ArticlePDF Available

Regulation of the proprotein convertase subtilisin/kexin type 9 in intestinal epithelial cells


Abstract and Figures

Proprotein convertase subtilisin/kexin type 9 (PCSK9) posttranslationally promotes the degradation of the low-density lipoprotein receptor (LDLr) in hepatocytes and increases plasma LDL cholesterol. It is not clear, however, whether PCSK9 plays a role in the small intestine. Here, we characterized the patterns of variations of PCSK9 and LDLr in fully differentiated Caco-2/15 cells as a function of various potential effectors. Cholesterol (100 microM) solubilized in albumin or micelles significantly downregulated PCSK9 gene (30%, P<0.05) and protein expression (50%, P<0.05), surprisingly in concert with a decrease in LDLr protein levels (45%, P<0.05). Cells treated with 25-hydroxycholesterol (50 microM) also displayed significant reduction in PCSK9 gene (37%, P<0.01) and protein (75% P<0.001) expression, whereas LDLr showed a decrease at the gene (30%, P<0.05) and protein (57%, P<0.01) levels, respectively. The amounts of PCSK9 mRNA and protein in Caco-2/15 cells were associated to the regulation of 3-hydroxy-3-methylglutaryl-CoA reductase and sterol regulatory element binding protein-2 (SREBP-2) that can transcriptionally activate PCSK9 via sterol-regulatory elements located in its proximal promoter region. On the other hand, depletion of cholesterol content by hydroxypropyl-beta-cyclodextrin upregulated PCSK9 transcripts (20%, P<0.05) and protein mass (540%, P<0.001), in parallel with SREBP-2 protein levels. The addition of bile acids (BA) taurocholate and deoxycholate to the apical culture medium lowered PCSK9 gene expression (25%, P<0.01) and raised PCSK9 protein expression (30%, P<0.01), respectively, probably via the modulation of farnesoid X receptor. Furthermore, unconjugated and conjugated BA exhibited different effects on PCSK9 and LDLr. Altogether, these data indicate that intestinal PCSK9 is highly modulated by sterols and emphasize the distinct effects of BA species.
Content may be subject to copyright.
Regulation of the proprotein convertase subtilisin/kexin type 9 in intestinal
epithelial cells
Franc¸ois Leblond,
Nabil G. Seidah,
Louis-Philippe Pre´court,
Edgard Delvin,
Michel Dominguez,
and Emile Levy
Departments of
Nutrition and
Biochemical Neuroendocrinology, Clinical Research Institute of Montre´al;
of Biochemistry, Research Center, CHU Sainte Justine, Universite´ de Montre´al, Montre´al, Que´bec, Canada
Submitted 10 July 2008; accepted in final form 26 January 2009
Leblond F, Seidah NG, Pre´court LP, Delvin E, Dominguez
M, Levy E. Regulation of the proprotein convertase subtilisin/
kexin type 9 in intestinal epithelial cells. Am J Physiol Gastrointest
Liver Physiol 296: G805–G815, 2009. First published January 29,
2009; doi:10.1152/ajpgi.90424.2008.—Proprotein convertase sub-
tilisin/kexin type 9 (PCSK9) posttranslationally promotes the deg-
radation of the low-density lipoprotein receptor (LDLr) in hepato-
cytes and increases plasma LDL cholesterol. It is not clear,
however, whether PCSK9 plays a role in the small intestine. Here,
we characterized the patterns of variations of PCSK9 and LDLr in
fully differentiated Caco-2/15 cells as a function of various poten-
tial effectors. Cholesterol (100 M) solubilized in albumin or
micelles significantly downregulated PCSK9 gene (30%, P0.05)
and protein expression (50%, P0.05), surprisingly in concert
with a decrease in LDLr protein levels (45%, P0.05). Cells
treated with 25-hydroxycholesterol (50 M) also displayed signif-
icant reduction in PCSK9 gene (37%, P0.01) and protein (75%
P0.001) expression, whereas LDLr showed a decrease at the
gene (30%, P0.05) and protein (57%, P0.01) levels,
respectively. The amounts of PCSK9 mRNA and protein in Caco-
2/15 cells were associated to the regulation of 3-hydroxy-3-
methylglutaryl-CoA reductase and sterol regulatory element bind-
ing protein-2 (SREBP-2) that can transcriptionally activate PCSK9
via sterol-regulatory elements located in its proximal promoter
region. On the other hand, depletion of cholesterol content by
hydroxypropyl--cyclodextrin upregulated PCSK9 transcripts
(20%, P0.05) and protein mass (540%, P0.001), in parallel
with SREBP-2 protein levels. The addition of bile acids (BA)
taurocholate and deoxycholate to the apical culture medium low-
ered PCSK9 gene expression (25%, P0.01) and raised PCSK9
protein expression (30%, P0.01), respectively, probably via the
modulation of farnesoid X receptor. Furthermore, unconjugated
and conjugated BA exhibited different effects on PCSK9 and
LDLr. Altogether, these data indicate that intestinal PCSK9 is
highly modulated by sterols and emphasize the distinct effects of
BA species.
enterocyte; PCSK9; cholesterol; bile acids; farnesoid X receptor;
FAMILIAL HYPERCHOLESTEROLEMIA is characterized by elevated
plasma low-density lipoprotein (LDL) and deposition of LDL
in arteries, leading to premature cardiovascular events (15).
This genetically dominant hypercholesterolemia is related to
molecular aberrations in the LDL receptor (LDLr) or its ligand
apolipoprotein B. Recently, mutations in a third gene, propro-
tein convertase subtilisin/kexin type 9 (PCSK9), were associ-
ated to this disease (1). Human PCSK9 gene is 22 kb long,
comprising the promoter region and 12 exons, and it is located
on chromosome 1p32 (49). The gene produces an mRNA of
3,636 bp encoding a 692-aa protein. Although it is proposed
that secreted PCSK9 interacts with LDLr, enters the endocytic
recycling pathway with LDLr, and impairs LDLr pathway by
increasing the degradation of the receptor (5, 37), the precise
molecular basis has yet to be determined.
PCSK9 is mainly expressed in the liver. It is thought to be
involved in liver regeneration, neuronal differentiation/apopto-
sis, and cortical neurogenesis (49), but its primary function
apparently consists in negatively regulating LDLr, likely by a
posttranscriptional manner, e.g., degradation of LDLr in acidic
subcellular compartments, possibly endosomes/lysosomes (23,
30, 49, 50). Even though PCSK9 has been found expressed
substantially in intestine (49), studies have been mostly re-
stricted to the liver (23, 30, 50). However, maintaining cho-
lesterol homeostasis in the body requires accurate metabolic
cross talk between hepatic and intestinal processes to ade-
quately cope with large fluctuations in dietary cholesterol
intake (58), whereas imbalance may lead to elevated LDL
cholesterol levels and increased risks for coronary heart
disease, the main cause of death in Western societies (56).
Notably, the intestine plays a key role in cholesterol balance
in animals and humans (55), constitutes the only site for
absorption of dietary sterols (57), quantitatively represents
the single active location for cholesterogenesis (53, 59), and
remains the second most important organ for the uptake and
degradation of circulating LDL (52). Whether PCSK9 re-
duces the number of LDLr in the enterocyte basolateral
membrane is not known. This information is crucial since
LDLr behavior in absorptive cells does not necessarily
reflect that in hepatocytes.
Poor information is available about the regulation of PCSK9
even though it seems to be an attractive target for lowering
LDL cholesterol (1). In mice, PCSK9 is downregulated by
dietary cholesterol. Conversely, PCSK9 gene expression is
upregulated in mice overexpressing sterol regulatory element
binding proteins (SREBP)-1and SREBP-2 (two transcription
factors activated by low levels of intracellular cholesterol)
(24). The nutritional status also modulates PCSK9 expression.
For example, 24-h fasting in mice dramatically decreased
hepatic PCSK9 mRNA and protein levels, which were progres-
sively restored by carbohydrate refeeding (9). Similarly, statins
have been shown to lower LDL by inducing SREBP-2, which
enhances LDLr expression. Moreover, the peroxisome prolif-
Address for reprint requests and other correspondence: E. Levy, GI-Nutri-
tion Unit, CHU Sainte-Justine, 3175 Coˆ te Ste-Catherine, Montreal, Quebec,
Canada, H3T 1C5 (e-mail:
The costs of publication of this article were defrayed in part by the payment
of page charges. The article must therefore be hereby marked “advertisement
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Am J Physiol Gastrointest Liver Physiol 296: G805–G815, 2009.
First published January 29, 2009; doi:10.1152/ajpgi.90424.2008.
0193-1857/09 $8.00 Copyright ©2009 the American Physiological Society G805
erator-activated receptor (PPAR)-agonist fenofibrate down-
regulates PCSK9 gene expression and protein synthesis in
controls and not, as expected, in PPAR--deficient mice (31).
Most of the work on PCSK9 regulation has been performed in
relation to the liver, but it must be extended to the small
intestine, an important site for cholesterol homeostasis. There-
fore, the purpose of the present study was to examine the
regulation of PCSK9 in an established Caco-2/15 enterocyte
cell line. An additional aim of the present work was to
determine the expression profile of LDLr and various nuclear
transcription factors.
Cell culture. Caco-2/15 cells (American Type Culture Collection,
Rockville, MD) were grown at 37°C with 5% CO
in minimal
essential medium (MEM) (GIBCO-BRL, Grand Island, NY) contain-
ing 1% penicillin-streptomycin and 1% MEM nonessential amino
acids (GIBCO-BRL) and supplemented with 10% decomplemented
fetal bovine serum (FBS) (Flow, McLean, VA). Caco-2/15 cells
(passages 30 40) were maintained in T-75-cm
flasks (Corning Glass
Works, Corning, NY). Cultures were split (1:6) when they reached
70 –90% confluence, by use of 0.05% trypsin-0.5 mM EDTA
(GIBCO-BRL). For individual experiments, cells were plated at a
density of 1 10
cells/well on 24.5-mm polycarbonate Transwell
filter inserts with 0.4-m pores (Costar, Cambridge, MA), in MEM
(as described above) supplemented with 5% FBS. The inserts were
placed into six-well culture plates, permitting separate access to the
upper and lower compartments of the monolayers. Cells were cultured
for various periods, including 21 days, at which the Caco-2/15 cells
are highly differentiated and appropriate for lipid synthesis (16, 34,
48). The medium was refreshed every second day.
