Discussion

This study of the metabolic behavior in plasma of size-defined HDL subfractions provides new insights into the physiology of an insufficiently understood lipoprotein. Strengths of the study include its applicability because it was done in free-living humans who had typical phenotypes associated with low or high HDLc, complete dietary control using controlled feeding of a commonly eaten diet, and the extensive testing by compartmental modeling of pathways directly implicated by the experimental results. The central finding is that the 4 HDL size subfractions are all secreted into plasma and circulate mainly within the secreted size for 1 to 4 days before they are removed from the circulation. These results are not consistent with stepwise size expansion and contraction as major pathways for HDL metabolism and especially not with a common metabolic framework that considers very small discoidal HDL as the main nascent particle that is a precursor of the larger cholesterol-rich HDL.^10,13,32 Still, the complex peaks of the tracer enrichment curves in apoA-I in HDL size subfractions, most clearly evident in the smallest discoidal HDL, and small but real differences among the subfractions in peak time are evidence for interconversion of size subfractions but as minor pathways. Therefore, we propose that the processes of cholesterol uptake and transfer into and out of HDL occur mainly within rather than between size subfractions.

The tracer appeared in plasma apoA-I of 4 HDL sizes at similar times, the tracer enrichment time plots intersected the time zero origin, and the major peaks of the apoA-I tracer enrichment curves did not progress in time from small to large HDL. These observations together provide a strong case for a considerable degree of secretion into plasma of all 4 HDL size-defined subfractions and metabolism that occurs mainly within the secreted size. These tracer enrichments in apoA-I are strikingly unlike those in kinetic studies of apoB lipoproteins in which the peaks of the enrichment curves follow one another in order of size from large very low–density lipoprotein to small very low–density lipoprotein, intermediate-density lipoprotein, and large and small low-density lipoprotein, and the main component of the low-density lipoprotein apoB tracer enrichment curve starts several hours after tracer enrichment appears in very low–density lipoprotein, thus demonstrating dominance of precursor-product metabolism of apoB lipoproteins according to lipoprotein size.^24–26 Results of previous tracer studies of apoA-I in light and dense HDL are also discordant with a major precursor-product relation of HDL size subfractions in plasma.^21–23

Cells that have ATP-binding cassette A1, including hepatocytes, produce nascent HDL with considerable heterogeneity of size from 7.0 to 12 nm, similar to the range of sizes in the present study. In baby hamster kidney cells, apoA-I solubilizes membrane cholesterol and phospholipid when ATP-binding cassette A1 is available, producing nascent HDL with variable lipid content.³³ The variability in the ratio of membrane cholesterol to phospholipid in the microdomain that engages in HDL formation is responsible for the heterogeneity in size of the nascent HDL, a high cholesterol to phospholipid ratio correlating with large size.³³ Specific acyl chains of the membrane phospholipid also affect the size of nascent HDL. In cultured primary mouse hepatocytes, cholesterol loading of the cells and treatment with an LXR agonist increased formation of all sizes of nascent HDL from 7 to 12 nm, especially the larger ones.³⁴ ApoA1 is able to acquire phospholipid and unesterified cholesterol intracellularly and from the plasma membrane.³⁵ Accordingly, primary mouse hepatocytes transfected with the human apoA1 gene secrete lipidated apoA1 that contains both phospholipid and cholesterol and is in the common size range of HDL2 and HDL3.^36,37

In human embryonic kidney cells expressing ATP-binding cassette A1, the nascent HDL particles that were produced from exogenous apoA-I were mostly spheroidal or spherical in shape from <6 nm to 12 nm in diameter, and 90% of the cholesterol was unesterified.³⁸ Kinetic studies of HDL formation in this cell system³⁹ and previous evidence in J774 macrophages⁴⁰ and human fibroblasts⁴¹ showed that each size subfraction is formed simultaneously at the cell surface and released into the culture media with no evidence of conversion of small to large HDL. All told, this considerable body of evidence on HDL formation and secretion in cultured hepatocytes and other cells showing direct large and small HDL production gives important mechanistic support to the results of our in vivo kinetic study.

After a nascent HDL detaches from the hepatocyte surface, in vivo, LCAT in the Space of Disse, or in hepatic sinusoids, pulmonary circulation, or systemic arterial circulation, can rapidly convert unesterified to esterified cholesterol to create the cholesterol ester–filled core of plasma HDL,⁴² which may protect HDL from abnormally rapid renal catabolism. It is possible that LCAT action may not affect the size of HDL particles but rather their shape^38,43,44 and that the usual size distribution and spherical shape of plasma HDL mostly may be established in nascent HDL before it reaches the systemic venous circulation where it is sampled.

