Metabolic heterogeneity in very low-density lipoproteins

Nletabottc heterogeneity lipoproteins

in very tow-density

James Shepherd, M.D., Ph.D., and Christopher Glasgow, United Kingdom

J. Packard, PbD.

Apolipoprotein B (apo B) is a major structural protein component of human chylomicrons, very low-density (VLDL), intermediate-density (IDL), and low-density (LDL) lipoproteins. In fact, it is the only constituent of these particles that retains its association with them throughout their lifetime in the circulation and, as such, can usefully be employed as a tracer of their metabolism. Early studies based on this approach showed that VLDL apo B was processed through IDL to LDL, a concept that was formalixed in a mathematic model published by Berman et al.’ in 1978. The model describes a delipidation cascade within VLDL (Fig. 1, A) in which triglyceride is removed stepwise from the particles as they transfer through the IDL density interval into LDL; new protein enters the spectrum at the level of the largest, least dense, most triglyceride-rich VLDL. Such a concept held sway for a long time and is adequate to explain the process of VLDL catabolism when the particle spectrum is considered as a single pool. However, anomalies arose in hypertriglyceridemia because in this circumstance more VLDL apo B was generated than was required for LDL production.2 Consequently it was necessary to postulate that such patients catabolixe some VLDL directly from their plasma instead of converting it to LDL. Fisher et al9 investigated this problem in depth by quantifying the secretion of B protein along the length of the VLDL spectrum. They concluded that particles did not appear at a single port of entry but rather were secreted throughout the entire lipoprotein distribution (Fig. 1, B). This concept naturally raised the possibility that there might be “metabolic channeling” within VLDL.’ That is, B protein might be fed into alternate, noncommunicating pathways that run in parallel through the VLDL delipidation cascade (Fig. 1, From the University Department of Pathological Biochemistry, Royal Inihwy. Reprint requests: James Shepherd, M.D., Department of Pathological Biochemistry, Royal Infirmary, University of Glasgow, Glasgow G4 OSF, United Kingdom.

C). Such an idea could be used to formulate a general model embracing apo B metabolism in all subjects, whether normal or dyslipidemic (Fig. 2). We set out to test this model, but to do so it was necessary to subdivide VLDL -into fractions of welldefined flotation rate that could then be subjected to metabolic study. The approach of Fisher et al3 to that problem had been to use molecular sieve chromatography. We selected an alternative and more easily manageable system based on the cumulative flotation procedure of Lindgren et al5 that permits sequential separation of multiple fractions of VLDL from small quantities of plasma. The following describes our experiences using this method to examine the metabolism of VLDL subfractions in normal and dyslipidemic individuals. METABOLIC PROPERTIES OF LARGE (SVEDSERG FLOTATION UNIT 100 TO 400) VLDL IN NORMOLIPEMK SUBJECTS

Apo B, trace labeled with T, disappeared from the S, 100 to 400 interval of normal human plasma in a monoexponential fashion and with a fractional clearance rate of 10 to 20 pool~/day.~ The radiolabeled protein was transferred quantitatively in the process into the intermediate (S, 12 to lOWdensity interval. Its clearance from there was considerably slower and could be represented by a biexponential decay proflle, indicating that the process involved more than one mechanism. Little of this apo B, originally derived from large VLDL, ultimately reached LDL. The majority was removed directly from the circulation by an unknown process. We investigated the mechanism involved’ by taking advantage of the knowle&e,$hat modikation of arginine residues on ape B with 1,2 cyclohexanedione interferes with its interaction with cell membrane receptors without materially a%cting the physicochemical properties of its parent lipoprotein. When tracers of native and cyclohexanedione-treated large VLDL were injected into the bloodstream of normolipemic individuals, 503

504

February 1987 American Heart Journal

Shepherd and Packard

;

A.

5

FLOTATION

RATE

5.

-(O-O-o-o]-

60

c.

t 1. Evolution of apo B metabolism. A, al.’ B, Multiple input channels proposed by

Fig.

a kinetic model describing VLDL Delipidation cascade of Berman et of Fisher et al? C, VLDL apo B Packard et aL6

@@I L

t 56 Flg.