Cell treatments. Cholesterol and 25-hydroxycholesterol (Sigma-
Aldrich Canada, Oakville, ON) were dissolved in chloroform and
dried under nitrogen. Acetone (0.5 ml) was then added to dissolve the
dried sterol. This solution was slowly added with continuous stirring
to 3 ml of an albumin solution (250 mg of fatty acid-poor BSA in 3
ml of 10% BSA pH 7.4). This mixture was placed under a stream of
nitrogen until the odor of acetone was no longer detectable. Micellar
cholesterol was prepared as described previously (45). The mixed bile
salt micelle, as a vehicle of cholesterol and 25-hydroxycholesterol,
contained 4.8 mM sodium taurocholate and 0.3 mM monoolein. The
micellar solution was warmed to 37°C and stirred vigorously before
use. Albumin- or micelle-bound sterols were mixed to MEM without
FBS (18 h) before being added to the apical compartment. Hy-
droxypropyl--cyclodextrin (Sigma-Aldrich Canada) was directly
dissolved in MEM at a 25 mM concentration and then added to the
apical compartment. Cholate (4.8 mM), chenodeoxycholate (250 M)
and conjugated forms (Calbiochem, Gibbstown, NJ), as well as
deoxycholate (250 M) (ICN Biomedicals, Aurora, OH) and conju-
gated forms (Calbiochem, Gibbstown, NJ) were dissolved in 95%
ethanol and evaporated under nitrogen before being mixed with MEM
at their final concentrations.
Antibodies against PCSK9. The specificity of the Abs (particularly
PCSK9) (43, 46) was evaluated by various methods, including West-
ern blotting following the incubation of the Abs in the presence or
Fig. 1. Influence of cholesterol (solubilized in
albumin) incubated for 24 h on gene and
protein expressions of proprotein convertase
subtilisin/kexin type 9 (PCSK9) and low-den-
sity lipoprotein receptor (LDLr) in intestinal
epithelial cells. Caco-2/15 cells were incu-
bated with fresh medium, allowed to grow and
differentiate, and tested for the effects of cho-
lesterol on transcript levels and protein mass
of PCSK9 and LDLr by RT-PCR and Western
blotting, respectively. The control cells were
incubated with albumin only. Values are ex-
pressed as means SE for 3 different exper-
iments. *P0.05; **P0.01; ***P
0.001 vs. controls.
AJP-Gastrointest Liver Physiol VOL 296 APRIL 2009
absence of specific antigens, the omission of the primary PCSK9 Ab
in Western blot, and the identification of the human PCSK9 sequences
following immunoprecipitation and SDS-PAGE.
Western blots. To assess the presence and regulation of PCSK9,
LDLR, and SREBP-2, Caco-2/15 cells were homogenized and ade-
quately prepared for Western blotting as described previously (33).
The Bradford assay (Bio-Rad) was used to estimate protein concen-
tration. Proteins were denatured in sample buffer containing SDS and
DTT (Thermo Scientific, Rockford, IL), separated on a 7.5% SDS-
PAGE and electroblotted onto Hybond-C extra nitrocellulose mem-
branes (Amersham Biosciences, Piscataway, NJ). Nonspecific binding
sites of the membranes were blocked with defatted milk proteins
followed by the addition of primary antibodies directed against the
targeted proteins: -actin (Sigma-Aldrich Canada), LDLr (Research
Diagnostic, Flanders, NJ), PCSK9 (amino acids 31-454) (kind gift of
G. Dubuc and J. Davignon, Clinical Research Institute of Montreal,
University of Montreal, Montreal, QC, Canada), and SREBP-2 (Cayman,
Ann Arbor, MI). The relative amount of primary antibody was
detected with species-specific horseradish peroxidase-conjugated
secondary antibody. Even if identical protein amounts of tissue
homogenates were applied, the -actin protein was used as a
reference protein. Molecular size markers were simultaneously
loaded on gels (data not shown on the figures). Blots were
developed and the mass of proteins were quantified by using an HP
Scanjet scanner equipped with a transparency adapter and soft-
ware. Importantly, the utilization of differential loading and quan-
tification suggested a dynamic range of densitometric measure-
RT-PCR. Experiments for mRNA quantification as well as for
GAPDH (as a control gene) were performed by using the Eppendorf
Mastercycler Gradient PCR machine (Eppendorf, Westbury, NY) as
reported previously (40, 48). Approximately 30 40 cycles of amplifica-
tion were used at 95°C for 30 s, 58°C for 30 s, and 72°C for 30 s. The
following primer sequences were used: farnesoid X receptor (FXR)
ward (5-GTCCACTGGCGTGTTCACCA-3) and reverse (5-
GTGGCAGTGATGGCATGGAC-3); 3-hydroxy-3-methylglutaryl
(HMG)-CoA reductase forward (5-ACCCTTAGTGGCTGAAACA-
verse (5-CGGGATTCCATGCTCCTTGACTTT-3). Amplicons were
visualized on standard ethidium bromide-stained agarose gels. Under
these experimental conditions relative to RT-PCR, 34 –36 cycles corre-
sponded to the linear portion of the exponential phase. Pilot and previous
studies in our laboratories showed that the RT-PCR, under our experi-
mental conditions, is quantitative employing different amounts of RNA.
Fold induction was calculated by using GAPDH (glyceraldehyde 3-phos-
phate dehydrogenase) as the reference gene.
Fig. 2. Effect of cholesterol (solubilized in bile
acid micelles) incubated for 24 h on gene and
protein expressions of PCSK9 and LDLr intes-
tinal epithelial cells. Caco-2/15 cells were incu-
bated with fresh medium, allowed to grow and
differentiate, and tested for the effects of micel-
lar cholesterol on transcript levels and protein
mass of PCSK9 and LDLr by RT-PCR and
Western blotting, respectively. The control cells
were incubated with bile acid micelles only.
Values are expressed as means SE for 3
different experiments. *P0.05 vs. micelles.
AJP-Gastrointest Liver Physiol VOL 296 APRIL 2009
Statistical analysis. All values were expressed as means SE. The
data were evaluated by ANOVA, where appropriate, and the differ-
ences between the means were assessed by the two-tailed Student’s
The first issue addressed by our studies was to explore
whether a difference could be noted in the expression of
PCSK9 under the influence of sterols. The incubation of fully
differentiated Caco-2/15 cells with various concentrations of
cholesterol solubilized in albumin resulted in a decrease in the
gene and protein expression of PCSK9. At 100 M cholesterol,
decreases of 25% (P0.001) and 55% (P0.05) were
significantly recorded in mRNA and protein mass levels, re-
spectively (Fig. 1A). When the regulation of LDLr was inves-
tigated, a lower protein expression (60%, P0.001) along
with significantly unchanged mRNA levels was noted (Fig.
1B). Since bile acid (BA)-facilitated cholesterol absorption
occurs in the small intestine in physiological conditions, solu-
tions containing either micelles alone or micelles together with
cholesterol were added to the apical media. Again, a micellar
cholesterol-mediated drop (30 –50%) was recorded in the gene
and protein expression of PCSK9 (Fig. 2A), accompanied with
a similar reduction in LDLr protein expression (Fig. 2B).
Finally, the substitution of cholesterol by 25-hydroxycholes-
terol produced a similar results on PCSK9 (Fig. 3A) and on
both mRNA and protein levels of LDLr (Fig. 3B).
In view of these findings, we assessed protein expression
of nuclear SREBP-2 with an antibody that distinguished the
truncated active nuclear form from its membrane-linked
precursor. Also, since LDLr and HMG-CoA reductase, the
limiting enzyme in cholesterol synthesis, are sensitive to
SREBP-2 and sterol in the small intestine, we determined
HMG-CoA reductase transcription. As illustrated in Fig. 4A,
the administration of micellar cholesterol or 25-hydroxy-
cholesterol to Caco-2/15 cells lowered HMG-CoA reductase
transcripts (30 and 55%, respectively). Moreover, measure-
ment of SREBP-2 revealed a lessening in its protein expres-
sion (Fig. 4B).
We next evaluated how depletion of cholesterol cell content
modulates PCSK9 and LDLr. The extraction of cholesterol was
achieved with hydroxypropyl--cyclodextrin that has been
shown to selectively eliminate cholesterol from the plasma
membrane, in preference to other lipids (28). As illustrated in
Fig. 5, Aand B, hydroxypropyl--cyclodextrin markedly in-
creased the gene and protein expression of PCSK9 and LDLr.
The expression of HMG-CoA reductase and SREBP-2 was
Fig. 3. Effect of 25-hydroxycholesterol (sol-
ubilized in albumin) incubated for 24 h on
gene and protein expressions of PCSK9 and
LDLr in intestinal epithelial cells. Caco-2/15
cells were incubated with fresh medium, al-
lowed to grow and differentiate, and tested for
the effects of 25-hydroxycholesterol on tran-
script levels and protein mass of PCSK9 and
LDLr by RT-PCR and Western blotting, re-
spectively. The control cells were incubated
with albumin only. Values are expressed as
means SE for 3 different experiments. *P
0.05; **P0.01; ***P0.001 vs. controls.
AJP-Gastrointest Liver Physiol VOL 296 APRIL 2009
upregulated as well (Fig. 5C). To determine whether this
impact is mainly due to cholesterol extraction, Caco-2/15 cells
were supplemented with a combination of hydroxypropyl--
cyclodextrin and micellar cholesterol. The mixture largely
restored the control levels.
Since BA play a significant role in cholesterol metabolism
and are increasingly being appreciated as metabolic integrators
and signaling factors, we subsequently tested their specific
effects on PCSK9. Taurocholate (4.8 mM) reduced mRNA
level of PCSK9 by 25% without affecting LDLr expression
(Fig. 6). On the other hand, the addition of deoxycholate (250 M)
enhanced the protein expression of PCSK9 by 30% (Fig. 7A). LDLr
was more responsive to deoxycholate by showing a 70% and
twofold increase in gene and protein expression, respectively
(Fig. 7B). As noted in Fig. 8, taurocholate slightly diminished
the transcripts of HMG-CoA reductase without affecting
SREBP-2 and FXR gene expression. On the other hand, de-
oxycholate decreased FXR gene expression, enhanced the
protein expression of SREBP-2, and remained without effect
on HMG-CoA reductase (Fig. 8).
