Lipolytically Modified Triglyceride-Enriched HDLs Are Rapidly Cleared From the Circulation
The precise biochemical mechanisms underlying the reduction of HDL levels in hypertriglyceridemic states are currently not known. In humans, we showed that triglyceride (TG) enrichment of HDL, as occurs in hypertriglyceridemic states, enhances the clearance of HDL-associated apolipoprotein A-I (apoA-I) from the circulation. In the New Zealand White rabbit (an animal model naturally deficient in hepatic lipase [HL]), however, TG enrichment of HDL is not sufficient to alter the clearance of either the protein or lipid moieties of HDL. In the present study, therefore, we determined in the New Zealand White rabbit the combined effects of ex vivo TG enrichment and lipolytic transformation of HDL by HL on the subsequent metabolic clearance of HDL apoA-I. Results of the in vivo kinetic studies (n=18 animals) showed that apoA-I associated with TG-enriched rabbit HDL modified ex vivo by catalytically active HL was cleared 22% more rapidly versus TG-enriched HDL incubated with heat-inactivated HL, and 26% more rapidly than fasting (TG-poor) HDL incubated with active HL (P<0.05 for both). Furthermore, a strong correlation was observed between the HDL TG content and apoA-I factional catabolic rate (0.59, P<0.05) in the combined active HL groups. These data establish that TG enrichment of HDL with subsequent lipolysis by HL enhances HDL apoA-I clearance, but neither TG enrichment of HDL without HL lipolysis nor HL lipolysis in the absence of previous TG enrichment of HDL is sufficient to enhance HDL clearance. These data further support the important interaction between HDL TG enrichment and HL action in the pathogenesis of HDL lowering in hypertriglyceridemic states.
There is a strong inverse correlation between plasma levels of HDL cholesterol (HDL-C) and the development of atherosclerotic cardiovascular disease.1,2⇓ Hypertriglyceridemia is one of the most frequent metabolic abnormalities observed in association with low HDL-C,1,3,4⇓⇓ although the mechanism accounting for this association is not entirely clear. We have previously shown in humans that triglyceride (TG) enrichment of HDL predisposes HDL particles to more rapid clearance of their apolipoprotein A-I (apoA-I) component.5 ApoA-I is the major protein component of HDL and the fractional catabolic rate (FCR) of apoA-I is a surrogate marker of HDL holoparticle clearance. Moreover, because the catabolism of apoA-I correlates closely with plasma HDL-C levels,6,7⇓ we surmised that the enhanced catabolism of apoA-I from TG-enriched HDL plays an important role in HDL-C lowering in hypertriglyceridemic states.5,8–10⇓⇓⇓ In contrast to our observation in humans,5 in a recent study in New Zealand White (NZW) rabbits, we found that TG enrichment of rabbit HDL per se did not enhance the FCR of HDL apoA-I nor HDL cholesteryl ester (CE).11 We hypothesized that the natural deficiency of hepatic lipase (HL) in the NZW rabbit could explain the lack of enhanced clearance of TG-enriched HDL in this model.11
Several lines of evidence indicate that HL plays a prominent role in mediating HDL metabolism. It has been observed in humans that postheparin HL activity is inversely associated with plasma levels of HDL-C.12–14⇓⇓ Similarly, overexpression of HL in several animal models resulted in marked reductions in HDL-C.15–17⇓⇓ HL possesses both a lipolytic function and a non-lipolytic function, both of which play a role in mediating HDL catabolism, but appear to mediate different HDL metabolic pathways. In adenovirus transfection experiments overexpressing HL in HL-deficient mice, the ligand function of HL enhanced selective HDL-CE uptake and clearance, whereas the lipolytic function of HL was required to enhance HDL apoA-I clearance.17,18⇓ Nevertheless, the effect of HL lipolytic action on the in vivo clearance of TG-enriched HDL has not previously been examined. Similarly the effect of a physiologically relevant level of HL lipolysis ex vivo on HDL catabolism in vivo has not been investigated, particularly in an animal model naturally deficient in HL.
In the present study, we investigated the effects of TG enrichment and HL-mediated lipolysis of HDL on the subsequent rate of clearance of HDL apoA-I in the NZW (ie, wild type) rabbit, an animal model deficient in HL. Our specific aim was to determine whether TG enrichment of HDL, similar to that observed in hypertriglyceridemic states, followed by ex vivo hydrolysis of the HDL by HL, would enhance the clearance of HDL apoA-I to a greater extent than TG enrichment without lipolysis, or lipolysis of HDL particles that were not TG-rich. We compared the metabolic clearance of TG-rich HDL incubated ex vivo with catalytically active HL to (1) the rate of clearance of TG-rich HDL incubated ex vivo with heat-inactivated HL and (2) the clearance of fasting (TG-poor) HDL incubated with active HL.
