(Received for publication, April 28, 1995; and in revised form, May 15, 1995 )
From the
Plasma phospholipid transfer protein mediates the net movement
of phospholipids between lipoproteins and between lipid bilayers and
high density lipoprotein. In this study, the mouse phospholipid
transfer protein cDNA was cloned by reverse transcription polymerase
chain reactions based on the cDNA sequence of human phospholipid
transfer protein. The predicted amino acid sequence of mouse
phospholipid transfer protein shows the protein to be 476 amino acids
long and to have a sequence identity of 83% with that of human
phospholipid transfer protein. Mouse plasma phospholipid transfer
protein activity is 1.5-2 times that of human plasma phospholipid
transfer protein activity. As in humans, mouse peripheral tissues
displayed a higher abundance of phospholipid transfer protein mRNA than
observed in central organs. The order of phospholipid transfer protein
mRNA expression was as follows: lung > adipose tissue, placenta,
testis > brain > muscle, heart, liver. We examined the regulation
of phospholipid transfer protein expression by dietary cholesterol and
by bacterial lipopolysaccharide. A high fat, high cholesterol diet
caused a significant increase (35%) in plasma phospholipid transfer
protein activity and a significant increase (18%) in high density
lipoprotein phospholipids. This increased activity was accompanied by
Plasma phospholipid transfer protein (PLTP)
PLTP
and CETP, a protein that transfers phospholipids as well as cholesteryl
esters and triglycerides, each account for about 50% of the plasma
phospholipid transfer activities(9) . Addition of PLTP to a
cholesteryl ester transfer assay containing CETP results in a marked
enhancement of CETP activity (3) by a mechanism that is most
likely the enhanced binding of CETP to phospholipid-enriched
HDL(9) . CETP activity plays a central role in HDL metabolism,
and its transcription is up-regulated by dietary
cholesterol(17) . These two proteins are evolutionarily related
and are part of a gene family that also includes lipopolysaccharide
binding protein (LBP) and bactericidal/permeability increasing
protein(10, 11) . LBP and bactericidal/permeability
increasing protein modulate the organism's response to bacterial
infections by their ability to bind
lipopolysaccharide(12, 13) . Recent studies suggest
that CETP may also be involved in the acute phase response because it
also binds lipopolysaccharide,
Given the
similarities among the members of the lipid transfer/lipopolysaccharide
binding protein gene family, we investigated whether mouse PLTP is also
regulated by dietary cholesterol and lipopolysaccharide. We chose the
mouse as a model organism for two reasons. First, mouse plasma has no
measurable CETP mass and so its plasma phospholipid transfer activity
would be due solely to PLTP. This would simplify future studies on the
phospholipid metabolism of this animal. Second, in order to lay the
foundations of PLTP transgenesis and gene knock-out manipulations in
mouse we need to characterize the structure, pattern of expression, and
gene regulation of PLTP in the wild-type mouse. So, in this study we
have isolated and characterized PLTP cDNA from C57BL/6 mouse and
determined the tissue distribution of its mRNA. We indicate here that
PLTP has a tissue distribution that is distinct from that of the other
gene family member and that lipopolysaccharide and dietary cholesterol
regulate its expression.
The mouse PLTP cDNA was obtained by polymerase chain
reaction amplification of lung cDNA using degenerate primers based on
the human PLTP cDNA sequence(10) . The cDNA sequence and the
predicted amino acid sequence of mouse PLTP are shown in Fig. 1.
Both mouse and human PLTP leader sequences are 17 amino acids in length
with 9 identical amino acids, conforming to the pattern of similarity
among the signal sequences of PLTP, CETP, lipoprotein lipase, and
apolipoproteins A-I, and A-IV that was described
previously(10, 21) . Like human PLTP, the predicted
mature mouse PLTP is 476 amino acids long with an 83% amino acid
sequence identity (Fig. 1). At the nucleotide sequence level,
the identity in the translated regions of the human and mouse genes is
86%, while the 3`-untranslated regions are divergent.