Given the different properties of unconjugated and conju-
gated BA, we have examined their effects on the expression of
PCSK9 and LDLr. As noted in Table 1, cholate was more
powerful than taurocholate in reducing PCSK9 mRNA,
whereas glycocholate significantly enhanced it. No significant
modifications were observed in PCSK9 protein mass and LDLr
gene expression following the incubation of Caco-2/15 cells
with cholate, taurocholate, and glycocholate. Cholate was also
able to downregulate LDLr protein mass. Although some
alterations were noted in FXR and SREBP2, they never
reached statistical significance. We also assessed the influence
of chenodeoxycholate, taurochenodeoxycholate, and glycoche-
nodeoxycholate on the expression of PCSK9, LDLr, FXR, and
SREBP2 and could not record significant alterations, except for
glycochenodeoxycholate, that increased LDLr and FXR
mRNA. Finally, we evaluated the impact of the two conjugated
forms of deoxycholate. Taurodeoxycholate and glycodeoxy-
cholate were both capable of reducing PCSK9 mRNA levels.
However, only taurodeoxycholate was able to downregulate
LDLr gene expression. In fact, deoxycholate was more effec-
tive in modulating gene and protein expressions of PCSK9,
LDLr, and SREBP-2.
Despite valuable advances, additional studies are clearly
warranted to understand the complex molecular mechanisms
that orchestrate cholesterol homeostasis in the small intes-
tine. A tremendous opportunity is offered by the recent
discovery of PCSK9, which displays various functions,
Fig. 4. Impact of micellar cholesterol and 25-
hydroxycholesterol incubated for 24 h on 3-hy-
droxy-3-methylglutaryl (HMG)-CoA reductase
and SREBP-2 expression in intestinal epithelial
cells. Caco-2/15 cells were incubated with fresh
medium, allowed to grow and differentiate, and
tested for the effects of cholesterol and 25-hy-
droxycholesterol on transcript levels of HMG-
CoA reductase by RT-PCR and sterol regulatory
element binding protein-2 (SREBP-2) by Western
blot. The control cells were incubated with bile
acid micelles. Values are expressed as means
SE for 3 different experiments. *P0.05; **P
0.01; ***P0.001 vs. micelles.
AJP-Gastrointest Liver Physiol VOL 296 APRIL 2009
including hepatic LDLr degradation. In view of the small
intestine’s high capacity to absorb lipids and elaborate most
of the major lipoprotein classes and considering the well-
known actions of a number of nutrients and hormones on
lipid metabolism and transport at the intestinal level, the
lack of knowledge about the modulation of PCSK9 in the
gut is puzzling. For the first time, the present work at-
tempted to detail the modulation of PCSK9 in intestinal
cells. According to our data, there is a discrete regulation of
PCSK9 from stimuli, originating from apical media, such as
sterols, BA, and cyclodextrin. Apparently, PCSK9 and
LDLr are stimulated or downregulated in a similar fashion
via SREBP-2 transcription factor. Furthermore, in our ex-
periments, we have supplemented Caco-2/15 cells with
sterols or caused the depletion of cholesterol cell content
using hydroxypropyl--cyclodextrin, an approach that is
both rapid and highly efficient (18, 19). In addition, hy-
droxypropyl--cyclodextrin is entirely surface acting, so,
apart from removing cholesterol, it has a minimal effect on
cell membranes (25). Our findings clearly showed that
provision of sterols to enterocytes downregulated the gene
and protein expression of PCSK9. Conversely, the lessening
Fig. 5. Regulation of PCSK9, LDLr, HMG-
CoA reductase, and SREBP-2 expression by
hydroxypropyl--cyclodextrin in intestinal
epithelial cells. Caco-2/15 cells were incu-
bated with fresh medium, allowed to grow and
differentiate, and tested for the effects of hy-
droxypropyl--cyclodextrin on transcript lev-
els and protein mass of PCSK9 and LDLr by
RT-PCR and Western blotting, respectively.
The transcripts of HMG-CoA reductase and
protein mass of SREBP-2 were also assessed.
Values are expressed as means SE for 3
different experiments. *P0.05; **P
0.01; ***P0.001 vs. controls. †0.05;
0.01 vs. cyclodextrin.
AJP-Gastrointest Liver Physiol VOL 296 APRIL 2009
of cholesterol cell content enhanced PCSK9 transcription. In
similar fashion, PCSK9 expression was highly downregu-
lated by dietary cholesterol in mouse liver (38). This is also
consistent with the demonstration that PCSK9 expression
was strongly induced by statins in a dose-dependent manner
in HepG2 cells and in human primary hepatocytes, likely a
result of the cholesterol-lowering effect of the drug, and that
this induction was efficiently reversed by mevalonate (11).
Altogether, these studies pointed out that 1) the regulation
of PCSK9 expression is dependent on the presence or
absence of sterols not only in hepatocytes, but also in
intestinal epithelial cells, and 2) the modulation of PCSK9
by sterols, which are supplied either exogenously or endo-
genously, is achieved at the transcription level.
It has been reported that gain-of-function mutations in
PCSK9 have a 23% decreased level of cell surface expression
of LDL receptors and a 38% decrease in internalization of
LDL, whereas loss-of-function mutations are associated with a
16% increased level of cell surface LDLr and a 35% increased
internalization of LDL (7). Apparently, the catalytic activity of
PCSK9 appears not to be required for LDLr degradation, but it
is essential for activation and secretion of PCSK9 (10, 22, 39).
In our work, the protein expression of LDLr showed similar
changes to PCSK9 in Caco-2/15 cells with sterol supplemen-
tation or under the sterol-depleted conditions. Considering that
the function of PCSK9 is to promote the degradation of LDLr
(23), as noted before (3), the parallel change in LDLr to
PCSK9 by sterols is surprising. However, previous studies
have reported a similar behavior of the two proteins in HepG2
cells (26). One might consider that these cells lack a compo-
nent or machinery necessary for the coordination of the ex-
pression of these proteins, but other research groups have not
found a reciprocal regulation of PCSK9 and LDLr. For exam-
ple, inhibition of squalene synthase upregulates PCSK9 and
LDLr expression in rat liver (4) and there was no reverse
relation of PCSK9 and LDLr in rat liver cell line (17). PCSK9
was identified as one of the genes that are regulated by
SREBPs (24, 38). The SREBPs are members of the basic
helix-loop-helix leucine zipper family of transcription factors
that regulate the expression of the target genes by binding to
the sterol-regulatory elements (SRE) in their promoter regions
(6). Dubuc et al. (11) reported that the transcription of PCSK9
was increased by statins, most likely through SREBP-2 acti-
vation in primary human hepatocytes, and proposed the impor-
tance of conserved SRE in the promoter region of mouse, rat,
and human PCSK9 genes. Thereafter, the expression of PCSK9
was found dependent on the absence or presence of sterols via
SRE in the minimal promoter region of the human PCSK9 gene
by both SREBP-1 and SREBP-2 in HepG2 cells, and it was
proposed that the predominant transcriptional regulator of
PCSK9 by cholesterol is SREBP-2 in vivo (26). Accordingly,
our data on intestinal epithelial cells showed that the adminis-
tration of sterols reduced SREBP-2 transcripts while decreas-
ing PCSK9 gene and protein expression. On the other hand,
depletion of cholesterol content in Caco-2/15 cells by cyclo-
dextrin raised the levels of mRNA and protein mass of PCSK9
Fig. 6. Modulation of PCSK9 and LDLr gene
and protein expression by 4.8 mM taurocholate
incubated for 24 h in intestinal epithelial cells.
Caco-2/15 cells were incubated with fresh me-
dium, allowed to grow and differentiate, and
tested for the effects of taurocholate on tran-
script levels and protein mass of PCSK9 and
LDLr by RT-PCR and Western blotting, re-
spectively. Values are expressed as means
SE for 3 different experiments. *P0.01 vs.
AJP-Gastrointest Liver Physiol VOL 296 APRIL 2009
and LDLr. As expected, the changes of SREBP-2 and PCSK9
in Caco-2/15 cells paralleled the alterations in mRNA encoding
HMG-CoA reductase, the key cholesterol biosynthetic enzyme.
These results might suggest that PCSK9 would be under the
control of SREBP-2 in Caco-2/15 cells, as is the case for LDLr
and HMG CoA reductase (12–14).
BA are amphiphilic molecules synthesized from cholesterol
exclusively in the liver, represent the major route for removal
of excess cholesterol from the body, and are essential for
effective transport of dietary fat (29). In addition to their
traditional role in dietary lipid absorption and cholesterol
homeostasis, it has become clear now that BA constitute
versatile signaling molecules endowed with systemic endo-
crine functions. In fact, BA are ligands for G protein-coupled
receptors and modulate several nuclear hormone receptors (27,
35, 36). Through activation of these diverse signaling path-
ways, BA have been shown to regulate not only their own
synthesis and enterohepatic recirculation, but also triglyceride,
cholesterol, and glucose homeostasis (51). Given the broad
relationship of BA with multiple pathways and their recent link
with PCSK9 in hepatocytes (32, 44), we decided to investigate
their impact on PCSK9 in the intestine. In the present experi-
ments, we employed taurocholate and deoxycholate, two pow-
erful BA. According to our data in Caco-2/15 cells, tauro-
cholate brought about a decrease in the gene expression of
PCSK9, whereas deoxycholate enhanced that of PCSK9 and
LDLr along with a lessening of FXR mRNA. The distinct
effects of BA on PCSK9 gene expression were also noted in
another study on human hepatocytes that showed a repression
by chenodeoxycholic acid, an induction by deoxycholic acid,
and no influence with cholic acid and ursodeoxycholic acid
(32). Further work is needed to explore whether these separate
impacts on PCSK9 may be due to the potential activation by
BA of other nuclear receptors such as pregnane X receptor,
constitutive and rostane receptor, and vitamin D receptor in
addition to FXR (54, 60).
Chenodeoxycholate and cholate are the two primary BA in
humans and are conjugated mainly to glycine and taurine (21).
