1. MASTER’S DEGREE IN SCIENCES, TECHNOLOGIES AND HEALTH
Sciences, Health Management and Engineering
Cellular and Molecular Biosignaling & Physiopathology
From Animal to Man: Analysing and Managing Health and Food Risks
2nd-year Master’s Thesis
EFFECTS OF STATINS AND PCSK9 ON LDLR PATHWAY
AND GLUCOSE-STIMULATED INSULIN SECRETION OF
HUMAN PANCREATIC β-CELLS
Thesis defense: September 28th
, 2016
Magdalena DIAZ-OVALLE
UMR 1280 PhAN Physiologie des Adaptations Nutritionnelle
CHU Hôtel Dieu Nantes 1 place Alexis Ricordeau 44093 Nantes, France
Internship supervisor: Dr. Aurelie Thedrez, Dr. Patricia Parnet
Jury:
External Committee Member: Dr. Michel Krempf, professor of endocrinology and metabolic disease,
Université de Nantes.

3. MASTER’S DEGREE IN SCIENCES, TECHNOLOGIES AND HEALTH
Sciences, Health Management and Engineering
Cellular and Molecular Biosignaling & Physiopathology
From Animal to Man: Analysing and Managing Health and Food Risks
2nd-year Master’s Thesis
EFFECTS OF STATINS AND PCSK9 ON LDLR PATHWAY
AND GLUCOSE-STIMULATED INSULIN SECRETION OF
HUMAN PANCREATIC β-CELLS
April - September 2016
Magdalena DIAZ-OVALLE
UMR 1280 PhAN Physiologie des Adaptations Nutritionnelle
CHU Hôtel Dieu Nantes 1 place Alexis Ricordeau 44093 Nantes, France
Internship supervisor: Dr. Aurelie Thedrez, Dr. Patricia Parnet
Jury:
External Committee Member: Dr. Michel Krempf, professor of endocrinology and metabolic disease,
Université de Nantes.

4. ANTI -PLAGIARISM AGREEMENT
I, the undersigned, Magdalena DIAZ OVALLE
Declare being aware that plagiarism of documents, or parts of documents, published on all types of
formats, including the Internet, constitutes a breach of copyright, as well as deliberate fraud.
Consequently, I undertake to quote all sources that I have used in order to produce my written works
(reports, thesis, slide presentations, etc.)
Nantes, September the 18th
, 2016
Signature:

5. ACKNOWLEDGEMENTS
I would like to thank Pr. Dominique Darmaun for welcoming me in the 1280 INRA PhAN
laboratory.
I would like to thank Dr. Gilles Lambert for allowing me to be part of his research team.
I would like to thank Dr. Aurélie Thedrez for having the patience and dedication to introduce me to
the world of cellular culture and research, for carefully but firmly guiding me through every step of
the way and teaching me about a vast amount of subjects.
I would like to thank all the staff at the laboratory for welcoming me into their workspace and
sharing with me this unique experience.
I would like to thank the Man-Imal team for the beautiful job they do: the accomplishment of an
international master, which brings together people, culture and knowledge for the enrichment and
gain of us all.
And finally, I would like to thank my classmates: Begoña, Niki, Michelle, Simon, Tanveer, Ioannis,
Jessica, Delphine, Richard and specially my friend Sofía, for making this experience even more
enjoyable and rewarding.

6. ABSTRACT
Introduction: Statins, common drugs used to treat hypercholesterolemia, slightly increase the risk
of type 2 diabetes (T2D). It is unknown whether new treatments with monoclonal antibodies
directed against Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9), an inhibitor for the
recycling of the LDL receptor (LDLR), may enhance this risk. Here, we wanted to evaluate how
statins and PCSK9 modulate the LDLR pathway of human pancreatic β-cells and how these
modulations can alter their glucose-stimulated insulin secretion (GSIS).
Materials and methods: EndoC-βH1 human pancreatic β cells were starved in glucose and treated
with or without mevastatin and with or without recombinant PCSK9. Bodipy-LDL or unlabeled-
LDL were added in some experiments. Cell surface LDLR expression was measured by flow
cytometry. Bodipy-LDL uptake was analyzed by flow cytometer and confocal microscopy. PCSK9
secretion in culture supernatants was quantified by ELISA. GSIS assays were performed in Krebs-
Ringer buffer with glucose increasing concentrations (2.8 to 15 mM) with or without exendin-4 or
phosphodiesterase inhibitor IBMX, with or without PCSK9, and/or an excess of LDL. Insulin was
measured in supernatants and cell lysates by ELISA.
Results: Cell surface LDLR expression and LDL uptake were significantly increased with
mevastatin whereas decreased with PCSK9. However, mevastastin and PCSK9 treatments did not
seem to alter GSIS of cells supplemented or not with LDL. Interestingly, EndoC-βH1 cells secreted
significant amounts of PCSK9 (5.27 ng/million of cells) that was increased with mevastatin
treatment but not affected by LDL supplementation.
Conclusions: Our in vitro data shows that PCSK9 and mevastatin modulate LDLR function and
expression in human pancreatic β-cells without apparently altering their GSIS, and, for the first
time, that human β-cells are able to secrete PCSK9.
Keywords: Statins, PCSK9, LDL, hypercholesterolemia, type 2 diabetes.

