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Cattedra and Servizio Autonomo di Endocrinologia, University of Messina, School of Medicine, 98125 Messina, Italy, and Genetics and Biochemistry Branch, NIDDK, National Institutes of Health, Bethesda, Maryland 20892-1587
Address all correspondence and requests for reprints to: Jacob Robbins, M.D., NIDDK, Genetics and Biochemistry Branch, Building 10, Room 6C 201A, 10 Center Drive, MSC 1587, Bethesda, Maryland 20892-1587.
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Abstract |
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Of the three classes of lipoproteins (VLDL, LDL, and HDL), HDL is the major carrier of thyroid hormones. While LDL delivers cholesterol (and T4) to cells, HDL is the scavenger of cholesterol. We thus hypothesized that HDL could also facilitate thyroid hormone exit from cells. This hypothesis was tested on two human cell lines: skin fibroblasts and hepatocytes (Hep G2), using physiological concentrations of HDL or, as control, physiological concentrations of LDL, HSA, TTR, and TBG or buffer. Because cell surface receptors for HDL are regulated by intracellular cholesterol in a manner opposite to that of LDL receptors, we evaluated the effect of HDL (and other proteins) in three states: normal, high, and low intracellular cholesterol content (i.e. normal, high, and low expression of HDL receptors).
In both cell lines and with either T4 or T3, we found that: 1) HDL as well as the other proteins tested increased the efflux and augmented both the initial rate of exit and the equilibrium value. 2) The efflux did not saturate over a wide range of protein concentrations. 3) The effect of HDL, LDL, and the other proteins on the fractional efflux rate of thyroid hormones remained the same irrespective of the intracellular cholesterol content (and, therefore, irrespective of the expression of either LDL or HDL receptors). 4) HSA, TTR, and TBG were, on a mass basis, equipotent and more efficient than lipoproteins. However, the effect of lipoproteins – whose Ka for T4 is comparable to that of HSA – was disproportionately high. On a molar basis, LDL (about 80% of the weight being accounted for by lipids) was more effective than HDL2 (about 60% lipids) and HDL2 was more effective than HDL3 (about 40% lipids), suggesting that the disproportionate effect of lipoproteins was due to transfer of the lypophylic thyroid hormones to the lipid moiety of lipoproteins. 5. A mixture of HDL and LDL gave the same efflux rate as a mixture of HSA, TTR, and TBG.
The data indicate that the efflux of T4 and T3 from cells is rapid and appears not to be mediated by a particular lipoprotein. The disproportionately large effect of lipoproteins, which are low affinity thyroid hormone carriers, compared with nonlipoprotein carriers, and the greater effect of LDL compared with HDL, might indicate that the lipoproteins establish a nonspecific physical contact with the plasma membrane and that their hydrophobic nature favors the release of the similarly hydrophobic thyroid hormones.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cells are bathed by solutions of proteins such as plasma, interstitial fluid and cerebrospinal fluid. Circulating proteins bind thyroid hormones and these proteins include thyroxine-binding globulin (TBG), transthyretin (TTR), albumin (HSA), and lipoproteins (4). Previously, we demonstrated that low-density lipoproteins (LDL), which carry <1% of total circulating T4, facilitate T4 entry into LDL receptor competent human fibroblasts (5). The effect was selective in that none of the three major plasma transporters of thyroid hormones (TBG, TTR, HSA) mimicked LDL (5). The effect of these three proteins – as well as of HDL (S. Benvenga, unpublished) – was the opposite: they prevented T4 entry into fibroblasts in a dose-dependent fashion. Because LDL receptors are ubiquitously expressed in the body, the implication was that LDL delivers T4, in addition to cholesterol, to peripheral cells.
The major lipoprotein carriers for thyroid hormones in human plasma are high density lipoproteins (HDL) which account, on the average, for 91% and 78% of the total lipoprotein binding of T4 and T3, respectively (6). The role of HDL in cholesterol homeostasis is 2-fold: delivery of cholesteryl esters to steroidogenic tissues via selective transfer to the cells (7), and removal of cholesterol from cells by accepting the free cholesterol that desorbs from the cell membranes (8, 9). This process is poorly understood even after the identification of several HDL receptors (or binding sites) on the plasma membrane of several cell types (7, 9). We, therefore, wished to evaluate whether HDL could also facilitate thyroid hormone efflux and tested this hypothesis in two cell lines used both in thyroidology and lipidology: human skin fibroblasts and Hep G2 cells. Fibroblasts are a typical model of peripheral tissue, whereas Hep G2 cells are a typical model of hepatocytes, and liver is an organ central to both hormone and lipid homeostasis. Fibroblasts are bathed by interstitial fluid, while hepatocytes are bathed by plasma because of the fenestrated endothelial lining in liver. The effect of HDL was compared with that of the other plasma carriers of thyroid hormones.