Given their hydrophilic-hydrophobic properties, unconjugated
and conjugated BA may differ in their various actions, includ-
ing membrane permeability, lipid solubilization, intracellular
signaling, and DNA synthesis (2, 8, 20, 35, 41, 47). We have
therefore assessed the modulation of unconjugated and conju-
gated BA to taurine or glycine. Our findings indicate the high
ability of cholate, taurodeoxycholate, and glycodeoxycholate
to alter the gene expression of PCSK9. Cholate and taurode-
oxycholate also diminished LDLr protein content and mRNA,
respectively, in Caco-2/15 cells. The effects of cholate were
superior to those of taurocholate and quite divergent from those
of glycocholate. On the other hand, chenodeoxycholate and tau-
rochenodeoxycholate were ineffective in modulating PCSK9 and
LDLr. Only the glycochenodeoxycholate displays a capacity to
Fig. 7. Modulation of PCSK9 and LDLr
gene and protein expression by 250 M de-
oxycholate incubated for 24 h in intestinal
epithelial cells. Caco-2/15 cells were incu-
bated with fresh medium, allowed to grow
and differentiate, and tested for the effects of
deoxycholate on transcript levels and protein
mass of PCSK9 and LDLr by RT-PCR and
Western blotting, respectively. Values are ex-
pressed as means SE for 3 different exper-
iments. *P0.05; **P0.01; ***P
0.001 vs. controls.
AJP-Gastrointest Liver Physiol VOL 296 APRIL 2009
upregulate LDLr and FXR mRNA. However, we were not able to
show LDLr mRNA upregulation by chenodeoxycholate, previ-
ously reported by Nakahara et al. (42), presumably because of the
difference in experimental techniques. Additional studies are nec-
essary to uncover novel mechanisms by which specific BA mod-
ulate PCSK9 and LDLr. This in turn may have far reaching
consequences in the control of the overall body metabolism
homeostasis and the multiorgan implications.
The authors thank Schohraya Spahis, Carole Garofalo, E
´milie Grenier, and
Genevie`ve Lalonde for helpful technical assistance.
This study was supported by the Canadian Institutes of Health Research
(Grant MOP 49433 to E. Levy and 36496 to N. G. Seidah).
1. Abifadel M, Varret M, Rabes JP, Allard D, Ouguerram K, Devillers
M, Cruaud C, Benjannet S, Wickham L, Erlich D, Derre A, Villeger
L, Farnier M, Beucler I, Bruckert E, Chambaz J, Chanu B, Lecerf
JM, Luc G, Moulin P, Weissenbach J, Prat A, Krempf M, Junien C,
Seidah NG, Boileau C. Mutations in PCSK9 cause autosomal dominant
hypercholesterolemia. Nat Genet 34: 154 –156, 2003.
2. Albalak A, Jackson AA, Donovan JM. Hepatobiliary Diseases: Basic
Research and Clinical Application. Dordrecht, The Netherlands: Kluwer
Academic, 1997.
Fig. 8. Modulation of HMG-CoA reductase,
farnesoid X receptor (FXR), and SREBP-2
expression by 4.8 mM taurocholate and 250
M deoxycholate incubated for 24 h in intes-
tinal epithelial cells. Caco-2/15 cells were
incubated with fresh medium, allowed to
grow and differentiate, and tested for the
effects of taurocholate and deoxycholate on
HMG-CoA reductase and FXR transcript lev-
els by RT-PCR, as well as on SREBP-2 pro-
tein mass by Western blot. Values are ex-
pressed as means SE for 3 different exper-
iments. *P0.05 vs. controls.
AJP-Gastrointest Liver Physiol VOL 296 APRIL 2009
3. Attie AD, Seidah NG. Dual regulation of the LDL receptor—some clarity
and new questions. Cell Metab 1: 290 –292, 2005.
4. Bedi M, Niesen M, Lopez D. Inhibition of squalene synthase upregulates
PCSK9 expression in rat liver. Arch Biochem Biophys 470: 116 –119,
5. Benjannet S, Rhainds D, Essalmani R, Mayne J, Wickham L, Jin W,
Asselin MC, Hamelin J, Varret M, Allard D, Trillard M, Abifadel M,
Tebon A, Attie AD, Rader DJ, Boileau C, Brissette L, Chretien M,
Prat A, Seidah NG. NARC-1/PCSK9 and its natural mutants: zymogen
cleavage and effects on the low density lipoprotein (LDL) receptor and
LDL cholesterol. J Biol Chem 279: 48865– 48875, 2004.
6. Brown MS, Goldstein JL. The SREBP pathway: regulation of cholesterol
metabolism by proteolysis of a membrane-bound transcription factor. Cell
89: 331–340, 1997.
7. Cameron J, Holla OL, Ranheim T, Kulseth MA, Berge KE, Leren TP.
Effect of mutations in the PCSK9 gene on the cell surface LDL receptors.
Hum Mol Genet 15: 1551–1558, 2006.
8. Combettes L, Dumont M, Berthon B, Erlinger S, Claret M. Effect of
the bile acid taurolithocholate on cell calcium in saponin-treated rat
hepatocytes. FEBS Lett 227: 161–166, 1988.
9. Costet P, Cariou B, Lambert G, Lalanne F, Lardeux B, Jarnoux AL,
Grefhorst A, Staels B, Krempf M. Hepatic PCSK9 expression is regu-
lated by nutritional status via insulin and sterol regulatory element-binding
protein 1c. J Biol Chem 281: 6211– 6218, 2006.
10. Cunningham D, Danley DE, Geoghegan KF, Griffor MC, Hawkins
JL, Subashi TA, Varghese AH, Ammirati MJ, Culp JS, Hoth LR,
Mansour MN, McGrath KM, Seddon AP, Shenolikar S, Stutzman-
Engwall KJ, Warren LC, Xia D, Qiu X. Structural and biophysical
studies of PCSK9 and its mutants linked to familial hypercholesterolemia.
Nat Struct Mol Biol 14: 413– 419, 2007.
11. Dubuc G, Chamberland A, Wassef H, Davignon J, Seidah NG,
Bernier L, Prat A. Statins upregulate PCSK9, the gene encoding the
proprotein convertase neural apoptosis-regulated convertase-1 implicated
in familial hypercholesterolemia. Arterioscler Thromb Vasc Biol 24:
1454 –1459, 2004.
12. Field FJ, Born E, Murthy S, Mathur SN. Regulation of sterol regulatory
element-binding proteins by cholesterol flux in CaCo-2 cells. J Lipid Res
42: 1687–1698, 2001.
13. Field FJ, Fujiwara D, Born E, Chappell DA, Mathur SN. Regulation of
LDL receptor expression by luminal sterol flux in CaCo-2 cells. Arterio-
scler Thromb 13: 729 –737, 1993.
14. Field FJ, Shreves T, Fujiwara D, Murthy S, Albright E, Mathur SN.
Regulation of gene expression and synthesis and degradation of 3-hy-
droxy-3-methylglutaryl coenzyme A reductase by micellar cholesterol in
CaCo-2 cells. J Lipid Res 32: 1811–1821, 1991.
15. Goldstein JL, Brown MS. The LDL receptor locus and the genetics of
familial hypercholesterolemia. Annu Rev Genet 13: 259 –289, 1979.
16. Grenier E, Maupas FS, Beaulieu JF, Seidman E, Delvin E, Sane A,
Tremblay E, Garofalo C, Levy E. Effect of retinoic acid on cell
proliferation and differentiation as well as on lipid synthesis, lipoprotein
secretion, and apolipoprotein biogenesis. Am J Physiol Gastrointest Liver
Physiol 293: G1178 –G1189, 2007.
17. Grozdanov PN, Petkov PM, Karagyozov LK, Dabeva MD. Expression
and localization of PCSK9 in rat hepatic cells. Biochem Cell Biol 84:
80 –92, 2006.
18. Hansen GH, Immerdal L, Thorsen E, Niels-Christiansen LL, Nystrom
BT, Demant EJ, Danielsen EM. Lipid rafts exist as stable cholesterol-
independent microdomains in the brush border membrane of enterocytes.
J Biol Chem 276: 32338 –32344, 2001.
19. Hansen GH, Niels-Christiansen LL, Thorsen E, Immerdal L, Danielsen
EM. Cholesterol depletion of enterocytes. Effect on the Golgi complex and
apical membrane trafficking. J Biol Chem 275: 5136 –5142, 2000.
20. Heuman DM, Pandak WM, Hylemon PB, Vlahcevic ZR. Conjugates of
ursodeoxycholate protect against cytotoxicity of more hydrophobic bile
salts: in vitro studies in rat hepatocytes and human erythrocytes. Hepa-
tology 14: 920 –926, 1991.
21. Hofmann AF. Chemistry and enterohepatic circulation of bile acids.
Hepatology 4: 4S–14S, 1984.
22. Homer VM, Marais AD, Charlton F, Laurie AD, Hurndell N, Scott R,
Mangili F, Sullivan DR, Barter PJ, Rye KA, George PM, Lambert G.
Identification and characterization of two non-secreted PCSK9 mutants
associated with familial hypercholesterolemia in cohorts from New Zea-
land and South Africa. Atherosclerosis 196: 659 – 666, 2008.
23. Horton JD, Cohen JC, Hobbs HH. Molecular biology of PCSK9: its role
in LDL metabolism. Trends Biochem Sci 32: 71–77, 2007.
24. Horton JD, Shah NA, Warrington JA, Anderson NN, Park SW,
Brown MS, Goldstein JL. Combined analysis of oligonucleotide mi-
croarray data from transgenic and knockout mice identifies direct SREBP
target genes. Proc Natl Acad Sci USA 100: 12027–12032, 2003.
25. Ilangumaran S, Hoessli DC. Effects of cholesterol depletion by cyclo-
dextrin on the sphingolipid microdomains of the plasma membrane.
Biochem J 335: 433– 440, 1998.
26. Jeong HJ, Lee HS, Kim KS, Kim YK, Yoon D, Park SW. Sterol-
dependent regulation of proprotein convertase subtilisin/kexin type 9
expression by sterol-regulatory element binding protein-2. J Lipid Res 49:
399 – 409, 2008.