Isolation of HDL, Incubation with Human VLDL and HL, and Radiolabeling
All procedures followed were in accordance with institutional guidelines. Blood was obtained via cardiac puncture from overnight-fasted male donor NZW rabbits sedated with ketamine and xylazine. HDL (density [d], 1.063 to 1.25) was isolated by sequential ultracentrifugation of whole rabbit serum11 and then dialyzed in Tris buffer (0.15 mol/L NaCl and 0.1 mol/L Tris-Base, pH 7.4). To prepare the TG-rich tracers, 12 mL of rabbit serum was incubated ex vivo with postprandial human VLDL for 6 hours, as we have previously described.11
In paired experiments, an equal amount of TG-rich HDL (2.4±0.1 mL; 11.1±2.0 mg HDL protein) was incubated with either catalytically active HL (n=6) or with HL that had previously been heat inactivated at 58°C for 1 hour (n=6). In separate experiments, a similar quantity of fasting HDL (2.1±0.1 mL; 7.4±0.5 mg HDL protein, n=6 incubations) was incubated with active HL. Included in each mixture was bovine serum albumin at a final concentration of 2.5% and 60 U/mL of heparin sodium (Organon Teknika). The incubations with active HL were performed at 34°C for 4 hours, whereas those with inactive HL were performed at 4°C for 4 hours. HDL from each incubation was reisolated by ultracentrifugation at d=1.25 mg/dL, and dialyzed overnight in Tris-EDTA buffer (0.15 mol/L NaCl, 0.01 mol/L Tris-Base, and 5 mmol/L EDTA, pH 7.4).
The reisolated HDL preparations (1.5±0.4 mg) were iodinated by a modification of the iodine monochloride method of McFarlane19 by using 500 μCi of 131I (NEN). Cold HDL carrier protein, 1.5±0.4 mg, was added to the iodinated HDL. The HDL tracers were then washed at d=1.25 g/mL at 4°C, followed by dialysis in Tris-EDTA buffer.
Analysis of Tracer Composition and Size
The chemical composition, size (as determined by 4% to 30% non-denaturing polyacrylamide gradient gel electrophoresis), and integrity of the HDL tracers were analyzed as previously described.11,20,21⇓⇓ The size distribution of the labeled HDL tracers over time was also monitored. Briefly, each HDL tracer prepared, as described above, was incubated with rabbit serum at 37°C. Serum was then collected from the incubation mix at 2 minutes, 10 minutes, 30 minutes, 1 hour, 2 hours, and 4 hours. For the fasting (TG-poor) HDL+active HL tracer, the distribution of radioactivity between size fractions was also monitored in vivo, after injection of the tracer into the rabbit. HDL isolated from serum was then analyzed for size on polyacrylamide gradient gel electrophoresis gels. A radius of 5.5 nmol/L was arbitrarily chosen as the separation point for small and large particle size. A 5.5-nmol/L peak radius was previously shown to be the size of a population of TG-enriched HDL particles in hypertriglyceridemic subjects postprandially.22 The relative concentration of small and large particles was determined by multiplying the size of each band by its fractional area.23 Gel slices of the small and large HDL were then counted for radioactivity.
Isolation and Assay of HL
Purification of HL from postheparin human plasma was achieved by heparin sepharose affinity chromatography essentially as previously described by using a linear sodium chloride gradient24,25⇓ applied to HiTrap heparin sepharose affinity columns (Amersham Pharmacia Biotech). Purified HL was detected by Western blotting analysis (by using a monoclonal antibody, a gift from Dr. J. Hill, University of British Columbia, Vancouver, Canada). HL activity was measured as previously described with 14C-triolein as substrate in the presence of 1 mol/L NaCl.13 The activity of the active HL added to the incubation mix was similar for the TG-rich HDL+active HL and fasting HDL+active HL experiments (52.4±9.7 μmol free fatty acid (FFA)/h, n=12; P=not significant between TG-rich and fasting experiments).
HDL Turnover Study
NZW male rabbits, 4.5 to 5.2 kg in weight, were used for the HDL kinetic experiments (6 animals in each of 3 groups for a total of 18 animals).