The shallow decline in the apoA-I tracer enrichment curves after their peaks required delay compartments that represent extravascular residence before entry into the circulating HDL compartment, as reported previously in nonhuman primates.^16,17,45 The extravascular compartments metabolized a variable percentage of the apoA-I secretion from minimal to 37%. These nonsampled delay pools could represent the hepatic lymphatics or sinusoids, other lymphatics, or the pulmonary or systemic small vessels and capillaries and adjacent tissue, such as vascular intima. The time of appearance of a late peak of tracer enrichment in apoA-I of large HDL of 5 to 20 hours is consistent with estimates of residence time of HDL in human lymphatics.^46,47 Remodeling of nascent HDL could occur in these extravascular spaces before it appears in the systemic venous circulation where it is sampled.

According to the results of the present study, an HDL that appears in the systemic venous circulation in a specific size range remains mainly or entirely in that range throughout its lifetime of 1 to 4 days. This is especially so for large HDL with diameter between 8.2 and 9.5 nm, which is not enlarged to very large HDL, 9.6 to 12.0 nm, or contracted to medium HDL, 7.0 to 8.2 nm, and for very large HDL which does not contract to a smaller size category during circulation. Only a small percentage of medium HDL is converted to large HDL, 35.0% in the low HDL and 1.6% in the high HDL group, and no medium HDL is converted to very large HDL. This suggests that HDL particles have structural stability while they circulate. Recent studies of native human HDL favor a trefoil model having 4 apoA-I molecules per particle in most spherical HDL sizes and 5 in the largest subfraction, HDL2b, which corresponds to very large HDL.⁴⁸ The trefoil’s capacity for cholesterol ester can decrease by twisting and increase by untwisting. The trefoil of a medium HDL can untwist enough to produce a large HDL. However, fully untwisted, the 4-apoA-I trefoil of large HDL may not be able to accommodate sufficient additional cholesterol ester molecules to enlarge to very large HDL unless it acquires a fifth apoA-I. However, the incorporation of extra apoA-I molecules to an already stable large HDL may be energetically unfavorable in vivo because it would require disruption of the bonds between 2 of the 4 apoA-I molecules in the trefoil.

The maintenance of size of spherical HDL subfractions as they circulate does not mean that reverse cholesterol transport is not occurring. Flux of cholesterol into HDL from cells during circulation may be balanced by flux of cholesterol transferred to apoB lipoproteins by CETP and by selective uptake of cholesterol ester by the liver. Thus, reverse cholesterol transfer could be very active but mainly within rather than among the HDL sizes, as constrained by trefoil untwisting and twisting.

Although expansion of large to very large HDL may not be common under normal metabolic circumstances, as suggested by the results of our study, we speculate that it could become important in genetically based CETP deficiency or pharmacological inhibition of CETP, in which HDL is especially large. The load of cholesterol ester accumulating in HDL could overcome the energy required to accommodate more apoA-I molecules to convert large to very large HDL. In addition, or alternatively, an increase in HDL size could result from expansion of large HDL that has 4 apoA-I molecules and very large HDL which has 5 apoA-I molecules to their maximum sizes.

Metabolism of HDL size subfractions in vivo has been studied in 2 distinct experimental models in nonhuman primates, vervet monkeys.^16,17 In one study, small-size lipoprotein A-I HDL was prepared by immunoaffinity chromatography, radiolabelled, reinjected, and its metabolism followed.¹⁶ Some of the small HDL was converted directly to large HDL and some to medium-size HDL. This conversion took place in a noncirculating extravascular compartment. The medium-size HDL formed from small HDL was not itself converted to large HDL. The size of medium HDL in the vervet study is similar to large (α-2) HDL in the present study. In the present study, large HDL is in part a product of medium HDL and is not converted to very large HDL, consistent with the findings in vervets. Furthermore, in these vervet monkeys, substantial amounts of medium and large-size HDL did not arise from small-size HDL.¹⁶ For example, 50% of large HDL in the monkeys was not accounted for by conversion from small or medium HDL and, thus, must be directly secreted into the circulation, supporting the finding in the present study that large HDL is secreted into plasma. In the other study, plasma HDL was partially delipidated to convert some of the large and very large HDL to small and medium HDL, and a large amount of the partially delipidated HDL was reinjected without prior labeling.¹⁷ Medium HDL was converted to large and very large HDL directly, whereas large HDL was not converted to very large HDL, analogous to the earlier tracer study and the present study. However, in contrast to the present study, in both nonhuman primate studies, small HDL was quantitatively converted to larger sizes.