7. Table of contents
List of illustrations..............................................................................................................................................................1
Figures .............................................................................................................................................................................1
Tables...............................................................................................................................................................................1
List of Used Abreviations...................................................................................................................................................2
I Introduction......................................................................................................................................................................4
1. Context of study............................................................................................................................................................4
2. Lipoprotein Metabolism...............................................................................................................................................4
3. LDLR and PCSK9 ........................................................................................................................................................6
4. Hypercholesterolemia ..................................................................................................................................................7
5. Statins and anti-PCSK9 as hypolipidemic treatments .................................................................................................8
6. Type 2 Diabetes, Statins and anti-PCSK9 .................................................................................................................10
II Personal study: .............................................................................................................................................................14
1. Materials and methods...............................................................................................................................................14
1.1 EndoC-βH1 human pancreatic cells ....................................................................................................................14
1.1.1 Matrigel Support ..........................................................................................................................................15
1.1.2 Culture medium............................................................................................................................................15
1.1.3 Thawing........................................................................................................................................................15
1.1.4 Viable cell counting .....................................................................................................................................15
1.1.5 Seeding and subculture.................................................................................................................................16
1.1.6 Cryopreservation ..........................................................................................................................................17
1.2 EndoC-BH1 cell seeding and culture for assay performing ................................................................................17
1.2.1 Day 0: Cell seeding ......................................................................................................................................17
1.2.2 Day 2: Deprivation of glucose and treatment with and without mevastatin (Except for GSIS assay) ........17
1.3 PCSK9 secretion assay and measurement of cell surface LDLR expression......................................................19
1.3.1 Day 3: Deprivation OF glucose and treatment with and without mevastatin with or without PCSK9........19
1.3.2. PCSK9 secretion assay................................................................................................................................19
1.3.2.1 Elisa test for PCSK9 (ELISA QUANTIKINE® R&D SYSTEMS).........................................................19
1.3.2.2 ELISA assay protocol................................................................................................................................19
1.3.3 Cell surface LDLR expression measurement...............................................................................................20
1.4 LDL uptake measurement ...................................................................................................................................20
1.5 Glucose stimulated insulin secretion (GSIS) assay .............................................................................................21
1.5.1 Recovering of culture supernatants..............................................................................................................22
1.5.2 Cell Lysates..................................................................................................................................................22
1.5.3 Measure of the secreted and produced insulin by ELISA (MERCODIA INSULINE ASSAY®) ..............22
1.5.4 Protein measurement content .......................................................................................................................23
1.6 Cell staining analysis by Flow Cytometry...........................................................................................................24
1.7 Statistical analysis................................................................................................................................................24
2. Results ........................................................................................................................................................................25
2.1 Statins and PCSK9 modulates LDL receptor expression in EndoC-βH1 cells ...................................................25
2.2 Statins and PCSK9 modulates LDL uptake by EndoC-βH1 cells.......................................................................27
2.3 Basal secretion of PCSK9 by EndoC-βH1cells and its modulation by statins....................................................29
2.4 Statins and PCSK9 do not apparently alter GSIS................................................................................................29
3. Discussion ..................................................................................................................................................................31
4. Conclusion .................................................................................................................................................................33
Bibliography......................................................................................................................................................................34

8. 1
LIST OF ILLUSTRATIONS
Figures
Figure 1: Lipoprotein metabolism pathway (exogenous and endogenous pathway)……………………..…...5
Figure 2: Internalization of LDL-C by LDLR……………………..……………….......……………………..6
Figure 3: Degradation of LDL-R mediated by PCSK9………………………………………………………..7
Figure 4: Summary diagram of the experimental procedure of producing the Endoc βH1-line………..…...14
Figure 5: EndoC-BH1 cells after 3 days being cultured in a support matrigel……….……………………...16
Figure 6: Diagram overview of the structure of the different procedures performed with the EndoC-ΒH1
cells...................................................................................................................................................................18
Figure 7: Statins and PCSK9 modulates LDL receptor expression in EndoC-βH1 cells……..……………..25
Figure 8 Statins and PCSK9 modulates LDL uptake by EndoC-βH1 cells ……………………....................27
Figure 9: Statins and PCSK9 modulate the capacity of Endoc-βH1 cells to internalize LDL…..……….27-28
Figure 10: Basal secretion of PCSK9 by EndoC-βH1cells and its modulation by statins...............................29
Figure 11: Statins and PCSK9 do not apparently alter GSIS...........................................................................30
Tables
Table 1: Concentration of the cells and volume of culture medium for each different support………...……17
Table 2: Components of the different solutions used in the Krebs-Ringer solution………..………………..21
Table 2: Components for Lysis cell solution (without protease)……………………..………………...……22
Table 3: Fluorochromes used in flow cytometry…………………………………………………………..…24

9. 2
LIST OF USED ABREVIATIONS
APC: AlloPhycoCyanine
Apo B-100: Apolipoprotein B-100
BODIPY: Bore-Dipyromethene
BSA: Bovine Serum Albumin
cAMP: Cyclic Adenosine Monophosphate
CVD: Cardio Vascular Disease
DAPI: 4',6-diamidino-2-phenylindole
DMEM: Dulbecco’s Modified Eagle Medium
DMSO: Dimethyl Sulfoxide
ECM: Extracellular Matrix
EGTA: Ethylene Glycol Tetra Acetic Acid
ELISA: Enzyme Linked Immuno-Sorbent Assay
FBS: Fetal Bovine Serum
FH: Familial Hypercholesterolemia
FSC: Forward Scatter (size parameter of cells in flow cytometry)
GLP1: Glucagon-Like Peptide 1
GOF: Gain of Function
GSIS: Glucose-Stimulated Insulin secretion
HDL: High Density Lipoprotein
HeFH: Heterozygous Familial Hypercholesterolemia
HEPES: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
HoFH: Homozygous Familial Hypercholesterolemia
hTERT: Human Telomerase Reverse Transcriptase
IDL: Intermediate Density Lipoproteins
LDL: Low Density Lipoprotein
LDL-C: Low Density Lipoprotein Cholesterol
LDLR: Low Density Lipoprotein Receptor
LPL: Lipoprotein lipase

