Clinical Biochemistry 36 (2003) Shirya Rashid, Ph.D., Takehiko Watanabe, M.D., Ph.D., Taro Sakaue, MD, Gary F. Lewis, M.D.

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1 Clinical Biochemistry 36 (2003) Mechanisms of HDL lowering in insulin resistant, hypertriglyceridemic states: the combined effect of HDL triglyceride enrichment and elevated hepatic lipase activity Shirya Rashid, Ph.D., Takehiko Watanabe, M.D., Ph.D., Taro Sakaue, MD, Gary F. Lewis, M.D., FRCPC* From the Department of Medicine, Division of Endocrinology and the Department of Physiology, University of Toronto, Toronto, Ontario, Canada Received 15 January 2003; received in revised form 9 May 2003; accepted 13 May 2003 Abstract Hypertriglyceridemia, low plasma concentrations of high density lipoproteins (HDL) and qualitative changes in low density lipoproteins (LDL) comprise the typical dyslipidemia of insulin resistant states and type 2 diabetes. Although isolated low plasma HDL-cholesterol (HDL-c) and apolipoprotein A-I (apo A-I, the major apolipoprotein component of HDL) can occur in the absence of hypertriglyceridemia or any other features of insulin resistance, the majority of cases in which HDL-c is low are closely linked with other clinical features of insulin resistance and hypertriglyceridemia. We and others have postulated that triglyceride enrichment of HDL particles secondary to enhanced CETP-mediated exchange of triglycerides and cholesteryl ester between HDL and triglyceride-rich lipoproteins, combined with the lipolytic action of hepatic lipase (HL), are driving forces in the reduction of plasma HDL-c and apoa-i plasma concentrations. The present review focuses on these metabolic alterations in insulin resistant states and their important contributions to the reduction of HDL-c and HDL-apoA-I plasma concentrations The Canadian Society of Clinical Chemists. All rights reserved. Keywords: Hepatic lipase; High density lipoprotein (HDL); Apolipoprotein A-I; Type 2 diabetes; Insulin resistance syndrome; Metabolic syndrome; Hypertriglyceridemia; Atherosclerotic cardiovascular disease; Cholesteryl ester transfer protein Approximately one quarter of the North American population has evidence of insulin resistance [1]. Resistance to the normal physiologic actions of insulin (i.e., insulin resistance), with its associated metabolic, inflammatory and coagulation abnormalities, is a major risk factor for Type 2 diabetes as well as atherosclerotic cardiovascular disease (ASCD) [2,3]. The typical dyslipidemia that is associated with insulin resistance is felt to play an important, although not exclusive, role in the accelerated ASCD in affected individuals [4 6]. Hypertriglyceridemia, low plasma concentrations of high density lipoproteins (HDL) and qualitative changes in low density lipoproteins (LDL) comprise the typical dyslipidemia of insulin resistant states [4 6]. In fact, a high triglyceride (TG)/HDL-cholesterol (HDL-c) ratio is felt by some investigators to be the single most characteristic feature of the insulin resistance syndrome, even more * Corresponding author. Tel.: ; fax: address: gary.lewis@uhn.on.ca (G.F. Lewis). highly predictive of insulin resistance than the presence of abdominal obesity [7]. Furthermore, the high TG/low HDL-c lipid phenotype is highly atherogenic in the setting of insulin resistance [4 6,8]. Low plasma concentrations of HDL-c and its major protein component, apolipoprotein A-I (apo A-I), are particularly powerful, independent risk factors for ASCD [9 11]. Consequently, there is great interest in determining the mechanisms responsible for reduced HDL-c and apo A-I plasma concentrations, particularly in insulin resistant states. Putative cardioprotective functions of HDL particles include a direct inhibition of pro-atherogenic processes at the arterial wall, including inhibition of LDL oxidation, prevention of monocyte adhesion and chemotaxis, reduction in macrophage formation, and inhibition of endothelial dysfunction and apoptosis [12]. The protective role of HDL against ASCD, however, is most widely attributed to its key role in mediating the reverse cholesterol transport from peripheral tissues to the liver for either reutilization or bile acid synthesis [13]; in this manner, HDL particles are be /03/$ see front matter 2003 The Canadian Society of Clinical Chemists. All rights reserved. doi: /s (03)00078-x

