enzyme activity (Fig. 5). In the light of the polyclonal nature of the antibodies used, these results implied that the total IgG fraction was capable of recognizing both active and some inactive form(s) of the enzyme. If this were the case, one would expect the inactive antigen to compete with mature (i.e. enzymatically active) form(s) of lipoprotein lipase for antibody recognition. Indeed, immunoprecipitation experiments reported in Fig. 7A show the existence of such competition. At any given concentration of immobilized IgG, the enzyme activity recovered in the supernatant is increased when homogenate from CCCP- treated cells is added to a known amount of mature active lipoprotein lipase. It can be concluded that an inactive molecular form of lipoprotein lipase does exist in the endoplasmic reticulum of CCCP-treated cells.

4430 Intracellular Activation of Lipoprotein Lipase

A

0

a

0 2 o U ) m o # ) INMOBILIZED ANTI-LPL IQ ( ~ 0 ) SOLUBLE ANTI-LPL ( ~ 0 )

FIG. 7. Differential binding of mature and endoplasmic reticulum-sequestered lipoprotein lipase antigens to anti-lipoprotein lipase antibodies. The results were obtained according to protocols described under “Experimental Procedures.” A , immunoprecipitation of mature lipoprotein lipase from homogenates of differentiated cells in the presence of homogenates from either undifferentiated cells (0, reference curve) or differentiated cells treated with 10 p~ CCCP for 45 min as described in Fig. 4 (V, competition curve). 100% activity corresponds to the activity values determined in the absence of Affi-Gel-bound antibodies. They were respectively 1.42 milliunits for the reference curve and 1.46 milliunits for the competition curve. B, immunoinhibition of mature lipoprotein lipase in the presence of homogenates of either undifferentiated cells (0) or CCCP- treated differentiated cells (A, V). 100% activity values correspond to 1.37 milliunits (-) and 1.55 milliunits (-----) for reference curves and to 1.42 milliunits and 1.5 milliunits when adding, respectively, a 1.25-fold (A) and a 2.5-fold excess (V) of proteins from homogenate of CCCP-treated cells over proteins from homogenate of untreated differentiated cells. Each curve in A and B is representative of two separate experiments.

In separate series of experiments shown in Fig. 7B, we were able to determine that the catalytically active enzyme pos- sesses at least one antigenic site which is not present on the inactive form of lipoprotein lipase. When immunoinhibition assays of lipoprotein lipase activity are performed with the soluble anti-lipoprotein lipase IgG fraction, no competition between active and inactive molecules can be detected. The inhibition of enzyme activity is solely due to a population of antibodies which saturates the antigenic sites present in the domain(s) involved directly, or indirectly, in the recognition and/or the catalysis of substrate; the saturation of other epitopes by (at least) one other population of antibodies does occur but does not interfere with such a recognition ( i e . enzymatic activity). Since no competition takes place, the results favor the absence of some catalytically active enzyme structure in CCCP-treated cells.

DISCUSSION

The transport pathway of membrane and secretory proteins has been the subject of numerous investigations in the past decade (see Refs. 17 and 30 for reviews). A secretory process for lipoprotein lipase of adipose cells has been postulated by different authors (32-35), as well as the existence of a precursor form of the enzyme both in adipocytes (8, 33) and in rat heart cells (36). If so, enzyme activation should take place on its way to or at the cell surface (1, 8). Previous data from our laboratory using Ob17 cells argue against an activation process occurring near or at the cell surface but pointed out the possibility of an intracellular activation (14). The presence of catalytically active molecules in monensin-treated cells, which show an apparent K,,, value (0.5 mM) for glycerol trioleate and a stimulation factor by apo-C2 identical to those determined for the heparin-releasable enzyme (not shown), suggests strongly that enzyme activation should take place before

enzyme molecules leave Golgi cisternae. Furthermore, since monensin is known in other cellular systems to block protein discharge from medial to trans-cisternae (24), it is possible that fully active molecules are already present in medial Golgi cisternae.