Figure 1:
Alignment of mouse and human PLTP cDNA
and mature protein. The amino acid sequence of the mature mouse PLTP
derived from cDNA sequencing. m-DNA, mouse PLTP cDNA; h-DNA, human PLTP cDNA; m-aa, mouse
PLTP amino acid sequence; h-aa, human PLTP amino acid
sequence.
Figure 2:
Northern blot analysis of
poly(A)
Figure 3:
RNase protection analysis of total RNA
from human adipose tissue and heart. Fifty µg of total RNA from
tissues was used for the RNase protection assay. See
``Experimental Procedures'' for
details.
Figure 4:
Regulations of PLTP activity and mRNA
levels by lipopolysacchride administration. Three µl of mouse
plasma was used for PLTP activity assay. Fifty µg of total RNA was
used for RNase protection assay. PLTP activity assay, p <
0.001; RNase protection assay for liver, adipose tissue, and lung total
RNA, p < 0.001, p < 0.01, p < 0.01,
respectively.
Figure 5:
Regulations of PLTP activity and mRNA
levels by high fat and high cholesterol diet. The PLTP activity assay
and RNase protection assay were same as Fig. 4. PLTP activity
assay, p < 0.02; RNase protection assay for liver RNA;
RNase protection assay for adipose tissue RNA, p =
0.09; RNase protection assay for lung RNA, p <
0.01.
We have shown in this study that mouse and human PLTP have
similar sequence and similar tissue distribution of mRNA production.
The plasma activity is 1.5-2 times human levels. These findings
suggest that the mouse will have utility as a model organism to study
the role of PLTP in lipid metabolism. In mouse, as in human,
extrahepatic tissues contribute to a large fraction of total PLTP mRNA
production. It is likely that production of PLTP in these tissues is
important in regulating phospholipid transfer to and from these
tissues. Lung, adipose tissue, and testis were the tissues with the
highest levels of PLTP expression. Although its PLTP expression ranks
low among the tissues studied here, the large mass of liver means that
this organ produces an appreciable amount of PLTP mRNA. Thus liver,
adipose tissue and lung likely contribute to most of the total
circulating PLTP mass.
PLTP belongs to LBP gene family(10) ,
but it has its own distinct structure, function, and regulatory
patterns. Although evolutionarily PLTP is more similar to LBP and
bactericidal/permeability increasing protein than to CETP(10) ,
functionally it is more similar to CETP in that both transfer
phospholipids between lipoproteins(9, 16) . In its
regulation by lipopolysaccharide, PLTP is also more similar to CETP
than to LBP and bactericidal/permeability increasing protein since this
agent down-regulates expression of both proteins. PLTP mRNA levels are
responsive to dietary cholesterol, but the -fold increase and
tissue-specific up-regulation are different from CETP (see below).
Unlike CETP, whose cholestyl ester transfer activity is inhibitable by
lipopolysaccharide(15) , PLTP activity is not inhibited by
lipopolysaccharide. The regulatory mechanisms of PLTP appear to have
differentiated during evolution from those of its family members so as
to adapt to this protein's unique function.
The 35% increase
in plasma PLTP activity in response to dietary cholesterol and fat is
not comparable to the 5-10-fold increase observed with CETP in
CETP transgenic mice(17) , but it is statistically significant (p < 0.02). A similar up-regulation is seen in rabbits in
which, after administration of a cholesterol (0.15%) diet, PLTP
activity is increased 12 and 50% depending on whether they are
hyporesponsive and hyperresponsive to cholesterol,
respectively(22) . Also different from CETP regulation by
cholesterol that occurs in most tissues(17, 23) , PLTP
mRNA level is up-regulated by cholesterol diet in lung but not in
adipose tissue and liver. This would suggest that cholesterol
up-regulatory transcription factors must have different interactions
with tissue-specific factors for these two genes.
