Clin Biochem, Vol. 23,343- 347, 1990
0009-9120/90 $3.00 ÷ .00 Copyright c 1990 The Canadian Society of Clinical Chemists.
Printed in Canada. All rights reserved.
Cellular and Secreted Lipoprotein Lipase Revisited GERARD Centre de Biochimie du CNRS,
AILHAUD
Faculte des Sciences,
Lipoprotein lipase (LPL) of adipose cells is present only in membrane compartments, mainly in the Golgi apparatus. LPL is a typical secretory protein which appears to be active as a homodimer. The process of LPL synthesis and maturation requires multiple steps. LPL is synthesized in the endoplasmic reticulum as an inactive monomer of M, 51,000; a high-mannose, inactive monomer of M, 55,500 is then formed. An active homodimer form, bearing two complex oligosaccharide chains per monomer of M r 58,000, forms in the Golgi apparatus. This mature form, present in secretory vesicles, can be secreted constitutively or after exposure to heparin. A model is proposed in which LPL is present in secretory vesicles in a potentially active, condensed, or "polymerized" form. This model, which applies to various LPL-containing tissues in different species -- including human -- would explain the "activation" of LPL.
KEY WORDS: lipoprotein lipase; cryptic enzyme; enzyme activation; Golgi apparatus; adipocytes; differentiation; heparin.
Parc Valrose, 06034
Nice Cedex,
France
chemical properties of the cells after adipose conversion (12,13). The comparative properties of differentiated cells and mature rodent adipocytes show that the specific activities of key lipolytic and lipogenic enzymes (including LPL), and different hormonal responses, are within the same range of magnitude in both cases (13). The validity of these cellular models is also strongly supported by the ability of undifferentiated 3T3-F442A cells (14) and Ob17 cells (15), to develop in vivo into unilocular adipocytes after injection into athymic mice. It must be stressed that adipose cells in culture are less fragile, remain multilocular, and have a low neutral lipid content compared to adipocytes in vivo; consequently, cultured adipose cells offer clear advantages over adipocytes, to study LPL as a secreted enzyme.
Introduction
S t a t u s of i n t r a c e l l u l a r l i p o p r o t e i n lipase
L
Studies of LPL localization by indirect immunofluorescence revealed that LPL antigen was mainly present in the Golgi apparatus of differentiated Ob17 cells (16), rat adipose cells (17), and h u m a n adipose cells (18). When examined in Ob17 adipose cells, no patent activity was detectable in gently homogenized cells (19). All latent activity (see below) could be unmasked by disrupting membrane structures with neutral detergents, such as Triton X-114. The sequestration of LPL in closed membrane structures was supported by experiments showing that the number of titratable antigenic sites of LPL increased dramatically upon unmasking of the activity; in addition, LPL activity then became sensitive to proteolysis by trypsin. Additional experiments using Triton X-114 indicated that LPL molecules are not amphipathic; thus, LPL cannot be considered as an integral membrane protein. These results (19) favor the localization of LPL in adipose cells as being typical for a secretory protein, and underline the absence of LPL in the cell cytoplasm.
ipoprotein lipase (LPL), synthesized in parenchymal cells of tissues of mesodermal origin, is the key enzyme responsible for the hydrolysis of plasma triglycerides from apolipoprotein CII-containing lipoproteins at the capillary endothelium (1). Rapid regulatory tissue-specific changes in its activity occur during fasting and refeeding, illustrating its inverse and coordinate expression in muscle and adipose tissue. This regulation allows fatty acids to be directed according to the metabolic requirements of the parenchymal cells (1-3). In response to nutritional changes, LPL activity at the endothelium can vary dramatically (4-6), whereas, it is not changed greatly in adipocytes (1,7). Numerous studies performed on intact fat pads, isolated adipocytes, and cells of preadipocyte clonal lines have documented the role of hormones and other factors in the control of LPL synthesis and expression in these cells (8). The validity of using preadipocyte clonal lines established from mouse, such as 3T3-L1 (9), 3T3-F442A (10), and Ob17 (11) for the study of LPL, relies primarily on the bio-
M a t u r a t i o n p r o c e s s o f l i p o p r o t e i n lipase in a d i p o s e cells Correspondence: Prof. G6rard Ailhaud, Centre de Biochimie du CNRS, Facult~ des Sciences, Parc Valrose, 06034 Nice C~dex, France. Manuscript received October 11, 1989; revised March 8, 1990; accepted March 13, 1990. CLINICAL BIOCHEMISTRY,VOLUME 23, OCTOBER 1990