An aliquot of each tracer containing 1.0±0.2 mg HDL protein and 4.3±1.0 (×107) cpm of 131I-HDL was injected into the right marginal ear vein. Blood samples (2 mL) were obtained during the next three days from a vein in the opposite ear at the following time intervals: 10 minutes and 1, 2, 3, 4, 6, 24, 27, 30, 48, and 51 hours. HDL (d1.063 to d1.25) was isolated (from ≈1 mL serum) by sequential ultracentrifugation at 4°C.11 131I radioactivity specifically associated with HDL apoA- I (isolated with 15% SDS-PAGE gels) was determined as previously described.21
The radioactivity die-away curves were analyzed by using a two-pool model as previously described20,21⇓ with the SAAM II program (SAAM Institute). The average coefficient of variation for apoA-l FCRs were 14±1% for TG-rich HDL+HL, 24±4% for TG-rich HDL, and 14±4% for fasting (TG-poor) HDL+HL.
Results are presented as mean±SEM. Paired t tests were performed to compare log-transformed FCR values between TG-rich HDL+active HL and TG-rich HDL+inactive tracers (because the same donor HDL was used for these two experiments) and to test differences in tracer composition and size. Unpaired t tests were performed to compare the log-transformed FCR, composition, and size between TG-rich HDL+active HL and fasting (TG-poor) HDL+active HL tracers (because donor HDLs from separate animals were used for these two experiments). Correlates of HDL TG content and apoA-I FCR were obtained by using Pearson correlation analysis.
The mean total serum TG, cholesterol, CE, and PL concentrations over the course of the experiments (n=18 animals) averaged 0.76±0.04, 0.75±0.04, 0.69±0.04, and 0.97±0.04 mmol, respectively, showing that the lipid parameters were in steady state. There were no significant differences in serum lipid levels in the animals receiving the TG-rich HDL+active HL, TG-rich HDL+inactive HL, and fasting (TG-poor) HDL+active HL tracers (n=6 animals in each group).
Table 1 presents the mean lipid compositions and the sizes of the rabbit HDL tracers. Lipolysis of the TG-rich HDL with active HL resulted in a 31% average decline in HDL TG content (P<0.001), a 29% increase in the relative mass of CE (P=0.07), and a 2-fold increase in the FFA content (P=0.06) compared with TG-rich HDL incubated with heat-inactivated HL. In addition, in comparison to fasting (TG-poor) HDL+active HL, the TG-rich HDL+active HL injectate contained a significantly greater %mass of TG (2-fold; P<0.05) and markedly less cholesterol, CE, and phospholipid (PL) contents (52%, 57%, and 38%, respectively; P<0.01, P<0.001, and P<0.05, respectively). The TG-rich HDL+active HL tracer was markedly reduced in peak size in comparison to TG-rich HDL+inactive HL and tended to be larger than fasting (TG-poor) HDL+active HL (peak radius, 5.09±0.21, 5.84±0.30, and 4.71±0.07 nm, respectively; P<0.01 and P=0.12, respectively).
The relative distribution of radioactive label in HDL tracers was also monitored in HDL size subfractions in rabbit serum in vitro (for all 3 HDL tracers) and in vivo (for the fasting [TG-poor] HDL+active HL tracer) (as discussed in Methods). The majority of HDL injectate 131I counts were found to be in the small-sized HDL subfraction for all 3 HDL tracers (70±6%). After incubation of the HDL tracers with rabbit serum in vitro and in vivo, the proportion of counts did not shift substantially between HDL size subfractions. Two hours after injection, 66±6% of 131I counts were in the small-sized HDL subfraction (in vitro and in vivo observations combined). This result reflects a similar exchange process of apoA-I in all 3 HDL tracers with the heterogeneous HDL pool in serum.
Effect of HL-Mediated Lipolysis of TG-rich HDL on the FCR of HDL ApoA-I
Figure 1A shows the die-away clearance curves of radiolabeled-HDL apoA-I from TG-rich HDL+active HL and TG-rich HDL+inactive HL from one representative animal in each group. The kinetic parameters (FCR) derived from the clearance curves are presented in Table 2. Results from the kinetic experiments showed a more rapid clearance of apoA-I associated with TG-rich HDL+active HL compared with apoA-I in TG-rich HDL+inactive HL in 5 of 6 paired experiments, with a 22% greater mean apoA-I FCR of TG-rich HDL+active HL versus TG-rich HDL+inactive HL (P<0.05).
