1. PCSK9 is expressed in pancreatic d-cells and does not alter insulin secretion
Cédric Langhi a,1
, Cédric Le May a,1
, Valéry Gmyr d,e
, Brigitte Vandewalle d,e
, Julie Kerr-Conte d,e
,
Michel Krempf a,b,c
, François Pattou d,e
, Philippe Costet a,c
, Bertrand Cariou a,b,c,*
a
INSERM U915, Nantes F-44000, France
b
Université de Nantes, Faculté de Médecine, Institut du Thorax, Nantes F-44000, France
c
CHU de Nantes, Clinique d’Endocrinologie, Institut du Thorax, Nantes F-44000, France
d
University Lille Nord de France, Lille, France
e
Diabetes Biotherapies, INSERM U859, Lille, France
a r t i c l e i n f o
Article history:
Received 14 October 2009
Available online 28 October 2009
Keywords:
Cholesterol
LDL receptor
Insulin secretion
Diabetes
Somatostatin
a b s t r a c t
PCSK9 (Proprotein Convertase Subtilisin Kexin type 9) is a proprotein convertase that plays a key role in
cholesterol homeostasis by decreasing hepatic low-density lipoprotein receptor (LDLR) protein
expression. Here, we investigated the expression and the function of PCSK9 in pancreatic islets. Immuno-
histochemistry analysis showed that PCSK9 co-localized specifically with somatostatin in human pancre-
atic d-cells, with no expression in a- and b-cells. PCSK9 seems not to be secreted by mouse isolated islets
maintained in culture. Pcsk9-deficiency led to a 200% increase in LDLR protein content in mouse isolated
islets, mainly in b-cells. Conversely, incubation of islets with recombinant PCSK9 almost abolished LDLR
expression. However, Pcsk9-deficiency did not alter cholesterol content nor glucose-stimulated insulin
secretion in mouse islets. Finally, in vivo glucose tolerance was similar in Pcsk9+/+
and Pcsk9À/À
mice under
basal conditions and following streptozotocin treatment. These results suggest, at least in mice, that
PCSK9 does not alter insulin secretion.
Ó 2009 Elsevier Inc. All rights reserved.
PCSK9 (Proprotein Convertase Subtilisin Kexin type 9), also
known as NARC1 (Neural Apoptosis-Regulated Convertase 1), is
the ninth member of the proprotein convertase (PC) family [1].
PCSK9 plays a key role in the regulation of cholesterol homeostasis
(for review, see [2]). PCSK9 is secreted by the liver and acts as a nat-
ural inhibitor of the low-density lipoprotein (LDL) receptor (LDLR)
pathway. PCSK9 promotes the down-regulation of hepatic LDLR in
a post-transcriptional manner [3,4]. In humans, ‘‘gain-of-function”
Pcsk9 mutationsare associatedto autosomal dominanthypercholes-
terolemia and premature atherosclerosis [5]. In contrast, ‘‘loss-of-
function” Pcsk9 mutations lead to low levels of LDL-cholesterol
(LDL-C) resulting in a protection against cardio-vascular disease
[6]. Thus, PCSK9 inhibition is thought to be a promising way to
achieve low-levels of LDL-C in combination with statins [2].
The LDLR is expressed in pancreatic b-cells in humans, rats [7]
and mice [8]. As a functional consequence, the atherogenic lipopro-
teins like VLDL and LDL are toxic for b-cells because they induce
necrosis [9] or apoptosis [8]. Recently, some studies have sug-
gested that cholesterol homeostasis in pancreatic b-cells plays a
crucial role in b-cell function [10,11]. To date, no study has ad-
dressed the presence and the functional significance of PCSK9 in
the endocrine pancreas. Hepatic PCSK9 expression was found to
be altered in rodent models of diabetes [12], and stimulated by
insulin in vivo in mice [13]. Moreover, it has been demonstrated
very recently that plasma PCSK9 levels were positively correlated
with fasting blood glucose in humans [14,15].
In view of this data, it can be suggested that PCSK9 may alter
cholesterol homeostasis and b-cell function in pancreatic islets.
Thus, we investigated the expression and function of PCSK9 both
in mouse and human isolated islets, as well as its functional role
in glucose homeostasis in vivo in mice.
