Lipoprotein lipase: recent contributions from molecular biology.

Critical Reviews in Clinical Laboratory Sciences, 29(3,4):243-268 (1992)

Lipoprotein Lipase: Recent Contributions from Molecular Biology Johan Auwerx, * Pascale Leroy, and Kristina Schoonjans

Critical Reviews in Clinical Laboratory Sciences Downloaded from informahealthcare.com by University of Otago on 12/31/14 For personal use only.

Laboratoire de Biologie des Regulations chez les Eucaryotes, Centre de Biochimie, UMR 134 du CNRS, Parc Valrose, 06108 Nice, Cedex 2, France Referee: Dr. John D. Brunzeli, Dept. of Medicine, Dlv. of Metabolism, Endocrinology and Nutrition, RG-26, Unlverslty of Washlngton, Seattle, Washington 98195.

* To whom all correspondence should be addressed.

ABSTRACT: Lipoprotein lipase (LPL) is a glycoprotein enzyme that is produced in several cells and tissues. LPL belongs to a large lipase gene family that includes, among others, hepatic lipase and pancreatic lipase. After secretion, LPL becomes anchored on the luminal surface of the capillary endothelial cells. There it hydrolyzes triglycerides in triglyceriderich lipoproteins, generating free fatty acids that can serve either as a direct energy source or can be stored. Through this action LPL plays a pivotal role both in energy and in lipoprotein metabolism. LPL production is regulated in a tissue-specific fashion by developmental, hormonal, and nutritional factors. The recent availability of the regulatory sequences of the LPL gene will greatly facilitate these regulatory studies in the future. In man, several mutations resulting in familial LPL deficiency have been delineated at a molecular level. The study of these mutations is not only very beneficial from a clinical point of view but also contributes in a major way to our understanding of the structure-function relationship of LPL and other lipases. In this review major attention is given to molecular studies relating to the regulation of LPL production, to the defects underlying LPL deficiency, and to structure-function relationship of the lipases. KEY WORDS: triglycerides, secretion, heparin, gene expression, inherited disorders, lipoprotein, cholesterol, atherosclerosis.

1. INTRODUCTION Despite the fact that milky lipemic plasma was already recognized approximately 2 centuries ago, it was not until 2 decades ago that it was shown to be associated with deficiency of the enzyme lipoprotein lipase (LPL). Since then, a wealth of knowledge regarding the structure, function, and regulation of this

1 O40-8363/92/$.50 0 1992 by CRC Press, Inc.

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enzyme has accumulated. Instead of giving an extensive review of older data concerning LPL, we focus on more recent results, which were mainly gathered through the help of molecular biology and genetic approaches. Therefore, we often refer to previous reviews for additional information (e.g., the reviews in the book “Lipoprotein Lipase” edited by Borensztajn, or reviews such as References 1 through 8).


II. SYNTHESIS AND SECRETION OF LPL LPL is a glycoprotein enzyme that, after secretion becomes anchored on the luminal surface of capillary endothelial cells (Figure l).9LPL has been described in several avian and mammalian species. The enzyme is synthesized in several differentiated tissues, such as skeletal and heart muscle, macrophages, adipose tissue, and the lactating mammary gland, but not in adult liver.’ Recently, however, the synthesis of LPL in liver of newborn rats was In all the tissues where LPL is found, it is synthesized as an inactive proenzyme in the endoplasmic reticulum and it becomes activated by consecutive glycosylation Up to 70 or 80% of processes occurring in the Golgi apparatus (Figure l).3*’2-14 the newly synthesized LPL is degraded in the lysosomes before it can be secreted;

LPL SYNTHESIS

PARENCHYMAL CELL

INTERSTITIAL SPACE

VASCULAR LUMEN

WanscrJption

TG-rich lipoprotein

proenzyme glycosylation

secretion

FIGURE 1. Lipoprotein lipase synthesis and secretion. Schematic representation of the different and often complex steps in LPL synthesis and secretion. The mature enzyme is depicted by the two circles attached to its glycosaminoglycan receptor on the endothelial cell wall.

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this amount can be reduced to 25% by heparin treatment of The fully synthesized and latent LPL is stored in secretory vesicle^,^^,^* translocated to the cell surface in a process stimulated by cAMP,I9 and released from the cell by an ~ can be retained active secretion process that is enhanced by heparin in v i 0 - 0 . LPL at the cell surface. Phosphatidylinositols play a role in this anchoring, but the mechanism is unclear at present .20*21 It has been shown that phospholipase C (PLC) treatment induces the release of cell-surface-bound LPL. 20,21 Two possible mechanisms can be invoked to explain this effect. On the one hand, it has been claimed that LPL, in the hypothesis that LPL is anchored to the cell surface via its phosphatidylinositol anchor, is released directly by PLC.20On the other hand, Chajek-Shaul et a1.21propose that LPL release by PLC is due to the release of heparan-sulfate to which the LPL is bound. Furthermore, this release of LPL from the cell surface by PLC could also be a regulated process because a regulated PLC has been described on the cell surface.22 LPL has a high affinity for heparin and related polysaccharides. In fact, a distinct region in the LPL molecule has been identified, which mediates this interaction (see below). Via this heparin-binding site, the secreted LPL, which has moved through the extracellular space, becomes anchored to glycosaminoglycans on the endothelial cell surface, thereby extending into the circulation where it acts upon triglyceride-rich lipoproteins. Recently, it has been shown that this anchoring is mediated by a specific heparan-sulfate proteoglycan receptor for LPL on the endothelial cells, which is actually also involved in recycling and transcytosis of LPL.23Therefore, the distribution of LPL on the endothelium is governed both by the sites where the enzyme is being synthesized and by the distribution of LPL receptors .24 Intravenous heparin administration can competitively displace LPL from the endothelial cell surface receptor, enabling the measurement of LPL activity and immunoreactivity in plasma. The LPL found on the endothelial surface usually occurs as a noncovalent homodimer. This homodimeric LPL is less susceptible to degradation and is stabilized by heparin. LPL has also been found in the circulation, a substantial portion being lipoprotein a s ~ o c i a t e dWhether .~~ all of it is associated with lipoproteins is not yet determined, but it is clear that feeding can induce some shifts in its association with certain lipoproteins. The exact function of the circulating LPL remains, however, to be determined. LPL is cleared from the circulation by the liver, which is ultimately responsible for the degradation of LPL and therefore is also partially responsible for determining the levels of LPL found in the

111. PHYSIOLOGY OF LPL LPL hydrolyzes triglycerides of exogenous and endogenous origin (Figure 2A). Like the two related lipases, pancreatic lipase and hepatic lipase (HL), LPL

hydrolyzes the 1,3 position in tri-, di-, and monoglycerides. For this activity, LPL requires the presence of apolipoprotein CII as a cofactor. This apolipoprotein

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Functions A

Hydrolysis of

VLDL

of

Lipoprotein Lipase

B

triglycerides

-0

% to d

Retention of

lipoproteins

hebat cell Modification of Storage or utilizatbn


extracellular

c

Secretion of

apo

nascent lipoproteins

D

B-containing particles modified liwwoteins

hepatocyte

I

Internalization of remnants

FIGURE 2. Functions of the LPL molecule. (A) LPL is involved in the hydrolysis of triglycerides in triglyceride-richlipoproteins, such as VLDL. It functions as a homodimer bound via a specific glycosaminoglycan receptor on the vascular side of endothelial cells. In the process, free fatty acids are generated, which are used for energy or storage. (B) LPL bound to the extracellular matrix can retain lipoproteins in the extravascular space, thereby making them more susceptible to different modifications (such as oxidation, depicted by lightning), which in turn facilitates the uptake of the resulting lipoproteins by scavenger cells like the macrophage. (C) LPL also regulates the production rates of apo-&containing lipoprotein particles. In fact, it reduces the secretion rate because it facilitates the reuptake of these freshly secreted particles by modifying them. These phenomena then suppress secretion of new particles. (D) LPL, bound on the surface of lipoproteins, functions as a ligand for the low density receptor related protein (LRP), thereby enhancing the uptake of chylomicron remnants and p-VLDL particles by this receptor in liver cells.

is present in excess in the plasma and occurs on the substrate lipoproteins of LPL.In contrast, apolipoprotein CIII acts as an inhibitor of LPL activity.26This inhibitory activity was illustrated dramatically by the occurrence of a marked hypertriglyceridemia in transgenic mice overexpressing the apo CIII gene.27 LPL, together with apo CII, is responsible for the hydrolysis of triglycerides in triglyceride-rich lipoproteins, which provide free fatty acids that are utilized for oxidation (for instance, as a source of energy for muscle) or storage (such as is the case for adipose tissue). Therefore, LPL seems to play a gatekeeper role for energy storage.Z8The lipoprotein remnant particles generated by the interaction of LPL upon chylomicrons or VLDL can either be further processed by means

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of hepatic lipase into LDL29 or removed from the circulation by the remnant receptor. Consequently, LPL plays a pivotal role not only in energy utilization and tora age^*,^^ but also in lipoprotein metabolism.2 LPL not only has a major role in the metabolism of lipoproteins in the circulation, but it also interacts with lipoproteins on a more localized basis (Figure 2). In the artery wall, it has been demonstrated recently that LPL increases the retention of LDL and VLDL particles to the subendothelial matrix, potentiating their conversion to more atherogenic forms (Figure 2B).3iOnce these lipoproteins are retained, LPL may act to hydrolyze triglycerides, resulting in particle modification. These altered particles are ultimately taken up more avidly by several cells in the microcosmos of the atherosclerotic plaque^.'^ Other LPL-independent mechanisms, such as conversion of the retained lipoproteins to more oxidized species, may also enhance the uptake of the particles into cells in the atherosclerotic plaque. A similar binding or retention of lipoproteins may also occur on the surface of several cells, such as Hep G2 cells or fibroblasts, where LPL acts as a bridging agent between proteoglycans on the plasma membrane and lipoprotein^.^^ These last authors also suggest that the cellular binding may be involved in receptor-independent removal of lipoproteins from the circulation. LPL may be implicated in both the control of the productiod4 and removal of lipoprotein^.^^ In fact, the mechanism by which LPL regulates the production of apo-B-containing particles suggests that the net secretion rate of apo-B-containing particles is reduced by enhanced reuptake of nascent lipoproteins, a mechanism facilitated after modification of the lipoproteins in the presence of LPL (Figure 2C).34The role of LPL in the removal of chylomicrons from the circulation and their subsequent hepatic uptake cannot be completely ascribed to the remodeling of triglyceride-rich lipoproteins but appears to be mediated by the LDL-receptorrelated protein or LRP (Figure 2D).35In an elegant set of experiments, it was demonstrated that in Hep G2 cells, LPL enhanced the uptake of chylomicron remnants or P-VLDL particles in an LDL-receptor-independent fashion. Furthermore, the authors showed that LPL could be directly cross-linked to the LRP protein, indicating that both LPL and apo E can act as ligands for the LRP. This role of LPL as a ligand for receptors had been suggested previously by Felts et al.36and Goldberg et al.25