their

initial

rates of conversion

O-18

t%

2. General model for apo B metabolism.

to intermediate

density-remnant particies were the same.However, subsequent catabolism of the arginine-modified tracer from the IDL fraction was 50% slower than that of the native material. This suggestedto us, by analogywith our earlier experiencesof LDL metabolism, that processingof remnant particles originating from large VLDL involves interaction of the lipoprotein with a receptor in a processdependent on the patencyof arginine residueson the B protein. These in vivo tidings correlated with in vitro observationamade by Bradley et al.& in Houston who noted that whereaslarge VLDL from normal subjects were unable to bind to receptorson cultured fibroblasta, small particies of Sr 20 to 60 did so and by a prm that required the participation of B protein. So, material initiaIly secretedinto the large VLDL flotation rangein normal subjectsappearsto be catabolizedin two stages.First, lipoprotein bpase rapidly removesthe majority of the core triglyceride and in so doing induces a conformaknal changein

the apo B on the particle surface,which makes it recognizableby cell membranereceptors.The latter, in the second metabolic event, are responsiblefor removing the particle from the plasma without generatingLDL in the process. CATABOLISM OF S, SD to 400 VLDL APO B IN TYPE Ill AND TYPE IV HYPERLlPOPROTEtMEWA

Subjects with hypertriglyceridemia commonly suffer from a combination

of VLDL

overproduction

coupledwith a delay in the clearanceof the lipoprotein from the ckdation. Reardon et aL2showed that during the catabolic process not all of the VLDL was transformed into LDL. That is, some VLDL particles were de&ined for direct elimination whereas the remainder were subject to lipolytic conversionto denserLDL. What these authors did not know waswhetherall VLDL particles sharedthe same probability of entering either pathway. It is

Metabolic heterogeneity Table I. Kinetic parameters hypertriglyceridemia

(mean

f 1 SD) of large VLDL VLDL apo B synthetic rate (mg . kg-’ . d-‘)

Subjects

Normal (n = 4)* Type IV hyperlipoproteinemia Control Taking bezafibrate (200 mg Type III hyperlipoproteinemia (n = 3)* Type III hyperlipoproteinemia Control Taking bezafibrate (200 mg 'BflOOto tS, 60 to

-

apo B in normal

subjects

VLDL apo B pool size (mg . dl-‘)

of

VLDL

505

and those with VLDL apo B fractional catabolic (pools . d-l)

-

rate

21.9 + 16.4

(n = 4)* t.i.d.1

10.6 f 4.7 9.25 + 2.2 -

3.7 + 2.2 1.25 3~ 0.86 -

9.8 -e 6.7 34.8 f 24.6 12.16 c 2.70

10.8 + 3.0 4.7 f 0.9

15.5 + 5.9 2.9 + 0.9

2.0 +- 0.9 4.5 +- 1.4

(n = 3)t t.i.d.1

400. 400.

known that most protein and all lipid components do exchangebetweenlipoproteins, but whether the particles reside in the circulation long enough to attain compositional and metabolic equilibrium is another matter. If we attribute any importance to the conformational change in apo B that is mentioned above,it is probable that they do not. We adopted the isolation and kinetic analysis proceduresthat had beenusedin normal subjectsto examine the metabolic defect in patients with type IV hyperlip0proteinemia.BThey had larger VLDL pool sixes and, as might be expected, cleared B protein from the St 60 to 400 range (Table I) more slowly than normal Administration of bezafibrate,a drug known to stimulate lipoprotein lipase,led to an increasein the fractional catabolism of this species. As we had already noted in normal subjecta, the majority of S, 60 to 400VLDL was convertedin the patients with hypertriglyceridemia to S, 12 to 60 remnants, which had a plasma residence time of about 1 day. Little of this material ultimately appearedin LDL. Presumably it was removed by a receptor-mediatedprocesssimilar to that seenin the normal group. It’ wa&of interest to notethat fibrate therapy had no effect on this particular catabolic mechanism.So, onecan postulate from these experiments that the difference in VLDL metabolism betweennormal subjects and patients with type IV hyperlipoproteinemia derives from overrepresentation of the large triglyceride-rich particles in the latter group rather than from a qualitative changein their handling of the lipoprotein. Patients with type IK hyperlipoproteinemia respondto bexafibratetreatment in a fashion similar to that of patients with type IV hyperlipoproteinemia. When autologouslarge VLDL was injected

into their bloodstream it was converted rapidly to smaller remnant particles whoseclearancefrom the plasma was very slow (residence time 2 days). Bexa6brat.eagain stimulated large triglyceride-rich VLDL clearancein these subjecta (Table I), doubling their fractional catabolism. So, again it appearsthat our two-stage model of VLDL apo B turnover is applicable, reinforcing the proposalthat within the Sr20to 400VLDL spectrum there are two particle types with substantially different catabolic rates.Both the nascent (large) and remnant (small) particles arecoisolatedif VLDL is treated asa single classand so any metabolic study basedon the useof total VLDL as tracer will generatekinetic data that representa composite of thesetwo processes. METABOLIC PROPERilES OF SMALL VLDL IN NORMAL SUBJECTS