Material and methods
Hormone immunoassays. Insulin content and secretion were as-
sessed in mice isolated islets by radioimmunoassay (Linco Research,
St. Charles, Missouri, USA). Plasma insulin levels in mice were mea-
sured by ELISA (Crystal Chem Inc., Chicago, Illinois, USA). Secreted
insulin and insulin content of human islets were assessed with an
immunoradiometric assay (Bi-Insulin IRMA, SANOFI Diagnostics
Pasteur, Marnes-la-Coquette, France). Somatostatin secretion of
isolated islets was measured using an EIA kit after a lyophilization
step (Phoenix Pharmaceuticals Inc., Burlingame, California USA).
Animals. Pcsk9+/À
mice were purchased from Jackson Laborato-
ries (Maine, USA) and interbred to produce Pcsk9À/À
and Pcsk9+/+
0006-291X/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved.
doi:10.1016/j.bbrc.2009.10.138
* Corresponding author. Address: CHU de Nantes, Clinique d’Endocrinologie,
l’Institut du Thorax, CHU Hôtel-Dieu, 1 Place Alexis Ricordeau, 44093 Nantes cedex,
France. Fax: +33 2 40 08 75 44.
E-mail address: bertrand.cariou@univ-nantes.fr (B. Cariou).
1
These authors contributed equally to this work.
Biochemical and Biophysical Research Communications 390 (2009) 1288–1293
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications
journal homepage: www.elsevier.com/locate/ybbrc

2. littermates. They were genotyped, as described later on [16].
Animals had free access to food and water under a 12-h light/12-
h dark cycle in a temperature-controlled environment. For the
multiple low doses of streptozotocin (STZ) model, male mice aged
8–10 weeks were injected i.p. for 5 consecutive days with STZ (Sig-
ma, France) at a concentration of 50 mg/kg body weight. For glu-
cose tolerance and in vivo insulin secretion tests, tail blood
samples were collected from 6 h-fasted mice after an i.p. injection
of 2 g/kg glucose. Plasma glucose levels were measured using an
automatic glucose monitor (Accu-check Active, Roche, Germany).
All animal studies were approved by the Unité de Thérapeutique
Expérimentale (Animal Facility Agreement No. BP44015).
Human and mouse islets isolation. Human pancreases were har-
vested from adult brain-dead donors in accordance with French
Regulations and with the local Institutional Ethical Committee.
Pancreatic islets were isolated after digestion of the tissue with
LiberaseÒ
(Roche Diagnostics, Meylan, France) according to the
method of Ricordi et al. [17]. Semi-purification was achieved with
continuous density gradients using a COBE 2991 cell separator. Is-
let number was determined on samples of each preparation after
dithizone staining and expressed as equivalent number of islets
(IE). Pancreatic islets from 8- to 12-week-old male PCSK9À/À
or
PCSK9+/+
mice were isolated by collagenase type V digestion (Sig-
ma, France), as previously described [18].
Immunohistochemistry. Human and mouse pancreases were
fixed overnight at 4 °C in 4% paraformaldehyde (PAF) solution
and then embedded in paraffin and sectioned (5 lm) before pro-
cessing. In some experiments, cell pellets were included in biolog-
ical glue (TissucolÒ
, Roche), fixed in 4% PAF and further processed
for paraffin embedding. Antibody specificities and dilutions are
listed in Supplemental Table 1 lists. Deparaffinized sections were
incubated with specific antibodies for 2 h at room temperature
PCSK9/Cyclophylin
mRNArelativelevels
Mouse Liver Mouse Islet
0.2
0.4
0.6
0.8
1.0
1.2
1.4
Insulin / PCSK9 / DAPI Glucagon / PCSK9 / DAPI
Somatostatin / PCSK9 / DAPI
merged
Human isolated islets
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
2.0
Human islets Panc1
PCSK9 / βactin
mRNArelativelevels
0.00.0
Human islets
medium
IHH
medium
6h 24h Overnight-cultured
PCSK9
0.0
0.2
0.4
0.6
0.8
1.0
1.2
PCSK9+/+ PCSK9-/-
Ex-vivo Somatostatin
secretion
Arbitraryunits
Fig. 1. PCSK9 is expressed in human pancreatic d-cells. (A) All PCSK9 mRNAs levels were measured by a Q-PCR analysis in liver and isolated pancreatic islets from PCSK9+/+
mice. The values are normalized to cyclophilin and are expressed relatively to those of liver mice arbitrarily set at 1. (B) All PCSK9 mRNAs levels were measured by a Q-PCR
analysis in human isolated pancreatic islets and Panc1 pancreatic carcinoma cell line. All values are normalized to b-actin and are expressed relatively to those of human islets
set at 1. (C) Immunostaining and confocal imaging of a representative pancreatic islet from human lean donors showed co-localization of PCSK9 (red) and somatostatin
expressing d-cells (green, merged in yellow). No co-staining of insulin expressing b-cells (green) or glucagon expressing a-cells (green) was observed. The nuclei are
counterstained with DAPI (blue) (magnification 40Â). (D) Secreted somatostatin levels from Pcsk9+/+
and Pcsk9À/À
mice isolated islets. Data represent a pool of three
independent experiments (with 120 islets/experiments). (E) An immunoblot analysis of PCSK9 secreted in the media of human islets and Immortalized Human Hepatocytes
(IHH).