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Materials and Methods |
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The tested concentrations of proteins took into account the nature of the extracellular fluid that bathes the two types of cells and the mean concentration of each individual protein in this fluid. On the average, concentrations in human plasma for HSA, TTR, TBG, LDL, and HDL (HDL2 and HDL3) are 40 g/liter (597 µM), 200 mg/liter (3.6 µM), 20 mg/liter (0.36 µM), 300 to 550 mg/liter (0.54 to 1.0 µM) and 1100 mg/liter (9.1 µM) (HDL3 = 780 mg/liter or 7.8 µM; HDL2 = 300 mg/liter or 1.5 µM).
Concentrations in the interstitial fluid were derived from the skin interstitial fluid/plasma ratios reported by Sloop et al. (11) and are 12 g/liter (177 µM), 60 mg/liter (1.1 µM), 6 mg/liter (0.11 µM), 40–88 mg/liter (0.087–0.16 µM) and 270 mg/liter (2.2 µM) (HDL3 = 187 mg/liter or 1.87 µM; HDL2 = 72 mg/liter or 0.36 µM). It should be noted that the ratios for TTR and TBG were not reported (11); thus, we assumed they were equivalent to those reported for HSA, viz. 0.3.
Cell cultures
HepG2 cells were from ATCC (Rockville, MD) and human skin
fibroblasts (GM 002674) were from the Human Genetic Mutant Repository
(Camden, NJ). Stock cultures in 75 cm2 Falcon flasks were
maintained in a humidified CO2 incubator at 37 C. Both cell
lines were fed the same medium, except that, as recommended, the final
concentration of FBS was 10% for Hep G2 cells and 20% for
fibroblasts. The medium was DMEM supplemented with FBS, 0.25 µg/ml
fungizone, 100 U/ml penicillin and 100 µg/ml streptomycin (Advanced
Biotechnologies, Columbia, MD). For experiments, fibroblasts were used
between passages 10 and 19.
Cells of either line were seeded in six-well plates (Costar, Cambridge, MA), fed the indicated medium and maintained in an incubator at 37 C, and were at or near confluency when incubation procedures were started. At 24 h before experiments, the medium was replaced by fresh medium.
Incubation procedures
The procedure to allow uptake of thyroid hormones was the same
for both cell lines and has been published in detail elsewhere (5).
Briefly, cells in each well were washed three times at room temperature
with HBSS buffered with 15 mM HEPES and then preincubated
with 2 ml HBSS-HEPES for 45 min at 37 C in the incubator. Then each
well-received 1 ml of "uptake incubation mixture" or solution A
(see below). One plate served to evaluate the equilibrium (1 h) uptake
of labeled hormone. (Initially, the uptake was evaluated on three
plates that were placed at the beginning, middle, and end of the
experiment. Given the excellent reproducibility of the uptake,
subsequent experiments were conducted with one plate only). Each of
the three top wells of this plate received solution A (25
pM 125I-T4 [DuPont NEN, Boston,
MA; S.A. 4400 Ci/mmol) or 125I-T3 [Dupont NEN;
S.A. 2200 Ci/mmol], buffer for proteins and Hanks-HEPES to achieve a
final volume of 1 ml). Each of the three bottom wells of the same plate
received 1 ml of solution B (the same as solution A plus 10
µM unlabeled L-T4 or L-T3) to
measure nonsaturable uptake. All the other plates of the experiment
were treated similarly until efflux solutions were added (see below).
After incubation for 60 min at 37 C in the incubator, the plates were
placed in an ice bath, the incubation mixtures A (top wells) and B
(bottom wells) were removed and each well was washed in rapid
succession with 2 ml ice-cold DMEM. Cells of the Uptake Plate were
solubilized in 1 ml 0.1 N NaOH at room temperature
under gentle shaking and a 0.75 aliquot/well was removed for counting
radioactivity. Total uptake of T4 (expressed in femtomols
per million cells) was approximately 6 (about 60% of which was
nonsaturable) for HepG2 cells and approximately 20 (30% nonsaturable)
for fibroblasts. The corresponding values for T3 were
approximately 12 (20% nonsaturable) for HepG2 cells and approximately
45 (15% nonsaturable) for fibroblasts.