2 422 S. Rashid et al. / Clinical Biochemistry 36 (2003) lieved to prevent the accumulation of cholesterol, foam cells, and fatty lesions in cells of the arterial intima [13]. Although isolated low plasma HDL-c can occur in the absence of hypertriglyceridemia or any other features of insulin resistance (associated with abdominal obesity, glucose intolerance or Type 2 diabetes), it is a less prevalent clinical occurrence and may be attributed to the presence of rare genetic disorders that affect HDL metabolism [14,15]. A few examples in which low plasma HDL-c and apoa-i concentrations do not seem to occur as a direct consequence of insulin resistance, include defects in the uptake of free cholesterol from peripheral cells (mutations in ABC-A1 are one such example) [16], defects in the esterification of free cholesterol (LCAT deficiency) [15], mutations of lipoprotein lipase or mutations in the apoa-i gene that may either affect the synthesis or catabolism of HDL [17,18]. The majority of cases of low HDL-c, however, are closely linked with other clinical features of insulin resistance and hypertriglyceridemia [10]. We and other investigators have postulated that hypertriglyceridemia, which is a frequent occurrence in insulin resistant conditions, combined with the action of hepatic lipase (HL), are driving forces in the reduction of HDL-c and apoa-i plasma concentrations [8,19,20]. The present review focuses on these metabolic alterations in insulin resistant states and their important contributions to the reduction of HDL-c and HDL-apoA-I plasma concentrations. cause-and-effect relationship, but they do suggest that the variables are closely linked either genetically, metabolically or in some other ill-defined manner. In addition to the above clinical observations, further evidence that the metabolism of TG-rich lipoproteins and HDL are closely linked has been provided by physiologic studies that have investigated the regulation of HDL metabolism. These studies have shown the existence of dynamic interactions between TG-rich lipoproteins and HDL in plasma and demonstrated that TG-rich lipoproteins impact on the metabolism of HDL in at least two important ways. One point of interaction is mediated by the lipolytic enzyme lipoprotein lipase (LPL) [20,34]. The lipolysis of TG-rich lipoproteins by LPL results in the formation of redundant surface materials, which are then transferred to HDL particles, thereby impacting on the maturation of HDL in the circulation and ultimately on plasma HDL lipid and protein concentrations [34,35]. Second, cholesteryl ester transfer protein (CETP) mediates a heteroexchange of core neutral lipids between lipoprotein classes and between lipoproteins and various tissues. In particular, CETP mediates the transfer of TGs from TG-rich lipoproteins to HDL (particularly HDL 2A ) in exchange for HDL cholesteryl ester (CE) [36,37]. This produces HDL particles that are TG-enriched within the particle core, and also relatively CE depleted [38]. These two processes may impact both HDL-c as well as apoa-i plasma concentrations as discussed further below. Hypertriglyceridemia in Insulin Resistant States and the Interaction between Triglyceride (TG)-rich Lipoproteins and HDL Hypertriglyceridemia is perhaps the most common lipid abnormality in insulin resistance and is primarily due to increased production of very low density (VLDL) particles [21,22]. In addition there is a well described elevation of chylomicrons and VLDL in the postprandial state, in large part secondary to competition for a saturable removal pathway for these TG-rich lipoproteins [23 25]. More recently we have described elevated production rates of intestinally derived lipoproteins in insulin resistance, which may also contribute to the hypertriglyceridemia [26]. Detailed discussion of the mechanisms of the hypertriglyceridemia in insulin resistance is beyond the scope of this review. Interested readers are referred to three recent reviews on this topic [21,22,27]. Several investigators have proposed that the lowering of HDL levels observed in insulin resistant states is largely a consequence of the fasting and postprandial hypertriglyceridemia commonly occurring in these states [19,28 30]. Significant negative correlations have in fact been demonstrated between postprandial plasma TG excursion, HDL TG content and fasting plasma HDL-c and apoa-1 concentrations in humans [8,28,31 33]. Correlations or inverse correlations between variables, of course, do not prove a Reduced LPL Activity and Enhanced Transfer of Neutral Lipids contribute to the Lowering of Plasma HDL-c in Insulin