To study the maturation process of LPL in differentiated Ob17 cells, drug-induced perturbation of the intracellular transport of the enzyme was provoked. Within different subcellular compartments known to be involved in the process of protein 343
AILHAUD
secretion, prior treatment of the cells with cycloheximide and heparin led to enzyme depletion, as shown by activity measurement and immunofluorescence microscopy. The repletion phase was then studied in the presence of monensin or carbonyl cyanide mchlorophenylhydrazone (CCCP), ionophores known to affect the intracellular transport of membrane and secretory proteins. Monensin-treated cells were found to synthesize fully active LPL. Under these conditions, the antigen accumulated in the Golgi apparatus and the heparin-stimulated enzyme release was extensively reduced. CCCP-treated cells did not contain any enzyme activity, but they showed detectable antigen which accumulated in the endoplasmic reticulum. Competition for binding to immobilized anti-LPL antibodies of mature and endoplasmic reticulum-sequestered antigens was observed. CCCP removal was rapidly followed by a transient burst of enzyme activity and a redistribution of the antigen in the different subcellular compartments. Therefore, these results showed that the activation of LPL is an intracellular event occurring after the enzyme exits from the endoplasmic reticulum, and before it reaches the t r a n s - G o l g i cisternae (16). The data reported above, coupled with reports indicating that LPL is a glycoprotein (20-22), suggested that the trimming of oligosaccharide chains, which occurs in c i s - and m e d i a l - G o l g i cisternae, may be an important step for LPL activation. To better understand the role of oligosaccharide chains in the activation of newly synthesized LPL, the cells were first depleted of any activity and enzyme content by cycloheximide treatment, and of activated sugar precursors of oligosaccharide chains by tunicamycin. The repletion of LPL content was studied in these cells maintained in the presence of tunicamycin after cycloheximide removal. During the repletion phase, the ECso values of inhibition by tunicamycin ( - 0 . 2 ~g/mL) of the incorporation of labeled glucose, mannose, or galactose into trichloracetic acid-insoluble material were identical. Under these conditions, the rate of protein synthesis was maximally decreased by 30%. Clearly, the recovery in LPL activity paralleled the recovery in hexose incorporation: no activity was recovered in the absence of glycosylation. An inactive form of LPL from tunicamycin-treated cells was detected by competition experiments with mature active LPL for the binding to immobilized anti-LPL antibodies, and by immunofluorescence staining (23).
Biosynthesis and turnover of lipoprotein lipase in adipose cells More recently, the biosynthesis and turnover of LPL have been investigated in differentiated 3T3F442A cells labeled with [35S]methionine. Pulsechase experiments, endo-~-N-acetylglucosaminidase H treatment, and analysis by SDS-PAGE indicate that LPL is synthesized in the endoplasmic reticu344
lum as a glycoprotein of Mr = 55,500, bearing two N-oligosaccharide side chains of the high-mannose type. This precursor form of LPL is transported within 10 min to the Golgi apparatus; this event is accompanied by the formation of a mature species of M r = 58,000. Treatment of the Mr = 58,000 species with glycopeptidase F yielded a M r = 51,000 protein similar to that observed after treatment of the M r = 55,500 precursor form, or after inhibition of N-glycosylation in tunicamycin-treated cells. The precursor form of LPL of Mr = 55,500 does not accumulate in the cells because after a labeling period of 2 h, only the M r = 58,000 species is detected. Only 20% of the newly synthesized molecules of M r = 58,000 are constitutively secreted, whereas 80% are degraded, most likely in lysosomes, as indicated by the inhibitory effect of leupeptin upon the degradation process. Under heparin stimulation, quantitative secretion of the mature form of LPL occurs; whereas, the intracellular degradation is arrested. Heparin mobilizes intracellular LPL without changing the rate of LPL export from the ER to the cell surface. Sucrose gradient centrifugation of the material from intracellular cisternae showed that the Mr = 55,500 precursor form is present as a monomer (s = 4.1S); whereas, the Mr = 58,000 mature form is present as a homodimer (s = 6.8S), to which LPL activity is associated (24). Figure 1 shows the model of LPL synthesis and transport from endoplasmic reticulum to cell surface in Ob17 and 3T3-F442A cells. An inactive monomer of Mr = 51,000 is in agreement with the molecular weight calculated from the nucleotide sequence of mouse LPL cDNA ( M r = 50,314) (25); whereas, the existence of secretory vesicles is indirectly supported by recent observations of their presence in rat cardiac cells (26).