27. Kawamata Y, Fujii R, Hosoya M, Harada M, Yoshida H, Miwa M,
Fukusumi S, Habata Y, Itoh T, Shintani Y, Hinuma S, Fujisawa Y,
Fujino M. A G protein-coupled receptor responsive to bile acids. J Biol
Chem 278: 9435–9440, 2003.
28. Kilsdonk EP, Yancey PG, Stoudt GW, Bangerter FW, Johnson WJ,
Phillips MC, Rothblat GH. Cellular cholesterol efflux mediated by
cyclodextrins. J Biol Chem 270: 17250 –17256, 1995.
29. Kuipers F, Stroeve JH, Caron S, Staels B. Bile acids, farnesoid X
receptor, atherosclerosis and metabolic control. Curr Opin Lipidol 18:
289 –297, 2007.
30. Lambert G. Unravelling the functional significance of PCSK9. Curr Opin
Lipidol 18: 304 –309, 2007.
31. Lambert G, Jarnoux AL, Pineau T, Pape O, Chetiveaux M, Laboisse
C, Krempf M, Costet P. Fasting induces hyperlipidemia in mice over-
expressing proprotein convertase subtilisin kexin type 9: lack of modula-
tion of very-low-density lipoprotein hepatic output by the low-density
lipoprotein receptor. Endocrinology 147: 4985– 4995, 2006.
Table 1. Influence of unconjugated and conjugated bile acids on PCSK9, LDLr, FXR, and SREBP2
Bile Acids
PCSK9 Expression LDLr Expression FXR SREBP-2
Gene Protein Gene Protein Gene Protein
Cholate 0.550.29 0.170.05
Taurocholate 0.720.04 0.990.05 1.030.10 0.880.07 0.960.25 1.370.47
Glycocholate 1.450.071.230.09 1.270.07 1.050.13 1.930.13 1.000.04
Chenodeoxycholate 0.990.01 1.070.05 0.980.02 0.760.09 1.230.13 0.890.03
Taurochenodeoxycholate 1.060.03 1.110.13 1.120.05 0.930.16 1.540.12 0.780.08
Glycochenodeoxycholate 1.260.11 0.920.04 1.320.110.710.06 2,180.180.760.00
Deoxycholate 1.130.09 1.510.11* 1.830.22* 1.980.150.660.16 1.670.20
Taurodeoxycholate 0.370.160.990.26 0.580.14* 0.590.26 0.720.33 0.870.17
Glycodeoxycholate 0.600.051.200.06 1.010.16 1.100.24 1.120.18 1.070.24
Data are expressed as the ratio between the gene of interest proprotein convertase subtilisin/kexin type 9 (PCSK9), low-density lipoprotein receptor (LDLr),
farnesoid X receptor (FXR), sterol regulatory element binding protein-2 (SREBP2)and the internal reference gene GAPDH (for mRNA) and -actin (for protein
mass). Values were compared to respective controls equivalent to 1 and are expressed as means SE for 3 different experiments. *P0.05; P0.01;
P0.001 vs. control.
AJP-Gastrointest Liver Physiol VOL 296 APRIL 2009
32. Langhi C, Le MC, Kourimate S, Caron S, Staels B, Krempf M, Costet
P, Cariou B. Activation of the farnesoid X receptor represses PCSK9
expression in human hepatocytes. FEBS Lett 582: 949 –955, 2008.
33. Levy E, Menard D, Suc I, Delvin E, Marcil V, Brissette L, Thibault L,
Bendayan M. Ontogeny, immunolocalisation, distribution and function of
SR-BI in the human intestine. J Cell Sci 117: 327–337, 2004.
34. Levy E, Stan S, Delvin E, Menard D, Shoulders C, Garofalo C, Slight
I, Seidman E, Mayer G, Bendayan M. Localization of microsomal
triglyceride transfer protein in the Golgi: possible role in the assembly of
chylomicrons. J Biol Chem 277: 16470 –16477, 2002.
35. Marin JJ, Barbero ER, Herrera MC, Tabernero A, Monte MJ. Bile
acid-induced modifications in DNA synthesis by the regenerating perfused
rat liver. Hepatology 18: 1182–1192, 1993.
36. Maruyama T, Miyamoto Y, Nakamura T, Tamai Y, Okada H,
Sugiyama E, Nakamura T, Itadani H, Tanaka K. Identification of
membrane-type receptor for bile acids (M-BAR). Biochem Biophys Res
Commun 298: 714 –719, 2002.
37. Maxwell KN, Breslow JL. Adenoviral-mediated expression of Pcsk9 in
mice results in a low-density lipoprotein receptor knockout phenotype.
Proc Natl Acad Sci USA 101: 7100 –7105, 2004.
38. Maxwell KN, Soccio RE, Duncan EM, Sehayek E, Breslow JL. Novel
putative SREBP and LXR target genes identified by microarray analysis in
liver of cholesterol-fed mice. J Lipid Res 44: 2109 –2119, 2003.
39. McNutt MC, Lagace TA, Horton JD. Catalytic activity is not required
for secreted PCSK9 to reduce low density lipoprotein receptors in HepG2
cells. J Biol Chem 282: 20799 –20803, 2007.
40. Montoudis A, Seidman E, Boudreau F, Beaulieu JF, Menard D,
Elchebly M, Mailhot G, Sane AT, Lambert M, Delvin E, Levy E.
Intestinal fatty acid binding protein regulates mitochondrion beta-oxida-
tion and cholesterol uptake. J Lipid Res 2008.
41. Moseley RH, Ballatori N, Smith DJ, Boyer JL. Ursodeoxycholate
stimulates Na
exchange in rat liver basolateral plasma membrane
vesicles. J Clin Invest 80: 684 – 690, 1987.
42. Nakahara M, Fujii H, Maloney PR, Shimizu M, Sato R. Bile acids
enhance low density lipoprotein receptor gene expression via a MAPK
cascade-mediated stabilization of mRNA. J Biol Chem 277: 37229 –
37234, 2002.
43. Nassoury N, Blasiole DA, Tebon OA, Benjannet S, Hamelin J, Poupon
V, McPherson PS, Attie AD, Prat A, Seidah NG. The cellular trafficking
of the secretory proprotein convertase PCSK9 and its dependence on the
LDLR. Traffic 8: 718 –732, 2007.
44. Nilsson LM, Abrahamsson A, Sahlin S, Gustafsson U, Angelin B,
Parini P, Einarsson C. Bile acids and lipoprotein metabolism: effects of
cholestyramine and chenodeoxycholic acid on human hepatic mRNA
expression. Biochem Biophys Res Commun 357: 707–711, 2007.
45. Peretti N, Delvin E, Sinnett D, Marcil V, Garofalo C, Levy E.
Asymmetrical regulation of scavenger receptor class B type I by apical and
basolateral stimuli using Caco-2 cells. J Cell Biochem 100: 421– 433,
46. Poirier S, Mayer G, Benjannet S, Bergeron E, Marcinkiewicz J,
Nassoury N, Mayer H, Nimpf J, Prat A, Seidah NG. The proprotein
convertase PCSK9 induces the degradation of low density lipoprotein
receptor (LDLR) and its closest family members VLDLR and ApoER2.
J Biol Chem 283: 2363–2372, 2008.
47. Rao YP, Stravitz RT, Vlahcevic ZR, Gurley EC, Sando JJ, Hylemon
PB. Activation of protein kinase C alpha and delta by bile acids: corre-
lation with bile acid structure and diacylglycerol formation. J Lipid Res
38: 2446 –2454, 1997.
48. Sane AT, Sinnett D, Delvin E, Bendayan M, Marcil V, Menard D,
Beaulieu JF, Levy E. Localization and role of NPC1L1 in cholesterol
absorption in human intestine. J Lipid Res 47: 2112–2120, 2006.
49. Seidah NG, Benjannet S, Wickham L, Marcinkiewicz J, Jasmin SB,
Stifani S, Basak A, Prat A, Chretien M. The secretory proprotein
convertase neural apoptosis-regulated convertase 1 (NARC-1): liver re-
generation and neuronal differentiation. Proc Natl Acad Sci USA 100:
928 –933, 2003.
50. Seidah NG, Prat A. The proprotein convertases are potential targets in the
treatment of dyslipidemia. J Mol Med 85: 685– 696, 2007.
51. Shen H, Zhang Y, Ding H, Wang X, Chen L, Jiang H, Shen X.
Farnesoid X receptor induces GLUT4 expression through FXR response
element in the GLUT4 promoter. Cell Physiol Biochem 22: 1–14, 2008.
52. Spady DK, Bilheimer DW, Dietschy JM. Rates of receptor-dependent
and -independent low density lipoprotein uptake in the hamster. Proc Natl
Acad Sci USA 80: 3499 –3503, 1983.
53. Spady DK, Dietschy JM. Sterol synthesis in vivo in 18 tissues of the
squirrel monkey, guinea pig, rabbit, hamster, and rat. J Lipid Res 24:
303–315, 1983.
54. Staudinger JL, Goodwin B, Jones SA, Hawkins-Brown D, MacKenzie
KI, LaTour A, Liu Y, Klaassen CD, Brown KK, Reinhard J, Willson
TM, Koller BH, Kliewer SA. The nuclear receptor PXR is a lithocholic
acid sensor that protects against liver toxicity. Proc Natl Acad Sci USA 98:
3369 –3374, 2001.
55. Thomson AB, Dietschy JM. Intestinal lipid absorption: major extracel-
lular and intracellular events. In: Physiology of the Gastrointestinal Tract,
edited by Johnson LR. New York: Raven, 1981, p. 1147–1220.
56. Turley SD. Cholesterol metabolism and therapeutic targets: rationale for
targeting multiple metabolic pathways. Clin Cardiol 27: III16 –III21,
57. Turley SD, Andersen JM, Dietschy JM. Rates of sterol synthesis and
uptake in the major organs of the rat in vivo. J Lipid Res 22: 551–569,
58. Turley SD, Dietschy JM. Sterol absorption by the small intestine. Curr
Opin Lipidol 14: 233–240, 2003.
59. Turley SD, Dietschy JM. Cholesterol metabolism and excretion. In: The
Liver: Biology and Pathobiology, edited by Arias I, Popper H, Schachter
D, and Shafritz DA. New York: Raven, 1982, p. 467– 492.