10. 3
mAbs: Monoclonal Antibodies
MFI: Mean Fluorescence Intensity
MTP: Microsomal Triglyceride Transfer Protein
NO: Nitric Oxide
PBS: Phosphate Buffered Saline
PCSK9: Proprotein Convertase Subtilisin/Kexin Type 9
PFA: Paraformaldehyde
SCID: Severe Combined Immunodeficient
SEM: Standard error of the mean
SOD: Superoxide dismutase
SREBP: Sterol regulatory element-binding protein
SSC: Side Scatter (granularity parameter of cells in flow cytometry)
T2D: Type 2 Diabetes
TGF: Transforming Growth Factor
VLDL: Very Low Density Lipoprotein
WT: Wild Type

11. 4
I INTRODUCTION
1. Context of study
Statins are currently the most widely used drugs to treat hypercholesterolemia, a condition
strongly associated to an increased risk of cardiovascular diseases (CVD). Generally, statins
treatment efficiently reduces LDL-cholesterol (LDL-C) level in plasma and therefore, the risk of
cardiovascular diseases (CVD). Its effect is predominantly mediated by an increased expression of
LDL receptor (LDLR) at the surface of cells (Stancu & Sima 2001). However, in some patients,
statins are unable to reach the LDL-C target levels despite being used with the highest tolerable
dose, or can induce serious side effects. Besides, recent studies have demonstrated that the sustained
use of statins slightly increases the risk of developing type 2 diabetes (T2D) (Preiss et al. 2011), a
disease characterized by an impaired insulin secretion of pancreatic β-cells and an insulin resistance
in the target tissues (Reinehr 2013).
The discovery of Proprotein convertase subtilisin kexin type 9 (PCSK9) in 2003, a natural
inhibitor for the recycling of LDLR (Lambert et al. 2012), provides new possibilities to develop
complementary or alternative treatments to statins. Monoclonal antibodies directed against PCSK9
have since emerged as robust cholesterol-lowering therapy and have been recently approved as a
new complementary or alternative treatment for hypercholesterolemic patients at high risk of
developing CVD who do not respond adequately to statins (Latimer et al. 2016). However, if long-
term treatment with anti-PCSK9 increases the risk of T2D, remains to be determined.
2. Lipoprotein Metabolism
Lipids are hydrophobic particles that require lipoproteins to transport fat within a water-soluble
medium. Lipoproteins contain both lipids and proteins and circulate through the blood until their
content is taken by peripheral tissues or they are cleared by the liver. Lipoproteins differ in size and
density; there is high density lipoproteins (HDL), low density lipoproteins (LDL), intermediate
density lipoproteins (IDL), very low density lipoproteins (VLDL) and chylomicrons (Goldberg
2015b).
There are mainly two pathways in lipoprotein metabolism: exogenous and endogenous
(Figure 1). Exogenous pathway is linked to chylomicrons, which comes from intestinal absorption
of dietary fats. Intestinal cells synthetize apolipoprotein (Apo) B-48 to incorporate it to triglycerides
that passes to the lymph and to circulation in the form of Chylomicrons. Lipoprotein lipase (LPL),
which is attached to the luminal of capillary endothelial cells, hydrolyzes the fatty acids of
triglycerides allowing them to be taken by muscles or adipose tissues, or to be bound and

12. 5
transported in circulation to other tissues, including the liver, by albumin (Moffatt & Stamford
2005).
The endogenous pathway starts with the circulation of VLDL from the liver to the rest of the
body. VLDL released from the liver contains Apo B100, cholesterol, cholesterol esters and
triglycerides, as well as Apo C-I, C-II, C-III and E. When it goes into the bloodstream, VLDL picks
up Apo CII (essential for LPL’s operation) and Apo E given by HDL, becoming a mature molecule
that can get to peripheral adipose tissue and muscles to deliver its content by hydrolysis reaction
with LPL. The composition of VLDL changes and it becomes IDL that can be hydrolyzed further
by hepatic LPL, to form LDL cholesterol (LDL-C). This LDL-C in circulation can go back to the
liver or be absorbed by the cells in different tissues. The absorption of LDL-C in tissues occurs due
to LDLR presence at cell surface. LDLR is a protein that binds to Apo B-100 and takes LDL-C
from circulation (Moffatt & Stamford 2005).
Figure 1: Lipoprotein metabolism, endogenous and exogenous (Crawford M.H. Current Diagnoses & Treatment:
Cardiology, 3rd
Edition). Exogenous pathway includes dietary absorption of fat by the small intestine into circulation in
the form of chylomicrons; and endogenous pathway showing biosynthesis of fats and cholesterol in the liver for sending
VLDL to peripheral tissues as well as absorbing remnants chylomicrons and LDL back into the liver. LDL absorption in
the cell is mediated by LDLR.

13. 6
3. LDLR and PCSK9
LDLR is a single chain transmembrane glycoprotein that specifically binds lipoproteins
containing Apo B-100 or the active form of Apo E to its class A domain. This specific domain
contains calcium ion and six cysteine residues that participate in the formation of a disulphide bond
between the LDLR and LDL-C (Yamamoto et al. 1984). For the endocytosis process, the LDLR is
located in membrane pits coated with protein clathrins. The pits invaginate to form coated endocytic
vesicles that fuse to form endosomes. The LDLR dissociates from LDL and returns to the cell
surface in a recycling process (Goldstein & Brown 1987) (Figure 2).
Figure 2: Internalization of LDL-C by LDLR. Namrata Chhabra 2016. http://i0.wp.com/www.namrata.co/wp-
content/uploads/2012/11/cholesterol-metabolism.jpg?resize=628%2C484
The Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) is a serine endoproteinase that
plays a crucial part in LDL-cholesterol (LDL-C) metabolism due to its role in the degradation of the
LDLR. PCSK9 was first described when a family with Familial Hypercholesterolemia (FH) was
found to have a mutation for PCSK9, which was later described as a “gain of function” mutation,
meaning an increase in the PCSK9 activity (Abifadel et al. 2003). PCSK9 cuts itself to become an
escort protein for LDLR, leading it into lysosomes for degradation. This interaction prevents LDLR
to adopt a closed conformation in the endosome, thus becoming a target for lysosome degradation
(Figure 3) (Mbikay et al. 2013). Produced mainly in the liver, PCSK9 can also be synthesized in the
intestine, brain and kidneys and its levels are measurable in human plasma. By downregulating
LDLR, PCSK9 decreases hepatic clearance of LDL-C (Druce et al. 2015). The congenital or
induced increase of this function can lead to hypercholesterolemia; its decrease causes
hypercholesterolemia.