Relationships between intracellular and secreted LPL The study of LPL secretion is complicated by the fact that inactivation of LPL molecules occurs after secretion. Depending upon the cellular model under investigation, spontaneous secretion of LPL was found to be nil (Ob17 cells), or was quite significant (3T3-F442A cells and rat adipose cells), whereas heparin-stimulated secretion occurred almost iramediately after addition of the sulfated glycosaminoglycan (27). In both cases, the activity of secreted LPL showed a first-order process of inactivation. This constant rate of inactivation, coupled with a decreased rate of secretion, prevented an accurate determination of enzyme secretion. A perifusion system was devised, which minimized and controlled the rate of LPL inactivation. This allowed us to estimate the true secretion rate of LPL (28), and to determine if activation of LPL occurred upon secretion; it had been hypothesized that, independent of protein synthesis, a hormone-stimulated intracellular translocation of LPL, leading to its secretion, is
CLINICAL BIOCHEMISTRY,VOLUME 23, OCTOBER 1990
CELLULAR AND SECRETED LIPOPROTEIN LIPASE REVISITED
f
ENDOPLASMIC RETICULUM
Polypeptide Precursorform (hlgh-mRnuose) lmino
Acids
~51
kD ~
INACTIVE MONOMER
55.5kD
MEDIAL/TRANS GOLGI
Matureform (2 complex | ~llgosaccharldechA|nS)[ ~--~ 5 8 k D
INACTIVE MONOMER
ACTIVE HOMODIMER
l . ~
|
Seereti°n ~'58 kD f LPLfunctionalpool
] SECRETORY VESICLES
)
Lysosomal degradatlon
J F i g u r e 1 - - M a t u r a t i o n process of LPL. This scheme indicates only the M r of the LPL monomer for the various forms Isee
text for comments). accompanied by an increase in its catalytic activity (7,29,30). Recently, using polyclonal antibodies raised in rabbits, against homogeneous LPL from mouse adipose cells, the secretion potential ~ of LPL has been thoroughly examined in differentiated 3T3F442A cells (31). The secretion potential ~, taken as the ratio of total releasable activity or antigen to initial intracellular activity or antigen, was determined in cells treated with heparin and cycloheximide, and shown to be equal to 1 for LPL antigen, but significantly greater than 1 for LPL activity; no intracellular LPL is actually degraded under these conditions (31). The existence of intracellular inhibitors of LPL activity has been excluded, as has the possibility that LPL becomes more active upon secretion into the medium in the presence of heparin (31). Moreover, under steady-state conditions of [35S]methionine incorporation into LPL protein, the inactive precursor form of LPL of Mr = 55,500 is not detectable, thus, excluding the existence of a reservoir of catalytically inactive LPL molecules which could explain a secretion potential greater than 1 for LPL activity. An important clue was obtained when a dramatic enhancement of the intracellular activity by a mere dilution of detergenttreated cell lysates was observed with no change in LPL antigen; however, the activity of secreted LPL was not affected by dilution. The total cellular activity reached a plateau which now became identical to that obtained in the medium of cells exposed to heparin and cycloheximide. These results, which indicate a systematic underestimation of intracellular LPL activity, have been extended to mouse, rat, and h u m a n adipose tissues, and to rat skeletal and
CLINICAL BIOCHEMISTRY, VOLUME 23, OCTOBER 1990
cardiac muscle (A. Pradines-Figu~res et al., unpublished data). To explain these results, it is postulated that LPL is present in a cryptic form within intracellular cisternae of the Golgi apparatus and post-Golgi vesicles (Figure 2). A first interaction is proposed between LPL (present as homodimers of 2 × 58 kD) and some intravesicular molecules, resulting in a specific complex. A second interaction between these complexes is proposed which results in a network. In other words, LPL molecules would be present within this network in a potentially active condensed or "polymerized" form. Condensation of LPL molecules probably occurs in the presence of heparin sulfate proteoglycans and sulfated glycosaminoglycans. A mere dilution in the presence of either heparin or heat-denaturated LPL (31) as competitors, would disrupt interactions between LPL molecules and the sulfated macromolecules and, by the law of mass action, would lead to the complete recovery of LPL as active homodimers (~ = 1 for LPL activity, see above). Therefore, in vivo, the process of exocytosis and secretion of LPL molecules into a large extracellular volume from LPL-containing vesicles (26), with a small intravesicular volume would be equivalent to a dilution phenomenon obeying the law of mass action, and leading to the secretion of active homodimers. This hypothesis, depicted in Figure 2, would explain the "activation" of LPL. Our findings also question the significance of intracellular LPL activity, as this is probably minimal or absent in intact cells. In vivo, the phenomenon of masking the activity of LPL is advantageous for the cell, resulting from the phospholipase A1 activity of the enzyme on glycerophospholipids (2).
345
AILHAUD Polymer
Polymer ,.:~'!:~"
•::Y
/N VITRO
% Vesicle
"::::i!!~.
~i:.