Resistant, Hypertriglyceridemic States LPL activity measured in adipose tissue biopsies indicate that this enzymes may in fact be elevated in obese, hyperinsulinemic but insulin resistant individuals, whereas the normal insulin-mediated stimulation of LPL activity, such as occurs in the postprandial state, has been shown to be blunted in this condition [39,40]. In Type 2 diabetes, particularly when glycemic control is poor and in patients who are relatively insulin deficient, LPL activity may be reduced [41]. Humans with either homo- or heterozygous LPL deficiency have low plasma HDL concentrations. The reduction in LPL activity could reduce the maturation of HDL particles that occurs as a result of the shedding of lipids and apolipoproteins from the surface of TG-rich lipoproteins during LPL-mediated lipolysis [42]. Thus, this theory holds that HDL production is reduced in hypertriglyceridemic states as a result of the reduced LPL activity. Kinetic studies in humans, however, have shown that hypertriglyceridemic individuals with low HDL-c have a significantly increased fractional catabolic rate (FCR) of apoa-i but no reduction in apoa-1 production rates, in comparison with normolipidemic subjects [43 47]. Similarly, in individuals with type 2 diabetes, hypertriglyceride-

3 S. Rashid et al. / Clinical Biochemistry 36 (2003) mia was shown to be associated with increased HDL FCR [48]. In contrast, the apoa-i FCR in a group of subjects with low HDL-c levels, but with normal TG concentrations, was found not to differ significantly from control subjects [49]. In hypertriglyceridemic individuals, the increase in apoa-i FCR, and not an alteration in apoa-i production, demonstrated significant positive correlations with the level of plasma TG, and strong negative correlations with HDL-c and apoa-i concentrations [43,50,51]. Since the major abnormality in HDL metabolism in hypertriglyceridemic states (which are frequently associated with underlying insulin resistance) is an enhancement in the clearance of HDL apoa-i rather than a reduction in HDL apoa-i production, reduced production of nascent HDL from a defective LPL-mediated lipolysis of TG-rich lipoproteins, while perhaps playing a role, is not likely to be the major mechanism whereby HDL-c and apoa-i plasma concentrations are lowered in hypertriglyceridemic, insulin resistant states. An alternate theory accounting for reduced HDL-c and HDL apoa-i plasma concentrations in hypertriglyceridemic, insulin resistant states include a major role for the ultimate metabolic consequence of the hetero-exchange of TG and CE between TG-rich lipoproteins and HDL that is mediated by CETP. The mass transfer of these neutral lipids is highly dependent on the concentration (pool size) of TG-rich lipoproteins in the plasma [36,37,52]. Hypertriglyceridemia that occurs in insulin resistance and type 2 diabetes has been shown to be associated with greater transfer of TG from the expanded pool of TG-rich lipoproteins (primarily large, CE enriched VLDL 1 particles) into HDL particles, and a concomitant increase in the transfer of CE out of HDL into the TG-rich lipoproteins [53,54]. Thus, such a CETP-mediated lipid exchange process can per se reduce plasma HDL-c concentrations, but not apoa-i concentrations, unless it is coupled with an additional process that increases the loss of apoa-i from the TG-rich/CE-depleted particles or increases HDL holoparticle uptake by tissues. Thus, CETP-mediated removal of CE from HDL particles cannot in and of itself explain the reduction in HDL protein (apoa-i) levels and enhanced clearance of HDL apoa-i that has been observed in hypertriglyceridemic humans, nor is it likely to entirely explain the reduction of HDL-c in this condition. Instead, as several investigators have proposed, it may be that the compositional changes induced in HDL by CETP may predispose HDL to greater subsequent catabolism, which can better explain the reduction in HDL-c and apoa-i plasma concentrations [4,20,34,55]. The Effect of TG Enrichment of HDL on HDL Particle Catabolism We and other investigators have postulated that the increase in HDL core TG content that occurs as a result of increased neutral lipid exchange between TG-rich lipoproteins and HDL in hypertriglyceridemic, insulin resistant states plays an important role in the decline in HDL particle numbers and cholesterol content, potentially by predisposing HDL particles to enhanced catabolism [4,8,34,36,56]. Studies have specifically examined the effect of