C. Langhi et al. / Biochemical and Biophysical Research Communications 390 (2009) 1288–1293 1289

3. (PCSK9, LDLR) followed by an overnight incubation at 4 °C (insulin,
glucagon, somatostatin). Antibodies were revealed with streptavi-
din–biotin FITC and Alexa fluor 594 (Molecular probes, In Vitro-
gen). Nuclei were counterstained with DAPI.
Western blot. Protein extraction and Western blots were per-
formed, as described later on [13]. The nature and origin of the
antibodies are detailed in Supplemental Methods.
Realtime quantitative PCR. Human (3000 IE per condition) or mice
islets were lysed in a 1% b-mercaptoethanol-containing buffer ob-
tained from an RNA extraction kit (Macherey Nagel, Hoerdt, France).
Real time quantitative PCR (Q-PCR) was performed as previously de-
scribed [13]. The primers used are detailed in Supplemental Data.
Statistical analysis. Each experiment is representative of at least
two independent experiments with a minimum of triplicates per
condition. All values are reported as means ± SEM. Statistical sig-
nificance was analyzed using a student’s unpaired t test. The values
of p < 0.05 were considered significant.
Results and discussion
PCSK9 is expressed in human pancreatic d-cells
First, we verified the expression of PCSK9 mRNA in isolated pan-
creatic islets from Pcsk9+/+
male mice. Real time Q-PCR analysis
showed that the expression of PCSK9 in mouse isolated islets
was about 30% of that detected in mouse liver (Fig. 1A). Next, we
investigated the expression of PCSK9 in the human pancreas. While
PCSK9 mRNA was detected in human islets from pancrease donors
(n = 3), PCSK9 was not expressed in the human pancreatic carci-
noma cell line Panc1 (Fig. 1B).
To further confirm the expression of PCSK9 at the protein level,
we performed an immunostaining of a paraffin embedded human
pancreas. Surprisingly, we found that PCSK9 co-localized with
somatostatin expressing d-cells, as shown in Fig. 1C. In contrast,
neither co-localization of PCSK9 with insulin expressing b-cells
nor with glucagon-expressing a-cells was observed. In addition,
no PCSK9 immunostaining was detected in the exocrine tissue.
Unfortunately, immunohistochemistry analysis failed to detect
PCSK9 in mouse pancreas sections due to a non-specific binding
with all the antibodies we tested (data not shown). As highlighted
recently with the characterization of somatostatin-deficient mice,
somatostatin exerts a paracrine regulatory action on the islet func-
tion [19]. We found that Pcsk9-deficiency did not alter the basal
secretion of somatostatin in isolated islets (Fig 1D). In accordance
with this observation, gross examination revealed that immuno-
staining for somatostatin was similar in pancreases from Pcsk9+/+
and Pcsk9À/À
mice (data not shown).
PCSK9 is secreted by the hepatocytes and circulates in the blood
stream at hormonal concentrations (i.e., 33–2988 ng/ml)
[14,15,20,21]. A Western blot analysis of a culture medium from
1 0.08 0.09 0.02*** 0.03 0.02***
LDLR
β-Actin
CTRL
purified PCSK9
(10 μg/ml)
purified PCSK9
(5 μg/ml)
Human isolated islets
PCSK9+/+ PCSK9-/-
LDLR
β-Actin
PCSK9+/+ PCSK9-/-
1.0 0.06 2.81 0.28**
PCSK9+/+ PCSK9-/-
1.0 0.25 1.63 0.80
derutluc-h42detalosiylhserF
LDLR / DAPI LDLR / DAPI
Fig. 2. PCSK9 antagonizes LDLR protein expression in pancreatic islets. (A) Immunostaining and confocal imaging of a representative pancreatic islet from 12-week-old
Pcsk9+/+
and Pcsk9À/À
mice for LDLR (red). (B) An immunoblot analysis of LDLR and actin expression in freshly isolated (on left part) and 24 h-cultured (on right part) islets
isolated from Pcsk9+/+
and Pcsk9À/À
mice. (C) LDLR content quantification human islets cultured for 6 h with the indicated amounts of purified human recombinant PCSK9.