The remaining plates, after the third wash with DPBS in the ice bath, received the efflux solutions (1 ml/well). The three top wells of the control plate received solution C (protein buffer brought to the final volume of 1 ml with Hanks-HEPES) while the three bottom wells of this plate and "protein plates" received solution D (the individual protein under study brought to the final desired concentrations with buffer and Hanks-HEPES in a final volume of 1 ml). Efflux was evaluated over 240 min at 37 C in the incubator after the internalization of the labeled hormone had been terminated by placing the plate in the ice-bath and efflux solutions had been added. Efflux at any given time point was also terminated by placing the control or protein plates in the ice bath. An aliquot (0.75 ml) of the medium (i.e. solution C or D) was removed and radioactivity counted.
In some experiments, we evaluated whether either favoring or decreasing interaction of LDL or HDL with the respective cell surface receptor would alter thyroid hormone efflux. Because cell surface binding sites (or receptors) for HDL are up-regulated by the free cholesterol concentration inside the cells, a classical method to obtain such up-regulation is to feed cells cholesterol. Typically, 24 h before experiment, the growth medium was removed from each well. After three subsequent washes with DPBS, each well received 2 ml DMEM containing 2 g/liter of fatty acid-free BSA (Sigma) and 50 mg/liter cholesterol. This manipulation results in an approximately 2- to 4-fold increase of free cholesterol concentration within the cell (12, 13) and a 2- to 4-fold increase in the number of HDL binding sites without changes in the affinity for HDL (12). Cholesterol loading also results in down-regulation of the LDL receptors, again without change in the affinity.
In contrast, HDL receptors are down-regulated by removing cholesterol from the medium and, therefore, from the cells. Cells depleted of cholesterol, on the other hand, overexpress LDL receptors (14). To deplete cells of cholesterol, we also followed the classical method (15). The day before the experiment, the growth medium was removed, and, after 3 washes with DPBS, each well received 2 ml of the same growth medium but containing 10% (Hep G2) or 20% (fibroblasts) lipoprotein-deficient (and, therefore, cholesterol deficient) human serum (LDS; Sigma) instead of the equivalent percentage of FBS. In the plates where no manipulation of the intracellular cholesterol took place, the growth medium was replaced with 2 ml/well of the same growth medium. While in cholesterol-depleted cells, the 1 h uptake of either hormone did not change significantly, cholesterol loaded cells of either cell line internalized about two times more T4 or T3 than in the standard conditions (no switch of medium). The explanation for this difference is unknown.
In our cell lines, cholesterol-depleted fibroblasts (15.2 ± 4.9 µg cholesterol/mg cell protein) had a binding capacity of sites for HDL equal to 52 ± 16 ng HDL protein/mg cell protein; in contrast, cholesterol-loaded fibroblasts had a cholesterol content and Bmax equal to 44 ± 9.3 µg cholesterol/mg cell protein and 217 ± 45 ng HDL protein/mg cell protein, respectively. The corresponding values for HepG2 cells were 23.1 ± 2.7 and 39 ± 4.5 µg cholesterol/mg cell protein and 41 ± 11 and 73 ± 19 ng HDL protein/mg cell protein. LDL receptors (in ng/mg cell protein) for cholesterol-depleted vs. cholesterol-loaded fibroblasts were 314 ± 51 vs. 52 ± 26, and for cholesterol-depleted vs cholesterol-loaded HepG2 cells, 207 ± 48 vs. 90 ± 31.
Handling of data
Unless stated otherwise, results are expressed as fractional
hormone efflux; viz., total T4 or T3 released
divided by T4 or T3 internalized and
multiplying the ratio times 100. Data are mean ± SD of at
least three experiments. The statistical significance was assessed by
the two-tailed Student’s t test and the level of
significance was set at P < 0.05.
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Results |
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. Efflux of both T4 and
T3 from both cell lines was linear for the first 10 min,
possibly representing the unidirectional outward movement of hormone.
Because the intracellular hormone pools were not measured, it is not
possible to calculate the quantity of hormone released. Unless
otherwise stated, therefore, data will be expressed as fractional
hormone efflux at 10 min. Efflux of T4 and T3
were similar, and both exited more rapidly from fibroblasts than from
hepatocytes (approximately 30% compared with 17%). After the first 10
min, labeled hormone in the medium approached a plateau, representing
equilibration of intracellular and extracellular hormone. At 240 min,
65–75% of both hormones had exited from fibroblasts and 40–50% from
hepatocytes. Because the in vivo intracellular pool of
T4 is much greater than that of T3, however,
more T4 than T3 would be exiting in
quantitative terms.