alterations in HDL TG content on HDL catabolism. In a series of in vitro experiments, Liang et al. found that incubation of HDL with CETP and VLDL (to TG-enrich HDL) produced a time-dependent dissociation of apoa-i from HDL [57]. We further investigated whether TG enrichment of HDL directly increases HDL catabolism in vivo. In healthy male subjects, the clearance of apoa-i associated with HDL isolated from the fasting state was compared to the clearance of HDL that had been TG enriched in vivo using a synthetic TG emulsion (intralipid) [58]. We observed that a mean twofold physiological increase in the TG content of HDL (from 3% to 6% of HDL mass) resulted in a significant, 26% increase in the FCR of HDL apoa-i [58]. Furthermore, the increase in HDL TG content correlated strongly and significantly with the increase in apoa-i catabolism [58]. In contrast, other changes in HDL composition, similar to those observed after a high fat meal (changes in phospholipids, cholesterol, apoe, apoc-iii, and apoc-i), were not strongly associated with changes in HDL apoa-i FCR [58]. Overall, this study demonstrated in humans that TG enrichment of HDL directly increases the catabolism of HDL apoa-i, particularly in LpA-I particles [58]. A potential mechanism for the enhanced catabolism of TG-enriched HDL was provided by Sparks et al. [59]. In a series of in vitro experiments with reconstituted LpA-I particles, the investigators calculated that a decrease in the CE/TG ratio in LpA-I particles (as when the particles are TG-enriched) decreases the thermodynamic stability and structural integrity of the particles [59]. This would tend to alter the surface charge and structure of apoa-i, making it more likely to dissociate from the particle [59]. The authors therefore contended that the dissociation of apoa-i from LpA-I particles having a low CE/TG ratio, as when the particles are TG-enriched, would be enhanced in hypertriglyceridemic states [59]. Whether this mechanism also applies specifically to human TG-rich HDL in vivo remains to be determined. Does TG Enrichment of HDL Per SE Enhance HDL Catabolism in the Absence of Lipolytic Modification of the Particles? Overall, the above findings indicated that variations in the TG content of HDL can have a significant impact on HDL catabolism. The question still remained, however, as to whether TG enrichment of HDL per se destabilizes HDL particles sufficiently to enhance the subsequent catabolism of HDL in vivo or if TG enrichment merely predisposes HDL to greater interaction with, and remodelling by, plasma factors which regulate HDL metabolism that is with intravascular lipolytic enzymes (or lipases). Indeed, in the study of Lamarche et al. (described above) in which the

4 424 S. Rashid et al. / Clinical Biochemistry 36 (2003) FCR of TG-rich vs. fasting HDL was examined in humans, we surmised that a sufficient quantity of lipases would be expected to be present in the normal human study subjects to modify and potentially enhance the metabolism of the TG-rich HDL injected in vivo [58]. Support for this view can also be found in a study by Horowitz et al. using isolated perfused rabbit kidneys (the kidney is a major site of HDL apoa-i degradation and catabolism in vivo) [45]. The investigators showed that the renal clearance of HDL apoa-i was not significantly enhanced unless the TG enriched HDL was subsequently treated with partially purified lipases [45]. Most in vitro studies on the subject also support the idea that both TG enrichment of HDL and hydrolysis by lipolytic enzymes are required to destabilise HDL particles to a significant extent. Clay et al., for example, compared the composition of HDL isolated from normolipidemic human plasma with HDL isolated from plasma that had been incubated with CETP and VLDL (to TG enrich the HDL) in the presence and absence of the lipolytic enzyme hepatic lipase (HL) [60]. Incubation of human plasma with CETP and VLDL resulted in a marked increase in HDL TG, a decline in HDL CE, but only minimal changes in apoa-i content vs. HDL isolated from plasma alone [60]. The addition of HL to the incubation mixture, however, resulted in loss of virtually all of the acquired TG, a further loss in CE, and a major loss of apoa-i from HDL ( 30%) [60]. We wished to further determine the roles of HDL TG enrichment and lipolytic modification of the particle on HDL catabolism in an in vivo setting. We investigated whether TG enrichment of HDL per se is sufficient to enhance HDL catabolism, as some