IV. STRUCTURE OF LPL Complementary DNA clones for LPL from several species, including man,37*38 guinea pig,4o and are currently available. In man, two mRNA species of approximately 3350 and 3750 bp are produced by alternate polyadenylation sites. The mRNA predicts that the preprotein contains 475 residues, from which a 27 aa (amino acid) prepeptide is removed to give rise to the 448 aa mature LPL protein. Taking this into account, one would predict 50,394 mol wt for the unmodified protein, which, after glycosylation, would translate to approximately 55,000 mol wt for the mature monomeric form of LPL. This 247


estimated molecular weight is consistent with values obtained after SD PAGE. It is unclear why these values differ from the values of 41,70043and 48,3004 that have been obtained by sedimentation equilibrium ultracentrifugation. The active form of LPL consists of a noncovalent h o r n ~ d i m e r .This ~ . ~ hom~ odimer is in equilibrium with the monomeric form, which, in contrast to the homodimer, is inactivated more rapidly. Based on the primary sequence and on the homology to human pancreatic lipase (PL), for which the three dimensional crystallographic structure has been determined,46several structural domains have been proposed in LPL. Strong sequence homology between LPL and PL, as well as a highly conserved positioning of disulfide bridges, suggests that the amino acids 132ser,Is6asp, and "'his, present in the central conserved region (which forms P-pleated sheets), forms the catalytic triad of the enzyme. Another highly conserved region on primary sequence analysis (Table 1) is located between aa 125 and 142 of human LPL. This domain is suggested to function as the interfacial lipid-binding d ~ m a i n . ~Although ~ * ~ ' the study of genetic mutants (see below) confirms that this is a crucial region for LPL to retain activity, other sites located in the C-terminal portion of LPL also appear to be important in determining interfacial lipid r e c ~ g n i t i o n . ~In ~ *human ~* PL, a lid or flap structure, which covers the access to the active site, has been suggested as being important for substrate positioning. Similarly, the region between aa 216 and 239, which contains a disulfide bridge, may function as a flap in LPL. Limited proteolytic digestion of LPL49has been suggested to remove part of this flap and alter kinetics of LPL activity in a major fashion.s0The region of LPL involved in the interaction with its cofuctor apolipoprotein CZZ has been defined to reside in the first 314 aa of the LPL molecule. Yang et al.51suggested that a region rich in positively charged amino acids around 14*lysand 1491ysis important for this interaction. The heparin-binding properties of LPL and HL were initially suggested to be confined

TABLE 1 Partial Amino Acid Sequence and, Homology Comparison of the Interfacial Lipid Binding Site of LPL from Various Species Amino acid sequence Rat Mouse Human Bovine Guinea pig

LEEEFNYPLDNVHLLGSLGAHAAGVAGS M......... .................. M........................I.. MAD ......G...............I.. M. D ..K. SV.

..................

Note: The aa sequence around the interfacial lipid-bindingregion of rat,47mouse:2 human,37 and guinea pig" LPL were aligned. Only amino acids differing from the rat sequence are indicated. The interfacial lipid-binding domain with the essential serine-residue (bold; residue 132) is underlined.

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to a stretch of basic amino acids between residues 290 and 300.40Studies with chimeric lipase defined the heparin-binding region to the COOH terminal part of LPL (between amino acid residues 312448).51aCareful analysis of LPL mutants, however, suggests that missense mutations far outside this site, such as '76ala and 188gly,also affect LPL interaction with heparin as well. Therefore, we suggest that the interaction of LPL with heparin is also highly dependent on its tertiary structure. ~ ~ . ~ ~is due Mature LPL contains approximately 7 to 8% c a r b ~ h y d r a t e ,which to N-glycosylation of two sites. 1651.53-56 Independent examinations of the LPL protein revealed N-glycosylation at residues asn43 and asn359 in guinea pig,53 residues am44 and asn361 in COW,^' and asn45 in chicken.54Furthermore, chicken LPL appears to contain a sulfate moiety associated with a complex oligosachar ride.^^ In human; no direct data on glycosylation are available but, by utilizing site-specific modification, it has been shown that residue asn43 is the most important glycosylation site in human LPL because mutation of this asn to an ala prevents the occurrence of intracellular or secreted LPL activity.57 Two other experiments further underscored the importance of glycosylation in the secretory process. First, the accumulation of intracellular enzyme when 3T3-Ll adipocytes were incubated with the glycosylation inhibitor t ~ n i c a m y c i nand, ~.~~ second, mice with the cldlcld mutation have catalytically inactive enzyme due to abnormal glycosylation, despite the presence of a normal amino acid s e q ~ e n c e . ~It~ .is~ * hypothesized that glycosylation is important in LPL activity because it is involved in determining the conformation or the assembly of the active enzyme. The protein and DNA sequences of all species, for which LPL cDNA clones are available at present, show a remarkable degree of conservation, which is highest in the region containing the interfacial lipid binding region (see Table 1). LPL itself belongs to a conserved family of related serine esterases (reviewed in Hide et al.59), to which hepatic lipase and pancreatic lipase belong (Figure 3). Interestingly, three dipteran proteins, the Drosophila yolk proteins 1, 2, and 3 (or vitellogenins), which show no lipolytic activity but act as lipid transporters, also share some sequence similarity .50@-'-63 The similarity between the different

,

Pancreatic lipase

Ancestral Lipase

f

/

LPL

Lipoprotein-metabolizing Lipaso

Primordial Gene HL Yolk Proteins

FIGURE 3. Model for the evolutionary relationship of the lipase superfamily and the Drosophila yolk proteins.

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members of the lipase gene family also extends to the three-dimensional strucThe genomic structure of the human LPL gene, which spans approximately 30 kb, has been determined by several group^.^^.^^ It was shown that, like the gene for hepatic lipase, the LPL gene contains 10 exons interrupted by 9 introns. The pancreatic lipase gene, on the contrary, has 13 exons and 12 introns, suggesting a divergence of digestive lipase and lipoprotein metabolizing lipase by gene duplication from a common ancestral lipase (reviewed in Hide et al. ,59 see Figure 3). Another duplication event then allowed the lipoprotein metabolizing lipase to further specialize into LPL and hepatic lipase, two enzymes that catalyze different but complimentary steps in lipoprotein metabolism. The multiple functional domains in LPL are postulated to be confined to specific exons. The asnlinked glycosylation site required for the expression of enzyme activity is assigned to exon 2, the catalytic serine and the lipid-binding domain to exon 4,the highly conserved central domain to exon 5 , and the heparin-binding site to exon 6.37,s7-s8*63.6s.66 The locus for the human LPL gene is located on chromosome 8.67

V. REGULATION OF LPL PRODUCTION The synthesis, processing, and secretion of LPL is regulated in a complex fashion during development and in response to dietary or hormonal changes. At least part of the regulation of LPL activity appears to be linked to alterations in transcription rates. Developmental changes are usually associated with important changes in LPL expression. In rats, for example, adipose tissue and heart LPL activity both increase during development. Whereas heart LPL activity reaches its maximum approximately at day 20, adipose tissue activity reaches a peak at day 40,after which it decreases to adult levels.68The change in heart LPL activity can be attributed to an increased transcription because LPL mRNA levels also increase strongly in the first month after birth.69No direct data are available for the expression of adipose tissue LPL mRNA during development, but upon differentiation of mouse fibroblast 3T3-Ll or Ob 1771 cells to adipocytes one also sees an increase in LPL gene t r a n s ~ r i p t i o n This . ~ ~ ~increase, ~~ translated to the in vivo situation, may correspond to the postnatal increase in adipose tissue LPL activity. Similarly, in monocytes where no LPL mRNA or activity is present, an induction of LPL mRNA is detected upon their differentiation into macroThe liver forms a notable exception to the rule that LPL expression is induced during development and differentiation. In this organ, LPL mRNA gradually disappears upon development in a fashion reminiscent of the decrease in a-fetoprotein mRNA.41,74 Fasting and feeding have major influences on the regulation of LPL activity, and the mechanisms of regulation apparently change when one looks at different regulatory events. In several species (including human, rat, mouse, guinea pig, and chicken), adipose-tissue LPL activity increases during feeding, whereas muscle and heart LPL activity have a tendency to decrease (reviewed in Ecke130).