14. 7
Interestingly, LDLR and PCSK9 are both transcriptionally regulated by the sterol regulatory
element-binding protein-2 (SREBP-2), a transcription factor that activates many genes involved in
cholesterol metabolism; therefore, as more LDLR is made, more PCSK9 is also produced. By this
mechanism, PCSK9 can prevent disproportionate cholesterol accumulation inside the cell by
averting the recycling of LDLR to the cell surface. This makes PCSK9 an important target to
inactivate in the treatment of hypercholesterolemia. Nevertheless, the side effects of this
inactivation have not yet been completely studied (Mbikay et al. 2013).
Figure 3: Degradation of LDLR mediated by PCSK9. The complex made out of LDL-C and LDLR is endocytosed
by cell to be after degraded in lysosomes (Lambert et al. 2012)
4. Hypercholesterolemia
Hypercholesterolemia is a condition that shows an increase of LDL-cholesterol in the plasma,
which can contributes to increase the risk developing cardiovascular disease (CVD).
Hypercholesterolemia can be classified as primary and secondary, according to their causes. Among
the primary causes it is possible to find single or multiple gene mutations. Among these gene
mutations, there are defects in the LDLR which diminished LDL clearance, a condition inherence of
codominant or complex multiple genes that can be heterozygous (1/200 to 1/500), or homozygous
(1/1 million); familial defective Apo B-100, a dominant condition that is present in 1/700 of the
population and it also leads to a diminished clearance of LDL. It is also possible to find PCSK9
gain of function (GOF) mutations, where PCSK9 presents an augmented function; LPL deficiency
which causes an endothelial LPL defect, and Apo C-II deficiency which also leads to LPL
deficiency. These two last conditions are recessive and very rare, but present worldwide. As
secondary causes, the most important in developed countries is a sedentary lifestyle accompanied

15. 8
by a high dietary intake of fat. Other common secondary causes are diabetes, hypothyroidism,
alcohol overuse, etc. (Goldberg 2015a).
Familial Hypercholesterolemia (FH) is a genetic condition characterized by high plasmatic
levels of LDL-C due to the different mutations mentioned before. It is present worldwide but with
an increased rate among French Canadian, Lebanese and south African populations. Untreated
adults show low-density lipoprotein cholesterol (LDL-C) levels >190 mg/dL (>4.9 mmol/L) or total
cholesterol levels >310 mg/dL (>8 mmol/L) (Youngblom & Joshua 2014). Heterozygous FH
(HeFH) can have a prevalence of 1:200 to 1:500, making it a rather common disease. These patients
can present two to threefold higher than normal LDL-C levels in blood. On the other hand,
homozygous FH (HoFH) is a more rare condition, affecting one in 300,000–1,000,000 individuals.
These patients can present three to sixfold higher than normal LDL-C levels, which can lead to
early development of cardiovascular disease or other pathologies related to high level of LDL-C
(Krähenbühl et al. 2016).
Diagnosis of FH is made by genetic testing considering three different genes for LDLR, ApoB
and PCSK9, which can present mutations, being LDLR mutation known as the most common with
60 to 80% of the FH cases (Krähenbühl et al. 2016). Around 5 to 10% of the patients with FH
present mutations in the ligand-binding domain for Apo B in LDLR (Lambert et al. 2012).
Combined mutations of these genes can also be found (Krähenbühl et al. 2016).
5. Statins and anti-PCSK9 as hypolipidemic treatments
Statins: Statins are the first choice of drugs for the treatment of hypercholesterolemia. Reducing
LDL-C levels in circulation with statins treatment has shown a positive correlation with the
reduction of CVD. Statins are currently the most powerful drug used for cholesterol-lowering,
decreasing LDL-C levels by 20 to 50% (Baigent et al. 2010).
The mechanism of action of statins involves the inhibition of HMG CoA reductase, the enzyme
that converts HMG-CoA into mevalonic acid, a cholesterol precursor. The inhibition of HMG-CoA
reductase determines the reduction of intracellular cholesterol. This reduction is mediated by the
activation SREBP-2, increasing LDLR expression, leading to an augmentation of the clearance of
LDL-C, but also lead to an increase of PCSK9 plasmatic levels (Horton et al. 2007).
Statins can also reduce hepatic synthesis of Apo B-100, leading to a decrease in the synthesis
and secretion of triglyceride rich lipoproteins and an increase of receptors production for
apolipoproteins B/E. This can explain why statins are capable of decreasing LDL-C in patients with
HoFH, where LDLR is not functional (Taylor et al. 2011).