Dilution
Homodimers
Detergent treatment
%
..~? ~;~: Inactive LPL ( p o t e n t i a l l y active)
Inactive LPL (potentially active)
Polymer
Plasma
A c t i v e LPL a f t e r dilution
.....
Exocytosls
::i m e m b r a n e
.'{!~i:.i%x
Homodimers
Dilution ...:.::.~
IN VIVO %
::~:"'"'"~::: :~,~
Inactive LPL ( p o t e n t i a l l y active)
Y
Secretion
Vesicle Depolymerizing LPL
.::: :
A c t i v e LPL after s e c r e t i o n
Figure 2--"Activation" process of LPL. This scheme indicates the condensed or "polymerized" state of LPL; it does not show the interactions between LPL molecules and sulfated glycosaminoglycans and heparan sulfate proteoglycans (see text for comments}.
To conclude, the proposed existence of a putative and important reservoir of inactive LPL molecules in cultured adipose cells appears to be related to a systematic underestimation of its in{racellular activity. Whether such a reservoir of LPL molecules of M r = 55,500 is indeed present in mature adipocytes isolated from adipose tissue, remains an open question.
Acknowledgements The author wishes to express his deep gratitude to Drs. C. Vannier, E. Amri, and A. Pradines-Figu~res for their outstanding contribution to this study.
References 1. Cryer A. Tissue lipoprotein lipase activity and its action in lipoprotein metabolism. Int J Biochem 1981; 13: 525-41. 2. Nilsson-Ehle P, Garfinkel AS, Schotz MC. Lipolytic enzymes and plasma lipoprotein metabolism. Annu Rev Biochem 1980; 49: 667-93. 3. Robinson DS. Assimilation, distribution and storage. Section C: the function of the plasma triglycerides in fatty acid transport. In: Florkin M, Schotz EH, eds. Comprehensive biochemistry, Vol. 18. Pp. 51-116. Amsterdam: Elsevier, 1970. 4. Olivecrona T, Bengtsson G, Marklund SE, Lindahl U, H65k M. Heparin-lipoprotein lipase interactions. Fed Proc Fed A m Soc Exp Biol 1977; 36: 60-5. 5. Jansen H, Stam H, Kalkman C, Hulsmann WC. On the dual localization of lipoprotein lipase in rat heart. Studies with a modified perfusion technique. Biochem 346
Biophys Res Commun 1980; 92: 411-6. 6. Semb H, Olivecrona T. Nutritional regulation of lipoprotein lipase in guinea pig tissues. Biochim Biophys Acta 1986; 876: 249-55. 7. Spencer IM, Hutchinson A, Robinson DS. The effect of nutritional state on the lipoprotein lipase activity of isolated fat cells. Biochim Biophys Acta 1978; 530: 375-84. 8. Eckel RH. Adipose tissue lipoprotein lipase. In: Borensztajn J, ed. Lipoprotein lipase. Pp. 79-132. Chicago: Evener Publishers, Inc., 1987. 9. Green H, Meuth M. An established pre-adipose cell line and its differentiation in culture. Cell 1974; 3: 127-33. 10. Green H, Kehinde O. Spontaneous heritable changes leading to increased adipose conversion in 3T3 cells. Cell 1976; 7: 105-13. 11. N~grel R, Grimaldi P, Ailhaud G. Establishment of preadipocyte clonal line from epididymal fat pad of ob/ob mouse that responds to insulin and to lipolytic hormones. Proc Natl Acad Sci USA 1978; 75: 6054-8. 12. Green H. Adipose conversion: a program of differentiation. In: Ailhaud G, ed. Obesity: cellular and molecularaspects. Pp. 15-24. Paris: INSERM Publications, 1979. 13. Ailhaud G. Adipose cell differentiation in culture. Moll Cell Biochem 1982; 49: 17-31. 14. Green H, Kehinde O. Formation of normally differentiated subcutaneous fat pads by an established preadipose cell line. J Cell Physiol 1979; 101: 169-72. 15. Gaillard D, Poli P, N6grel R. Characterization of ouabain-resistant mutants of the preadipocyte Ob17 clonal line. Adipose conversion in vitro and in vivo. Exp Cell Res 1985; 156: 513-27. 16. Vannier C, Amri E, Etienne J, N~grel R, Ailhaud G. Maturation and secretion of lipoprotein lipase in culCLINICAL BIOCHEMISTRY, VOLUME 23, OCTOBER 1990
CELLULAR AND SECRETED LIPOPROTEIN LIPASE REVISITED
17.
18.
19. 20.
21. 22.
23.