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. The initial efflux
rates were again linear for the first 10 min. HDL and HSA increased the
efflux of both T4 and T3 by a factor of 1.5 to
3, whereas the effect of LDL was considerably smaller. At equilibrium,
about 90% of each hormone accumulated extracellularly when the medium
contained either HSA or HDL. Repeated measurements of the initial
efflux rate are summarized in Table 1.
Efflux of both T4 and T3 was significantly more
rapid in the presence of HDL compared with LDL in both cell types. In
fibroblasts, HSA increased thyroid hormone efflux more than HDL, but in
hepatocytes the effect of HDL and HSA was not statistically
different.
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. In all cases, the efflux rate
increased linearly as the protein concentration was increased. With
hepatocytes, the highest concentrations tested ranged from 1 to 2.7
times the level in plasma for the individual lipoproteins; with
fibroblasts, the highest concentrations were 3.5 to 15 times the
estimated concentration of the individual lipoproteins in interstitial
fluid. Thus there was no evidence for saturation of a protein receptor
that might have been involved in hormone efflux. The data reported in
Fig. 2 also allow comparison of the efflux kinetics from the two cell
lines at the same concentrations of the tested proteins. In either cell
line, HSA started to give an appreciable effect (i.e.
statistically different from buffer) at 0.7–0.8 g/liter or 11.2
µM. At this concentration, LDL increased efflux by
1.4-fold (fibroblasts) to 1.5-fold (HepG2), HDL2 by
1.75-fold (fibroblasts) to 2-fold (HepG2), and HDL3 by
1.63-fold (fibroblasts) to 1.75-fold (HepG2). At this molar
concentration (11.2 µM), the superiority of lipoproteins
over HSA is much more striking. If the linearity is maintained for such
high concentrations of lipoproteins, the extrapolated increased
efflux of T4 from HepG2 cells promoted by HDL2 would be
3.6-fold more, and by HDL3, 2.1-fold more, whereas LDL would
deplete HepG2 cells of the hormone previously internalized. In
fibroblasts, HDL2 and LDL would cause the exit of all T4,
while HDL3 would increase efflux by 2.2-fold.
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, was unaffected by depletion or
enrichment of intracellular cholesterol when measured in the presence
of HDL3, LDL, or HSA.
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. The 10 min efflux into medium containing
the nonlipoprotein transport proteins, HSA, TTR, and TBG, was greater
than into medium containing HDL or LDL, and this was the case for both
types of cells. The efflux rates into medium containing the
nonlipoproteins were not statistically different for HSA, TTR, and TBG;
as a group, they increased thyroid hormone efflux more than the
lipoproteins.
To create a model for thyroid hormone efflux approaching that which
exists in vivo, mixtures of the lipoproteins and the other
transport proteins were tested. A portion of these data are presented
in Fig. 4. The 10 min efflux of
T4 and T3 from fibroblasts into medium
containing all five proteins was about 55%, was not significantly
different from that achieved with the individual nonlipoproteins, and
was slightly higher than the efflux into medium containing both
lipoproteins. The results with hepatocytes were similar except that the
maximal 10 min efflux was about 35%. In every case, LDL was the least
effective of the individual proteins.
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), and assuming that the same is true for TBG and TTR, this
was calculated by dividing the mean efflux rate in Table 1 by the
protein concentration in µM. These data are given in
Table 3 and they indicate the
following hierarchy: TBG > LDL > TTR > HDL(HDL2 > HDL3) >> HSA.
This indicates that the predominant factor determining a protein’s
effectiveness is its affinity for the hormone. A secondary factor,
which becomes operative for the lipoproteins and HSA, which have
similar affinities, is the lipid content which decreases in the order
LDL > HDL2 > HDL3 >> HSA. The greater effectiveness of the proteins
in fibroblasts compared with hepatocytes is striking when expressed in
these terms.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Because human skin fibroblasts internalize extracellularly preformed LDL-T4 complexes (5), but not HDL-T4 complexes (S. Benvenga, unpublished), we hypothesized that perhaps HDL, LDL, and T4 have the same relationship as HDL, LDL, and cholesterol, and that HDL serves as a scavenger of T4 exiting cells as they do for cholesterol (8, 9). Our experiments were patterned after the technique used by Mendel & Kunitake (12) to study cholesterol release from human skin fibroblasts. In keeping with other studies (20, 21) Mendel & Kunitake (12) reported a very slow rate of exit of cholesterol (fractional release about 0.6% per h), with a linear phase during the first 2 h. Cholesterol efflux from Hep G2 cells was slower than from fibroblasts (21).