In this study, we
found the HDL cholesterol and HDL phospholipid levels in mice treated
with lipopolysaccharide were significantly elevated. These changes were
due to an increase in HDL surface area rather than HDL particle number,
because HDL size was increased while apoA-I content was not altered.
HDL has been shown to be protective against endotoxic shock by binding
lipopolysaccharide(24) . This is likely an adaptive response
for the lipopolysaccharide-treated animal since larger HDL surface area
would have greater lipopolysaccharide binding capacity/HDL
particle(25) . Lipopolysaccharide treatment causes complex
changes in lipoprotein metabolism resulting in large changes in
lipoprotein profile. Many enzymes involved in HDL metabolism are
influenced by lipopolysaccharide treatment; there are increases in
serum amyloid protein (26) and probably hepatic lipase (27, 28) and decreases in lipoprotein
lipase(29) , lecithin cholesterol acyltransferase(27) ,
CETP (in animals with CETP activity(15) ) and, as shown in this
study, PLTP. Of these changes, none of them except that of PLTP could
possibly increase HDL phospholipid. Thus, the increased HDL
phospholipid that we observe here is likely due to decreased
phospholipid transfer by PLTP from HDL. The decreased PLTP levels
appear to play an important role in the altered quantities, sizes, and
lipid distribution of lipoproteins during acute phase.
It is
interesting to note that a dietary cholesterol and lipopolysaccharide
treatment similarly regulate PLTP in mice and CETP in CETP transgenic
mice(15, 17) . Similarly, these two proteins are
induced in obese people by 15 and 35%, respectively, relative to
nonobese people(30) . Also, it has been reported that cigarette
smoking increases CETP and PLTP activities by 18 and 8%,
respectively(31) . The correlation in CETP and PLTP activities
may be due to the fact that removal of either cholesterol or
phospholipid from a lipid membrane would disrupt an optimal
phospholipid/cholesterol ratio. This disruption caused by removal of
phospholipid by PLTP can be corrected either by a concomitant removal
of cholesterol (which would result in decreased surface area) or an
influx of phospholipid from another source. One mechanism by which
cholesterol removal could accompany phospholipid transfer is the
coordinate action of lecithin cholesterol acyltransferase and CETP.
Another possible reason for the correlation of PLTP and CETP activities
may be that processes that lead to a change in the surface area of
lipoproteins would need to be coupled to mechanisms that change the
core volume, either by neutral lipid transfer by CETP or by lipolysis.
Whichever of these mechanisms is operating, phospholipid transfer can
be expected to be interrelated with phospholipid and cholesterol
metabolism. Since mice and rats do not have significant CETP activity
it should be of interest to determine what alternative mechanisms of
cholesterol removal are coupled to PLTP activity in these animals.
It is interesting to find that adipose tissue express PLTP mRNA at
high levels in mice and humans. Adipose tissue also is a major site for
CETP synthesis(32) . In hamster, a strong inverse correlation
of adipose tissue CETP mRNA levels with the concentration of HDL
cholesteryl esters suggests a role for locally synthesized CETP in the
metabolism of HDL cholesteryl esters(32) . Locally synthesized
PLTP in adipose tissue may also be involved in HDL metabolism, since
CETP and PLTP activities are correlated in obese humans(30) .
Adipose tissue is also a major site for lipoprotein lipase
synthesis(33) . In vitro studies showed that PLTP
mediates the transfer and exchange of phospholipids between
triglyceride-rich lipoproteins and HDL during lipolysis(5) ,
suggesting a possible coordinated action of lipoprotein lipase and
PLTP. Since PLTP, CETP, and lipoprotein lipase are highly expressed in
the same tissues and together they play important roles in lipoprotein
remodeling, their coordinated action in adipose tissue deserves further
study.