60. Zollner G, Marschall HU, Wagner M, Trauner M. Role of nuclear
receptors in the adaptive response to bile acids and cholestasis: pathoge-
netic and therapeutic considerations. Mol Pharm 3: 231–251, 2006.
AJP-Gastrointest Liver Physiol VOL 296 APRIL 2009
... основным источником PCSK9 являются гепатоциты, секретирующие его в кровоток. однако и другие клетки организма могут продуцировать и секретировать PCSK9, например, клетки кишечника [12], поджелудочной железы [13], жировой ткани, почек и мозга. известно, что биологические циклы циркуляции PCSK9 соответствуют суточным ритмам человека: концентрация протеазы повышается поздно ночью и снижается ближе к вечеру. ...
This review will present an analysis of the mechanisms of PCSK9 influence on lipid metabolism and its role in the development of cardiovascular pathology, an assessment of the effectiveness and safety of PCSK9 inhibitors, the place of PCSK9 inhibitors in the clinical recommendations of the European, Russian Cardiological Society and the American College of the Heart.
... Blood circulating PCSK9 protein is not only a reflection of the hepatic origin, but also may be attributed to the gut secretion (Levy et al. 2013). PCSK9 expression depends on presence or absence of sterols in hepatocytes and intestinal epithelial cells (Leblond et al. 2009). The intestine is one of the major sites of PCSK9 expression and PCSK9 was almost exclusively expressed in the epithelial barrier of intestinal epithelial and goblet cells in the human duodenum and ileum (Le May et al. 2009). ...
Full-text available
Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a member of the proprotein convertase family of proteins that activate other proteins in cells. Functionally, PCSK9 binds to the receptor of low-density lipoprotein particles (LDL) to regulate cholesterol metabolism and lipoprotein homeostasis in human body. PCSK9 inhibition is a novel pharmacological strategy to control hypercholesterolemia and cardiovascular diseases. Recently accumulating evidence realizes that PCSK9 possesses other roles in cells, such as regulation of tissue inflammatory response, intratumoral immune cell infiltration, and tumor progression. This review discussed the advancement of PCSK9 research on its role and underlying mechanisms in tumor development and progression. For example, PCSK9 inhibition could attenuate progression of breast cancer, glioma, colon tumor, hepatocellular cancer, prostate cancer, and lung adenocarcinoma and promote apoptosis of glioma, prostate cancer, and hepatocellular cancer cells. PCSK9 deficiency could reduce liver metastasis of B16F1 melanoma cells by lowering the circulating cholesterol levels. PCSK9 gene knockdown substantially attenuated mouse tumor growth in vivo by activation of cytotoxic T cells, although PCSK9 knockdown had no effect on morphology and growth rate of different mouse cancer cell lines in vitro. PCSK9 inhibition thus can be used to control human cancers. Future preclinical and clinical studies are warranted to define anti-tumor activity of PCSK9 inhibition.
... Hepatocytes are the primary source of PCSK9 that is secreted into the circulation. However, also other cells can produce and secrete PCSK9, like cells in the intestines [3,4], pancreas [5], adipose tissue [6], kidneys [7] and brain [8]. Interestingly, the circulating levels of PCSK9 biologically increases in the late night, and decreases in the late afternoon, following a diurnal rhythm [9]. ...
Full-text available
Pro-protein convertase subtilisin/kexin type 9 (PCSK9) is secreted mostly by hepatocytes and to a lesser extent by the intestine, pancreas, kidney, adipose tissue, and vascular cells. PCSK9 has been known to interact with the low-density lipoprotein receptor (LDLR) and chaperones the receptor to its degradation. In this manner, targeting PCSK9 is a novel attractive approach to reduce hyperlipidaemia and the risk for cardiovascular diseases. Recently, it has been recognised that the effects of PCSK9 in relation to cardiovascular complications are not only LDLR related, but that various LDLR-independent pathways and processes are also influenced. In this review, the various LDLR dependent and especially independent effects of PCSK9 on the cardiovascular system are discussed, followed by an overview of related PCSK9-polymorphisms and currently available and future therapeutic approaches to manipulate PCSK9 expression.
... PCSK9 и CD36 (FAT). Помимо клеток печени, PCSK9 обильно экспрессируется в клетках кишечника, однако долгое время сведения о функции PCSK9 в этом органе были неизвестны [42,43]. Группой ученых под руководством E. Levy было проведено исследование, направленное на изучение роли PCSK9 в гомеостазе холестерола и транспорте липидов через стенку кишечника [43]. ...
9 (PCSK9) в регуляции транспорта липопротеинов. Нарушение транспорта, характеризующееся избыточным содержанием в сыворотке крови холестерола и атерогенных липопро-теинов низкой плотности, является ключевым фактором риска развития атеросклероза и сердечно-сосудистых заболеваний. В связи с тем, что PCSK9 вызывает деградацию рецепторов липопротеинов низкой плотности и повышает уровень атерогенных липопротеинов низкой плотности, этот фермент стал новой мишенью для разработки терапевтических препаратов при лечении и профилактики сердечно-сосудистых заболеваний. В то же время в обзорных статьях, посвященных PCSK9, недостаточно вни-мания уделяется дополнительной роли PCSK9 в регуляции транспорта липопротеинов. В связи с этим в представленном обзоре обсуждается влияние PCSK9 на другие рецепторы, участвующие в обмене липидов, дальнейшее изучение которых в перспекти-ве имеет важное практическое значение. Ключевые слова: PCSK9, регуляция, транспорт липопротеинов, липопротеины низкой плотности, рецепторы липопротеинов низкой плотности, рецепторы липопротеинов очень низкой плотности, рецептор аполипопротеина Е, лектиноподобный рецептор 1-го типа для окисленных липопротеинов низкой плотности, кластер дифференцировки 36. Для цитирования: Чаулин А.М., Дупляков Д.В. Роль PCSK9 в регуляции транспорта липопротеинов (обзор литературы). Вопросы биологической, медицинской и фармацевтической химии. 2021;24(1):42−45. https://doi. В связи с тем, что нарушение липидного об-мена является одним из ключевых факторов риска высокораспространенных сердечно-сосудистых за-болеваний (ССЗ), поиск новых молекул, регулиру-ющих липидный обмен, и изучение данных меха-низмов является важным научно-исследователь-ским направлением. Благодаря открытию в 2003 г. девятого члена семейства пропротеиновых конвер-таз-пропротеиновой конвертазы субтилизин-кексинового типа 9 (PCSK9) [1, 2], наметились зна-чительные положительные сдвиги в подходах к ле-чению и диагностики ССЗ. Установление важной роли PCSK9 в регуляции концентрации холестеро-ла и липопротеинов низкой плотности (ЛПНП) в плазме крови привело к разработке новых классов гиполипидемических препаратов и появились воз-можности использования PCSK9 в качестве нового биомаркера для ранней диагностики ССЗ [3, 4]. Липопротеины низкой плотности являются атерогенными липопротеинами и одними из основ-ных факторов риска развития атеросклероза, по-этому изучение механизмов, вызывающих повы-шение ЛПНП в плазме крови является перспектив-ным в плане разработки новой противоатероскле-ротической терапии [5, 6]. Пропротеиновая конвер-таза субтилизин-кексинового типа 9-это серино-вая протеаза, участвующая в регуляции поглоще-ния аполипопротеин В (апоВ)-содержащих атеро-генных ЛПНП гепатоцитами опосредованно через усиление деградации рецептора липопротеинов низкой плотности (рЛПНП). Благодаря данному механизму действия происходит регуляция концен-траций ЛПНП и холестерола в плазме крови [6]. Плазменные ЛПНП выводятся из плазмы кро-ви главным образом при помощи рЛПНП, распо-ложенных на поверхности гепатоцитов. После того как частица ЛПНП связывается с рЛПНП, образо-вавшийся комплекс рЛПНП-ЛПНП интернализует-ся в покрытые клатрином ямки и доставляется внутрь клетки, где данный комплекс диссоциирует
... Intestinal PCSK9 has been shown to be regulated by sterols and bile acids. 33 According to our knowledge, this is the first study showing that intestinal PCSK9 gene expression is regulated by glucose. ...
Cholesterol uptake and chylomicron synthesis are promoted by increasing glucose concentrations in both healthy and diabetic individuals during the postprandial phase. The goal of this study was to test whether acute inhibition of glucose uptake could impact cholesterol absorption in differentiated human intestinal Caco-2 cells. As expected, high glucose upregulated intestinal cholesterol metabolism promoting its uptake and incorporation in lipoproteins. This was accompanied by an increase in the gene expression of Niemann-Pick C1 Like 1 and proprotein convertase subtillisin/kexin type 9. Cholesterol uptake was attenuated by acute inhibition of glucose absorption by cytochalasin B, by a chamomile extract and by one of its main constituent polyphenols, apigenin 7-O-glucoside; however, chylomicron secretion was only reduced by the chamomile extract. These data support a potential indirect role for bioactives in modulating intestinal lipid pathways through effects on intestinal glucose uptake. This working hypothesis warrants further testing in an in vivo setting such as in hypercholesterolaemic or prediabetic individuals.
... In this context, we cannot exclude that the hypothesis where PCSK9 stimulates TRL apoB-48 secretion via enhanced intestinal cholesterol absorption may be more fully manifested in the fed state. Finally, a previous report from Caco-2 cells suggested that PCSK9-associated reduction of intestinal LDLR abundance stimulates endogenous cholesterol synthesis (52). On the one hand, studies conducted with evolocumab in insulin-sensitive healthy humans have reported only modest effects of PCSK9 on cholesterol synthesis (9,47). ...