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How this increase in adipose-tissue LPL is mediated is, however, less clear. Generally, it was believed that the response to feeding is mediated via altered LPL synthetic rates in adipose tissue. Consistent with this hypothesis, upon fasting, g ~ i n e a - p i gand ~ ~ chicken76have been reported as showing a decrease in LPL mRNA levels and synthetic rates. In rat and human adipose tissue, it was, however, reported that LPL mRNA levels and LPL synthesis did not change upon feeding.77-79These authors attribute the increased activity to a posttranslational phenomenon resulting in a higher LPL a ~ t i v i t y . Whether ~ ~ - ~ ~ these differences can be attributed to differences between various species or to differences in study design is unclear, but more studies on this subject are definitely needed. Note that fasting in the guinea pig did not induce an alteration in LPL mRNA levels in heart,75 although in the earlier literature fasting in several species has been associated with increased LPL activity in heart and skeletal muscle (reviewed in Borensztajnso);however, the mechanisms causing this increase are still undefined. In obesity, elevated adipose-tissue LPL activity has been reported (reviewed in Ecke130).Furthermore, it was shown that weight loss of very obese subjects, after hypocaloric regimens, was associated with elevations in adipose-tissue LPL activity and LPL mRNA levels.8' This enhanced LPL production may ultimately be the element that maintains the adipocyte volume constant and helps to preserve adipose tissue mass. Although an induction of adipose-tissue LPL activity has often been noted after administration of insulin, a key hormone governing the metabolic response to feeding, there is still controversy whether the effect of insulin can be ascribed to a direct effect on transcription or to a posttranscriptional p h e n o m e n ~ n .Improved ~.~~ diabetic control in diabetes patients, achieved either by insulin or with glyburide, resulted in increased adipose-tissue LPL activity and synthetic rates without alterations in LPL mRNA,82 suggesting that better insulin supplies do not affect transcription in vivo. In vitro, insulin increases the net amounts of LPL secreted by cells (reviewed in Ecke130); however, there are two different mechanisms described by which this is attained, depending upon the system used. First, insulin has been shown to increase LPL activity and synthetic rates in isolated primary rat adipocytes through an increase in mRNA which is caused by an altered stability of the LPL mRNA rather than through transcriptional i n d u ~ t i o n .Second, ~~ similar studies performed by Semenkovich et al .70 in differentiated mouse 3T3-Ll cells confirmed increased levels of LPL activity in the three compartments measured (intracellular, cell surface, and medium) but failed to detect a corresponding increase in LPL mRNA or transcription rates. Whether this controversy can be attributed to the differences in cell culture systems used awaits further study. The above-mentioned regulation by insulin appears to be specific for adipose tissue because only limited effects were detected in muscle LPL (reviewed in Borensztajnso). Other hormones that have been shown to play a major role in the regulation of LPL production are ligands of the steroidlthyroid hormone receptor supergene family. Glucocorticoid hormones have been shown to alter LPL in a number of cells and tissues. Although in the earlier literature there was some controversy

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regarding the effects of glucocorticoidson adipose tissue LPL (reviewed in EckeP), Ong et al.85recently reported a decrease in adipocyte LPL synthetic rates, secretion, and LPL mRNA levels both in vivo in rats and in vitro in cultured adipocytes. A similar decrease in LPL mRNA and LPL activity has been reported in the neonatal liver74where it was demonstrated that glucocorticoid administration enhanced the disappearance rates of LPL mRNA upon development. Glucocorticoids have, however, an inductive effect on LPL mRNA and activity level in the monocyte/macrophage.86Similarly, muscle LPL activity appears to be induced by glucocorticoid administration (reviewed in Borensztajnaoand CryeF’). Further studies are needed to sort out whether these effects are mediated by the same or different glucocorticoid responsive elements (GRE). Sex steroids are also noted for their effects on lipoprotein and lipid metabolism. The effects of sex steroids on gene expression are known to be highly species specific. For example, in the rat it has been shown that estrogens decrease LPL activity in adipose tissue (reviewed in Ecke130). However, we recently demonstrated that this decrease was due to lowered LPL mRNA levels and was specific for adipose tissue because no effects of estrogens on heart LPL mRNA levels or activity could be detected (Pernado-Onsurbe et al., unpublished data). The situation is completely different in rabbits, where a strong increase in adipose-tissue LPL mRNA and activity after administration of ethinyl estradiol was detected.a8In humans, finally, results are conflicting. Either no effect of estrogens on adipose-tissue LPL or on postheparin plasma LPL activitya9or a decrease in LPL activityg0has been reported. In rats, administration of high concentrations of androgens has been reported to decrease adipose tissue LPL (reviewed in Ecke130). We, on the contrary, could not demonstrate any significant effects of castration and subsequent administration of testosterone, dihydrotestosterone, or nandrolone administration upon adiposetissue LPL activity or mRNA levels in male rats (Pernado-Onsurbe et al., unpublished data). Progesterone has been reported to increase LPL activity in rat adipose tissue in an estrogen-dependent way (reviewed in Ecke130), while on a cellular level progesterone increases cellular LPL activity in 3T3-Ll adipocytes.91 In neonatal liver cell lines, such as BWTG3, progesterone can induce LPL gene transcription, resulting in a net increase in LPL secretion in the medium.92Thyroid hormone is also reported to affect LPL production. In rats, thyroid hormone does not regulate adipose-tissue LPL transcription rates and mRNA levels, but it nevertheless results in an increased LPL activity, mass, and synthetic rate in adipose tissueg3or in heart muscle (reviewed in Borensztajnao).In humans, hypothyroidism is usually characterized by accumulation of lipoprotein remnants, and a decreased muscle and adipose-tissue LPL activity, although no change in LPL activity is detected in hyperthyroid states (reviewed in E~kel,~O Borensztajn,80and CryeP7). In cell culture, it is very hard to distinguish between a direct effect of thyroxine on LPL expression and an effect of thyroxine on adipocyte cell differentiati~n.~~ Other polypeptide hormones and growth factors also appear to have an effect on LPL production. Mucrophuge colony -stimulating factor induces LPL mRNA and LPL activity in monocytes and macro phage^.^^ In the breast, LPL is a crucial enzyme because it allows hydrolysis of plasma triglycerides for milk formation. 252


During pregnancy, mammary gland has a low LPL activity but this changes drastically around parturition. First of all, suckling appears to be a prerequisite for the maintainance of high LPL activity in the breast, but profactin is thought to be the crucial hormonal regulator because it has a strong inductive effect on mammary LPL production (reviewed in Scow and C h e r n i ~ k ~In~ )adipose . tissue, however, parturition and lactation are associated with a reciprocal decrease in LPL activity, which, it appears, is not caused by prolactin (reviewed in EckePO). Another peptide hormone, growth hormone, was also shown to increase LPL mRNA synthetic rate, and, consequently, LPL production in adipocyte cell lines.97 In contrast to those hormones that activate nuclear receptors, several of these polypeptide hormones use classic signal transduction pathways to activate cells. The breakdown of membrane phospholipids, initiated by cellular activation by hormones or growth factors, results in the activation of the protein kinase C (PKC) / Ca2+signal transduction pathway. Pradines et al. demonstrated that part of the effects of growth hormone could be ascribed to a stimulation of the PKC signal transduction pathway.97Previously, it was also shown that in the macrophage-like THP-1 cell line, stimulation of the PKC / Ca2+ signal transduction system resulted in the induction of LPL activity and mRNA level^.^*.^^ Agents that increase intracellular CAMP concentrations, such as epinephrine, choleratoxin, or ACTH, also have an important regulatory effect on LPL activity. In muscle and heart, such agents generally increase LPL activity when studied in vivo.*O In cultured muscle cells, conflicting results are reported: these substances have been shown to have either no effect,99to cause a decrease,2’.lmor to cause an increase in LPL activity.lO’Elevation of CAMPlevels in adipose tissue or in adipose-tissue pads and cultured adipocytes results in decreased LPL production. More recently, two new and apparently unrelated factors (i.e., tumor necrosis factor and fibrates) have been shown to influence LPL production. However, the fact that they both have important effects on cell proliferation and differentiation may provide a common link between them. Tumor necrosisfuctor was shown to specifically decrease adipose tissue LPL production by inhibition of LPL gene transcription,7 I 75* 103- 108 an effect potentially related to the anti-adipogenic and dedifferentiating properties of TNF on adipose tissue. Because TNF inhibits the expression of several adipogenic genes, the ultimate effect is a loss of morphologically differentiated adipose tissue and the occurrence of cachexia. Io3 In the liver of the adult rat, TNF administration has the opposite effect because a sharp induction of LPL mRNA levels (not normally present in adult liver) is detected after TNF, demonstrating once more the important tissue-specific differences in the regulation of LPL expre~sion.’~ Whether this reinduction of liver LPL can be explained by the regression of the liver to a more undifferentiated state awaits further study, but our recent data usingfibrutes and experiments in neonates support the hypothesis that liver differentiation may be involved in the regulation of LPL t r a n ~ c r i p t i o n .Fibrates ~ ~ * ~ ~ are potent hypolipidemic drugs that induce peroxisomal proliferation and, upon prolonged administration to rodents, can induce proliferation of liver cells,”O resulting in hepatoma formation. In 309102

I

253


fact, when we administered fibric acid derivatives to adult rats, a reappearance ~' of LPL transcription and of LPL mRNA in the liver could be i n d ~ c e d . This effect was specific for the liver because transcription rates did not change in adipose tissue or muscle. Therefore, we suggest that the neonatal state, fibrate administration, and TNF administration are all characterized by an enhanced proliferative capacity of the hepatocytes, such as is seen in the undifferentiated state. It is not clear if the hepatic reinduction of LPL by fibrates can also be seen in humans, but it is interesting to note that treatment with fibrates augments LPL activity in postheparin plasma. ' I 2

VI. REGULATORY SEQUENCES AND TRANSCRIPTION FACTORS Enormous amounts of work will be necessary, however, to determine which potential cis-acting sequences and corresponding transcription factors are functionally implicated in the fine tuning of LPL production in vivo. The availability of the genomic sequences, including the upstream regulatory sequences (URS), make the detailed mapping of cis-acting sequences and the study of proteins involved in transcriptional regulation now feasable. Within the reported 5 ' URS of the human LPL gene, several potential binding sites for transcription factors have been identified: a TATA box is present at position - 30, two CAAT motifs are located at - 65 and - 506 and may represent potential binding sites for NF1, two potential C/EBP sites are located at - 68 and - 509, a potential CAMP responsive element may be located at - 306, two potential fat-specific elements are found at positions - 630 and - 360, and, finally, there are three potential binding sites for octamer (Oct) transcription factors at -580, - 186, and -46.63,65J13 The human LPL 5' URS exhibits a striking degree of homology with the murine LPL gene, which is exemplified by the conservation of several potentially important cis-acting motifs. The first of the potential binding sites demonstrated to have a physiological importance in the human gene was the Oct site at position -46, which was shown to bind the Oct-1 and Oct-2 proteins."3 Deletion of this site caused a decreased expression of chimeric LPL 5' URS-CAT constructs upon transfection of either 3T3-Ll or Hep G2 cells, arguing for a role of this site in LPL gene expression. Also in the mouse 5' URS, this Oct-1 binding site was shown to be important for the expression of the LPL gene.Il4 In fact, it was shown that the Oct-1 binding site was situated in a domain that was hypersensitive to endonuclease digestion, suggesting that this site may be sufficient to alter the chromatin state, thereby rendering it more accessible to other transcription factors.Il4 Furthermore, several groups identified a region in the LPL promotor that is involved in suppressing LPL transcription rates. 1 1 3 . 1 1 5It was postulated that this region may be important in determining LPL production rates in several tissues and is responsible for the LPL extinction that occurs in adult liver (see above). In close proximity to this region, we were recently able to identify a functional GRE. This element binds bacterially produced glucocorticoid receptors and has been shown to mediate the inductive response of the LPL gene to glucocorticoids upon transient transfection experiments, 155 (Devos, Schoonjans, Auwerx, unpublished data). 254