In mice and humans, lung is a major tissue expressing PLTP
mRNA. This suggests an important function of PLTP in the lung, most
likely in surfactant production and metabolism. It was recently
reported that HDL and LDL could stimulate primary cultures of type II
pneumocytes to secrete phospholipids(34) . PLTP may facilitate
uptake of secreted phospholipid by HDL in the interstitial fluid of the
lung, as well as subsequent deposition of phospholipid into the
alveolar membrane. Because lung was the only tissue among those
examined that increased PLTP expression (
It has been
suggested that PLTP is involved in the provision of substrate for
lecithin cholesterol acyltransferase and/or transferring
phospholipid-rich surface fragments from triglyceride-rich lipoproteins
to HDL during catalyzed lipolysis(5) , i.e. that PLTP
promotes HDL formation or increases HDL-phospholipid concentration. On
the other hand, there is evidence from rats suggesting that PLTP
enables either an increased uptake of HDL-phospholipid by tissues or an
increased clearance of HDL particles(8) . Thus, whether PLTP
activity leads to a net transfer into or out of HDL seems to depend on
the physiological context. Our findings in this study also suggest the
same, because although HDL phospholipid and HDL cholesterol increased
in both lipopolysaccharide-treated animals and animals on a cholesterol
diet, we find that the levels of PLTP under these two treatments change
in opposite directions. Since the phospholipid contents of
LDL+VLDL were essentially unchanged, the changes in PLTP levels
must have influenced primarily the phospholipid fluxes to and from
tissues. In a high fat and cholesterol diet, it may be advantageous for
the animal to increase the phospholipid flow into tissues by increasing
PLTP levels. Conversely, with lipopolysaccharide treatment, it may be
more advantageous to limit phospholipid flow between HDL and tissues by
decreasing PLTP concentrations. Because lipopolysaccharide treatment
and dietary fat and cholesterol have complex effects, the understanding
of the role that PLTP plays in lipid metabolism will have to await the
study of animals whose PLTP expression is specifically altered.
However, it appears likely that HDL-phospholipid levels are determined
both by PLTP levels and by gradients of phospholipid concentration
between HDL and other phospholipid sources.
The nucleotide sequence(s) reported in this paper has been
submitted to the GenBank®/EMBL Data Bank with accession
number(s) U28960[GenBank® Link].
We thank Dr. Alan Tall for his invaluable guidance and
discussions which have made this work possible. The human heart and
human adipose tissue samples were generous gifts from Dr. Jie Wang.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
100% increase in phospholipid transfer protein mRNA in lung. After
lipopolysaccharide injection, plasma phospholipid transfer protein
activity was decreased by
66%. This decrease in activity was
associated with a similar decrease in phospholipid transfer protein
mRNA in lung, adipose tissue, and liver. The decrease in plasma
phospholipid transfer protein activity was also associated with a
significant increase (17%) in high density lipoprotein phospholipid
concentration. The opposite changes in phospholipids levels with
lipopolysaccharide treatment and dietary cholesterol despite similarly
increased high density lipoprotein phospholipids levels indicate that
high density lipoprotein phospholipids levels are likely determined
both by phospholipid transfer protein levels and by gradients of
phospholipids concentration between high density lipoprotein and other
phospholipids sources.
(
)transfers phospholipids among lipoprotein particles (1, 2, 3) and from lipid bilayers to high
density lipoproteins (HDL)(1, 4) . There is
accumulating evidence indicating that PLTP plays an important role in
the remodeling of lipoproteins. In vitro experiments show that
partially purified PLTP mediates both the transfer and exchange of
phospholipids between triglyceride-rich lipoproteins and HDL during
lipolysis(5) . PLTP can also cause conversion of HDL
to larger (10.9 nm) and smaller (7.8 nm) particles in a time- and
concentration-dependent fashion(6, 7) . PLTP activity
also modulates the activities of lecithin cholesterol acyltransferase
and cholesteryl ester transfer protein (CETP)(3, 9) .
In in vivo experiments using partially purified PLTP injected
into rats, the turnover rate of
[
C]phosphatidylcholine ether-labeled HDL
increases by 50%. This suggests that PLTP can facilitate the transfer
of radiolabeled phospholipid between HDL and tissues or that PLTP
increases the rate of HDL uptake by these tissues(8) .