investigators have contended, or whether this alteration in particle composition must also be accompanied by substantial lipolysis of HDL lipids [61]. We specifically compared the metabolic clearance of the apoa-i and CE components of TG-enriched and fasting, relatively TG-poor rabbit HDL in the wild-type New Zealand white (NZW) rabbit, an animal model naturally deficient in the lipolytic enzyme, hepatic lipase (HL) [61]. Whole rabbit HDL was first enriched with TG by incubating HDL ex vivo with human VLDL, isolated by ultracentrifugation, and labeled with 131 I and 3 H-cholesteryl oleyl ether tracers [61]. The tracers were then injected into recipient NZW rabbits. We achieved an 87% mean physiologic increase in the percentage mass of TG in the TG-rich HDL vs. control TG-poor HDL, and apart from the normal physiologic depletion of HDL CE associated with TG enrichment of HDL, other measured components of HDL (phospholipid, protein, and size) remained unaltered [61]. Nonetheless, we found no significant difference in the FCR of HDL apoa-i nor in the rate of selective HDL CE clearance between the tracers [61]. We postulated that HL deficiency in the rabbit model could account for the lack of observed difference between the clearance of TG-rich and fasting rabbit HDL [61]. In other words, we showed that TG enrichment of the particles, in the absence of HL activity, is not sufficient per se to enhance HDL particle clearance [61]. Increased postheparin plasma HL Activity is an Integral Feature of Insulin Resistant States and Plays an Important Role in the Catabolism of TG-rich HDL There are at least three primary lipases tethered to the vascular endothelium and active against plasma lipoproteins LPL, HL, and endothelial lipase (EL) [62]. The in vitro evidence to date suggests that HL plays an important role in promoting the catabolism of the TG-rich HDL prevalent in hypertriglyceridemic, insulin resistant states [60,63]. Although EL has been shown to have a major effect in altering HDL plasma concentrations by enhancing its catabolism, since EL is predominantly a phospholipase and has minimal TG lipase activity [64], it is unlikely to be the primary lipase mediating the enhanced catabolism of TG enriched HDL, although this has not specifically been examined. Conversely, incubation of TG enriched HDL with either LPL or HL in vitro have both been shown to mediate a reduction in HDL size and a loss of apoa-i from HDL [60,65]. In contrast to LPL, however, HL has a higher affinity for HDL than for VLDL or chylomicrons [66,67]. Furthermore, among the different subfractions of HDL, HL has been shown in vitro to selectively hydrolyze the relatively TGrich HDL 2 and HDL 1 subfractions [68,69]. HL is a 476 amino acid glycoprotein lipolytic enzyme that is synthesized by hepatocytes [70,71]. It is found localized at the surface of liver sinusoidal capillaries anchored by heparan sulfate proteoglycans [72,73]. In terms of its regulation, HL appears to be modulated by several different genetic and environmental factors such as gender and polymorphisms in the HL promoter (LIPC) locus [74]. Moreover, as demonstrated in numerous studies, an elevation in postheparin plasma HL activity is a particularly prevalent occurrence in insulin resistant states such as obesity and type 2 diabetes, and appears to be related to the low HDL levels in these states [75 79]. Despres et al., for instance, investigated the association between body fat composition, as determined by computed axial tomography, and postheparin plasma HL activity in a sample of 16 obese women [79]. The investigators demonstrated significant positive associations between intra-abdominal fat deposition and HL activity (p 0.66, p 0.005), which in turn correlated negatively with HDL 2 -c levels (p 0.66, p 0.05) [79]. Conversely, in another study of 21 healthy older men who underwent diet-induced weight loss, loss of intra-abdominal fat was found to be significantly correlated with a reduction in postheparin HL, which in turn was associated with increased HDL 2 -c levels [78]. Other investigators have observed similar correlations between HL and other indices of adiposity in addition to intra-abdominal fat content and further contended that the relation between HL and adiposity reflects the modulating