Similar results were obtained in two independent experiments. Data represent mean ± SEM. **
p < 0.01, ***
p < 0.001.
1290 C. Langhi et al. / Biochemical and Biophysical Research Communications 390 (2009) 1288–1293

4. human isolated islets failed to detect PCSK9 protein after 6 h and
24 h of culture, compared with immortalized human hepatocytes
(IHH) (Fig. 1E). Previous data showed that mice specifically defi-
cient for Pcsk9 in the liver have no detectable PCSK9 in the plasma
[22]. The absence of PCSK9 in the medium from pancreatic islets is
consistent with the observation that the liver is the main source of
circulating PCSK9.
Circulating PCSK9 reduces LDLR in pancreatic islets
The PCSK9-mediated LDLR degradation has been extensively
characterized, especially in the liver [2]. We assessed whether
PCSK9 was able to regulate the LDLR expression also in the pancre-
atic islets. Immunostaining pattern of LDLR clearly showed an in-
crease of LDLR content within islets from Pcsk9À/À
mice when
compared to Pcsk9+/+
mice (Fig. 2A). Coimmunostaining experi-
ments demonstrated that LDLR overexpression mainly occurred
in the b-cells from Pcsk9À/À
mice (data not shown). Accordingly,
Western blot analysis performed with freshly isolated islets
showed that LDLR protein content was increased by nearly 200%
in Pcsk9À/À
mice (Fig. 2B, left part), as reported before in other or-
gans such as the liver [16] and the small intestine [23]. To further
determine whether the increased LDLR expression in Pcsk9À/À
mice
could be attributable to a specific lack of PCSK9 in islets rather than
a global absence of circulating PCSK9, the same experiments were
carried out in islets cultured in vitro for 24 h. Under those condi-
tions, the LDLR protein content did not remain significantly in-
creased in the islets from Pcsk9À/À
mice (Fig. 2B, right part),
suggesting an initial action of circulating PCSK9 rather than an in-
tra-islet paracrine effect (Fig. 1D). Both parabiosis experiments and
in vivo infusions of physiological concentrations of human recom-
binant PCSK9 clearly demonstrate that circulating PCSK9 controls
hepatic LDLR expression [21,24]. In addition, circulating PCSK9 also
reduces LDLR in extra-hepatic tissues like lung, kidney, intestine,
and adipose tissue [25]. In contrast, PCSK9 infusion has no effect
on LDLR expression in the adrenals [24]. Incubation of human islets
for 6 h with 5 and 10 lg/ml of recombinant purified PCSK9 almost
abolished the LDLR protein expression (Fig. 2C). Similar results
were obtained with wild-type mouse islets treated with purified
PCSK9 (data not shown). Altogether, these results indicate that cir-
culating PCSK9 is able to modulate LDLR expression in pancreatic
islets.
PCSK9 does not alter cholesterol content and glucose-induced insulin
secretion in islets
Recent studies have focused on the potential relationship be-
tween cholesterol homeostasis and insulin secretion, with a delete-
rious effect of high intra-cellular cholesterol content on GSIS [11].