Full-text available
Intestinal TG-rich lipoproteins (TRLs) are important in the pathogenesis of atherosclerosis in insulin resistance (IR). We investigated the association of plasma proprotein convertase subtilisin/kexin type 9 (PCSK9) concentrations with apoB-48-containing TRL metabolism in 148 men displaying various degrees of IR by measuring in vivo kinetics of TRL apoB-48 during a constant-fed state after a primed-constant infusion of L-[5,5,5-D3]leucine. Plasma PCSK9 concentrations positively correlated with TRL apoB-48 pool size (r = 0.31, p = 0.0002) and production rate (r = 0.24, p = 0.008) but not fractional catabolic rate (r = -0.04, p = 0.6). Backward stepwise multiple linear regression analysis identified PCSK9 concentrations as a positive predictor of TRL apoB-48 production rate (standard β = +0.20, p = 0.007) independent of BMI, age, type 2 diabetes/metformin use, dietary fat intake during the kinetic study, and fasting concentrations of TGs, insulin, glucose, LDL cholesterol, or C reactive protein. We also assessed intestinal expression of key genes involved in chylomicron processing from duodenal samples of 71 men. Expression of PCSK9 and HMG-CoAR genes was positively associated (r = 0.43, p = 0.002). These results support PCSK9 association with intestinal secretion and plasma overaccumulation of TRL apoB-48 in men with IR.
... Although the contribution of the intestine to circulating PCSK9 levels is unknown, studies have shown that PCSK9 could be secreted by the basolateral compartment of intestinal cells, where it colocalizes with the LDL receptor (36). Furthermore, in intestinal cells, PCSK9 expression could be regulated by sterol and bile acids via the farnesoid X receptor (37). It was also recently suggested that the concentration of circulating bile acids was increased after RYGB surgery, independent of CR (38). ...
Full-text available
Context Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a key regulator of low-density lipoprotein cholesterol (LDL-C) concentrations. In patients with severe obesity, biliopancreatic diversion with duodenal switch (BPD-DS) surgery induces substantial weight loss and favourably influence lipoprotein metabolism. The impact of BPD-DS surgery on PCSK9 levels is unknown. Objectives To determine the acute and chronic impact of BPD-DS surgery on PCSK9 levels and whether the acute impact of BPD-DS could be explained by BPD-DS surgery-associated caloric restriction (CR). Design, Settings and Participants PCSK9 levels were measured in 20 men and 49 women (mean age; 41.5±11.1) with severe obesity before, 24 hours, 5 days and 6 and 12 months after undergoing BPD-DS and in a comparable control group (n=31) at baseline and at 6 and 12 months. PCSK9 levels were also measured during 3-day CR in patients (n=7) with severe obesity and type 2 diabetes. Results PCSK9 levels increased 13.4% after 24 hours (248.7±64.8 to 269.7±63.8 ng/ml, p=0,02) and decreased 9.5% at 12 months compared to baseline (217,6±43,0 ng/mL, p<0,0001). LDL-C levels decreased 36.2% after 24-hr (2,6±0,7 to 1,7±0,6 mmol/L, p<0,0001) and 30% at 12 months compared to baseline (1,7±0,5 mmol/L, p<0,0001). Compared to baseline levels, PCSK9 levels were lower at day 2, but not at day 1 or 3 after CR. Conclusion BPD-DS is associated with acute increases in PCSK9 levels that do not appear to be explained by CR but may be due to an acute response following surgery. BPD-DS induces chronic reductions in both PCSK9 and LDL-C levels.
... Although the number of significant genes in the gut samples was insufficient for GO analysis, it is intriguing to note that PCSK9, one of the differentially expressed genes, plays a key role in cholesterol and lipid homeostasis. In fact, cholesterol and various types of bile acids have been shown to suppresses PCSK9 mRNA expression in Caco2 intestinal cultures (Leblond et al., 2009). ...
Full-text available
A capability for analyzing complex cellular communication among tissues is important in drug discovery and development, and in vitro technologies for doing so are required for human applications. A prominent instance is communication between the gut and the liver, whereby perturbations of one tissue can influence behavior of the other. Here, we present a study on human gut-liver tissue interactions under normal and inflammatory contexts, via an integrative multi-organ platform comprising human liver (hepatocytes and Kupffer cells) and intestinal (enterocyte, goblet cells, and dendritic cells) models. Our results demonstrated long-term (>2 weeks) maintenance of intestinal (e.g., barrier integrity) and hepatic (e.g., albumin) functions in baseline interaction. Gene expression data comparing liver in interaction with gut, versus isolation, revealed modulation of bile acid metabolism. Intestinal FGF19 secretion and associated inhibition of hepatic CYP7A1 expression provided evidence of physiologically relevant gut-liver crosstalk. Moreover, significant non-linear modulation of cytokine responses was observed under inflammatory gut-liver interaction; for example, production of CXCR3 ligands (CXCL9,10,11) was synergistically enhanced. RNA-seq analysis revealed significant upregulation of IFNα/β/γ signaling during inflammatory gut-liver crosstalk, with these pathways implicated in the synergistic CXCR3 chemokine production. Exacerbated inflammatory response in gut-liver interaction also negatively affected tissue-specific functions (e.g., liver metabolism). These findings illustrate how an integrated multi-tissue platform can generate insights useful for understanding complex pathophysiological processes such as inflammatory organ crosstalk. This article is protected by copyright. All rights reserved.
... These results presumably translate to a decrease in LDLR degradation and increased uptake of apoB lipoproteins. Consistently, PCSK9 mRNA and protein expression in intestinal epithelial cells was decreased after incubation with cholesterol and 25-hydroxycholesterol (69). Conversely, a high-fat diet (47% calories) containing 3% cholesterol was reported to have no significant effect on plasma PCSK9 concentrations or hepatic LDLR mRNA expression. ...
Full-text available
Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a serine protease involved in the regulation of LDL receptor (LDLR) expression and apolipoprotein B lipoprotein cholesterol metabolism. Hepatic PCSK9 protein expression, activity, and secretion have been shown to affect cholesterol homeostasis. An upregulation of hepatic PSCK9 protein leads to increased LDLR degradation, resulting in decreased uptake of apoB lipoproteins and a consequent increase in the plasma concentration of these lipoproteins, including LDL and chylomicron remnants. Hence, PCSK9 has become a novel target for lipid-lowering therapies. The aim of this review is to outline current findings on the metabolic and dietary regulation of PCSK9 and effects on cholesterol, apoB lipoprotein metabolism, and cardiovascular disease (CVD) risk. PCSK9 gene and protein expression have been shown to be regulated by metabolic status and the diurnal pattern. In the fasting state, plasma PCSK9 is reduced via modulation of the nuclear transcriptional factors, including sterol regulatory element-binding protein (SREBP) 1c, SREBP2, and hepatocyte nuclear factor 1α. Plasma PCSK9 concentrations are also known to be positively associated with plasma insulin and homeostasis model assessment of insulin resistance, and appear to be regulated by SREBP1c independently of glucose status. Plasma PCSK9 concentrations are stable in response to high-fat or high-protein diets in healthy individuals; however, this response may differ in altered metabolic conditions. Dietary n-3 polyunsaturated fatty acids have been shown to reduce plasma PCSK9 concentration and hepatic PCSK9 mRNA expression, consistent with their lipid-lowering effects, whereas dietary fructose appears to upregulate PCSK9 mRNA expression and plasma PCSK9 concentrations. Further studies are needed to elucidate the mechanisms of how dietary components regulate PCSK9 and effects on cholesterol and apoB lipoprotein metabolism, as well as to delineate the clinical impact of diet on PCSK9 in terms of CVD risk.
Full-text available
Genetic defects in SAR1B GTPase inhibit chylomicron (CM) trafficking to the Golgi and result in a huge intraenterocyte lipid accumulation with a failure to release CMs and liposoluble vitamins into the blood circulation. The central aim of this study is to test the hypothesis that SAR1B deletion (SAR1B-/- ) disturbs enterocyte lipid homeostasis (e.g., FA β-oxidation and lipogenesis) while promoting oxidative stress and inflammation. Another issue is to compare the impact of SAR1B-/- to that of its paralogue SAR1A-/- and combined SAR1A-/- /B-/- To address these critical issues, we have generated Caco-2/15 cells with a knockout of SAR1A, SAR1B, or SAR1A/B genes. SAR1B-/- results in lipid homeostasis disruption, reflected by enhanced mitochondrial FA β-oxidation and diminished lipogenesis in intestinal absorptive cells via the implication of PPARα and PGC1α transcription factors. Additionally, SAR1B -/- cells, which mimicked enterocytes of CM retention disease, spontaneously disclosed inflammatory and oxidative characteristics via the implication of NF-κB and NRF2. In most conditions, SAR1A-/- cells showed a similar trend, albeit less dramatic, but synergetic effects were observed with the combined defects of the two SAR1 paralogues. In conclusion, SAR1B and its paralogue are needed not only for CM trafficking but also for lipid homeostasis, prooxidant/antioxidant balance, and protection against inflammatory processes.
Full-text available
Proprotein convertase subtilisin kexin type 9 (PCSK9) lowers the abundance of surface low-density lipoprotein (LDL) receptor through an undefined mechanism. The structure of human PCSK9 shows the subtilisin-like catalytic site blocked by the prodomain in a noncovalent complex and inaccessible to exogenous ligands, and that the C-terminal domain has a novel fold. Biosensor studies show that PCSK9 binds the extracellular domain of LDL receptor with Kd = 170 nM at the neutral pH of plasma, but with a Kd as low as 1 nM at the acidic pH of endosomes. The D374Y gain-of-function mutant, associated with hypercholesterolemia and early-onset cardiovascular disease, binds the receptor 25 times more tightly than wild-type PCSK9 at neutral pH and remains exclusively in a high-affinity complex at the acidic pH. PCSK9 may diminish LDL receptors by a mechanism that requires direct binding but not necessarily receptor proteolysis.
Full-text available
GLUT4, the main insulin-responsive glucose transporter, plays a critical role in maintaining systemic glucose homeostasis and is subject to complicated metabolic regulation. GLUT4 expression disorder might cause insulin resistance, and over-expression of GLUT4 has been confirmed to ameliorate diabetes. Here, we reported that farnesoid X receptor (FXR) and its agonist chenodeoxycholic acid (CDCA) could induce GLUT4 transcription in 3T3-L1 and HepG2 cells. Furthermore, CDCA could increase the GLUT4 protein amount in C57BL/6J mice sex-dependently. The following progressive 5'-deletion analysis and site-mutation investigation further suggested that FXR could induce GLUT4 expression through FXR response element (FXRE) in the GLUT4 promoter. EMSA and knock-down of retinoid X receptor (RXR) indicated that FXR binds to the GLUT4-FXRE as a monomer and RXR does not participate in the FXR stimulation of GLUT4 expression. In addition, we demonstrated that FXR does not interfere with insulin-induced GLUT4 translocation to plasma membrane. All these data thereby implied that FXR is a new transcription factor of GLUT4, further elucidating the potential role for FXR in glucose metabolism.