(S, 20 to SD)

Since the flux of material passing from large VLDL into LDL is not sufiicient to account for the production rate of the latter, we must concludethat particles secreted directly into the $ 20 to 60 interval are the progenitorsof LDL. Kinetic studies haveshown (Fig. 3) that when VLDL of S, 20 to 60 is labeled in its entirety, its metabolic behavior is substantially diEerent from that of the remnant particles generatedfrom large VLDL, even though both speciessharethe sameflotation ratea Directly radioiodinated Sr 20 to 60 VLDL apo I3 is traneferred rapidly through IDL to LDL at a rate that is sufilcient to account for LDL production. In an attempt to delineatemore accuratelythe immediate precursorof LDL, we subfractionatedthis spectrum into S, 20 to 40 and 40 to 60 particles.Even thesetwo closely related specieshad different metabolic properties,the former being more rapidly and completely

506

Shepherd

and Packard 1

A

! Sf 100-400 Sf 20 - 60

B

' Sf 20 -60

0 1,

.

% 01:

‘.

1.

J

I

I 100

0

\;\

_ 200

1

J 0

loo

0

HOURS

Fig. 3. Metabolism of S, 20 to 60 lipoproteins in type III hyperlipoproteinemia. A, Plasma decay curve of VLDL apo B derived from large S, 100 to 400 VLDL. B, Plasma decay profiles represent the clearance of total S, 20 to 60 VLDL apo B labeled ex vivo. Table

II. Effects of bezafibrate on S, 20 to 60 VLDL apo B metabolism in type III hyperlipoproteinemia VLDL apo B Subjects

synthetic (mg . kg-’

rate . d-*)

VLDL apo B

VLDL apo B fractional

pool size (mg . dl-‘)

catabolic rate (pools . d-l)

13.8 rt 1.2* 9.7 * 2.1

30.8 ” 8.1 14.6 ct 2.5

1.20 f 0.33 1.66 + 0.35


<0.02

NS

Type III hyperlipoproteinemia (n = 6) Control Taking bezafibrate (200 mg t.i.d.) ttest ‘Mean

+ 1 SD.

transferred to LDL. Notwithstanding, their propertiea overlapped substantially, indicating that ultracentrifugation does not provide the optimal approach for separating them into metabolically distinct species. S, 20 TO SO VLDL APO B METABOLIBM WITH TYPE Ill HYPERLIPOPROTB

IN PATIENTS

From the foregoing it appears that within VLDL there etit populations of particle8 that share many common physicochemical properties and yet remain metabolically distinct in teof both their origins and fates. The need to dissect these particles into homogeneous subgroups poees a challenge to our current isolation techniques. One approach to the problem is to examine individuals whwe B protein metabolism is perturbed for genetic reasons so that there is an imbalance in the d&&rib&ion of newly secreted small VLDL and simihirly sized remnants derived from large triglyceride-rich particle hydrolysic. Patients with type III hyperlipoprotememk, the primary structure of whose E pro&&n limita their abiity to clear VLDL remnants, are obvious candidataxt for such a study. We have examined the metabolic propertiea of small VLDL in such individual~ and have followed their response to bafibrate therapy. The drug normahzed their plasma lipid levels, although the compoeitional anomaly that we

noted in their YLDL per&ted. Interestingly, the low level of LDL that characterizes such subjecta did not increase’ in re8pon8e to treatment. So, what metabolic processes can be responsible for reducing their VLDL (Table II) without producing reciprocal increments in either IDL or LDL? Our understanding of theeffects of fibrates on pkma lipases led us to expect that there would be an increment in VLDL catabolism. However,. for tbe group as a whole, this clearly was not the CBB~.Only two of the six made such a response (unpublished result& The major action of the drug warmto inhibit the rate of B protein secretion into the VLDL density interval (Table II). Further kinetic analysis will be necessary before we can say whether the nascent or remnant VLDL particles were primarily affected. The failure of bezaflbrate to accelerate the catabolism of small VLDL in these patientswith type III hyperlipoproteinemia suggesta that the rate-limiting steps respon&le for their clearance are not primarily dependent on the lip&tic process. It is tempting to predict that apo E may play a role in this regard. kw~A@&lBH H

OF B#AtL@, 20 TO 60) V&L tw@UL Itwe!