Effect of HDL TG Enrichment, in the Presence of Active HL, on FCR of HDL ApoA-I
Figure 1B illustrates the die-away curves of TG-rich HDL+active HL and fasting HDL (TG-poor)+active HL from one animal in each group. The mean FCR of apoA-I in TG-rich HDL+active HL was 26% greater than fasting (TG-poor) HDL apoA-I. Linear regression analysis revealed a positive correlation between HDL apoA-I FCR and HDL TG content (TG-rich HDL+active HL and fasting (TG-poor) HDL+active HL groups combined; R=0.59, P<0.05; Figure 2).
We have shown in these experiments that the ex vivo action of catalytically active HL on TG-enriched HDL enhances the subsequent metabolic clearance of HDL apoA-I in NZW rabbits, a species naturally deficient in HL. Modification of TG-rich HDL by a physiologically relevant level of HL enzyme activity resulted in a 22% mean increase in the FCR of HDL apoA-I in comparison with TG-rich HDL incubated with heat-inactivated HL. HL modification of HDL that was not TG-rich (ie, fasting [TG-poor] HDL) did not result in a similar enhancement of HDL apoA-I clearance. In fact, the clearance of apoA-I associated with TG-rich HDL+active HL was 26% more rapid than apoA-I in fasting (TG-poor) HDL+active HL. A significant positive correlation was observed, moreover, between the %mass of TG within the HDL tracers and the corresponding HDL apoA-I FCR in the combined TG-rich and fasting (TG-poor) HDL+active HL groups (R=0.59, P<0.05).
Our results are consistent with in vitro studies showing that TG-enriched HDL is a better substrate for HL-mediated hydrolysis and particle destabilization than TG-poor HDL.26 In humans, we have previously shown that TG-lowering (with gemfibrozil treatment) significantly reduces TG enrichment of HDL and consequently reduces the lipolytic transformation of HDL to small HDL species.27 Moreover, it provides a mechanism explaining our previous findings in humans and the NZW rabbit.5,11⇓ Although TG-enrichment of HDL was found to directly enhance the metabolic clearance of HDL apoA-I in healthy human subjects,5 in HL-deficient NZW rabbits, we observed no difference in the clearance of TG-enriched versus native rabbit HDL apoA-I or CE.11 We postulated that a deficiency of HL in rabbits explained the contrasting effects of TG enrichment on HDL metabolism in the two species. The present study clearly supports this hypothesis. Overall, our findings emphasize the importance of TG-HL interactions in altering HDL apoA-I catabolism, and suggest that both TG enrichment and lipolysis by HL are required for the enhancement of HDL particle clearance.
HL has been shown in several experimental model systems to possess both a lipolytic function and a non-lipolytic (ligand) function, both of which play a role in mediating HDL metabolism.17,18⇓ The present studies were not designed, however, to investigate the non-lipolytic properties of HL, but to determine the effect of the lipolytic function of HL on the metabolism of TG-rich HDL. HL action on TG-rich HDL induced a reduction in HDL TG content and size and an increase in HDL FFA content, in comparison with TG-rich HDL+inactive HL; however, the relative proportion of other lipid components of HDL—cholesterol, CE, and PL—were not different between the tracers. Although HL has a well characterized phospholipase activity, much of the PL transfers occur between HDL subfractions and would tend to be masked in our study as we used total HDL.28
During the process of TG enrichment of HDL with VLDL in the present study, cholesterol esterification by lecithin cholesterol acyltransferase was not inhibited and would have added to the depletion of free cholesterol within the HDL injectates and limited the depletion of core CE within HDL during the process of TG loading by cholesteryl ester transfer protein. The HDL tracers in this study were generally in the size and lipid composition range previously reported for rabbit HDL (native rabbit HDL tends to be larger and more TG-enriched than human HDL);29 however, the TG-rich HDL particles incubated with heat-inactivated HL were slightly larger in size.
Remodeling of TG-enriched HDL by active HL resulted in enhanced HDL apoA-I clearance in five of six paired experiments conducted (P<0.05 for the group). We speculate that TG enrichment of HDL favors greater lipolytic modification of the particle by HL, resulting in the formation of remnant HL particles that are more rapidly removed from the circulation by receptor-mediated holoparticle uptake. In a previous in vitro study, Barrans et al28 had shown that the combination of TG-enrichment of HDL2, followed by HL-mediated hydrolysis of core TG, results in the formation of smaller α-HDL particles of reduced size, which they termed remnant HDL. ApoA-I associated with these remnant HDL particles showed greater high-affinity binding and uptake into human hepatoma cells compared with non-lipolyzed TG-rich HDL.30 Remnant HDL apoA-I also demonstrated enhanced clearance in rat liver perfusion studies.28 These findings suggested that remnant HDL undergoes greater holoparticle uptake at the liver. The present study, showing rapid in vivo clearance of apoA-I associated with lipolytically modified TG-rich HDL particles, provides in vivo support for this concept.