We found that the fractional efflux rates of T4 and T3 from both fibroblasts and HepG2 cells are much faster than the corresponding rates for cholesterol and that efflux of either hormone from HepG2 cells is slower than from fibroblasts. Although this in vitro system (monolayer cultures of cells with a static extracellular milieu) does not accurately reflect the in vivo situation, it can be used to assess the potential role of lipoproteins in thyroid hormone release from cells. To test for specificity of the postulated scavenger role of HDL, it was necessary to study (and compare) not only LDL, but also the three nonlipoprotein plasma carriers of thyroid hormones. To approach the in vivo situation, we also tested mixtures of these proteins. Evaluation of cholesterol efflux in the presence or absence of lipoproteins has been evaluated in a static milieu (for Refs., see 12) and it has been reported that early measurements (0–90 min) of this efflux represent the unidirectional movement from inside to outside the cell without any appreciable influx and with no interference by possible inhibition of reuptake (21, 22). This could have been theoretically important in the case of hepatocytes, but it has been demonstrated that only after 24 h do HepG2 cells secrete modest amounts of lipoproteins (21, 23), LDL more than HDL, and that their reuptake is quantitatively insignificant (23). De novo secretion of LDL has been quantified at <3 µg/ml (23), whereas that of HDL was not quantified but is less; therefore, our experiments with exogenously added LDL and HDL are not affected by this negligible secretion of lipoproteins.
Efflux in the presence or absence of several substances has been
studied in a static milieu (24, 25, 26) including ions (27) and drugs (28),
always looking at the initial, outward directed, unidirectional flux
and assuming no interference between the substance and the compound
whose efflux was being investigated. Hennemann et al. (2)
studied T4 and T3 efflux from monolayer
cultures of rat hepatocytes by changing the medium every 2 min to avoid
possible reuptake of the hormone. They described three exponential exit
rates, which were similar for T4 and T3 and had
half times of approximately 0.36, 1.6, and 8 min. The slowest, in terms
of the fractional rate, is approximately 0.09 per min. Ribeiro et
al. (3) studied T3 efflux from a poorly differentiated
rat hepatoma cell line by replacing the medium at 1, 5, 10, 30, and 60
min. The fractional exit rate of the slow component was 0.01 per min.
Both studies showed one or two faster rates attributed to release of
hormone from the cell membrane. The initial efflux rates that we
observed in HepG2 cells (
0.02 per min) and fibroblasts (0.03 per
min) are intermediate between the slow component in these two studies.
Because of the limited availability of most of the proteins we tested,
we did not change the efflux medium in our experiments; however, two
sets of data indicate that, irrespective of the hormone or the cell,
our measurement of hormone efflux over the first 10 min was not biased
by possible inhibition of reuptake due to proteins. First, efflux
maintained the same 10-min linearity (but at different rates) in
protein-containing and protein-free medium. This would not be expected
if proteins with either rapid (TBG, TTR, HSA) or slow (LDL, HDL)
equilibration with thyroid hormones and enormously different affinity
for the hormones (cf 10) were blocking reentry. Second, if proteins
were blocking reuptake of hormone over the first 10 min, then in the
experiments with protein mixtures (Fig. 4) addition of high affinity
sites (TBG, TTR) to low affinity sites (HSA, lipoproteins) would have
trapped a great deal of hormone extracellularly and, therefore, would
have increased the radioactivity measured in the medium. This was not
the case.
While our data demonstrate that at ambient concentrations HDL are
effective facilitators of thyroid hormone efflux from either cell line
their action is not specific. Indeed, ambient concentrations of TBG,
TTR, or HSA were as effective (Hep G2) or slightly more effective
(fibroblasts). Ambient concentrations of LDL, however, were uniformly
less effective. This and the following pieces of evidence militate
against a mechanism mediated by cell-surface HDL receptors. First, the
efflux curves of T4 or T3 did not show
saturation at HDL concentrations as high as 1 g/liter (i.e.