IN LEWA

Homozygous familial hypercholeaterolemia~ is another condition in which the spectral diitribution of apo B-containing particles is perturbed in the

Volume 113 Number 2. Part 2

plasma. The disorder was originally thought to derive purely from a defect in LDL metabolism but it is .now clear that there are also repercussions affecting both VLDL and IDL, inasmuch as the concentrations of the latter are increased in the circulation of affected individuals. This is perhaps not surprising in light of recent evidence (see above) that particles in the VLDL and IDL density intervals also interact with the LDL. receptor. We examined the metabolic fate of small VLDL (S, 20 to 60) in a group of patients with homozygous familial hypercholesterolemia and confirmed that the concentrations of both small VLDL and IDL were increased in their plasma. Moreover, the VLDL (both large and small) was enriched in cholesteryl esters relative to triglyceride suggesting that remnants were accumulating in these density intervals (unpublished results), analogous to the situation in type III hyperlipoproteinemia. Examination of apo B plasma decay curves confirmed that the catabolism of VLDL (S, 20 to 60) was retarded. In consequence, the rate of transfer of B protein into IDL and LDL was reduced so that in some subjects up to 9 days elapsed before peak radioactivity values were attained in LDL. This is by far the slowest conversion rate that we have ever recorded in any kinetic study of this kind. Since we know. that the defect in familial hypercholesterolemia is specifically located at the level of the LDL receptor gene, our results pose intriguing questions about the relationships between receptor activity and VLDL metabolism. These questions have already been raised in a studylo that employed the Watanabe (WHHL) rabbit model of LDL receptor deficiency. In normal rabbits most VLDL is removed from the plasma by receptors before it can become LDL. However, the lack of LDL receptors in the WHHL mutant delays the direct catabolism of VLDL and enhances its conversion to LDL. These results have been extrapolated to humans with the suggestion that LDL oversynthesis, which characterizes familial hypercholesterolemic homozygotes, is the result of more complete lipolytic conversion of VLDL. However, this proposal is still unproved and indeed the work of Soutar et all’ raises the suggestion that the accumulated LDL in the plasma of familial hypercholesterolemic homozygotes comes more from direct production of this species than from enhanced conversion from VLDL. The issue is complex because we must take into account not only the metabolic heterogeneity within the VLDL spectrum but also the evidence that familial hypercholesterolemia represents the expression of a number of closely related disorders rather than a single point mutation in the human genome.

Metabolic VLDL APO B METABOLISM

heterogeneity

of

VLDL

507

IN PROSPECT

The ,development of our understanding of the processes involved in VLDL apo B metabolism as depicted in Figs. 1 and 2 is beginning now to be limited by methodologic restrictions. If it is true that the metabolic properties of a VLDL particle are dependent on its ,origin, we will be required to be able to define clearly the nature of the particle spectrum that is secreted by the human liver under physiologic and pathologic conditions. The current approach to kinetic analysis using ex vivo radioiodinated tracers suffers from the inherent weakness that synthetic rates are extrapolated from catabolic data. In any population of VLDL particles, those that are. removed slowly will be present in greatest mass and will be overrepresented after iodination. It is possible to conceive that in this situation there may be a subpopulation of rapidly metabolized VLDL particles that escape detection by the application of conventional kinetics, even though they may be of major significance in the overall metabolic scheme. To some extent, by fractionating VLDL into narrower density intervals we tend to overcome this limitation, but in the end there will be no alternative but to investigate the de novo generation of lipoprotein particles. Some studies of this kind are already in the literature and have yielded interesting information. Large-scale implementation of this approach will require the application of new technology, possibly based on the use of stable isotopes. We acknowledge the expert secretarial help of Joyce Pollock. REFERENCES

1. Berman M, Levy RI, Eisenberg S, Bilheimer DW, Phair RD, Goebel RH. Metabolism of apo B and apo C lipoproteins in man. Kinetic studies in normal and hyperlipoproteinemic subjects. J Lipid Res 1978;19:38. 2. Reardon MF, Fidge NH, Nestel PJ. Catabolism of very low densitv linonrotein B armprotein in man. J Clin Invest _1978+%3&* 3. Fisher WR, Zech LA, Bardalaye P, Warmke G, Berman M. The metabolism of anolinoprotein B in subiects with hwertriglyceridemia and polydisperse LDL. J “Lipid Res i980, 21:766. 4. Fisher WR. Apoprotein B kinetics in man. In: Berman M, Grtmdy SM, Howard BV, eds. Lipoprotein kinetics and modelling. New York: Academic Press Inc, 198243. 5. Lindgren FJ, Jensen LC, Hatch FT. The isolation and quantitative analysis of serum lipoproteins. In: Nelson GJ, ed. Blood lipids and lipoproteins: quantitation, composition and metabolism. New York: John Wiley & Sons Inc 1972: 181. 6. Packard CJ, Munro A, Lorimer AR, Gotto AM, Shepherd J. Metabolism of apolipoprotein B in large, triglyceride-rich very low density lipoproteins of normal and hypertriglyceridemic subjects. $ Clin Invest 1984,74:2178. 7. Packard CJ. Boas DE. Cleee RJ. Bedford D. Shenherd J. Effects of 1,2 cyclihexr&edi&e modification on the-metabolism of very low density apoprotein B: potential role of