5 S. Rashid et al. / Clinical Biochemistry 36 (2003) effect of secondary factors associated with increased adiposity namely hyperinsulinemia. Nie et al., for example, investigated the relation between body mass index (BMI) and HL activity in a large sample of healthy men stratified according to genetically defined differences in HL activity [80]. Results of the study demonstrated highly significant (p 0.001) associations between BMI and HL activity in this cohort [80]. The investigators further observed that mean BMI tended to be similar in men with varying HL promoter genotypes, indicating that the relation between BMI and HL activity was not due to an effect of HL activity on adiposity, but rather adiposity, or factors associated with adiposity, on HL activity [80]. More specifically, the authors of this study surmised that the correlation between BMI and HL activity reflected an increase in circulating plasma insulin levels, which tends to accompany increases in BMI [80]. In support of this theory, a site at the promoter region of the HL gene has recently been identified as being similar in sequence to a motif involved in the binding of insulin-responsive transcription factors [74,81], although the functional significance of this site is not known. HL activity, however, unlike LPL, is not upregulated in a clear cut fashion by insulin. Studies investigating the effect of insulin on HL activity have shown contradictory results. For example, while some studies in patients with type 2 diabetes have shown an increase in HL activity with hyperinsulinemia [75], others have shown a decline [82]. It is more likely that instead of hyperinsulinemia per se, insulin resistance at the liver secondary to obesity or type 2 diabetes in some fashion induces the increase in HL activity in these states. Consistent with this idea, studies in normal and diabetic rats have shown that increases in liver HL activity are induced by chronic, but not acute insulin administration [83]. The authors of the study concluded that chronic alterations in metabolic status that occur in response to prolonged hyperinsulinemia and insulin resistance induced the increase in liver HL activity in the animals [83]. Several more recent studies in humans have reported direct correlations between HL activity and indices of insulin resistance, such as the plasma insulin response to oral glucose [78,78,84]. Very recently, we have shown that HL activity is increased in fructose-fed Syrian golden hamsters, an animal model of insulin resistance, and reduced with rosiglitazone treatment, an insulin sensitizer with PPAR agonist activity (unpublished observations). The Effect of HL on HDL Catabolism in the Context of TG Enrichment of HDL While alterations in HL activity are widely believed to play an important role in HDL metabolism, studies investigating the interaction between HL and HDL in humans have shown conflicting results. For instance, a common single nucleotide polymorphism in the promoter region (position 514) of the HL gene locus (LIPC), that has been Fig. 1. Schematic design of the proposed mechanisms of HDL lowering in insulin resistant, hypertriglyceridemic states. In insulin resistant, hypertriglyceridemic states there is a greater mass transfer of TG from the increased pool of apo B-containing, TG-rich lipoproteins, with heteroexchange of CE from HDL to TG-rich lipoproteins. This process results in the formation of HDL particles that are relatively TG enriched and CE depleted, in itself contributing to the lowering of plasma HDL cholesterol concentration. We propose that the TG-rich HDL particles thus formed are also cleared more rapidly from the circulation than non-tg-rich HDL due to one or all of the following three mechanisms: 1.) TG-rich, CE-depleted HDL particles have been shown to be thermodynamically less stable, having their apoa-i in a more loosely bound form; 2.) TG-rich HDL are more readily lipolyzed by HL, thereby reducing HDL size, and resulting in free apo A-I or lipid-poor pre- 1 particles (containing apoa-i together with a small amount of lipid) being shed from the particles; 3.) the HDL remnant particles that have been reduced in size may themselves be more readily cleared from the circulation. The above processes may then enhance HDL apoa-i FCR (fractional catabolic rate). The end result is a lowering of HDL-c and apoa-i levels in plasma. It should be noted that in addition to being irreversibly cleared from the circulation, lipolytically modified HDL or apo A-I that is shed from the particles can also undergo recycling to spherical HDL by the re-acquisition of lipid (indicated by the dashed lines). shown to significantly impact on HL activity levels in humans, has been