16. 9
Furthermore, the inhibition of the HMG-CoA reductase pathway results in the subsequent
inhibition of the production of specific prenylated proteins. This may be involved in the
improvement of endothelial and immune function, and other pleiotropic cardiovascular benefits of
statins. High levels of LDL-C in plasma can decrease the capacity of endothelial cells to produce
Nitric Oxide (NO), most likely due to the reduced availability of L-arginine, a physiological
substrate of NO synthase, which leads to an increased reduction of NO. The reduction of cholesterol
by statins can lead to an increase of the endothelial function. Some statins can induce the
transcriptional activation of eNOS, a gene in human endothelial cells. Additionally, statins can
inhibit tumor cells growth and enhance intracellular calcium mobilization (Stancu & Sima 2001).
Since the early 1990, different reviews on the effects of the statins have been published,
highlighting its benefits, particularly in reducing the risk of CVD, regardless the sex or age of
patients (Taylor et al. 2011).
When treatment with statins is performed, the results obtained will depend on the extent of
LDL-C lowering to reach a target level. To decide whether or not using statins, is necessary to
evaluate the risk of CVD for each patient, together with the history of acute myocardial infarction
or stroke, percentage of reduction of LDL-C required to achieve the target level, and choosing a
statin treatment that can provide this specific reduction. Since the response to a statin treatment can
vary from one patient to another, the doses adjustment to reach target levels is mandatory. When the
treatment with statins by itself fails, drug combination treatment should be considered. Target levels
of LDL-C are defined by the patient’s cardiovascular risk. According to the European guidelines,
the presence of atherosclerotic cardiovascular disease, for example, a history of acute myocardial
infarction or stroke, defines an LDL-C target 1.8 mmol/l (70 mg/dL). In primary prevention of
patients with diabetes mellitus, FH, or multiple risk factors leading to the estimation of high
cardiovascular risk, an LDL-C level 2.6 mmol/l (100 mg/dL) should be targeted. If these absolute
treatment goals are not reached, LDL-C levels should be at least halved (Reiner et al. 2011).
Nevertheless, in patients with HeFH, LDL-C target levels are frequently not reached via statins
alone because the baseline levels are very high. In patients with HoFH, the scarcity or even
complete absence of any functional LDLR makes the treatment with statins completely ineffective,
therefore an alternative or supplementary therapies are usually required (Krähenbühl et al. 2016).
Anti-PCSK9: SREBP-2 increases LDLR expression, increasing even more the clearance of
LDL-C, but also leads to an increase in the secretion of PCSK9 (Horton et al. 2007). For this
reason, the treatment with monoclonal antibodies (mAbs) anti-PCSK9 consists in a new strategy
that is beginning to show some promising results. There are currently three different monoclonal
antibodies for PCSK9: evolocumab, alirocumab, and bococizumab that are now being tested in

17. 10
clinical trial programs and appear to be very effective at reducing LDL-C levels, achieving an
additional 60–75 % reduction in patients treated with statins (Dorey 2015).
In 2015, evolocumab and alirocumab have received marketing authorization in the EU and the
USA. Currently, the cost of monoclonal antibodies may limit its use to patients with very high
LDL-C, patients intolerant to statins, or where statins are not efficient enough to reach target levels,
like in patients with HoFH with no residual LDLR function. The considerable potential health
benefits and savings in healthcare costs from preventing CVD events will need to be weighed and
compared against the likelihood of adverse effects of long-term statin therapy and the costs of
alternative treatments (Krähenbühl et al. 2016).
Other alternative treatments to hypercholesterolemia include mipomersen, an apoB synthesis
inhibitor, and lomitapide, a microsomal triglyceride transfer protein (MTP) inhibitor. This drugs act
preventing the production of chylomicrons, VLDL and LDL. The use of this treatment can lead to
an accumulation of hepatic fat, thus its use is currently authorized for the treatment of homozygous
FH only (Krähenbühl et al. 2016).
In a recent study, the use of alirocumab produced an important reduction in LDL-C levels, when
administrated with placebo or in combination with ezetimibe, another lowering-lipid drug that
reduces the absorption of cholesterol in the small intestine. Alirocumab showed a maximal
reduction of 47% when combined with placebo, and 54 to 57% when combined with non-statins
lowering lipid treatment such as ezetimibe and fenofibrate (Rey et al. 2016).
The viability of mAbs anti-PCSK9 as a therapy now depends on their safety profile. The use of
this therapy, chronically exposing the patient to mAbs has concerns regarding hypersensitivity and
immune response (Rallidis & Lekakis 2016).
6. Type 2 Diabetes, Statins and anti-PCSK9
T2D is a disease characterized by defects in GSIS and insulin action. Problems in the function
of pancreatic β-cells that can no longer compensate insulin resistance in the target cells, lead to the
development of hyperglycemia in individuals at high risk of TD2, especially in those with a family
history and genetically predisposed. Excessive rates of dyslipidemia and high LDL-C rates have
been shown to precede the development of T2D in predisposed subjects (Kashyap et al. 2003).
T2D and statins: statin therapy has been recently associated with a slightly enhanced risk of
T2D (Sattar et al. 2010). In a systematical review conducted by Taylor et al, with data collected
from two different statin-based trials, it was shown that out of 12 202 participants, only 2.8% of the