VII. MEASUREMENT OF LPL ACTIVITY, MASS, AND mRNA LPL activity can either be assayed in postheparin plasma or in tissue preparations. LPL measurements in plasma are relevant in the context of evaluations of lipoprotein metabolism. The administration of an IV bolus of heparin (50 to 100 Uikg of a heparin preparation with a molecular weight of 12,000 to 18,000 Da) releases several lipolytic activities (including LPL and HL), which result in hydrolysis of plasma triglycerides. The release of LPL is rapid, with a maximum of circulating LPL activity 5 to 10 min after the bolus, after which the enzymatic activity returns to baseline values within 2 h. Plasma LPL activity remains stable if immediately refrigerated, quickly frozen, and stored at - 80°C until analysis. Direct measurements of LPL activity in different organs are an alternative to the measurement of LPL activity in postheparin plasma. This procedure is not only helpful in evaluating local LPL production but also to detect LPL deficiency. Furthermore, the tissues most frequently analyzed (muscle and adipose tissue) do not produce HL, a confounding lipase activity in plasma. The methods employed to analyze LPL activity in tissues are widely adapted for use on small specimens obtained through needle biopsy of adipose tissue and skeletal muscle. LPL activity can then either be measured in acetone-ether preparations or in heparin eluates of the respective tissue^.^^*''^ Modern lipase activity assays are relatively easy to carry out and excellent reviews describing the measurement of lipase activity were written by Iverius and Ostl~nd-Lindqvist~~ and Nilsson-Ehle. 'I6 Typically, these assays involve the production of an oil-in-water emulsion by sonication of radiolabeled substrate triglycerides (3H or I4C triolein are used) with an emulsifier. The purity of the substrate is extremely important to ensure a reproducible high-quality assay. For the emulsifiers, one has a choice between phospholipids, synthetic detergents, and certain polysaccharides. A fatty acid acceptor (like albumin) and the LPL cofactor apo CII (usually in the form of serum) are added to this oil-in-water emulsion. The addition of albumin as fatty acid acceptor is extremely important because LPL is very susceptible to product inhibition, necessitating the need for efficient removal of fatty acids produced during the incubation. Pooled human serum is the most frequently used source of the apo CII activator protein. To minimize variation between different batches and to destroy lipolytic activity it is advisable to heat this serum to 56°C for 1 h. Serum can, however, be substituted for with purified apo CII. The incubation of enzyme (sample) with the substrate is carried out at an alkaline pH (in view of the alkaline pH optimum of LPL) at 27 to 37°C for a time interval consistent with zero-order kinetics, an adequate signal-to-noise ratio, and short enough to prevent enzyme denaturation. Consecutively, the reaction products (fatty acids) are extracted, isolated, and quantitated. Activity is then usually expressed in units, corresponding to the number of moles of fatty acid produced per minute and per milliliter. LPL activity in postheparin plasma has to be differentiated from HL activity, which is released simultaneously. The specificity of enzymatic measurement is therefore often established by the original physicochemical characteristics of LPL: 255


inhibition by I M NaCl, serum stimulation, and alkaline pH optimum. The use of specific antibodies to block either HL or LPL is a more efficient and accurate way to distinguish between these two lipolytic activities in postheparin plasma. Other techniques to obtain specificity for LPL are the separation of the two enzymes before activity assay by chromatography on heparin-~epharose,~' use of chemical inhibitors for HL (such as SDS), or use of specific substrates.'"j Recently, newer technologies allowed more detailed analysis of LPL both in the clinical and more basic setting. The availability of antibodies against LPL allowed the construction of several irnmunoassays specific for LPL immunoreactive muss. 117-121 These assays are not only easier and more accurate to perform but they cany the added advantage that they also allow the detection of inactive LPL imrnunoreactive material in the plasma. The availability of LPL irnrnunoassays was especially beneficial in the exploration of LPL deficiency. It enabled Babirak et al. l Z 1 to define the heterozygote state of LPL deficiency and, furthermore, was crucial in the analysis of LPL defects in LPL-deficient subjects. 1 2 2 ~ 1 2 3 Also, several more cellular- and molecular-oriented investigations were aided by the exploitation of antibody-based techniques, such as immunoprecipitations, ELISA's, or Western blotting. Furthermore, thanks to mRNA quantification techniques, we are currently capable of assaying LPL mRNA levels and production rates in humans in a clinical setting. Indeed, in contrast to several other proteins, LPL is produced in tissues that can be easily and almost harmlessly sampled. Needle biopsy, for instance, is sufficient to obtain adipose or muscle tissue to determine LPL mRNA levels.81 Determination of LPL mRNA can, in these samples, be combined with determination of LPL activity and/or mass, allowing a more complete picture of the LPL production pathway to be formed. Furthermore, a simple venipuncture can give a sufficient amount of macrophages to analyze macrophage LPL mRNA.

VIII. LPL DEFICIENCY AS A TOOL FOR STUDYING LPL STRUCTURE-FUNCTION RELATIONSHIP

A. Clinical Manifestations of LPL Deficiency The clinical hallmarks of familial LPL deficiency were recently and excellently summarized by Brunzell,z and therefore we will limit ourselves to a brief summary of the most important characteristics. Familial LPL deficiency is characterized in its homozygous form by severe fasting hypertriglyceridernia due to accumulation of chylomicrons. It is a relatively rare autosomal recessive disorder in the general population (frequency: 1 in lo5 or lo6) but in certain populations The carrier or the frequency can be as high as 1 in 5,000 to 1 in 10,000.124 heterozygote state of LPL deficiency in some areas is therefore as (or even more) prevalent as the heterozygote state of familial hypercholesterolemia. Iz4 Clinically, the homozygote form of this disease is usually detected in children by the occurrence of repeated bouts of colicky abdominal pain, pancreatitis, eruptive xan256


thomathosis (present in about half of the subjects), and hepatosplenomegaly. The plasma has a milky appearance (with plasma triglycerides usually above 2000 mg/dl), and LPL activity is absent in postheparin plasma in these subjects. Often, memory loss is also noted in these patients and, if subjects present with a triglyceride level above 4000 mg/dl, lipemia retinalis can be detected by fundoscopy . Secondary causes of hypertriglyceridemia, such as diabetes mellitus, alcohol intake, estrogen therapy, paraproteinemic disorders, and therapy with certain antihypertensive drugs, need to be excluded before the diagnosis of familial chylomicronemia can be established. Familial chylomicronemic syndromes are mostly due to familial LPL deficiency but can also be caused by familial deficiency of apo CII, a rare autosomal recessive disease in which apo CII, a cofactor of LPL, is deficient (reviewed in Santamarina-Fojo8), or by the extremely rare autosomal dominant occurrence of an inhibitor of LPL. 125 Utilizing both LPL activity and immunoreactivity determinations, Babirak et al. 12' were able to identify a heterozygote state of LPL deficiency characterized by LPL activity and mass levels intermediate between normal and LPL-deficient states. Furthermore, often a mild variable hyperlipidemia (of the familial combined hyperlipidemia type) was detected in relatives of probands with LPL deficiency. The relatives with abnormal lipid levels in such families were almost all heterozygotes for LPL deficiency. This led Babirak et a1.l2'jto suggest that some of the patients with familial combined hyperlipidemia could be classified as heterozygotes for LPL deficiency. Interestingly, Williams et al. 34 proposed a mechanistic explanation for the role of LPL in familial combined hyperlipidemia, a disease characterized by increased secretion of apo-B-containing particles. These investigators showed that the net secretion rate of apo-B-containing particles is reduced by enhanced reuptake of nascent lipoproteins, a mechanism facilitated after modification of the lipoproteins in the presence of LPL.34 Furthermore, several recent studies demonstrate a role for LPL in the clearance of lipoproteins via receptor-dependent (LRP)35or -independent mechanisms. 3 3

6. Molecular Defects in LPL Deficiency Through application of molecular techniques, major advances have been made in the molecular diagnosis of LPL deficiency. Development of specific antibodiesl 17-121 and their use in ELISA'21,123 or the use of radioimmunoassays to determine LPL mass were of great value for deciphering the various defects of the LPL protein, and this enabled classification of LPL protein defects. 122 Through the use of molecular genetic techniques, and the widespread application of the polymerase chain reaction in the amplification of cDNA or genomic DNA fragments in patient samples, several LPL mutations were mapped at a molecular level (reviewed in Lalouel et al.' and Santamarina-Fojo8).This provided us with useful information regarding structure-function relationships in the LPL molecule. Although at first sight LPL deficiency appears to be a homogeneous disorder, closer analysis of the LPL protein at a molecular level shows that this group oi 257 '


disorders can be subdivided into three large subgroups (Table 2). ‘22 The combined measurements of LPL activity, and pre- and postheparin LPL protein, with an ELISA, enabled the distinction of three different abnormalities. Class Z patients have no detectable LPL immunoreactivity in plasma, consistent with the absence or major alterations in the LPL protein. The complete absence of LPL may be explained by a putative “null allele’’ induced by a major defect in the LPL gene. The class ZZ subjects have no immunoreactive LPL material in preheparin plasma but have an increase in LPL immunoreactive material after heparin administration. Their LPL can also bind normally to heparin-Sepharose. It is hypothesized that these patients produce a catalytically inactive LPL in which the heparin-binding domain is still intact. In contrast, patients with a class ZZZ defect have LPL immunoreactive material both in pre- and postheparin plasma, with no further increase in LPL immunoreactive material upon heparin administration. The LPL immunoreactive material of these subjects, furthermore, does not bind to heparinSepharose, suggesting that the molecular defect in these subjects may include the heparin-binding site as well as the catalytic site. These studies at the protein level were quickly complemented by molecular identification of mutations at the gene level. These variants include insertions, deletions, frameshift mutations, splicing defects, and nonsense and missense mutations (summarized in Figure 4). Some of the protein defects resulting from several of the mutations so far identified at the genetic level could be fitted into the classification resulting from previous immunological work (see Table 3). There are, however, difficulties in classifying some of the compound heterozygotes because insufficient protein data are available at present. Class I mutations, which result in a null allele, were usually caused by severe mutations such as deletions, insertions, or nonsense mutations. Only one missense mutation (i.e., I4*gly to glu) located in the interfacial lipid-binding site (aa 125 to 142)37gave rise to a null allele. Note that, so far, none of the genetically identified mutations belong to the class I11 subgroup of protein defects, which also have defective heparin binding. This might suggest that class I11 mutations are caused by amino

TABLE 2 Classification of LPL Mutations According to Protein Defects LPL immunoreactive mass Class I I1 111

LPL activity

Preheparin

Postheparin

Proposed defect

Absent Absent Absent

0 0

0

Null allele Defect in catalytic site Defect in heparin binding and in catalytic site

.

t t t t

t t

Based on findings reported in Auwerx J, Babirak SP, Fujimoto WY, et al. fur J Clin lnvest 1989; 19: 433-7.