We found that very small discoidal (prebeta-1) HDL plays a minor role in the formation of larger spherical HDL, as indicated by the major peak tracer enrichment of very small discoidal apoA-I occurring at or after the peaks in the larger sizes. This is surprising because very small discoidal HDL is often thought of as the major secreted type of HDL that serves as the precursor for the larger spherical HDL formed during circulation. In fact, only 8% of total apoA-I secretion into plasma is the very small discoidal pool. Very small discoidal HDL was not converted to medium HDL in either group, and it contributed a small percentage of the production of large HDL, 14% and 4%, and of the production of very large HDL, 14% and 0.2%, in the low and high HDLc groups, respectively. However, we speculate that a large infusion of prebeta HDL could stimulate its conversion into large HDL sizes, as found in the HDL partial delipidation experiments in vervets¹⁷ and in humans after infusion of apoA-I phospholipid disks.⁴⁷

The main synthetic pathway for plasma very small discoidal HDL in the high HDL group is from medium HDL: 60% of small discoidal HDL synthesis comes from medium HDL, compared with 17% in the low HDL group. In the high HDL group, most of the small discoidal HDL is then cleared from the circulation. This suggests that formation of small discoidal HDL from medium HDL followed by clearance of the resulting small discoidal particle(s) is a potentially more meaningful pathway in the high than the low HDL group (11% of total apoA-I flux compared with <1%). Generation of small discoidal HDL from medium HDL could occur by LCAT- or phospholipid transfer protein–mediated fusion of medium HDL to form a larger HDL while shedding small discoidal HDL during remodeling.⁴⁹ CETP activity can trigger HDL fusion, with a reduction in the size of preexisting HDL and formation of new, smaller particles.⁵⁰ Joint CETP and hepatic lipase activity can lead to shedding of free or lipid-poor apoA-I from alpha HDL, which promptly becomes small discoidal HDL.⁵¹ Smaller spherical HDL, like medium HDL in our study, are more dynamic and unstable and tend to participate in this fusion process more than larger HDL.⁵² This is consistent with our kinetic findings that medium but not large or very large HDL are metabolized to small discoidal HDL.

We found that the most salient metabolic characteristics of individuals with low HDLc are the significantly faster clearance rate from plasma of large and very large HDL and the markedly and significantly slower clearance of small discoidal HDL. Secretion rates of apoA-I into plasma of each size of HDL and of total apoA-I were not significantly different between the groups. These results extend the findings in prior studies that had demonstrated a higher clearance rate of total HDL apoA-I in people who have low HDL levels, obesity, and the metabolic syndrome.^27–31

The proposed model of HDL physiology, motivated by the results of the present study, integrates structure, cell biology, and human metabolism (Figure 6). This model of HDL metabolism applies to participants who have low or high HDLc. Major enlargement and contraction of HDL size has been considered to typify the process of reverse cholesterol transport, a concept that may need to be reconsidered. More so, phenomena taking place within each size subfraction may be responsible for the biological properties of HDL.

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Figure 6.

Integrated proposal for the in vivo metabolism of plasma high-density lipoprotein (HDL) in humans. Apolipoprotein A-I (ApoA-I) is secreted by hepatocytes variably lipidated with phospholipid and unesterified cholesterol and interacts with ABCA1 to take up more unesterified cholesterol and form nascent HDL in a range of sizes. Exposure to LCAT in the hepatic lymphatics and sinusoids esterifies the cholesterol to form an HDL core of cholesterol ester, although the size of most of the HDL does not enlarge enough to become a larger subfraction. HDL in these discrete sizes enters the systemic circulation and then escapes to the interstitial space or lymphatic system. In these extravascular–extrahepatic spaces, very small, medium, large, and very large HDL take up and esterify free cholesterol from cells. Simultaneously or right after HDL goes back to the systemic circulation, CETP catalyzes the exchange of recently formed cholesterol esters for triglycerides present in apoB lipoproteins. Circulating HDL may come in contact with SRB1 on hepatocytes, which selectively takes up cholesterol ester from HDL. HDL particles do not experience major changes in size because the influx of cholesterol from cells is balanced by the efflux of cholesterol esters from each HDL particle by the CETP and SRB1 pathways. Hepatic lipase, acting on the triglyceride from the CETP reaction, also may maintain stability of size. This cycle of HDL circulation between plasma and the extravascular space can be repeated many times over several days. In the vascular or extravascular space, small discoidal HDL can, in a minor pathway, take up and esterify cholesterol from cells, becoming large or very large HDL. In a process facilitated by plasma PLTP, HL, CETP, and LCAT, medium-size HDL particles experience fusion to generate new small discoidal HDL and perhaps new medium or large HDL. All HDL size subspecies can be cleared from circulation via holoparticle uptake. Reverse cholesterol transport can be accomplished by coupling cholesterol uptake from cells with CETP-mediated cholesterol ester transfer to apoB lipoproteins and SRB1-mediated selective CE uptake by the liver, without transforming the size of the individual HDL. ABCA1 indicates ATP-binding cassette transporter A1; CE, cholesterol esters; CETP, cholesterol ester transfer protein; FC, free cholesterol; HL, hepatic lipase; L, large; LCAT, lecithin cholesterol acyltransferase; M, medium; PLTP, phospholipid transfer protein; SRB1, scavenger receptor B1; TG, triglycerides; VL, very large size HDL subfractions; and VS, very small discoidal.