18. 11
statins-treated patients developed diabetes, compared to 2.4% participants on control or placebo,
with a relative risk of developing diabetes of 1.18 (95% CI 1.01 to 1.39) (Taylor et al. 2011).
According to this review, this increased incidence of diabetes seems to be linked to baseline fasting
glucose levels and metabolic syndrome among participants randomized to statins. The effects of
statins and the presentation of the disease can also vary according to statin dose and type, sex and
pre-existing conditions. Some clinical studies have shown a relationship between the use of statins
and the incidence of diabetes with a dose-dependent association. In absolute terms, there were two
additional cases of diabetes per 1000 patient-year between those getting high dose therapy vs.
patients in moderate therapy (Preiss et al. 2011).
Statins can be classified according to tissue selectivity in hydrophilic and lipophilic. Both types
of statins have shown different effects on insulin resistance in several studies (Lim et al. 2014).
Hydrophilic statins, such as pravastatin and rosuvastatin are known to have fewer side effects; in
contrast, lipophilic statins, such as simvastatin, can cause unfavorable pleiotropic effects such as
reductions in insulin secretion and increase on insulin resistance. In an in vitro study from Yada et
al. simvastatin altered GSIS of murine pancreatic islets, whereas pravastatin did not (Yada et al.
1999). Lipophilic statins inhibit the synthesis of isoprenoids and suppress ubiauinone/coenzyme
Q10 biosynthesis, thus delaying the formation of adenosine triphosphate by pancreatic β-cells,
leading to impaired insulin secretion (Lim et al. 2014). Interestingly, an in vivo study in Otsuka–
Long–Evans–Tokushima Fatty (OLETF) rat, a diabetic strain that develops T2D after 24 weeks of
age, suggests that pravastatin exerts a preventive and therapeutic effect on the progression of TD2
by pleiotropic effects. Pravastatin enhances the activity of superoxide dismutase (SOD) in the
pancreas when it is given long-term treatment, also it downregulates the expression levels of
transforming growth factor (TGF β-1) and tumor necrosis factor (TNF-α), decreasing the proportion
of fibrosis in the pancreas of OLETF rat, thus showing anti-oxidative, anti-inflammatory and anti-
fibrotic effects supporting pravastatin clinical use in T2D patients and in chronic pancreatitis (Otani
et al. 2010). Nevertheless, in a recent study from Lorza-Gil et al., it was demonstrated that
pravastatin treatment for a period of at least two months in hypercholesterolemic mice model can
disturb glucose homeostasis and cause reductions on insulinemia and in ex-vivo GSIS of isolated
pancreatic islets (Lorza-Gil et al. 2016). This prolonged pravastatin treatment impaired the function
of the exocytosis machinery and increased β-cells death most likely as a result of the oxidative
stress, suggesting that prolonged use of statins may have a diabetogenic effect (Lorza-Gil et al.
2016).
The JUPITER Study revealed that there is a potential sex difference for incidence of diabetes
under statins. Sex stratification in this study revealed that the diabetes risk was increased by 49% in

19. 12
women and only 14% in men. This points out the importance of analyzing and reporting efficacy
and safety data in both men and women, both separately and combined (Mora et al. 2010).
High body weight is one of the most important risks factors in the developing insulin resistance
and the subsequent T2D. Statin treatment can increase bodyweight in some patients, which might
partly explain the higher risk of T2D in statin-treated patients (Swerdlow et al. 2015).
Cholesterol overloading in pancreatic β-cells that could be induced with statin treatment could
also be implicated. Indeed, the addition of LDL at a physiological concentration to cultured mice
islets has been associated to an impaired GSIS and an increase β-cells apoptosis (Rütti et al. 2009)
(Kruit et al. 2010).
Apparently, LDL seems to affect only the secretory machinery and not the insulin production
inside the cells, since there was no observable difference on the total insulin content or insulin
mRNA level between LDL-treated and untreated islets. In addition to this, a study of Bogan et al.
indicates that the excess of cholesterol in pancreatic β-cells tends to accumulate in the insulin
granules, turning them into the major site of cholesterol accumulation in β-cells. The excess
cholesterol modifies granule size and interferes with membrane remodeling, which might explain
the impairment in insulin secretion (Bogan et al. 2012). This effect seems to be reliant on LDLR,
because LDL did not impair maximal insulin secretion of islets from knockout mice for LDLR,
suggesting a key role of the LDLR in LDL cholesterol-induced β-cell dysfunction. Furthermore,
there is epidemiological data that indicates that FH patients, carrying LDLR mutations have lower
risk of developing T2D. This could raise the probability of an underlying relationship between the
transmembrane carriage of cholesterol, regulated by LDLR and T2D (Besseling et al. 2015)
T2D and anti-PCSK9: For the impact of PCSK9 and, therefore, of PCSK9 inhibitors on the
development of T2D, the evidence is still unclear. An assay performed by Mbikay et al, made in
PCSK9-null male mice suggests that PCSK9 deficiency may have an impact in glucose
homeostasis, with reduced pancreatic content and plasma levels of insulin (Mbikay et al. 2010). On
the other hand, a study performed by Langhi et al. shows that PCSK9 deficiency in PCSK9 null
mice did not alter in vitro GSIS in mouse islets, and, the glucose tolerance in vivo (Langhi et al.
2009).
Currently, two large clinical studies are being performed to evaluate the safety and effectiveness
of the treatment with anti-PCSK9 alirocumab and evolocumab (PROFICIO and ODYSSEY
respectively). Most recently, an analysis of Phase 3 studies from the ODYSSEY program showed
no evidence on the incidence between new onset diabetes and alirocumab treatment. Neither was
any evidence of any effect on transition from normo-glycaemia to pre-diabetes. This analysis was

20. 13
performed with the currently available data, in 3448 individuals without diabetes in a period of 6-18
months. Nevertheless, a bigger sample and longer follow-up are still needed to categorically dismiss
any effect of anti-PCSK9 (Colhoun et al. 2016).

21. 14
II PERSONAL STUDY:
Here, we would like to investigate how PCSK9 and statins modulate LDLR pathway in
human β-cells and how this can alter their GSIS. In order to do this, we performed a series of in
vitro analysis with human pancreatic beta cells, the EndoC-BH1 cells, previously treated with or
without mevastatin, and supplemented with or without PCSK9 and with or without LDL. First, we
analyzed the LDLR expression at the surface of cells. Then, we evaluated the LDLR functionality
by measurement of LDL uptake. Afterwards, we measured the capacity of secretion of PCSK9 by
EndoC-βH1 cells, and how a LDL overload can affect it. Finally we estimated the GSIS of EndoC-
BH1 cells in these conditions.
1. Materials and methods
1.1 EndoC-βH1 human pancreatic cells
EndoC-BH1 are human pancreatic β-cells immortalized by transgenes (figure 4)
(Endocells©). These cells secrete insulin in response to glucose stimulation, among other specific
β-cell markers, such as the glucose transporter SLC2A2, the insulin-processing enzyme PCSK1 and
genes that are associated with the β-cells secretory machinery. EndoC-BH1 cells are able to
maintain all the properties of primary human adult β-cells but, unlike them that proliferate really
poorly, EndoC-BH1 cells are continuously expanding. These cells were obtained from human fetal
pancreatic buds that were transduced using lentivirus. (Ravassard et al. 2011)
Figure 4: Schema of the experimental procedure of producing the Endoc βH1 cells: Step 1: transduction of human
fetal blanks with a lentiviral vector RIP-SV40LT. Step 2: transplantation of SCID mice transduced tissue. Formation of
insulinoma occurs after few months. Immunohistochemical analysis of a section of the insulinoma showing a large
number of β-cells (red). Step 3: insulinomas are collected and separated to transduce the cells with a new vector
expressing human telomerase reverse transcriptase (hTERT). These newly transduced cells are transplanted into a SCID
mouse and lead to the formation of a new insulinoma. Step 4: insulinoma is taken, dissociated and amplified in culture,
resulting in a pancreatic cell line.