The most convincing data arose from the phenotypic characteriza-
tion of the mice with a selective inactivation of Abca1in the b-cells
[10]. Despite normal plasma cholesterol levels, these mice had an
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
*
25 mM
Insulinsecretion(AU)
*
2.8 mM
0.2
0.4
0.6
0.8
1.0
1.2
PCSK9+/+ PCSK9-/-
PCSK9+/+
PCSK9-/-
FoldInduction(AU)
1
2
3
4
5
6
Islet Cholesterol
content
GSIS
1
2
3
4
5
6 *** ***
Relativeislet
cholesterolcontent(AU)
Relativeislet
cholesterolcontent(AU)
Islet Cholesterol
content
PCSK9+/+ PCSK9-/-
no LDL LDL 6.4mM no LDL LDL 6.4mM no LDL LDL 6.4mM no LDL LDL 6.4mM
PCSK9+/+
PCSK9-/-
Fig. 3. Pcsk9 deficiency does not alter cholesterol content and insulin secretion in mice isolated islets. (A) Cholesterol content was measured in freshly isolated islets from
Pcsk9+/+
and Pcsk9À/À
mice by fluorometric detection. (B) A Glucose-Stimulated Insulin Secretion (GSIS) was assessed in static incubations from Pcsk9+/+
and Pcsk9À/À
mouse
isolated islets. Data represent a pool of six independent experiments. The fold induction is the ratio of stimulated insulin secretion (25 mM glucose) over basal insulin
secretion (2.8 mM glucose). Data represent mean ± SEM. *
p < 0.05, **
p < 0.01, ***
p < 0.001 in comparison with 2.8 mM glucose condition. (C) Cholesterol content was measured
in islets from Pcsk9+/+
and Pcsk9À/À
mice cultured in LPDS 5% in the presence of 6.4 mM LDL for 24 h by fluorometric detection. (D) GSIS from Pcsk9+/+
and Pcsk9À/À
mouse
islets cultured in the presence or absence of 6.4 mM LDL for 24 h. The fold induction is the ratio of stimulated insulin secretion (25 mM glucose) over basal insulin secretion
(2.8 mM glucose). This figure is representative of three independent experiments. Data represent mean ± SEM. *
p < 0.05, **
p < 0.01, ***
p < 0.001 in comparison with 2.8 mM
glucose condition. §
p < 0.05 in comparison with 6.4 mM LDL.
C. Langhi et al. / Biochemical and Biophysical Research Communications 390 (2009) 1288–1293 1291

5. elevated total cholesterol content in islets due to an impaired cho-
lesterol efflux. Of functional relevance, these mice exhibited a de-
creased GSIS and an impaired glucose tolerance in vivo.
Considering the increase of LDLR expression in Pcsk9À/À
mice, we
determined whether Pcsk9-deficiency could alter cholesterol con-
tent in islets. We failed to detect any difference in cholesterol con-
tent in freshly isolated islets from Pcsk9+/+
and Pcsk9À/À
mice
(Fig. 3A). Then, we tested whether PCSK9 could alter the insulin
secretion. Glucose-stimulated insulin secretion (GSIS) tests were
performed ex vivo in isolated islets from Pcsk9+/+
and Pcsk9À/À
mice
(Fig. 3B). As expected, the insulin secretion was significantly in-
creased in response to high (i.e., 25 mM) glucose. However,
Pcsk9-deficiency did not alter basal nor glucose-induced insulin
secretion in accordance with a neutral effect of Pcsk9-deficiency
on islet cholesterol content. This apparent discrepancy between
the increased LDLR expression of freshly isolated islets and their
cholesterol content remains unclear. It cannot be ruled out that
Pcsk9-deficiency may increase the cholesterol efflux from the is-
lets. Alternatively, the consequences of the LDLR up-regulation in
the islets might be counteracted by the lower circulating LDL-C
levels in Pcsk9À/À
mice. A recent study showed that incubation of
mice isolated islets with a high LDL concentration impaired the
insulin secretion in an LDLR-dependent manner [26]. To assess
the role of PCSK9 upon lipotoxic conditions, the GSIS experiments
were performed in the presence of high LDL concentrations (i.e.,
6.4 mM) for 24 h. The measurement of cholesterol content be-
tween Pcsk9+/+
and Pcsk9À/À
mice islets demonstrated that LDL
incubation increased cholesterol content, but to a similar extent
between both genotypes (Fig. 3D). It should be noticed that such
LDL treatment did not increase cholesterol content in islets from
Time (min)
IPGTT before STZ
120
150
220
270
320
370
0 30 60 90 120
PCSK9+/+
PCSK9-/-
420
470
IPGTT after STZ
120
170
220
270
320
370
420
470
0 30 60 90 120
Time (min)
Bloodglucose(mg/dl)
PCSK9+/+
PCSK9-/-
0.10
0.20
0.30
0.40
0.50
0.60
0.70
-15 0 15 30
0.10
0.20
-15 0 15 30
0.30
0.40
0.50
0.60
0.70
Time (min)Time (min)
PCSK9+/+
PCSK9-/-
plasmainsulin(ng/ml)
Bloodglucose(mg/dl)plasmainsulin(ng/ml)
Days after last STZ injection
120
170
220
270
320
370
420
470
3 8 13 18 23 28 33 38 43 48 53 58 63 68
PCSK9+/+
PCSK9-/-
Bloodglucose(mg/dl)
PCSK9+/+
PCSK9-/-
Fig. 4. Pcsk9 deficiency does not alter insulin secretion in vivo in basal and streptozotocin-induced diabetes conditions. Pcsk9+/+
(n = 7) and Pcsk9À/À
(n = 7) male mice were
treated with five injections of low-dose streptozotocin (STZ, 50 mg/kg body weight). (A) Blood glucose levels were monitored on 6 h-fasted mice after the last injection of STZ.