258

Mutations in the LPL gene

+I t oa

.I


i

0 b 0 c

1‘

‘fa

3

2

I 1

5

4

JJJL

eeeeee

e e e e

3

8 910111213

14 1 5 1 6 1 7

0 + Thr

10 11

4 5 6 7

Nonsense mutations S W

b

Deletions

Slap

c

6 kb deklwn arm 3-5

st*224

d

3 kb delelion exon 9

Pap

e

Splicing Mutations

f

Spl~e dorm G

Q

Sp(reacccp1wAG + A A

3

207p,,

4

5

2 1 6 -D ~ ~ Ssr ~ 2 4 3 ~ ~ HIS ~

+ Glv

6

2 4 4 ~-D~ Thf ~ 15

3 8 2 ~ .+ ~ ~stap

1 5 7 ~ 1 0-D Alp

7

2SOkp -D PM

447ser

+ Thr

8

26ZTyr

188Gh -D Glu

9

156kp

176m

+

+ Hit

13 14

16

1OZThr

22ldG 262Tyr

-

+ .+

194!lC

10601,

V

a

2 0 4 h p -D Glu

-D Leu 12

Insertions

stop

2~

+ 1 4 2 ~ 1+ ~ Glu 1 5 6 + ~ ~kI~

0

a

1

1 3 6 ~ ~ Arg

pdrpb

11111 I J i1

eeeee

+ 6

6

1

-0 ~ ~

8

$ f-H ;”

2

Missense Mutations 9kp

3 kb

;

6 kb

L)

.+ stop

2 kb aplicalim erm 6

A

1

2

l7

FIGURE 4. Mutations in the LPL gene. Different mutations in the LPL gene are depicted by various symbols. Missense mutations are represented by a closed circle, nonsense mutations by an open circle, splicing mutations by a plus sign (+), insertions by a triangle, and deletions by a line representing the extent of the deletion. Notice the clustering of missense mutations in exons 4, 5, and 6. Following are Reference numbers describing the various mutations. Missense mutations: 1: 131-133; 2: 132, 133; 3: 132; 4: 134; 5: 127; 6: 127, 128; 7: 135; 8: 136; 9: 133, 137-140; 10: 141, 142; 11: 143; 12: 140, 144; 13: 127; 14: 141, 143; 15: 145; 16: 131, 133; 17: 131. Nonsense mutations: A: 143; B: 146; C: 133, 147; D: 132, 148; E: 149; F: 143; G: 129; 150. Insertion:151, 152. Deletions: 6 kb: 152; 3 kb: 153. Splicing mutations: 1: 143; 2: 145.

TABLE 3 LPL Mutations Classified by Resulting Protein Defects Kind of mutation

Exon

Ref.

Class

Mutation

Class I

2 kb duplication 221AG3 Stop,, loZThr-+ Stop l42GJy-+ Glu “’Tyr 3 Stop G-+A 6 kb deletion

Insertion Nonsense Nonsense Missense Nonsense Splice donor Deletion

3 4 3 2 I

151 132, 148 146 134 143 143 152

@ ,Asp ‘ Glu 24aArg-+ His lM11e 3 Thr 17”Ala3 Thr lB8Gly3 Glu

Missense Missense Missense Missense Missense

5 6 5 5 5

143 143 142 136 138, 139,147

Class II

259

I 5


acid changes affecting tertiary structure rather than by changes affecting the primary sequence, which is thought to contain the heparin-binding site (aa 314 to 448).s’s Missense mutations, in general, have been localized in exons 4, 5 , and 6, which provide crucial residues involved in catalytic function (see above). Derewenda and Cambil1aua suggest that this clustering is better understood if one interprets these data with the known tertiary structure of a related serine esterase, (i.e., human pancreatic l i p a ~ e in ~ ~mind. ) In analogy to the pancreatic lipase structural data, these authorsa suggest that the majority of clustered missense mutations lie either in the strands of the beta-pleated sheet or the loops neighboring. the catalytic site, These mutations may therefore directly affect the catalytic triad of LPL, I3%er,Is6asp,and 2 4 1 hi~, or play a major role in protein folding to establish a proper conformation of the hydrophobic core around the active site. Two different mutations affect amino acid 156a~p of the catalytic triad. 127m Through in vitro mutagenesis experiments, by changing the LPL serine residues followed by expression in cultured cells, Faustinella et al. Iz9 demonstrated that the 132ser residue (belonging to the catalytic triad) also was indispensible for LPL to obtain catalytic activity. Similar experiments involving mutagenesis of the entire catalytic triad of LPL supports the role of all three amino acids in enzyme function.130A nice approach to provide additional information about the structure-function of The first of LPL involved the construction of chimeric lipase enzymes such chimera generated a lipase containing the N-terminal 329 residues of rat hepatic lipase and the C-terminal 136 of LPL (HL-LPL),47whereas the reverse chimera contained the N-terminal 314 residues from human LPL and the Cterminal 147 residues of hepatic lipase (LPL-HL).48With this approach, it could be demonstrated that the catalytic properties of LPL were defined by the first 3 14 aa, because the LPL-HL chimera, in contrast to the HL-LPL chimera, required the apolipoprotein CII cofactor and could be inhibited by high salt concentrations.47.48.5 l a Furthermore, these results delineated the apo CII interaction site to the N-terminal part of LPL and showed that substrate specificity was, in part, determined by the C-terminal residues of the LPL molecule. .42.489s1a

IX. CONCLUSIONS The advances brought about by modern molecular biology have also affected the field of lipoprotein lipase in a major fashion. There has been a gain of indepth knowledge about LPL physiology and function, structure-function relationships are being unraveled at a high speed, and great progress is still expected in the field of molecular regulation of LPL. Some of these advances have already paid off in the clinical setting, most notably the facilitated genetic diagnosis of the disorder of lipoprotein lipase deficiency. Screening family members of LPLdeficient subjects may identify nonobligate heterozygote carriers, who are suggested to have an increased frequency of hyperlipidemia, most notably familial combined hyperlipidemia. 126 The implication of LPL deficits in hyperlipidemias, 260

other then the relatively rare familial hyperchylomicronemia, may be especially relevant when one takes the relative frequency of the camer state (which can reach 1 in 50 in certain regions) into account. The insights gained by studies on structure-function and regulation will undoubtedly become important to understand and develop new strategies for altering the efficiency of LPL-catalyzed hydrolysis of triglycerides and the accompanying effects.


ACKNOWLEDGMENTS This work is dedicated to Jenna and Matteo and has been made possible through grants from ‘‘Levenslijn; NFWO”, from ILSI, and from CNRS/ATIPE. Stimulating scientific discussions with Drs. G. Ailhaud, G. Verhoeven, S. Vilaro, M. Llobera, J. Peinado, M. Reina, S. Deeb, and A. Chait are gratefully acknowledged.

REFERENCES 1. Garfinkel AS, Schotz MC. Lipoprotein lipase. In: Gotto AM, ed. Plasma lipoproteins. Pp.

335-56. Amsterdam, The Netherlands: Elsevier Science, 1987. 2. Brunzell JD. Familial lipoprotein lipase deficiency and other causes of the chylomicronemia syndrome. In: Scriver CR, Beaudet AL, Sly WS, Valle D, eds. The metabolic basis of inherited disease, 6th ed. Pp. 1165-80. New York, NY: McGraw Hill, 1989. 3. Ailhaud G. Cellular and secreted lipoprotein lipase revisited. Clin. Biochem 1990; 23: 3437. 4. Olivecrona T, Bengtsson-Olivecrona G. Lipoprotein lipase and hepatic lipase. Curr Opin Lipidol 1991: 1: 222-30. 5. Bensadoun A. Lipoprotein lipase. Annu Rev Nutr 1991; 11: 217-37. 6. Kern PA. Lipoprotein lipase and hepatic lipase. Curr Opin Lipidol 1991; 2: 162-9. 7. Lalouel JM, Wilson DE, Iverius PH. Lipoprotein lipase and hepatic triglyceride lipase: molecular and genetic aspects. Curr Opin Lipid 1992; 3: 86-95. 8. Santamarina-Fojo S. Genetic dyslipoproteinemias:role of lipoprotein lipase and apolipoprotein CII. Curr Opin Lipid 1992; 3: 186-95. 9. Olivecrona T, Bengtsson-Olivecrona G. Lipoprotein lipase from milk - the model enzyme in lipoprotein lipase research. In: Borensztajn J, ed. Lipoprotein lipase. F‘p. 15-58, Chicago, IL: Evener Press, 1987. 10. Burgaya F, Peinado J, Vilaro S, et al. Lipoprotein lipase activity in neonatal rat liver cell types. Biochem J 1989; 259: 159-66. 11. Llobera M, Montes A, Herrera E. Lipoprotein lipase activity in liver of rat fetus. Biochem Biophys Res Commun 1979; 91: 272-7. 12. Chajek-ShaulT, Friedman G, Knobler H, et al. Importance of different steps of glycosylation for the activity and secretion of lipoprotein lipase in rat preadipocytes studied with monensin and tunicamycin. Biochim Biophys Acta 1985; 837: 123-34. 13. Amri EZ, Vannier C, Etienne J , et al. Maturation and secretion of lipoprotein lipase in cultured adipose cells. 11. Effects of tunicamycin on activation and secretion of the enzyme. Biochim Biophys Acta 1986; 875: 334-43.