24.
tured adipose cells. I. Intracellular activation of the enzyme. J Biol Chem 1986; 260: 4424-31. Deslex S, N~grel R, Ailhaud G. Development of a chemically defined serum-free medium for complete differentiation of rat adipose precursor cells. Exp Cell Res 1987; 168: 15-30. Deslex S, N~grel R, Vannier C, Etienne J, Ailhaud G. Differentiation of human adipocyte precursors in a chemically defined serum-free medium. Int J Obes 1986; 10: 19-27. Vannier C, Etienne J, Ailhaud G. Intracellular localization of lipoprotein lipase in adipose cells. Biochim Biophys Acta 1986; 875: 344-54. Bensadoun A, Ehnholm C, Steinberg D, Brown WV. Purification and characterization of lipoprotein lipase from pig adipose tissue. J Biol Chem 1974; 249: 2220-34. Fielding PE, Shore VG, Fielding CJ. Lipoprotein lipase: properties of the enzyme isolated from post heparin plasma. Biochemistry 1974; 13: 4318-23. Iverius PH, Ostland-Lindqvist AM. Lipoprotein lipase from bovine milk. Isolation procedure, chemical characterization, and molecular weight analysis. J Biol Chem 1976; 251: 7791-5. Amri E, Vannier C, Etienne J, Ailhaud G. Maturation and secretion of lipoprotein lipase in cultured adipose cells. II -- Effects of tunicamycin on activation and secretion of the enzyme. Biochim Biophys Acta 1986; 875: 33~ 43. Vannier C, Ailhaud G. Biosynthesis of lipoprotein lipase in cultured mouse adipocytes. II. Processing, subunit assembly, and intracellular transport. J Biol Chem 1989; 264: 13206-16.
CLINICAL BIOCHEMISTRY, VOLUME 23, OCTOBER 1990
25. Kirchgessner TG, Svenson KL, Lusis AJ, Schotz MC. The sequence of cDNA encoding lipoprotein lipase. A member of a lipase gene family. J Biol Chem 1987; 262: 8463-6. 26. Blanchette-Mackie EJ, Masuno H, Dwyer NK, Olivecrona T, Scow RO. Lipoprotein lipase in myocytes and capillary endothelium of heart: immunocytochemical study. A m J Physiol 1989; 256: E818-28. 27. Pradines-Figu~res A, Vannier C, Ailhaud G. Shortterm stimulation by insulin of lipoprotein lipase secretion in adipose cells. Biochem Biophys Res Commun 1988; 154: 982-90. 28. Vannier C, Ailhaud, G. A continuous flow method for the study of lipoprotein lipase secretion in adipose cells. Biochim Biophys Acta 1986; 875: 324-33. 29. Spooner PM, Chernick SS, Garrison MM, Scow RO. Development of lipoprotein lipase activity and accumulation of triacylglycerol in differentiating 3T3-L1 adipocytes. Effects of prostaglandin F2,,, 1-methyl-3 isobutylxanthine, prolactin, and insulin. J Biol Chem. 1979; 254: 1305-11. 30. Spooner PM, Chernick SS, Garrison MM, Scow RO. Insulin regulation of lipoprotein lipase activity and release in 3T3-L1 adipocytes. Separation and dependence of hormonal effects of hexose metabolism and synthesis of RNA and protein. J Biol Chem 1979; 254: 10021-9. 31. Vannier C, Deslex S, Pradines-Figu~res A, Ailhaud G. Biosynthesis oflipoprotein lipase in cultured mouse adipocytes. I. Characterization of a specific antibody. Relationships between the intracellular and secreted pools of the enzyme. J Biol Chem 1989; 264: 13199205.
347
0009-9120/90 $3.00 ÷ .00 Copyright c 1990 The Canadian Society of Clinical Chemists.
Printed in Canada. All rights reserved.
Cellular and Secreted Lipoprotein Lipase Revisited GERARD Centre de Biochimie du CNRS,
AILHAUD
Faculte des Sciences,
Lipoprotein lipase (LPL) of adipose cells is present only in membrane compartments, mainly in the Golgi apparatus. LPL is a typical secretory protein which appears to be active as a homodimer. The process of LPL synthesis and maturation requires multiple steps. LPL is synthesized in the endoplasmic reticulum as an inactive monomer of M, 51,000; a high-mannose, inactive monomer of M, 55,500 is then formed. An active homodimer form, bearing two complex oligosaccharide chains per monomer of M r 58,000, forms in the Golgi apparatus. This mature form, present in secretory vesicles, can be secreted constitutively or after exposure to heparin. A model is proposed in which LPL is present in secretory vesicles in a potentially active, condensed, or "polymerized" form. This model, which applies to various LPL-containing tissues in different species -- including human -- would explain the "activation" of LPL.
KEY WORDS: lipoprotein lipase; cryptic enzyme; enzyme activation; Golgi apparatus; adipocytes; differentiation; heparin.