We have previously shown that lipolytically modified, small human HDL particles (postprandial HDL isolated from humans, after intravascular administration of heparin) are cleared more rapidly from the rabbit circulation, in comparison with large unlipolyzed HDL (postprandial HDL isolated from humans before heparin administration),21 a finding that is completely in keeping with the findings of the present study. In the former study,21 the use of lipolytically modified human HDL in the rabbit raised concerns of cross-species physiological relevance. In addition, in that study the lipolysis was massive and nonspecific (ie, presumably because of a combination of HL, lipoprotein lipase, and endothelial lipase [EL]) and resulted in a marked increase in the FFA content of HDL. Nevertheless, the present study, which is far more physiologically relevant, supports our previous findings. Recently, EL, a new member of the lipase family, has been found to have a greater effect on HDL metabolism than HL when tested in vivo.31 Recombinant adenovirus overexpression of EL in apoA-I–transgenic mice resulted in an ≈50% greater decrease in HDL-C than an equivalent expression of HL.31 However, because EL exhibits predominantly phospholipase activity and comparatively less TG lipase activity, it likely plays less of a role in the metabolism of TG-rich HDL than HL.32 The relative effect of HL versus EL on TG-rich HDL remains to be determined.
In conclusion, results of the present study demonstrate that lipolysis of TG-rich HDL by HL action enhances the rate at which apoA-I is cleared from the circulation in comparison with (1) TG-rich HDL incubated with heat-inactivated HL and (2) fasting (TG-poor) HDL incubated with active HL. Our data indicate that the production of smaller HDL particles by HL enzyme action increases apoA-I clearance from TG-rich HDL. Because apoA-I FCR is a close correlate of HDL-C levels,6,7⇓ the processing of TG-rich HDL by HL can partly explain the deleterious reduction in HDL-C levels in hypertriglyceridemia. Equally important, we observed a strong correlation between the HDL TG content and apoA-I FCR in those studies in which the HDL tracers were subjected to HL lipolysis, which highlights the negative impact of progressively greater hypertriglyceridemia and HDL TG enrichment on HDL levels. Future studies are required to elucidate more precisely how HL processing affects the interaction between lipolytically modified HDL remnants and lipoprotein receptors to enhance HDL clearance in hypertriglyceridemic states.
This work was supported by an Operating Grant from the Heart and Stroke Foundation of Ontario (to Dr Lewis). Shirya Rashid is a PhD student supported by the Heart and Stroke Foundation of Canada. Dr Lewis is a Career Investigator of the Heart and Stroke Foundation of Canada and Canada Research Chair in Diabetes. Dr P. Hugh R. Barrett at the Department of Medicine, University of Western Australia, is supported by National Institutes of Health grant NCRR12609 and the Healy Medical Research Foundation. We thank Dr J. Hill for his gift of human hepatic lipase monoclonal antibody.
Received November 29, 2001; revision accepted January 7, 2002.
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- ↵Lewis GF, Rashid S, Uffelman K, Lamarche B. Mechanism of HDL lowering in insulin resistant states.In: Angel A, Dhalla N, Pierce G, Singal P, eds. Diabetes and Cardiovascular Disease: Etiology, Treatment and Outcomes. New York, NY: Klewer Academic/Plenum; 2001: 273–277.
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- ↵O’Meara NM, Cabana VG, Lukens JR, Loharikar B, Forte TM, Polonsky KS, Getz GS. Heparin-induced lipolysis in hypertriglyceridemic subjects results in the formation of atypical HDL particles. J Lipid Res. 1994; 35: 2178–2190.
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- ↵Clay MA, Newnham HH, Barter PJ. Hepatic lipase promotes a loss of apolipoprotein A-I from triglyceride-enriched human high density lipoproteins during incubation in vitro. Arterioscler Thromb. 1991; 11: 415–422.
- ↵Barrans A, Collet X, Barbaras R, Jaspard B, Manent J, Chap H, Perret B. Hepatic lipase induces the formation of pre-1 high density lipoprotein (HDL) from triacylglycerol-rich HDL2. J Biol Chem. 1994; 269: 11572–11577.
- ↵Maugeais C, Tietge UJ, Sun G, Marchadier DHL, Jaye M, Glick JM, Rader DJ. Comparison of endothelial lipase and hepatic lipase on HDL metabolism in vivo. American Heart Association Scientific Sessions. 2000; 102: II-596.Abstract.