261


14. Olivecrona T, Chernick SS, Bengtsson-Olivecrona G , et al. Synthesis and secretion of lipoprotein lipase in 3T3-Ll adipocytes: demonstration of inactive forms of lipase in cells. J Biol Chem 1987; 262: 10748-59. 15. Cupp M, Bensadoun A, Melford K. Heparin decreases the degradation rate of lipoprotein lipase in cultured adipocytes. J Biol Chem 1987; 262: 6383-88. 16. Vannier C, Ailhaud G. Biosynthesis of lipoprotein lipase in cultured mouse adipocytes. 11. Processing, subunit assembly, and intracellular transport. J Biol Chem 1989; 264: 1320616. 17. Vannier C, Deslex S, Pradines-Figueres A, et al. Biosynthesis of lipoprotein lipase in cultured mouse adipocytes. I. Characterization of a specific antibody and relationships between intracellular and secreted pools of enzyme. J Biol Chem 1989; 264: 13199-205. 18. Pradines-Figueres A, Vannier C, Ailhaud G. Lipoprotein lipase stored in adipocytes and muscle is a cryptic enzyme. J Lipid Res 1990; 31: 1467-76. 19. Friedman G, Chajek-Shaul T, Stein 0, et al. Beta-adrenergic stimulation enhances translocation, processing and synthesis of lipoprotein lipase in rat heart cells. Biochim Biophys A C ~ U1986; 877: 112-20. 20. Chan BL, Lisanti MP,Rodriguez-Boulan E, et al. Insulin-stimulated release of lipoprotein lipase by metabolism of its phosphatidylinositol anchor. Science 1988; 241: 1670-72. 21. Chajek-Shaul T, Halimi 0, Ben-Waim M , et al. Phosphatidylinositol-specificphospholipase C releases lipoprotein lipase from the heparin releasable pool in rat heart cell culture. Biochim Biophys Actu 1989; 1014: 178-83. 22. Ting AE, Pagano RE. Detection of a phosphatidylinositol-specificphospholipase C at the surface of Swiss 3T3 cells and its potential role in the regulation of cell growth. J B i d Chem 1990; 265: 5337-40. 23. Saxena U, Klein MG, Goldberg IJ. Identification and characterization of the endothelial cell surface lipoprotein lipase receptor. J B i d Chem 1991; 266: 17516-21. 24. Camps L, Reina M, Llobera M, et al. Lipoprotein lipase in lungs, spleen, and liver: synthesis and distribution. J Lipid Res 1991; 32: 1877-88. 25. Goldberg U,Kandel JJ, Blum CB, et al. Association of plasma lipoproteins with post-heparin activities. J Clin Invest 1986; 78: 1523-8. 26. Cardin AD, Jackson RL, Johnson JD. 5-Dimethylaminonapthalene1-sulfonyl3 aminotyrosyl apoprotein CIII. Preparation, characterization, and interaction with phospholipid vesicles. J Biol Chem 1982; 257: 4987-92. 27. Ito Y, Awolan N, O’Connell A, et al. Hypertriglyceridemia as a result of human apo CIII gene expression in transgenic mice. Science 1990; 249: 790-3. 28. Greenwood MRC. The relationship of enzyme activity to feeding behaviour in rats: LPL as the metabolic gatekeeper. Int J Obes 1985; 9 (Suppl. 1): 67-70. 29. Auwerx J, Marzetta C, Hokanson J, et al. Relationship between hepatic triglyceride lipase and low density lipoprotein characteristics. Arteriosclerosis 1989; 9: 3 19-25. 30. Eckel RH. Adipose tissue lipoprotein lipase. In: Borensztajn J, ed. Lipoprotein Lipuse. Pp. 79-132. Chicago, 11: Evener Press, 1987. 31. Saxena U, Klein MG, Vanni TM, et al. Lipoprotein lipase increases low density lipoprotein retention by subendothelial cell matrix. J Clin Invest 1992; 89: 373-80. 32. Lindqvist P, Ostlund-Lindqvist AM, Witztum JL, et al. The role of lipoprotein lipase in the metabolism of triglyceride-rich lipoproteins by macrophages. J Biol Chem 1983; 258: 908692. 33. Mulder M, Lombardi P, Jansen H, et a]. Heparan sulphate proteoglycans are involved in the lipoprotein lipase-mediated enhancement of the cellular binding of very low density and low density lipoproteins. Biochem Biophys Res Commun 1992; 185: 582-87. 34. Williams KJ, Petrie KA, Brocia RW, et al. Lipoprotein lipase modulates net secretory output of apolipoprotein B in vitro. A possible explanation for combined hyperlipidemia. J Clin Invest 1991; 88: 1300-6.

262


35. Beisiegel U, Weber W, Bengtsson-Olivecrona G. Lipoprotein lipase enhances the binding of chylomicrons to low density lipoprotein receptor-related protein. Proc Natl Acad Sci USA 1991; 88: 8342-6. 36. Felts JM, Itakura H, Crane RT. The mechanism of assimilation of constituents of chylomicrons, very low density lipoproteins and remnants - a new theory. Biochim Biophys Re5 Commun 1975; 66: 1467-75. 37. Wion KL, Kirchgessner TG, Lusis AJ, et al. Human lipoprotein lipase complementary DNA sequence. Science 1987; 235: 1638-41. 38. Auwerx J, Deeb S, Brunzell JD, et al. Transcriptional activation of the lipoprotein lipase and apolipoprotein E genes accompanies differentiation in some human macrophage-like cell lines. Biochemistry 1988; 27: 2651-5. 39. Senda M, Oka K, Brown WV, et al. Molecular cloning and sequence of a cDNA coding for bovine lipoprotein lipase. Proc Natl Acad Sci USA 1987; 84: 4369-73. 40. Enerback S, Semb H, Bengtsson-Olivecrona G, et al. Molecular cloning and sequence analysis of cDNA encoding lipoprotein lipase of guinea pig. Gene 1987; 58: 1-12. 41. Staels B, Auwerx J . Perturbation of developmental gene expression in rat liver by fibric acid derivatives: lipoprotein lipase and a-fetoprotein as models. Development 1992; 115: 1035- 1044. 42. Kirchgessner TG, Svenson KL, Lusis AJ, et al. The sequence of cDNA encoding lipoprotein lipase. A member of a lipase gene family. J Biol Chem 1987; 262: 8463-6. 43. Olivecrona T, Bengtsson G, Osborne JC. Molecular properties of lipoprotein lipase. Effects of limited trypsin digestion on molecular weight and secondary structure. Eur J Biochem 1982; 124: 629-33. 44.Iverius PH, Ostlund-Lindqvist AM. Lipoprotein lipase from bovine milk. Isolation procedure, chemical characterization and molecular weight analysis. J B i d Chem 1976; 251: 7791-5. 45. Osborne JC, Bengtsson-Olivecrona G, Lee NS, et al. Studies on inactivation of lipoprotein lipase. Role of the dimer to monomer dissociation. Biochemistry 1985; 24: 5606-1 1. 46. Winkler FK, D’Arcy A, Hunziker W. Structure of human pancreatic lipase. Nature 1990; 343: 771-4. 47. Wong, H, Davis RC, Nikazy J, et al. Domain exchange: characterization of a chimeric lipase of hepatic lipase and lipoprotein lipase. Proc Natl Acad Sci USA 1991; 88: 11290-4. 48. Dicheck HL, Parrott C, Brewer HB, et al. Functional characterization of a chimeric protein genetically engineered from human lipoprotein lipase and human hepatic lipase. Clin Re5 1992; 40: 321. 49. Bengtsson G, Olivecrona T. Lipoprotein lipase: modification of its kinetic properties by mild tryptic digestion. Eur J Biochem 1981; 113: 547-54. 50. Persson BH, Jornvall H, Olivecrona T, et al. Lipoprotein lipases and vitellogenins in relation to the known three-dimensional structure of pancreatic lipase. FEBS Lett 1991; 288: 33-6. 51. Yang CH, Gu Z , Yang HX, et al. Structure of bovine milk lipoprotein lipase. J . Biol Chem 1989; 264: 16822-7. 51a. Davis RC, Wong H, Mikazy J, et al. Chimeras of hepatic lipase and lipoprotein lipase. Domain localization of enzyme-specific properties. J Biol Chem 1992; 267: 21499-2 1504. 52. Iverius PH, Ostlund-Lindqvist AM. Preparation, characterization, and measurement of lipoprotein lipase. Methods Enzymol 1986; 129: 691-704. 53. Semb H, Olivecrona T. The relation between glycosylation and activity of guinea pig lipoprotein lipase. J Biol Chem 1989; 264: 4195-200. 54. Hoogewerf AJ, Bensadoun A. Occurrence of sulfate in an asparagine-linked complex oligosaccharide of chicken adipose lipoprotein lipase. J Biol Chem 1991; 266: 1048-57. 55. Masuno HC, Blanchette-Mackie EJ, Chemick SS, et al. Synthesis of inactive nonsecretable high mannose-type lipoprotein lipase by cultured brown adipocytes of combined lipasedeficient cld/cld mice. J Biol Chem 1991; 256: 1628-38. 56. Masuno HC, Schultz CJ, Park JW, et al. Glycosylation, activity and secretion of lipoprotein lipase in cultured brown adipocytes of newborn mice. Effect of tunicamycin, monensin, 1-deoxymannojirimycin, and swainsonine. Biochem J 1991: 277: 801-9.