associated with reduced HDL-c and apoa-i levels in some, but not in all populations studied [50,85 88]. Similarly, interventions that alter HL activity in humans for example, oral estrogen administration in womenare not consistently associated with variation in HDL-c levels [68]. These findings suggest that the effect of HL on HDL metabolism is not consistent in physiologically relevant settings, and is subject to modulation by metabolic, environmental, or genetic factors. Previous studies indicate that the hypertriglyceridemic, insulin resistant state is a clinical condition in which HL does exert a consistent, important effect on plasma HDL concentrations. Studies in humans investigating the role of HL on HDL metabolism in insulin resistant states, such as those described above, however, have only revealed associations between the two. They did not specifically determine mechanisms responsible for the decline in HDL levels occurring in association with increased HL activity in insu-

6 426 S. Rashid et al. / Clinical Biochemistry 36 (2003) lin resistance. We have postulated that the TG enrichment of HDL and the elevated HL lipolytic action that occur in these conditions act in concert to promote the increased HDL catabolism and decline in HDL-c levels in these states (illustrated in Fig. 1). To directly test this hypothesis, we investigated the combined effects of TG enrichment of HDL and lipolytic transformation of HDL by HL on the subsequent metabolic clearance of HDL apoa-i in the HL-deficient NZW rabbit [89,90]. More specifically, we compared the clearance of apoa-i associated with TG-rich and native, relatively TG-poor rabbit HDL tracers that had been lipolyzed by HL using two different methods (1) ex vivo lipolysis by purified human HL and [89] (2) in vivo lipolysis by adenovirus-mediated transfer of the human HL transgene (rhl-adv) [90]. While previous studies in several animal models have shown a decline in HDL levels with HL expression, these studies have generally examined this phenomenon in the presence of supraphysiological expression of HL [91 93]. Those studies also did not determine the role of HL action in the context of TG enrichment of HDL, which would more closely mimic the characteristic of HDL in insulin resistant, hypertriglyceridemic states. Our studies in the rabbit model, in contrast, investigated both of these physiologic processes and involved the use of moderate HL enzyme activity. Results of our in vivo studies in the rabbit showed that apoa-i associated with TG-enriched HDL, modified ex vivo by catalytically active HL, and reduced in size, was cleared 22% more rapidly vs. TG-enriched HDL incubated with heat-inactivated HL and 26% more rapidly than relatively TG-poor HDL incubated with active HL (p 0.05 for both) [89]. In rabbits injected with rhl-adv, HL activity increased 2- to sevenfold above endogenous levels. Moreover, there was a marked 50% enhancement in the metabolic clearance of TG-enriched HDL vs. TG-poor HDL in rhl- Adv rabbits (p 0.01) [90]. In contrast, in rabbits injected with the control rlacz-adv, there was no significant enhancement of apoa-i associated with TG-rich vs. native HDL [90]. Furthermore, in rabbits expressing human HL, but not in control rabbits, there was a significant decline in major HDL lipids, including HDL TG, phospholipid, and HDL-c, and a decrease in HDL particle size [90]. Overall, our studies in the HL-deficient rabbit model established that TG-enrichment of HDL, in the presence, but not in the absence, of physiologic levels of lipolytically active HL, is associated with enhanced HDL apoa-i clearance. Shedding of free apoa-1 or the formation of lipid poor small pre 1-HDL during intravascular lipolysis of TG-rich HDL by HL could enhance apoa-1 clearance from the circulation [94]. Alternatively, remnant HDL particles produced by this lipolytic process, may be more rapidly removed from the circulation by receptor-mediated uptake [95,96]. Both of these processes may play a role in the lowering of HDL-c and apoa-i in insulin resistant, hypertriglyceridemic states. Barrans et al. had previously demonstrated that HL-mediated lipolysis of TG enriched HDL 2 particles results in the formation of smaller, -HDL particles, which they termed remnant HDL [95]. They further demonstrated that apoa-i associated with these remnant HDL showed greater high affinity binding and uptake into HepG2 cells and isolated perfused liver compared to nonlipolyzed TG-rich HDL 2 [95,96]. Our studies in the