Parc Valrose, 06034
Nice Cedex,
France
chemical properties of the cells after adipose conversion (12,13). The comparative properties of differentiated cells and mature rodent adipocytes show that the specific activities of key lipolytic and lipogenic enzymes (including LPL), and different hormonal responses, are within the same range of magnitude in both cases (13). The validity of these cellular models is also strongly supported by the ability of undifferentiated 3T3-F442A cells (14) and Ob17 cells (15), to develop in vivo into unilocular adipocytes after injection into athymic mice. It must be stressed that adipose cells in culture are less fragile, remain multilocular, and have a low neutral lipid content compared to adipocytes in vivo; consequently, cultured adipose cells offer clear advantages over adipocytes, to study LPL as a secreted enzyme.
Introduction
S t a t u s of i n t r a c e l l u l a r l i p o p r o t e i n lipase
L
Studies of LPL localization by indirect immunofluorescence revealed that LPL antigen was mainly present in the Golgi apparatus of differentiated Ob17 cells (16), rat adipose cells (17), and h u m a n adipose cells (18). When examined in Ob17 adipose cells, no patent activity was detectable in gently homogenized cells (19). All latent activity (see below) could be unmasked by disrupting membrane structures with neutral detergents, such as Triton X-114. The sequestration of LPL in closed membrane structures was supported by experiments showing that the number of titratable antigenic sites of LPL increased dramatically upon unmasking of the activity; in addition, LPL activity then became sensitive to proteolysis by trypsin. Additional experiments using Triton X-114 indicated that LPL molecules are not amphipathic; thus, LPL cannot be considered as an integral membrane protein. These results (19) favor the localization of LPL in adipose cells as being typical for a secretory protein, and underline the absence of LPL in the cell cytoplasm.
ipoprotein lipase (LPL), synthesized in parenchymal cells of tissues of mesodermal origin, is the key enzyme responsible for the hydrolysis of plasma triglycerides from apolipoprotein CII-containing lipoproteins at the capillary endothelium (1). Rapid regulatory tissue-specific changes in its activity occur during fasting and refeeding, illustrating its inverse and coordinate expression in muscle and adipose tissue. This regulation allows fatty acids to be directed according to the metabolic requirements of the parenchymal cells (1-3). In response to nutritional changes, LPL activity at the endothelium can vary dramatically (4-6), whereas, it is not changed greatly in adipocytes (1,7). Numerous studies performed on intact fat pads, isolated adipocytes, and cells of preadipocyte clonal lines have documented the role of hormones and other factors in the control of LPL synthesis and expression in these cells (8). The validity of using preadipocyte clonal lines established from mouse, such as 3T3-L1 (9), 3T3-F442A (10), and Ob17 (11) for the study of LPL, relies primarily on the bio-
M a t u r a t i o n p r o c e s s o f l i p o p r o t e i n lipase in a d i p o s e cells Correspondence: Prof. G6rard Ailhaud, Centre de Biochimie du CNRS, Facult~ des Sciences, Parc Valrose, 06034 Nice C~dex, France. Manuscript received October 11, 1989; revised March 8, 1990; accepted March 13, 1990. CLINICAL BIOCHEMISTRY,VOLUME 23, OCTOBER 1990
To study the maturation process of LPL in differentiated Ob17 cells, drug-induced perturbation of the intracellular transport of the enzyme was provoked. Within different subcellular compartments known to be involved in the process of protein 343
AILHAUD
secretion, prior treatment of the cells with cycloheximide and heparin led to enzyme depletion, as shown by activity measurement and immunofluorescence microscopy. The repletion phase was then studied in the presence of monensin or carbonyl cyanide mchlorophenylhydrazone (CCCP), ionophores known to affect the intracellular transport of membrane and secretory proteins. Monensin-treated cells were found to synthesize fully active LPL. Under these conditions, the antigen accumulated in the Golgi apparatus and the heparin-stimulated enzyme release was extensively reduced. CCCP-treated cells did not contain any enzyme activity, but they showed detectable antigen which accumulated in the endoplasmic reticulum. Competition for binding to immobilized anti-LPL antibodies of mature and endoplasmic reticulum-sequestered antigens was observed. CCCP removal was rapidly followed by a transient burst of enzyme activity and a redistribution of the antigen in the different subcellular compartments. Therefore, these results showed that the activation of LPL is an intracellular event occurring after the enzyme exits from the endoplasmic reticulum, and before it reaches the t r a n s - G o l g i cisternae (16). The data reported above, coupled with reports indicating that LPL is a glycoprotein (20-22), suggested that the trimming of oligosaccharide chains, which occurs in c i s - and m e d i a l - G o l g i cisternae, may be an important step for LPL activation. To better understand the role of oligosaccharide chains in the activation of newly synthesized LPL, the cells were first depleted of any activity and enzyme content by cycloheximide treatment, and of activated sugar precursors of oligosaccharide chains by tunicamycin. The repletion of LPL content was studied in these cells maintained in the presence of tunicamycin after cycloheximide removal. During the repletion phase, the ECso values of inhibition by tunicamycin ( - 0 . 2 ~g/mL) of the incorporation of labeled glucose, mannose, or galactose into trichloracetic acid-insoluble material were identical. Under these conditions, the rate of protein synthesis was maximally decreased by 30%. Clearly, the recovery in LPL activity paralleled the recovery in hexose incorporation: no activity was recovered in the absence of glycosylation. An inactive form of LPL from tunicamycin-treated cells was detected by competition experiments with mature active LPL for the binding to immobilized anti-LPL antibodies, and by immunofluorescence staining (23).