57. Semenkovich CF, Luo CC, Nakanishi MK, et al. In virro expression and site-specific mutagenesis of the cloned human lipoprotein lipase gene. Potential N-linked glycosylation site asparagine 43 is important for both enzyme activity and secretion. J Biol Chem 1990; 265: 5429-33. 58. Oka K, Nakano T , Tkalcevic GT, et al. Molecular cloning of mouse hepatic triacylglycerol lipase gene expression in combined lipase deficient (cldkld) mice. Biochim. Biophys Acta 1991; 1089: 13-20. 59. Hide WA, Chan L, Li WH. Structure and evolution of the lipase superfamily. J Lipid Res 1992; 33: 167-78. 60. Baker ME. Is vitellogenin an ancestor of apolipoprotein B-100 of human low density lipoprotein and human lipoprotein lipase? Biochem J 1988; 255: 1057-60. 61. Terpstra P, Geert AB. Homology of Drosophila yolk proteins and the triacylglycerol lipase family. J Mol B i d 1988; 22: 663-5. 62. Persson BH, Bengtsson-Olivecrona G, Enerback S, et al. Structural features of lipoprotein lipase. Lipase family relationships, binding interactions, nonequivalence of lipase cofactors, vitellogenin similarities, and functional subdivisions of lipoprotein lipase. Eur J Biochem 1989; 179: 39-45. 63. Kirchgessner TG, Chuat JC, Heinzmann C, et al. Organization of the human lipoprotein lipase gene and evolution of the lipase gene family. Proc Natl Acad Sci USA 1989; 86: 9647-5 1 . 64. Derewenda ZS, Cambillau C. Effects of gene mutations in lipoprotein lipase as interpreted by a molecular model of the pancreatic triglyceride lipase. J Biol Chem 1992; 266: 231 129. 65. Deeb SS, Peng R. Structure of the human lipoprotein lipase gene. Biochemistry 1989; 28: 41 3 1-5. 66. Enerback S, Bjursell G. Genomic organization of the region encoding guinea pig lipoprotein lipase; evidence for exon fusion and unconventional splicing. Gene 1989; 84: 391-97. 67. Sparkes RS, Zollman S , Klisak I, et al. Human genes involved in lipolysis of plasma lipoproteins: mapping of loci for lipoprotein lipase to 8p22 and hepatic lipase to 15q21. Genomics 1987; 1: 138-44. 68. Chajek T , Stein 0, Stein Y.Pre- and postnatal development of lipoprotein lipase and hepatic triglyceride hydrolase activity in rat tissues. Atherosclerosis 1977; 26: 549-61. 69. Kirchgessner TG, LeBoeuf RC, Langner CA et al. Genetic and developmental regulation of lipoprotein lipase gene: loci both distal and proximal to the lipoprotein lipase structure gene control enzyme expression. J Biol Chem 1989; 264: 1473-82. 70. Semenkovich CF, Wims M, Noe L, et al. Insulin regulation of lipoprotein lipase activity in 3T3-Ll adipocytes is mediated at posttranscriptional and posttranslational levels. J Biol Chem 1989; 264: 9030-8. 71. Dani C, Amri EZ, Bertrand B, et al. Expression and regulation of pOb24 and lipoprotein lipase genes during adipose conversion. J Cell Biochem 1990; 43: 103-110. 72. Khoo JC, Mahoney EM, Witztum JL, Secretion of lipoprotein lipase by macrophages in culture. J . Biol. Chem 1981; 256: 7105-8. 73. Chait A, Iverius PH, Brunzell JD. Lipoprotein lipase secretion by human monocyte-derived macrophages. J Clin Invest 1982; 69: 490-3. 74. Peinado-Onsurbe J, Staels B, Deeb S, et al. Neonatal extinction of liver lipoprotein lipase expression. Biochim. Biophys. Acta 1992; 1131 : 281-6. 75. Cooper DA, Stein JC, Strieleman PJ, et al. Avian lipoprotein lipase cDNA sequence and reciprocal regulation of mRNA levels in adipose tissue and heart. Biochim Biophys Acta 1989; 1008: 92-101. 76. Enerback S, Semb H, Tavernier J, et al. Tissue-specific regulation of guinea pig lipoprotein lipase; effects of nutritional state and tumor necrosis factor on mRNA levels in adipose tissue, heart, and liver. Gene 1988; 64: 97-106.

264


77. Ong JM, Kern PA. The role of glucose and glycosylation in the regulation of lipoprotein lipase synthesis and secretion in rat adipocytes. J Biol Chem 1989; 264: 3177-82. 78. Ong JM, Kern PA. Effect of feeding on lipoprotein lipase activity, immunoreactive protein. and mRNA levels in human adipose tissue. J Clin Invest 1989; 84: 305-1 1. 79. Doolittle MH, Ben-Zeev 0, Elovson J, et al. The response of lipoprotein lipase to feeding and fasting. Evidence for posttranslational regulation. J Biol Chem 1990; 265: 4570-7. 80. Borensztajn J. Heart and skeletal muscle lipoprotein lipase. In: Borensztajn J, ed. Lipoprotein lipase. Pp. 133-48, Chicago, IL: Evener Press, 1987. 81. Kern PA, Ong JM, Saffari B, et al. The effects of weight loss on the activity and expression of adipose tissue lipoprotein lipase in very obese humans. N Engl J Med 1990; 322: 10539. 82. Simsolo RB, Ong JM, Saffari B, eta]. Effect of improved diabetes control on the expression of lipoprotein lipase in human adipose tissue. J Lipid Res 1992; 33: 89-95. 83. Ong JM, Kirchgessner TG, Schotz MC, et al. Insulin increases the synthetic rate and messenger RNA level of lipoprotein lipase in isolated rat adipocyes. J B i d Chem 1988; 263: 12933-8. 84. Raynolds MV, Awald PD, Gordon DF, et al. Lipoprotein lipase gene expression in rat adipocytes is regulated by isoproterenol and insulin through different mechanisms. Mol Endocrinol 1990; 4: 1416-22. 85. Ong JM. Simsolo RB, Saffari B, et al. The regulation of lipoprotein lipase gene expression by dexamethasone in isolated rat adipocytes. Endocrinology 1992; 130: 23 10-6. 86. Domin WS, Chait A, Deeb SS. Transcriptional activation of the lipoprotein lipase gene in macrophages by dexamethasone. Biochemistry 1991; 30: 2570-4. 87. Cryer A. Comparative biochemistry and physiology of lipoprotein lipase. In: Borensztajn J , ed. Lipoprotein lipase Pp. 277-327. Chicago, 11: Evener Press, 1987. 88. Demacker P, Staels B, Stalenhoef AFH, Auwerx J. Increased removal of P-VLDL after ethinyl estradiol is associated with increased mRNA levels for hepatic lipase, lipoprotein lipase, and the low density lipoprotein receptor in Watanabe heritable hyperlipidemic rabbits. Arteriosclerosis Thromb 1991; 11: 1652-9. 89. Applebaum-Bowden D, McLean P, Steinmetz A, et al. Lipoprotein, apolipoprotein, and lipolytic enzyme changes following estrogen administration in postmenopausal women. J Lipid Res 1989; 30: 1895-1906. 90. Iverius PH, Brunzell JD. Relationship between lipoprotein lipase and plasma sex steroid levels in obese women. J Clin Invest 1988; 82: 1106-12. 91. Spooner PM, Chernick SS, Garrison MM, et al. Insulin regulation of lipoprotein lipase activity and release in 3T3-LI adipocytes. J Biol Chem 1979; 254: 10021-9. 92. Peinado-Onsurbe J, Staels B, D e b S, and Auwerx J. Lipoprotein lipase expression in undifferentiated hepatoma cells is regulated by progesterone and protein kinase A. Biochemistry 1992; 31: 10121-10128. 93. Saffari B, Ong JM, Kern PA. Regulation of adipose tissue lipoprotein lipase gene expression by thyroid hormone in rats. J Lipid Res 1992; 33: 241-9. 94. Forest C, Grimaldi P, Czeucka D, et al. Establishment of a preadipocyte cell line from epididymal fat pad of the lean C57BU6J mouse: long-term effects of insulin and triiodothyronine on adipose conversion. In Vitro 1983; 19: 344-54. 95. Mori N, Gotoda T, Ishibashi S, et al. Effects of human recombinant macrophage colonystimulating factor on the secretion of lipoprotein lipase from macrophages. Arteriosclerosis Thromb 1991; 11: 1315-21. 96. Scow RO, Chernick SS. Role of lipoprotein lipase during lactation. In: Borensztajn J, ed. Lipoprotein lipase. Pp. 149-186. Chicago, 11: Evener Press, 1987. 97. Pradines-Figueres A, Barcellini-Couget S, Dani C, et al. Transcriptional control of the expression of lipoprotein lipase gene by growth hormone in preadipocyte Ob1771 cells. J Lipid Res 1990; 31: 1283-91.

265


98. Auwerx J, Deeb SS, Brunzell JD, et al. Lipoprotein lipase gene expression in THP-I cells. Biochemistry 1989; 28: 4563-7. 99. Cryer A, Choan P, Smith JJ. Effectors of lipoprotein lipase: secretion from isolated cardiac muscle cells incubated in vitro. Life Sci 1981; 29: 923-9. 100. Friedman G ,Chajek-Shaul T, Stein 0, et al. Modulation of lipoprotein lipase activity in cultured rat mesenchymal heart cells and preadipocytes by dibutyryl cyclic AMP, choleratoxin, and 3-isobutyl-I-methylxanthine. Biochim Biophys Actu 1983; 752: 106-17. 101. Palmer WK, Oscai LB, Bechtel PJ, et al. Dibutyryl CAMP-induced increases in triacylglycerol lipase activity in developing L8 myotube cultures. Can J Physiol Pharmucol 1990; 68: 68993, 102. Bensadoun A, Marita RA. Dibutyryl cyclic AMP decreases the rate of lipoprotein lipase synthesis in cultured adipocytes. Biochim Biophys Actu 1986; 879: 253-63. 103. Kawakami M, Pekala PH, Lane DL, et al. Lipoprotein lipase suppression in 3T3-Ll cells by an endotoxin-induced mediator from exudate cells. Proc Nut1 Acud Sci USA 1982; 79: 912-6. 104. Price SR, Olivecrona T, Pekala PH. Regulation of lipoprotein lipase synthesis by recombinant tumor necrosis factor; the primary regulatory role of the hormone in 3T3-Ll adipocytes. Arch Biochem Biophys 1986; 251: 738-46. 105. Price SR, Olivecrona T, Pekala PH. Regulation of lipoprotein lipase synthesis in 3T3-Ll adipocytes by cachectin. Biochem J 1986; 240: 601-4. 106. Semb H, Peterson J, Tavemier J, et al. Multiple effects of tumor necrosis factor on lipoprotein lipase in vivo. J Biol Chem 1987; 262: 8390-4. 107. Cornelius P, Enerback S, Bjursell G, et al. Regulation of lipoprotein lipase mRNA content in 3T3-Ll cells by tumor necrosis factor. Biochem J 1988; 249: 765-9. 108. Zechner R, Newman TC, Sheny B, et al. Recombinant human cachectin/tumor necrosis factor but not interleukin- l a downregulates lipoprotein lipase gene expression at the transcriptional level in mouse 3T3-Ll adipocytes. Mol Cell Biol 1988; 8: 2394-401. 109. Torti FM, Dieckman B, Beutler B, et al. A macrophage factor inhibits adipocyte gene expression; an in vitro model for cachexia. Science 1985; 229: 867-9. 110. Bars RGC, Elcombe CR. Enhanced longevity and phenotypic characterization of rat hepatocytes exposed to non-genotoxic carcinogens during long-term culture. In: Ciliberto G, Cortese R, Schibler U, Schutz G, eds. Gene expression during liver differentiation and diseuse. P. 218, Rome, Italy: IRBM Press, 1991. 1 1 1 . Green S. Receptor-mediated mechanisms of peroxisome proliferators. Biochem Pharmacol 1992; 43: 393-401. 112. Goldberg AP, Applebaum-Bowden DM, Bierman EL, et al. Increase in lipoprotein lipase during clofibrate treatment of hypertriglyceridemia in patients on hemodialysis. N Engi J Med 1979; 301: 1073-6. 113. Previato L, Parroa CL, Santamarina-Fojo S , et al. Transcriptional regulation of the human lipoprotein lipase gene in 3T3-Ll adipocytes. J Biol Chem 1991; 266: 18958-63. 114. Hua X , Enerback S, Hudson J, et al. Cloning and characterization of the promotor of the murine lipoprotein lipase-encoding gene: structural and functional analysis. Gene 1991; 107: 247-58. 115. Auwerx J, Staels B, Szpirer J, et al. Developmental regulation and extinction of liver lipoprotein lipase mRNA. Arteriosclerosis 1990; 10: 826a. 116. Nilsson-Ehle P. Measurements of lipoprotein lipase activity. In: Borensztajn J , ed., Lipoprotein lipase. Pp. 59-78. Chicago, Il: Evener Press, 1987. 117. Cheung AH, Bensadoun A, Cheng C. Direct solid-phase radioimmunoassay for chicken lipoprotein lipase. Anal Biochem 1979; 94: 346-57. 1 18. Olivecrona T, Bengtsson-Olivecrona G. Immunochemical properties of lipoprotein lipase; development of an immunoassay applicable to several mammalian species. Biochim Biophys Acru 1983; 752: 38-45.