rabbit provide in vivo support for this concept. It should be noted that our studies and those discussed immediately above have focused on the catabolic processes of HDL apoa-i clearance and cellular uptake, respectively. These studies did not investigate the process of HDL retroendocytosis and recycling described in several studies. Studies conducted in various primary cells and cell lines have indicated that a portion of HDL apoproteins bound and internalized by cells are resecreted or recycled via a retroendocytosis process [97 100]. Recycling of HDL and its apolipoproteins is thought to be mediated by the endosome recyling compartment [98] and has been postulated to involve the LDL receptor-related protein (LRP) [101], although this remains to be specifically tested. In future studies, the relative importance of HDL recycling in determining overall HDL levels will need to be examined in vivo across different animal models and in humans. In general, the etiology delineated in this review can explain, at least in part, the lowering of HDL levels in individuals demonstrating insulin resistance and frank hypertriglyceridemia, which constitute by far the majority of individuals with reduced HDL concentrations [10]. In individuals with low HDL in the absence of frank hypertriglyceridemia (i.e., those with isolated low HDL), however, HDL TG enrichment and elevated HL activity cannot explain the lowering of HDL levels. Subjects with isolated low HDL have been reported to be insulin resistant in some [102] but not all populations [103,104] and neither TG enrichment of HDL nor an elevation in HL activity has consistently been reported in these individuals [32,103]. Isolated low HDL has, in fact, been characterized as a metabolic disorder distinct from the hypertriglyceridemia-low HDL phenotype [10], and, as discussed above, has been attributed in many cases to rare familial syndromes [15]. Conclusions Overall, the interaction between HDL that is TG enriched and HL action plays an important role in the enhanced catabolism of HDL in insulin resistant, hypertriglyceridemic states, such as occurs in association with abdominal obesity and type 2 diabetes. It may be argued that the accelerated HDL clearance in these conditions contributes to reverse cholesterol transport, and may therefore not be pro-atherosclerotic. It is more logical to infer, however, that HL-mediated lipolysis of the TG enriched and CEdepleted HDL particles characteristic of these states will contribute quantitatively little to HDL CE uptake and reverse cholesterol transport [105]. Instead, the lowering of

7 S. Rashid et al. / Clinical Biochemistry 36 (2003) HDL plasma concentrations by this mechanism would result in fewer HDL particles to carry out antiatherosclerotic functions of HDL, including its inhibitory effects at the arterial wall on LDL oxidation, cellular proliferation, and macrophage formation. Thus, TG enrichment combined with HLmediated lipolysis of HDL likely contributes to the accelerated rate of atherosclerosis that is characteristic of insulin resistant conditions. In addition to being unfavourable, and likely pro-atherogenic, the TG enriched HDL-elevated HL phenotype is a prevalent, integral feature of insulin resistant, hypertriglyceridemic individuals. It is important to note that multiple pathways of HDL clearance have been identified to date, and the hypertriglyceridemic-hdl-lipolysis phenomenon may be only one such major pathway perturbed in insulin resistant individuals. Other mechanisms may also contribute substantially to HDL-c and HDL apoa-i lowering in insulin resistant/hypertriglyceridemic states. Future studies are required to elucidate more precisely the perturbations and contributions of the various HDL metabolic pathways in the lowering of HDL plasma concentrations in insulin resistant, hypertriglyceridemic states. References [1] Ford ES, Giles WH, Dietz WH. Prevalence of the metabolic syndrome among US adults: findings from the third National Health and Nutrition Examination Survey. JAMA 2002;287: [2] Reaven GM. Banting lecture Role of insulin resistance in human disease. [Review] [71 refs]. Diabetes 1988;37: [3] Reaven GM. Pathophysiology of insulin resistance in human disease. Physiol Rev 1995;75: [4] Lewis GF, Steiner G. Hypertriglyceridemia and its metabolic consequences as a risk factor for atherosclerotic cardiovascular disease in non-insulin-dependent diabetes mellitus. [Review] [204 refs]. 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