22. 15
1.1.1 Matrigel Support
The EndoC-BH1 cells are cultured on a matrigel composed by DMEM 4,5g/L of glucose
(11965092, Sigma-Aldrich®, Saint Louis MO, USA), supplemented with extracellular matrix
(ECM) (E1270, Sigma-Aldrich®, Saint Louis MO, USA) Penicillin/streptomycin 1% (Sigma-
Aldrich®, Saint Louis MO, USA), and fibronectin 2 µg/mL (F1141 Sigma-Aldrich®, Saint Louis
MO, USA). This homogenized solution is put on the wells covering all it surface and it is incubated
for a minimum of 1 hour at 37°C. Before seeding, the excess of coating medium must be eliminated
gently by aspiration.
1.1.2 Culture medium
The culture medium use to maintain EndoC-BH1 cells is composed of DMEM low glucose
5,6 mM/L (11885084 Sigma-Aldrich®, Saint Louis MO, USA), supplemented with 2% of
Albuminum Bovine Serum (BSA) fraction V (10775835001, Roche Applied Science, Mannheim,
Germany), 50 µM of 2-mercaptoethanol (60-24-2, Sigma-Aldrich®, Saint Louis MO, USA), 10mM
of nicotinamide (481907 VWR, Radnor, USA), 5,5 µg/mL of transferrin (T8158 Sigma-Aldrich®,
Saint Louis MO, USA), 6,7 ng/mL of sodium selenite (21,448-5 Sigma-Aldrich®, Saint Louis MO,
USA) and 100 µg/mL of Penicillin/streptomycin (Sigma-Aldrich®, Saint Louis MO, USA). The
medium is sterilized by filtration with a 0,22 µm filter and stored at 4°C.
1.1.3 Thawing
The EndoC-BH1 cells are cryopreserved in cryotubes at -80°C in fetal bovine serum (FBS)
(Biosera, Nuaille, France) with 20% dimethyl sulfoxide (DMSO) (Sigma-Aldrich®, Saint Louis
MO, USA). DMSO is used for avoiding water crystallization inside of the cells, which can lead to a
subsequent rupture when the cell is frozen. It is a toxic compound for cells at 37°C. For this
reasons, thawing must be done in the shortest time possible. Each cryotube is agitated in a bath at
37°C until a small ice crystal is detached. Then, the content of the cryotube is immediately
transferred to a falcon tube containing 9ml of culture medium previously warmed at 37° and
centrifuge for 5 minutes at 1800 rpm. After centrifugation, the supernatant is gently eliminated and
the cells remaining in the pellet are re-suspended in culture medium before being counted.
1.1.4 Viable cell counting
To perform cell counting, a Malassez glass is used. The Malassez is a counter grid made of
glass that has 10 columns and 10 lines. After well mixing the suspension of cells, 40 µL are taken
and mix with 40 µL of PBS 0.01% eosin used to exclude dead cells from the counting. Contrary to
viable cells, dead cells are permeable to eosin and therefore, colored in red upon its contact. Then

23. 16
10 µL of solution is drop between the Malassez slide and a coverslip before counting, under an
optical microscope, the sedimentary cells inside the grid. Finally, the concentration of viable cells in
the solution is calculated knowing that the volume contained in one column is 0.01 µL.
1.1.5 Seeding and subculture
To be maintained, the cells are seeded on matrigel support at a density of 70.000-75.000
cells/cm2
and in a final culture volume in accordance with the support that can be seed (Figure 5 and
Table 1). In the case of subculture of cells, it is necessary to detach them first from the matrigel
support. To achieve this, the monolayer of cells is washed with D-PBS 1X at 37°C and
subsequently incubated with a solution of trypsin (25300054, Life technologies®, Bleiswijk,
Netherlands) at 37°C and 5% CO2 for 10 minutes. Once the cells are detached, they are taken from
the recipient, which can also be washed with culture medium to collect the greatest possible amount
of cells. A small sample is taken for counting as explained in the section 1.1.4. Then, the cell
suspension is centrifuged at 1200 rpm for 6 minutes, the supernatant is eliminated, and the cells are
resuspended in the appropriate volume of culture medium to be seeded in the previously described
conditions.
Figure 5: EndoC-BH1 cells after 3 days being cultured in a
support matrigel
100µm