266


119. Olivecrona T, Chernick SS, Bengtsson-Olivecrona G, et al. Combined lipase deficiency (cld/cld) in mice; demonstration that an inactive form of lipoprotein lipase is synthesized. J Biol Chem 1985; 260: 2552-7. 120. Goers JWF, Pedersen ME, Kern PA, et al. An enzyme-linked immunoassay for lipoprotein lipase. Anal Biochem 1987; 166: 27-35. 121. Babirak S, Iverius PH, Fujimoto WY, et al. Detection and characterization of the heterozygote state for lipoprotein lipase deficiency. Arteriosclerosis 1989; 9: 326-34. 122. Auwerx J, Babirak SP, Fujimoto WY, et al. Defective enzyme protein in lipoprotein lipase deficiency. Eur J Clin Invest 1989; 19: 433-7. 123. Kern PA, Martin RA, Carty J, et al. Identification of lipoprotein lipase immunoreactive protein in pre- and post-heparin plasma from normal subjects and patients with type I hyperlipoproteinemia. J Lipid Res 1990; 31: 17-26. 124. Gagne C, Brum LD, Julien P, et al. Primary lipoprotein lipase deficiency; clinical investigation of a French Canadian population. Can Med Assoc J 1989; 140: 405-1 1. 125. Brunzell JD, Miller NE, Alaupovic P, et al. Familial chylomicronemia due to a circulating inhibitor of lipoprotein lipase activity. J Lipid Res 1983; 24: 12-194. 126. Babirak SP, Brown BS, Brunzell JD. Familial combined hyperlipidemia and abnormal lipoprotein lipase. Artherosclerosis Thrombosis 1992; 12: 1 176-1 183. 127. Ma Y, Kastelein J, Wilson B, et al. Two missense mutations at the first and second base of codon Asp156in the proposed catalytic triad of lipoprotein lipase support the major role of AsplS6in catalytic activity. Arreriosclerosis Thromb 1991; 11 (Abstr.): 1416. 128. Faustinella F, Chang A, Van Biervliet JP, et al. Catalytic triad residue mutation ( A ~ p ' ~ ~ + G l y ) causing familial lipoprotein lipase deficiency. J Biol Chem 1991; 266: 14418-24. 129. Faustinella F, Smith L, Semenkovich CF, et al. Structural and functional roles of highly conserved serines in human lipoprotein lipase. J Biol Chem 1991; 266: 9481-5. 130. Emmerich J, Beg OU, Peterson J, et al. Human lipoprotein lipase; analysis of the catalytic triad by site directed mutagenesis of ser-132, asp-156, and his-241. J Biol Chem 1992; 267: 4161-5. 131. Lohse P, Lohse P, Beg 0, et al. Familial chylomicronemia: identification of a unique patient homozygote for two separate mutations in the LPL gene. Arteriosclerosis Thromb 1991; 11 (Abstr.): 1415. 132. Deeb SS, Reina M, Peterson J, et al. Gene mutations in patients with lipoprotein lipase deficiency. Arteriosclerosis Thromb 1991; 11 (Abstr.): 1418. 133. Ishimura-Oka K, Semenkovich CF, Faustinella F, et al. Identification of compound heterozygotes for lipoprotein lipase deficiency in three unrelated families. Arteriosclerosis Thromb 1991; 11 (Abstr.): 1415. 134. Ameis D, Kobayashi J, Davis RC, et al. Familial chylomicronemia (type I hyperlipoproteinemia) due to a s'ingle missense mutation in the lipoprotein lipase gene. J Clin Invest 1991; 87: 1165-70. 135. Bruin T , Kastelein JJP, Van Diermen D, et al. A missense mutation Pro157 Arg in lipoprotein lipase (LPL Nijmegen) resulting in loss of catalytic activity. Eur J Biochem 1992; 208 26772. 136. Beg OU, Meng MS, Skarlatos SI, et al. Lipoprotein lipase Bethesda: a single amino acid substitution (Ala176+Thr) leads to abnormal heparin binding and loss of enzymatic activity. Proc Natl Acad Sci USA 1990; 87: 3474-8. 137. Emi M, Wilson DE, Iverius PH, et al. Missense mutation (Gly -,Gly188)of human lipoprotein lipase imparting functional deficiency. J Biol Chem 1990; 265: 5910-6. 138. Monsalve MV, Henderson H, Roederer G, et al. A missense mutation at codon 188 of the human lipoprotein lipase gene is a frequent cause of lipoprotein lipase deficiency in persons of different ancestries. J Clin Invest 1990; 86: 728-34. 139. Paulweber B, Weibusch H, Miesenboeck G, et al. Molecular basis of lipoprotein lipase deficiency in two Austrian families with type I hyperlipoproteinemia. Atherosclerosis 1991; 86: 239-50.

267


140. Julien P, Normand T, Bergeron J, et al. Prevalence and origin of familial lipoprotein lipase deficiency in the French-Canadian population of Quebec. Arteriosclerosis Thromb 1991; 11 (Abstr.): 1416. 141. Dichek HL, Fojo SS, Beg OU, et al. Identification of two separate allelic mutations in the lipoprotein lipase gene of a patient with the familial hyperchylomicronemia syndrome. J Biol Chem 1991; 266: 473-7. 142. Henderson HE, Ma Y, Hassan MF, et at. Amino acid substitution Thr) in exon 5 of the lipoprotein lipase gene causes lipoprotein lipase deficiency in three unrelated probands: support for a multicentric origin. J Clin Invest 1991; 87: 2005-1 1. 143. Gotoda T, Senda M, Gamou T, et al. Nucleotide sequence of human cDNA coding for lipoprotein lipase (LPL) cloned from placental cDNA library. Nucl Acid Res 1989; 17: 235 1. 144. Ma Y, Henderson HE, Ven Murthy MR, et al. A mutation of the human lipoprotein lipase gene as the most common cause of familial chylomicronemia in French Canadians. N Engl J Med 1991; 324: 1761-6. 145. Hata A, Emi M, Luc G , et al. Compound heterozygote for lipoprotein lipase deficiency: Ser to ThrZa and transition in 3’ splice site of intron 2 (AG to AA) in the lipoprotein lipase gene. Am J Hum Genet 1990; 47: 721-6. 146. Henderson HH, Devlin R, Peterson J, et al. Frameshift mutation in exon 3 of the lipoprotein lipase gene causes a premature stop codon and lipoprotein lipase deficiency. Mol Biol Med 1990; 7: 511-17. 147. Emi M, Hata A, Robertson M, et al. Lipoprotein lipase deficiency resulting from a nonsense mutation in exon 3 of the lipoprotein lipase gene. Am J Hum Genet 1990; 47: 107-1 1. 148. Takagi A, Ikeda Y, Tsutsumi 2, et al. Molecular studies on primary lipoprotein lipase deficiency; one base deletion (G916) in exon 5 of LPL gene causes no detectable LPL protein due to the absence of LPL mRNA transcript. J Clin Invest 1992; 89: 581-91. 149. Funke H, Wiesbusch H, Paulweber B, et al. Identification of the molecular defect in a patient with type I hyperlipidemia. Arteriosclerosis 1990; 10 (Abstr.): 830. 150. Hata A, Robertson M, Emi M, et al. Direct detection and automated sequencing of individual alleles after electrophoretic strand separation: identification of a common nonsense mutation in exon 9 of the human lipoprotein lipase gene. Nucl Acid Res 1990; 18: 5407- 11. 151. Devlin RH, Deeb SS, Brunzell JD, et al. Partial gene duplication involving exon-alu interchange results in lipoprotein lipase deficiency. Am J Hum Genet 1990; 46: 112-19. 152. Langlois S, Deeb SS, Brunzell JD, et al. A major insertion accounts for a significant proportion of mutations underlying human lipoprotein lipase deficiency. Proc Nut1 Acad Sci USA 1989; 86: 948-52. 153. Benlian P, Loux N, De Gennes JL, et al. A homozygous deletion of exon 9 in the lipoprotein lipase gene causes type I hyperlipoproteinemia. Arteriosclerosis 1991; 11 (Abstr.): 1465.

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