(
)and its
transcription is down-regulated by lipopolysaccharide
treatment(15) . However, unlike CETP, LBP is markedly
up-regulated by lipopolysaccharide treatment(14) .
Mouse PLTP cDNA Cloning
First strand cDNA was
synthesized from mouse lung mRNA using oligo(dT) as a primer. The mouse
PLTP cDNA was amplified from total cDNA by primer extension and
polymerase chain reactions (21) using rTth DNA polymerase
(Perkin Elmer) and several degenerate primers based on the human PLTP
cDNA sequence(10) . The amplified products were cloned into
Bluescript KS- (Stratagene) and sequenced according to standard
procedures.
RNA Isolation, Northern Blot Analysis, and RNase
Protection Assay
Total RNA was isolated from various tissues
using the lithium chloride/urea extraction method(18) . The
integrity and quality of all RNA samples was determined by
electrophoresis on agarose/borate gels before mRNA isolation.
Polyadenylated RNA was isolated from total RNA by batch isolation using
oligo(dT)-cellulose. Northern blot analysis of mRNA was performed as
described previously(19) . A fragment of the human PLTP cDNA
(112 base pairs, 1544-1655) was cloned into Bluescript SK+
(Stratagene) and used to prepare radiolabeled cRNA probes for the RNase
protection assay, which was performed as described
previously(17) .
PLTP Activity Measurement
Phospholipid transfer
activity was measured with
[H]dipalmitoylphosphatidylcholine (DPPC; DuPont
NEN) -containing vesicles that were prepared as described
previously(20) . Briefly, 10 µmol of egg
phosphatidylcholine containing 10 nmol of [
H]DPPC
was dried under a stream of nitrogen, resuspended in 1 ml of a solution
of 10 mM Tris, 150 mM NaCl, 1 mM EDTA, pH
7.4, was probe-sonicated, and then was centrifuged to remove lipid
aggregates. Transfer of radiolabeled phospholipids was measured by
incubating an aliquot of 100 µl of each sample with
[
H]DPPC vesicles (125 nmol of
phosphatidylcholine) and HDL
(250 µg of protein) in a
final volume of 400 µl at 37 °C for 1 h. Vesicles were
subsequently precipitated by the addition of 300 µl of a solution
of 500 mM NaCl, 215 mM MnCl
, 445 units/ml
heparin, and the radioactivity of a 500-µl aliquot of the
supernatant was measured.
Dietary Study
C57BL/6 mouse (males, 25-30 g, n = 6) had free access to food and water. They were fed
either the high cholesterol, high fat diet (20% coconut oil, 1%
cholesterol from Research Diets Inc. New Bruswick, NJ) or a standard
laboratory diet (Purina Laboratory rodent chow 5001) for 1 week before
being sacrificed.
Lipopolysaccharide Administration Study
C57BL/6
mouse (males, 25-30 g, n = 6) had free access to
food and water. They were fed a standard laboratory diet (Chow 5001).
Lipopolysaccharide (Escherichia coli 0127:B8, from Difco) was
suspended in sterile saline. Stock lipopolysaccharide was sonicated and
diluted just before use, at a dosage of 50 µg/animal. After 15 h,
the animals were sacrificed.
Statistical Analysis
Differences between groups
were tested by Student's t test. All analyses were
performed with the Statworks® software. Significance levels given
are those for the two-tailed test. Data are presented as mean ±
S.D. Significant results were with p < 0.01 unless
otherwise stated.
PLTP Gene Expression in Mouse
Although mice have negligible or no CETP activity, their PLTP
activity is present. We measured PLTP activity in mice of three
different strains. The PLTP activity in C57BL/6, C3H, and BALB/C mice
were 220% (±15%), 205% (±10%), and 155% (±20%) of
human plasma activity, respectively (n = 6 for each
group).