Evidence for an intracellular activation process is supported by experiments using CCCP as a proton ionophore, under conditions where protein synthesis levels are significant (Figs. 5-7). NO enzyme activity is present in CCCP-treated cells whereas inactive molecules are immunologically detectable and also titrable. After CCCP removal, the recovery of enzyme activity is faster than in control cells (Fig. 6). This observation would indicate that the inactive form(s) of lipoprotein lipase could represent a precursor form(s) of the enzyme. This proenzyme-enzyme relationship would be in agreement with the fact that this higher rate of reemergence of active molecules is a transient phenomenon (Fig. 6A). I t would also be in agreement with the appearance of the antigen in the Golgi apparatus after CCCP removal from CCCP-treated cells, showing the resumption of enzyme transport (Fig. 4K). It could be argued that the restoration of lipoprotein lipase activity is merely due to the restoration of ATP levels, which in turn allow recovery of the synthesis of activated precursors of the high-mannose oligosaccharide whose transfer onto polypeptide chains might be required for enzyme activation. This hypothesis is not very likely since this transfer, that is, the first glycosylation step in the endoplasmic reticulum, is not inhibited when intracellular ATP levels are decreased after exposure to CCCP in murine plasmablasts (25) and in Chinese hamster ovary cells infected by vesicular stomatitis virus (30). It could be also argued that exposure of adipose cells to CCCP prevents an ATP-dependent phosphorylation of the enzyme which may be necessary for activity. This hypothesis can likely be excluded since (i) no proof of phosphorylation has been yet reported and (ii) the only effect of exposing adipocytes to catecholamines, which triggers phosphorylation of hormone-sensitive lipase (37,38), is a decrease in lipoprotein lipase activity (9).

Regarding the degradation process in cycloheximide-treated cells (Figs. 1A and 4E), the decrease in enzyme activity observed in the absence of heparin (tI l2 -30 min) is a true reflection of a decrease in enzyme content. In contrast, when heparin is present, the pool of enzyme molecules escapes degradation. Thus, it appears that, in the presence of such an “inducer” of enzyme release, lipoprotein lipase molecules are- not directed into a compartment (e.g. lysosomal) where degradation could occur. In order to delineate this process, further experiments in the presence of monensin were carried out. As shown in Fig. 3, cycloheximide was added at different times during the repletion phase to cells treated, or not treated, with monensin. As expected, the rate of enzyme degradation in untreated cells increases as a function of time, that is, as a function of increasing amounts of intracellular lipoprotein lipase. However, in monensin-treated cells, the rate of enzyme degradation after cycloheximide addition remains slow. This slow degradation process is not due to decreased enzyme content since monensin-treated cells accumulate, at any given time, higher amounts of lipoprotein lipase than untreated cells (Figs. 2 and 3). Altogether, the results suggest that the enzyme degradation is a post-Golgi event. At least two hy- potheses could be put forward to explain the lack of enzyme degradation in monensin-treated cells: (i) intralysosomal pH could be raised, affecting in turn proteolysis (39), or (ii) lipoprotein lipase molecules could remain in the Golgi apparatus and thereby could not enter another compartment able to deliver its content to lysosomes. In favor of the second

Intracellular Activation of Lipoprotein Lipase 443 1

hypothesis, preliminary experiments reveal in Ob17 cells the existence of storage vesicles for lipoprotein lipase. These vesicles disappear in monensin- and heparin-treated cells whereas lipoprotein lipase antigens remain detectable in the Golgi apparatus. Thus, it is likely, but not proven, that the degradation of lipoprotein lipase takes place after discharge of enzyme molecules from trans-Golgi cisternae into transport vesicles.

In conclusion, lipoprotein lipase is synthesized in a precursor form in the endoplasmic reticulum of adipose cells. I t is activated either during transport to and/or within the proxi- mal cisternae (cis- and medial) of the Golgi apparatus. The molecular basis of lipoprotein lipase activation is still un- known but is presently under investigation. After activation, degradation of mature molecules can be prevented during stimulation of enzyme secretion by heparin. It is thus tempt- ing to postulate that Ob17 cells synthetize and degrade lipoprotein lipase molecules in a rather “constitutive” manner and that this steady-state situation is not affected unless appropriate stimuli direct some cellular compartment toward secretion.

Acknowledgments-Thanks are due to M. Rossignol and L. Noe (Paris) for efficient technical help and to Drs. E. Van Obberghen- Schilling and L. Austin for careful reading of the manuscript. We are indebted to G. Oillaux for expert secretarial assistance.

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