Biosynthesis and turnover of lipoprotein lipase in adipose cells More recently, the biosynthesis and turnover of LPL have been investigated in differentiated 3T3F442A cells labeled with [35S]methionine. Pulsechase experiments, endo-~-N-acetylglucosaminidase H treatment, and analysis by SDS-PAGE indicate that LPL is synthesized in the endoplasmic reticu344
lum as a glycoprotein of Mr = 55,500, bearing two N-oligosaccharide side chains of the high-mannose type. This precursor form of LPL is transported within 10 min to the Golgi apparatus; this event is accompanied by the formation of a mature species of M r = 58,000. Treatment of the Mr = 58,000 species with glycopeptidase F yielded a M r = 51,000 protein similar to that observed after treatment of the M r = 55,500 precursor form, or after inhibition of N-glycosylation in tunicamycin-treated cells. The precursor form of LPL of Mr = 55,500 does not accumulate in the cells because after a labeling period of 2 h, only the M r = 58,000 species is detected. Only 20% of the newly synthesized molecules of M r = 58,000 are constitutively secreted, whereas 80% are degraded, most likely in lysosomes, as indicated by the inhibitory effect of leupeptin upon the degradation process. Under heparin stimulation, quantitative secretion of the mature form of LPL occurs; whereas, the intracellular degradation is arrested. Heparin mobilizes intracellular LPL without changing the rate of LPL export from the ER to the cell surface. Sucrose gradient centrifugation of the material from intracellular cisternae showed that the Mr = 55,500 precursor form is present as a monomer (s = 4.1S); whereas, the Mr = 58,000 mature form is present as a homodimer (s = 6.8S), to which LPL activity is associated (24). Figure 1 shows the model of LPL synthesis and transport from endoplasmic reticulum to cell surface in Ob17 and 3T3-F442A cells. An inactive monomer of Mr = 51,000 is in agreement with the molecular weight calculated from the nucleotide sequence of mouse LPL cDNA ( M r = 50,314) (25); whereas, the existence of secretory vesicles is indirectly supported by recent observations of their presence in rat cardiac cells (26).
Relationships between intracellular and secreted LPL The study of LPL secretion is complicated by the fact that inactivation of LPL molecules occurs after secretion. Depending upon the cellular model under investigation, spontaneous secretion of LPL was found to be nil (Ob17 cells), or was quite significant (3T3-F442A cells and rat adipose cells), whereas heparin-stimulated secretion occurred almost iramediately after addition of the sulfated glycosaminoglycan (27). In both cases, the activity of secreted LPL showed a first-order process of inactivation. This constant rate of inactivation, coupled with a decreased rate of secretion, prevented an accurate determination of enzyme secretion. A perifusion system was devised, which minimized and controlled the rate of LPL inactivation. This allowed us to estimate the true secretion rate of LPL (28), and to determine if activation of LPL occurred upon secretion; it had been hypothesized that, independent of protein synthesis, a hormone-stimulated intracellular translocation of LPL, leading to its secretion, is
CLINICAL BIOCHEMISTRY,VOLUME 23, OCTOBER 1990
CELLULAR AND SECRETED LIPOPROTEIN LIPASE REVISITED
f
ENDOPLASMIC RETICULUM
Polypeptide Precursorform (hlgh-mRnuose) lmino
Acids
~51
kD ~
INACTIVE MONOMER
55.5kD
MEDIAL/TRANS GOLGI
Matureform (2 complex | ~llgosaccharldechA|nS)[ ~--~ 5 8 k D
INACTIVE MONOMER
ACTIVE HOMODIMER
l . ~
|
Seereti°n ~'58 kD f LPLfunctionalpool
] SECRETORY VESICLES
)
Lysosomal degradatlon
J F i g u r e 1 - - M a t u r a t i o n process of LPL. This scheme indicates only the M r of the LPL monomer for the various forms Isee