We determined
the tissue distribution of PLTP mRNA in mouse (strain C57BL/6) by
Northern blot analysis (Fig. 2). Hybridization of a PLTP probe
to poly(A) mRNA isolated from various mouse tissues
indicated the existence of a single transcript of approximately 1.8
kilobases, similar in length to that of human(10) . In another
set of analyses, we used the RNase protection assay to measure PLTP
mRNA concentration in lung, adipose tissue, and testis (Fig. 2C). The order of PLTP mRNA abundance is as
follows: lung > adipose tissue, placenta > testis > brain >
muscle > heart > kidney > liver > spleen.
RNA from various human tissues. Two
µg/lane of poly(A)
RNA from various mouse tissues
was probed with a 749-base pair fragment (nucleotides 50-798 in Fig. 3) that was random primer-labeled. A and B, two set of Northern blot experiments; C, RNase
protection assay for three tissues (30 µg of total RNA was used)
using mouse 3`-PLTP cRNA as probe. For details see ``Experimental
Procedures.''
Because of the
striking levels of PLTP mRNA expression in mouse adipose tissue, we
investigated whether PLTP was also expressed in human adipose tissue.
Human adipose tissue (from the heart) shows approximately 5 times as
much PLTP mRNA expression as that seen in the heart (Fig. 3).
Regulations of PLTP Activity and mRNA Levels
Lipopolysaccharide Regulation
In order to
evaluate the effects of lipopolysaccharide on PLTP gene expression,
lipopolysaccharide was injected into mice at 50 µg/animal. This
resulted in a marked decrease in plasma PLTP activity and profound
reductions of PLTP mRNA in lung, adipose tissue, and liver (Fig. 4). In order to evaluate the effects of altered PLTP gene
expression on the plasma lipoprotein response to lipopolysaccharide, we
measured plasma lipid and lipoprotein concentrations.
Lipopolysaccharide-treated animals had significantly higher HDL
phospholipids and HDL cholesterol than controls (Table 1).
Analysis by fast protein liquid chromatograph gel filtration of pooled
mouse (n = 6) plasmas confirmed these observations
(data not shown). By nondenaturing gradient gel electrophoresis, the
HDL particles isolated by ultracentrifugation were increased in size
(from 10.2 to
10.9 nm). However, apoA-I levels in such HDL
from lipopolysaccharide-treated and control animals were unchanged
while apoE was increased, as determined by SDS-polyacrylamide gel
electrophoresis (data not shown). Although there was an increase of
plasma triglyceride concentration, this did not reach statistical
significance probably because of the low lipopolysaccharide dose used.
The effect of lipopolysaccharide on PLTP plasma activity was not due to
an interaction of lipopolysaccharide with PLTP because in an in
vitro assay with mouse plasma there was no direct inhibition of
transfer activity at lipopolysaccharide concentrations up to 0.3
µg/µl. We also verified that the amount of lipopolysaccharide
used in the assay system did not influence the lipoprotein
precipitability by heparin-Mn
.
Dietary Regulation
In order to determine whether
there are alterations in PLTP activity and mRNA levels in response to
dietary fat and cholesterol, mice were fed for 1 week a diet containing
20% coconut oil and 1% cholesterol, which has previously been shown to
significantly increase CETP activity and mRNA in CETP transgenic mice (17) . In this study, plasma PLTP activity was significantly
increased (35%) in response to the high fat and cholesterol diet (Fig. 5), but the increase of PLTP mRNA was observed only in
lung, not in liver and adipose tissue (Fig. 5). The
HDL-phospholipids and HDL-cholesterol were increased 18 and
50% respectively, while the cholesterol and phospholipid levels of
the very low density and low density lipoprotein fraction was increased
300 and
60%, respectively (Table 1).
100%) in response to a
high fat and cholesterol diet and this was accompanied by a significant
increase (
30%) in plasma PLTP activity, this organ must be
contributing significant amounts of plasma PLTP.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.