February

Shepherd and Packard receptors in intermediate density lipoprotein catabolism. J Lipid Res 1985;26:1058. 8. Bradley WA, Hwang S-LC, Karlin JB, et al. Low density lipoprotein receptor binding determinants switch from apolipoprotein E to apolipoprotein B during conversion of hypertriglyceridemic very low density lipoprotein to low density lipoproteins J Biol Chem 1984;259:14728. 9. Shepherd J, Packard CJ, Stewart JM, et al. Apolipoprotein A and B (S, 100-400) metabolism during bezafibrate therapy in hypertriglyceridemic subjects. J Clin Invest 1984;74:2164.

American

1987

Heart Journal

10. Kita T, Brown MS, Bilheimer DW, Goldstein JL. Delayed clearance of very low density and intermediate density lipoproteins with enhanced conversion to low density lipoprotein in WHHL rabbits. Proc Nat1 Acad Sci USA 1982; 795693. 11. Soutar AK, Myant NB Thompson GR. Simultaneous measurement of apolipoprotein B turnover in very low and low density lipoproteins in familial hypercholesterolemia. Atherosclerosis 1977;28:247.

Y. Antero Kesliniemi, M.D., M. Fiirkkihi, M.D., K. Kervinen,* P. Koivisto, M.D., M. Vuoristo, M.D., and T. A. Miettinen, M.D. Helsinki,

Finland

Low-density lipoprotein (LDL) is probably the most atherogenic lipoprotein in human plasma. LDL is also the lipoprotein that carries most of the cholesterol transported in the plasma, and apolipoprotein (apo) B is almost the only structural apoprotein in LDL. Thus there is a need to understand the mechanisms that regulate the formation and catabolism of LDL apo B and the association of LDL with the liver-intestine axis, the only route for elimination of cholesterol from the body. This article will focus on the factors that regulate LDL levels in humans.

are removed rapidly by the liver.” This uptake probably occurs to a lesser extent in humans. The process seems to be mediated by a specific receptor for apo B-100 and is facilitated by ape E.’ In humans a significant fraction of VLDL is .converted to LDL, which is then removed from the circulationby either the liver or the peripheral tissues. A major proportion of this removal is mediated by a receptor for apo B-100,2 the same receptor that mediates the-uptake of VLDL remnants. The remainder is cleared by the nonreceptor mechanism.

CURRENTCONCEPTOFLDLAPOBFORMATlONAND CATABOLISM

The receptors for LDL apo B-100 play a key role in the clearance of LDL from the plasma, as has been demonstrated in patients with a conspicuously elevated LDL level in familial hypercholesterolemia (FH).3 In these patients LDL is cleared slowly because of LDL receptor deficiency. Thus in a girl with homozygous FH, the fractional catabolic rate (FCR) for LDL is only 0.131 day-’ (Fig. 2, left panel), resulting in an elevated LDL cholesterol level of 484 mg/dl. However, her father, who is heterozygous for FH, has an LDL clearance with an FCR of 0.213 day-’ and remains at an LDL level of 310 mg/dl (Fig. 2, middle panel). His reeeptormediated pathway of LDL clearance can be determined by the simultaneous injection of native and glucosylated LDL;2 The latter cannot be cleared by the LDL receptor pathway. The di@erence between the catabolism of native LDL (total catabolism) and

It is believed that the liver secretes very lowdensity lipoprotein (VLDL) containing apo B-100 as its major structural protein (Fig. 1). VLDL particles interact with lipoprotein lipase and, following lipolysis, are converted to smaller lipoproteins, VLDL remnants. In many animals most VLDL remnants

From the Second Department of Medicine, University of Helsinki. Supported by grants from the Sigrid Juselius Foundation, the Finnish Alcohol Research Foundation, the Finnish Heart Foundation, the Finnish Foundation for Medical Research, the Paavo Nurmi Foundation, and the Medical Research Council of the Academy of Finland. Studies done under a contract with the Finnish Life Insurance Companies. Reprint requests: Y. Antero Kesiiniemi, M.D., Department of Medicine, University of Oulu, Kajaanintie 50, SF-90220 Oulu, Finland. *Medical Student.

ROLEOFTHELDLRECEPTORPATHWAY