We performed an intra-peritoneal glucose tolerance test (2 g/kg body weight, IPGTT) before (B,D) and 20 days after the last injection of STZ (C,E) on 12 h-fasted mice. Blood
glucose (B,C) and plasma insulin (D,E) levels were monitored at the indicated times. Data represent mean ± SEM.
1292 C. Langhi et al. / Biochemical and Biophysical Research Communications 390 (2009) 1288–1293

6. LDLR deficient mice, confirming the integrity of this pathway in
Pcsk9À/À
mice islets (data not shown). Such an absence of intra-cel-
lular content variation could be explained by the progressive loss
of LDLR upregulation in Pcsk9À/À
mice islets cultured during 24 h
(Fig. 2B, right part). In accordance with a deleterious effect of high
intra-cellular cholesterol content [25], the treatment of islets with
high LDL concentrations significantly reduced the GSIS (Fig. 3E).
However, there was no difference between both genotypes, sug-
gesting that PCSK9 has a neutral effect on insulin secretion mea-
sured ex-vivo.
Pcsk9-deficiency does not alter in vivo glucose homeostasis in mice
To further assess whether PCSK9 is involved in the control of
whole-body glucose homeostasis, we performed dynamic tests in
Pcsk9À/À
and Pcsk9+/+
mice. Since PCSK9 has been suspected to play
a role in apoptosis and cellular regeneration [1,21], we tested the
hypothesis that PCSK9 may alter b-cell survival. We generated a
model of multiple low doses of STZ-induced diabetes in Pcsk9+/+
and Pcsk9À/À
mice. Random fed blood glucose levels raised in a
similar manner in both genotypes (Fig. 4A). Intra-peritoneal glu-
cose tolerance tests (IPGTT) were performed before (Fig. 4B) and
20 days after the last injection of STZ (Fig. 4C). Pcsk9+/+
and
Pcsk9À/À
mice had similar glucose tolerance and became equally
glucose intolerant following STZ injections. In order to assess the
insulin secretion in vivo, we measured the first-phase insulin re-
sponse 2 min after a glucose challenge (2 g/kg body weight). Under
basal condition, the plasma insulin levels were increased similarly
in Pcsk9+/+
and Pcsk9À/À
mice (Fig. 4D). The two phases of insulin
secretion following the glucose challenge were abolished after
STZ injections in a similar manner in both genotypes (Fig. 4E). Alto-
gether, these results indicate that Pcsk9-deficiency did not alter
glucose homeostasis under basal conditions and after STZ treat-
ment in mice.
Regarding the association between circulating PCSK9 levels and
fasting plasma glucose concentrations [14,15], a potential link be-
tween PCSK9 and metabolic diseases such as diabetes mellitus
could be evoked. Our findings suggest, at least in mice, that
Pcsk9-deficiency did not impact insulin secretion ex-vivo and
in vivo.
Acknowledgments
This work was supported by Agence Nationale de la Recherche
(«Physiopathologies humaines 2006 R0651ONS»), Fondation de
France, Fondation Cœur et Artères, and ALFEDIAM. C. Langhi is a re-
cipient of a fellowship from the Nouvelle Société Française d’Athé-
rosclérose. C. Le May was supported by a grant from the Fondation
pour la Recherche Médicale. P. Costet and B. Cariou are titulars of a
Contrat d’interface INSERM-CHU de Nantes. We acknowledge Dr.
Philippe Lefebvre (INSERM U545) for providing us the Panc1 cell
line. We are indebted to Dr. Nan Ying (Merck, Rahway) for giving
us human recombinant PCSK9.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bbrc.2009.10.138.
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