text for comments). accompanied by an increase in its catalytic activity (7,29,30). Recently, using polyclonal antibodies raised in rabbits, against homogeneous LPL from mouse adipose cells, the secretion potential ~ of LPL has been thoroughly examined in differentiated 3T3F442A cells (31). The secretion potential ~, taken as the ratio of total releasable activity or antigen to initial intracellular activity or antigen, was determined in cells treated with heparin and cycloheximide, and shown to be equal to 1 for LPL antigen, but significantly greater than 1 for LPL activity; no intracellular LPL is actually degraded under these conditions (31). The existence of intracellular inhibitors of LPL activity has been excluded, as has the possibility that LPL becomes more active upon secretion into the medium in the presence of heparin (31). Moreover, under steady-state conditions of [35S]methionine incorporation into LPL protein, the inactive precursor form of LPL of Mr = 55,500 is not detectable, thus, excluding the existence of a reservoir of catalytically inactive LPL molecules which could explain a secretion potential greater than 1 for LPL activity. An important clue was obtained when a dramatic enhancement of the intracellular activity by a mere dilution of detergenttreated cell lysates was observed with no change in LPL antigen; however, the activity of secreted LPL was not affected by dilution. The total cellular activity reached a plateau which now became identical to that obtained in the medium of cells exposed to heparin and cycloheximide. These results, which indicate a systematic underestimation of intracellular LPL activity, have been extended to mouse, rat, and h u m a n adipose tissues, and to rat skeletal and
CLINICAL BIOCHEMISTRY, VOLUME 23, OCTOBER 1990
cardiac muscle (A. Pradines-Figu~res et al., unpublished data). To explain these results, it is postulated that LPL is present in a cryptic form within intracellular cisternae of the Golgi apparatus and post-Golgi vesicles (Figure 2). A first interaction is proposed between LPL (present as homodimers of 2 × 58 kD) and some intravesicular molecules, resulting in a specific complex. A second interaction between these complexes is proposed which results in a network. In other words, LPL molecules would be present within this network in a potentially active condensed or "polymerized" form. Condensation of LPL molecules probably occurs in the presence of heparin sulfate proteoglycans and sulfated glycosaminoglycans. A mere dilution in the presence of either heparin or heat-denaturated LPL (31) as competitors, would disrupt interactions between LPL molecules and the sulfated macromolecules and, by the law of mass action, would lead to the complete recovery of LPL as active homodimers (~ = 1 for LPL activity, see above). Therefore, in vivo, the process of exocytosis and secretion of LPL molecules into a large extracellular volume from LPL-containing vesicles (26), with a small intravesicular volume would be equivalent to a dilution phenomenon obeying the law of mass action, and leading to the secretion of active homodimers. This hypothesis, depicted in Figure 2, would explain the "activation" of LPL. Our findings also question the significance of intracellular LPL activity, as this is probably minimal or absent in intact cells. In vivo, the phenomenon of masking the activity of LPL is advantageous for the cell, resulting from the phospholipase A1 activity of the enzyme on glycerophospholipids (2).
345
AILHAUD Polymer
Polymer ,.:~'!:~"
•::Y
/N VITRO
% Vesicle
"::::i!!~.
~i:.
Dilution
Homodimers
Detergent treatment
%
..~? ~;~: Inactive LPL ( p o t e n t i a l l y active)
Inactive LPL (potentially active)
Polymer
Plasma
A c t i v e LPL a f t e r dilution
.....
Exocytosls
::i m e m b r a n e
.'{!~i:.i%x
Homodimers
Dilution ...:.::.~
IN VIVO %
::~:"'"'"~::: :~,~
Inactive LPL ( p o t e n t i a l l y active)
Y
Secretion
Vesicle Depolymerizing LPL
.::: :
A c t i v e LPL after s e c r e t i o n
Figure 2--"Activation" process of LPL. This scheme indicates the condensed or "polymerized" state of LPL; it does not show the interactions between LPL molecules and sulfated glycosaminoglycans and heparan sulfate proteoglycans (see text for comments}.
To conclude, the proposed existence of a putative and important reservoir of inactive LPL molecules in cultured adipose cells appears to be related to a systematic underestimation of its in{racellular activity. Whether such a reservoir of LPL molecules of M r = 55,500 is indeed present in mature adipocytes isolated from adipose tissue, remains an open question.
Acknowledgements The author wishes to express his deep gratitude to Drs. C. Vannier, E. Amri, and A. Pradines-Figu~res for their outstanding contribution to this study.
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