500 times more than the kDa for HDL receptors). In addition, an
appreciable effect of HDL on thyroid hormone exit started at
concentrations of 40 mg/liter HDL protein (i.e. 40-fold
greater than the kDa for HDL receptors). Similar reasoning excludes a
role for LDL receptors in thyroid hormone efflux (Fig 2). In contrast,
we showed saturability and an unequivocal role for LDL receptors in the
entry of LDL-T4 complexes into cells (5). Additional
evidence is that the fractional efflux rates of T4 or
T3 from either cell line did not show changes when HDL
receptors or LDL receptors were up-regulated or down-regulated.
Our data agree with those of Mendel & Kunitake (12) on
cholesterol efflux. They found that the effect of HDL was nonspecific
in that it was independent of the expression of HDL receptors resulting
from manipulations of the cholesterol content of fibroblasts, it was
mimicked by HSA, and it was not saturable even at high concentrations
of either protein (HDL, 1 g/liter and HSA, 80 g/liter). In addition,
they showed that the same fractional efflux of cholesterol from
fibroblasts was elicited by a concentration of HDL (based on mass) that
was about 20-fold lower than that of HSA. Inspection of our Fig. 2
shows that the same is true for thyroid hormone efflux from either cell
line.
Having ruled out the intervention of cell receptors, the facilitated
efflux of thyroid hormones promoted by lipoproteins can be attributed
to a "sink" effect. Lipoproteins would trap the hormone that is
being released and, by doing so, they maintain the gradient from inside
to outside the cells which drives thyroid hormone efflux. Some of the
data, however, suggest that facilitation might occur via a nonspecific
physical contact of the acceptors with the plasma membrane. A major
interest of our investigation was to compare the effect of HDL with LDL
and of both lipoproteins with nonlipoprotein thyroid hormone carriers.
The comparable effectiveness of HDL and TBG is striking considering the
three to four orders of magnitude difference in their Ka for thyroid
hormones. On a molar basis, the order of hierarchy for efflux from
either cell line was TBG > LDL > TTR > HDL (HDL2 > HDL3) >> HSA.
One has, therefore, to conclude that the predominant factor determining
a protein’s effectiveness is its affinity for the hormone. A secondary
factor, which becomes operative for the lipoproteins and HSA, which
have similar affinity, is the lipid content that decreases in the order
LDL > HDL2 > HDL3 >> HSA. We speculate that, in being released from
cells, thyroid hormone, just as cholesterol, desorbs from a lipoprotein
structure (the plasma membrane) to lipoprotein solubilized particles
(the lipoproteins). Because of the hydrophobicity of thyroid hormones,
they will be accepted better by particles more enriched in lipids if
this transfer includes a physical contact between the plasma membrane
and the acceptors. The difference between fibroblasts and hepatocytes
(summarized in Tables 1 and 3) is presumably related to intracellular
binding differences and is in keeping with the greater release from
fibroblasts in protein-free medium. An intrinsic cell-difference is
also supported by the difference remaining constant (i.e. 4-
to 6-fold more in fibroblasts vs. Hep G2) irrespective of
the acceptor protein (Table 3).
In conclusion, HDL do facilitate thyroid hormone exit from hepatocytes and fibroblasts in a manner resembling cholesterol efflux. Unlike the specificity of the LDL receptor-mediated entry of the LDL-T4 complexes (5), the action of HDL in promoting thyroid hormone efflux is nonspecific and nonreceptor mediated. However, the mechanism may imply a physical contact with the plasma membrane. Indeed, a number of studies (29, 30, 31) indicate that cholesterol efflux is relatively apolipoprotein-independent and, instead, the result of lipid-lipid interactions between the plasma membrane and the lipid moiety of the lipoproteins, either phospholipids or cholesterol. As a result, free cholesterol diffuses passively between the plasma membrane and lipid acceptor particles through the unstirred water layer around the cells (22). In a similar manner, by accepting the hormone that is being released, lipoproteins could maintain the gradient from inside to outside the cells that drives thyroid hormone efflux. At ambient, physiological concentrations of proteins, the magnitude of the effect of HDL is comparable to that of the "major" plasma transport proteins (TBG, TTR, HSA), even when these three proteins are combined. In addition to the evidence for LDL-mediated entry of T4 into cells, this is a second indication that lipoproteins may be more important for thyroid hormone physiology than appears to be the case from their minor role as carriers of thyroid hormone in plasma.
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Acknowledgments |
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Received January 22, 1998.
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