Full-text available
To investigate whether, and by what mechanisms, luminal (dietary) cholesterol regulates cholesterol synthesis in human intestinal cells, HMG-CoA reductase activity, gene expression, synthesis, and degradation were investigated in CaCo-2 cells exposed to taurocholate micelles containing cholesterol. In cells incubated with cholesterol solubilized in 5 mM taurocholate and 30 microM monoolein, HMG-CoA reductase activity was decreased. 25-Hydroxycholesterol, delivered to the cells in the same manner as native cholesterol, was significantly more potent in inhibiting reductase activity and was used, therefore, to investigate mechanisms for sterol regulation. Cells incubated with taurocholate micelles without cholesterol lost cellular cholesterol into the medium causing an increase in HMG-CoA reductase activity and enzyme mass. Although steady-state levels of HMG-CoA reductase mRNA were increased under conditions of cholesterol efflux, synthesis rates of reductase protein were not increased. An increase in activity and enzyme mass in cells incubated with micelles alone, however, was accompanied by a significant decrease in the rate of degradation of reductase protein. In contrast, sterol influx from taurocholate micelles was associated with a marked decrease in HMG-CoA reductase activity and mass without altering mRNA levels except at high concentrations of the polar sterol which did decrease reductase mRNA levels by 50%. The absorption of apical sterol resulted in a significant decrease in the translational efficiency of reductase mRNA and a modest increase in the rate of degradation of the enzyme. Thus, although the primary function of the enterocyte is to transport luminal (dietary) cholesterol to other tissues of the body, apically derived cholesterol enters metabolic pools within the cell which regulates its own cholesterol synthesis. Dietary cholesterol, therefore, will regulate the contribution to the total body cholesterol pool of endogenously derived cholesterol from the intestine. The mechanism for this regulation of intestinal HMG-CoA reductase by luminal cholesterol occurs primarily at the post-transcriptional level.
Full-text available
Na+:H+ and Cl-:HCO3- exchange are localized, respectively, to basolateral (blLPM) and canalicular (cLPM) rat liver plasma membranes. To determine whether these exchangers play a role in bile formation, we examined the effect of a choleretic agent, ursodeoxycholate (UDCA), on these exchange mechanisms. 22Na (1 mM) and 36Cl (5 mM) uptake was determined using outwardly directed H+ and HCO3- gradients, respectively. Preincubation of blLPM vesicles with UDCA (0-500 microM) resulted in a concentration-dependent increase in initial rates of amiloride-sensitive pH-driven Na+ uptake, with a maximal effect at 200 microM. UDCA (200 microM) increased Vmax from 23 +/- 2 (control) to 37 +/- 7 nmol/min per mg protein; apparent Km for Na+ was unchanged. Preincubation with tauroursodeoxycholate (200 microM), taurocholate (10-200 microM) or cholate, chenodeoxycholate, or deoxycholate (200 microM) had no effect on pH-driven Na+ uptake. UDCA (200 microM) had no effect on either membrane lipid fluidity, assessed by steady-state fluorescence polarization using the probes 1,6-diphenyl-1,3,5-hexatriene, 12-(9-anthroyloxy) stearic acid, and 2-(9-anthroyloxy) stearic acid (2-AS), or Na+,K+-ATPase activity in blLPM vesicles. In cLPM vesicles, UDCA (0-500 microM) had no stimulatory effect on initial rates of HCO3(-)-driven Cl- uptake. Enhanced basolateral Na+:H+ exchange activity, leading to intracellular HCO3- concentrations above equilibrium, may account for the bicarbonate-rich choleresis after UDCA infusion.
Full-text available
By using a constant infusion technique in the hamster, rates of uptake of [14C]sucrose-labeled hamster low density lipoprotein (hamLDL) and methylated hamster LDL (MehamLDL) were directly measured in 15 tissues. From these measurements the magnitude of LDL receptor-dependent and receptor-independent lipoprotein transport was calculated. The whole-animal clearance of hamLDL equaled 547 microliters/hr per 100 g of body weight. LDL clearance per g of tissue was highest in the liver (114 microliters/hr per g), ovary (43), spleen (36), adrenal gland (29), and intestine (24) and was lowest in fat (0.75), brain (0.35), and muscle (0.26). When adjusted for organ weight, the sum of the absolute clearance rates in all of the tissues examined equaled the rate of whole-animal LDL turnover. Liver accounted for 73%, and the jejunum and ileum combined accounted for 7% of whole-animal clearance. The 12 other tissues each accounted for only a minor portion of LDL clearance. Rates of uptake of Me-hamLDL were much less in many tissues and accounted for only 6-12% of the uptake of LDL in the liver, ovary, adrenal gland, lung, and kidney. However, this receptor-independent uptake was quantitatively more important in the intestine (44%) and spleen (72%) and accounted for essentially all LDL uptake in organs such as muscle, skin, and brain. Thus, in the hamster, most LDL is taken up and degraded by the liver. This uptake process is greater than 90% mediated by the LDL receptor and manifests saturation kinetics. Finally, cholestyramine feeding increases receptor-mediated LDL transport in the liver but in no other tissue studied.
Full-text available
This study was undertaken to measure and compare the rates at which digitonin-precipitable sterols (DPS) were synthesized in vivo in the major organs of five different animal species. These rates were assessed by measuring the velocity at which [3H]water was incorporated into DPS in the intact animal. The animals used were chosen to include species that carried most of their plasma cholesterol either predominantly in high (rat, hamster) or low (guinea pig) density lipoproteins (HDL and LDL, respectively) or more evenly distributed between the LDL and HDL fractions (monkey and rabbit). Whole animal sterol synthesis was much higher in the rat (16.1 mumol/hr) than in the other four species (2.9-4.6 mumol/hr) when normalized to a constant body weight of 100 g. This uniquely high rate of sterol synthesis could be attributed predominantly to an extremely high rate of incorporation of [3H]water into DPS by the liver of the rat. When expressed per g of tissue, the highest content of newly synthesized sterol in all species was found in tissues such as adrenal gland, ovary, and gastrointestinal tract. However, the content of [3H]DPS in the liver varied markedly from a high of 2279 nmol/hr per g in the rat to a low of only 109 nmol/hr per g in the guinea pig. Consequently, when expressed as a percentage of total body synthesis, the whole liver of the rat contained 51% of the [3H]DPS while this figure was much lower in the monkey (40%), hamster (27%), rabbit (18%), and guinea pig (16%). Thus, in all species except the rat, the major sites for sterol synthesis appeared to be the gastrointestinal tract, carcass (predominantly the muscle), and skin. In addition, even though the content of newly synthesized sterol per g of adrenal gland was higher than in nearly any other tissue in all of the species examined, it was further demonstrated that in the rat most of this [3H]DPS was derived from the blood (and, therefore, ultimately from the liver) whereas in the other species it was largely synthesized locally within the gland. Thus, these studies demonstrated that in many species the liver is quantitatively far less important as a site for sterol synthesis than previously believed and, as a correlate of this, most sterol utilized by extrahepatic tissues is largely synthesized locally within those tissues.
A brief review is given of the chemistry of bile acids, emphasizing the relationship between chemical structure, physical properties and enterohepatic cycling of the major primary and secondary bile acids. Features of the enterohepatic circulation of primary and secondary bile acids in man are summarized. The effects of bile acid feeding on the composition of the enterohepatic circulation in man are reviewed. Methods for characterizing the enterohepatic circulation of bile acids in man are tabulated.
Intraduodenal infusion of hydrophobic bile salts to bile-fistula rats leads within hours to severe hepatocellular necrosis and cholestasis; simultaneous administration of conjugates of ursodeoxycholate, either intraduodenally or intravenously, reduces or prevents liver injury. To evaluate the short-term protective effects of ursodeoxycholate at the cellular level, we incubated primary monolayer cultures of adult rat hepatocytes or freshly isolated washed human erythrocytes for 1 to 240 min with varying defined concentrations of different bile salts in the presence or absence of ursodeoxycholate. Cytolysis was quantified by measuring the release into the medium of cytosolic lactate dehydrogenase (hepatocytes) or hemoglobin (erythrocytes). In both systems, cytolysis increased sigmoidally with increasing bile salt concentration, and the relative toxicity of different bile salts proceeded in the following order: tauroursodeoxycholate was less toxic than taurocholate, which was less toxic than taurodeoxycholate. Taurochenodeoxycholate was more toxic to erythrocytes than taurodeoxycholate; the two were equally toxic to rat hepatocytes. Unconjugated bile salts were more toxic than their conjugates. The addition of tauroursodeoxycholate to taurochenodeoxycholate or taurodeoxycholate led to time-dependent and concentration-dependent reduction or elimination of the toxicity of the more hydrophobic component. Protection was evident within minutes. With respect to hemolysis, at pH 8.5 glyco was less protective than tauroursodeoxycholate, and free ursodeoxycholate was only minimally protective. We conclude that the hepatocytotoxicity of hydrophobic bile salts at millimolar concentrations is markedly reduced in the presence of tauroursodeoxycholate. Conjugates of ursodeoxycholate also prevented disruption of erythrocytes by bile salts, suggesting that protection does not depend on liver-specific pathways of bile salt uptake, compartmentation, transport or metabolism.(ABSTRACT TRUNCATED AT 250 WORDS)
Neomycin was used to assess the involvement of Ins (1,4,5)P3 in the Ca2+ release from the endoplasmic reticulum induced by the bile acid taurolithocholate. In saponin-permeabilized rat hepatocytes, neomycin via its ability to bind Ins (1,4,5)P3 abolished the release of Ca2+ induced by added Ins (1,4,5)P3. In contrast, it did not alter the Ca2+ release initiated by the bile acid. In intact cells, neomycin had no effect on the [Ca2+]i rises promoted by taurolithocholate and vasopressin. It is suggested that the effect of taurolithocholate in liver is not mediated by Ins (1,4,5)P3 but results from a primary action on endoplasmic reticulum.