24. 17
Table 1: Concentration of the cells and volume of culture medium for each different kind of support
cm2
70.000/cm2
75.000 cm2
Volume of
culture medium
T80 80 5.600.000 6.000.000 15ml
T25 25 1.750.000 1.875.000 5ml
6 well plate 9.6 672.000 720.000 2ml
12 well plate 3.5 245.000 252.500 1ml
24 well plate 1.9 133.000 142.500 0.5ml
Millicel 8 0.7 49.000 52.500 200µl
1.1.6 Cryopreservation
The cryopreservation of the cells can be performed at any time to preserve the cells that
were amplified by culture to be used after. Once the cells have been collected and counted they are
centrifuged at 1200 rpm for 6 minutes. The supernatant is eliminated and cells are re-suspended in
FBS cold (put at 4°C). The cryotubes must also be previously cold at 4°C. FBS 20% DMSO cold
must be added after, drop by drop, in equal quantity to the suspension to have a final concentration
of FBS 10% DMSO (1 ml per cryotube in total). Each cryotube can contain 9 to 10 million cells.
The cryotubes containing the cells must immediately be putted on the -80°C fridge inside a
polystyrene box. The use of polystyrene box will slow down the freezing speed to reduce cellular
death.
1.2 EndoC-BH1 cell seeding and culture for assay performing
1.2.1 Day 0: Cell seeding
A flat bottom 96-well plate or a Millicell EZ-slide from Millipore® (Merck Millipore,
Merck Life Science, Darmastadt, Germany ) for the confocal microscopy analysis, are previously
prepared with coating medium (as described in section 1.1.1). The cells are detached and counted as
described before (section 1.1.5). Then, the cells are seeded at a concentration of 70000 cells in 100
µL of culture medium per well, and incubated for 48 hours at 37°C and 5% of CO2 atmosphere
(Figure 6a).
1.2.2 Day 2: Deprivation of glucose and treatment with and without mevastatin (Except for
GSIS assay)
Excepted for GSIS assays, culture medium is replaced after 48 hours of incubation with 100
µL glucose starving medium composed of DMEM no glucose (11966025, Sigma-Aldrich®, Saint
Louis MO, USA) supplemented with 2,8 mM of glucose, 2% of BSA, 50 µM of 2-mercaptoethanol,

25. 18
10mM of nicotinamide, 5,5 µg/mL of transferrin; 6,7 ng/mL of sodium selenite and 100 µg/mL of
Penicillin/streptomycin (figure 6a). In the case of GSIS, culture medium is replaced after 48 hours
of incubation with 100 µL of fresh culture medium (figure 6b). In both cases, depending on the
conditions of the assay, the medium is supplemented with mevastatin (10µg/ml) or with DMSO (the
vehicle in which mevastatin is solubilized) for controls. Cells are next incubated for 16 hours at
37°C and 5% of CO2 atmosphere (Figure 6a).
a)
b)
Figure 6: a) Schema of the different procedures performed with the EndoC-ΒH1 cells. b) Schema showing the
treatment for GSIS assay, where starving medium is changed the day 3;

26. 19
1.3 PCSK9 secretion assay and measurement of cell surface LDLR expression
1.3.1 Day 3: Deprivation OF glucose and treatment with and without mevastatin with or
without PCSK9
On day 3, after 16 hours of incubation in glucose deprived medium with or without
mevastatin, culture medium is replaced with 50 µL of fresh glucose deprived medium with and
without mevastatin (10µg/ml) and with or without recombinant PCSK9 WT (6000ng/ml) or PCSK9
gain of function D374Y (PCSK9 GOF) (150 or 600ng/ml), which has commonly an affinity 10 fold
higher than the wild type variety. Cells are then incubated for 4 hours at 37°C and 5% of CO2
(Figure 6a).
1.3.2. PCSK9 secretion assay
For PCSK9 secretion assays, an excess of LDL (200 µg/ml) was added or not during the last
3 hours of incubation (Figure 6a). After this period of time, the supernatant is recovered and stored
at -20°C for subsequent quantification of secreted PCSK9 by ELISA (Quantikine ® ELISA; R&D
Systems, Minneapolis, USA)
1.3.2.1 Elisa test for PCSK9 (ELISA QUANTIKINE® R&D SYSTEMS)
This assay uses the quantitative sandwich enzyme immunoassay technique. The microplates
included on the assay have been coated previously with a specific antibody for human PCSK9.
Standards and samples are pippeted into the wells and the immobilized antibody bounds any
PCSK9 present. After washing away any unbound substances, an enzyme-linked polyclonal
antibody specific for human PCSK9 is added to each well. After another wash to remove any
unbound antibody-enzyme reagent, a substrate solution for the enzyme is added to the wells.
Finally, reaction is stopped by addition of an acid stop solution. The presence of enzyme and
substrate will induce a quantifiable coloration that can be read by an optical reader.
1.3.2.2 ELISA assay protocol
All the reagents in the PCSK9 ELISA must be put at room temperature before performing
the assay. For generating the frame of the assay, it is necessary to reconstitute the Human PCSK9
standard using the Calibrator Diluent RD5P. This reconstitution produces a stock solution of
40ng/mL. The stock solution is used to produce a dilution series where each tube has half
concentration than the precedent. Calibrator diluent RD5P is used for each dilution and is also used
as the zero standard. A 100 µL of Assay Diluent RD1-9 is added into each well. Then, 50 µL of

27. 20
each dilution prepared before and the samples are put into the wells. The plate is covered with a
plastic strip is incubated for 2 hours at room temperature on an agitator. After this time, a wash is
performed four times with wash buffer solution in the autowasher, programed previously with the
correct amount of wash per well and repetitions. After the wash, it is important to eliminate any trail
of wash buffer with paper towels. Afterwards 200 µL of human PCSK9 Conjugate (the enzyme-
linked polyclonal antibody specific for human PCSK9) is added to each well, cover with adhesive
strip and leave for incubation for 2 hours a room temperature. The previous washing procedure
described is repeated once again after the incubation period. Finally, 200 µL of Substrate Solution
are added to each well and incubated until a reaction is visualized (around 15 minutes or less).
During this period, the plate must be protected from the light. After the incubation period is finished
and reaction is observed, 50 µL of Stop solution is added to each well. This produces a change in
the color of the wells, turning them from blue to yellow. The optical density of each well must be
measure within 30 minutes, using a microplate reader set at 450 nm. The concentration of PCSK9 in
the samples is established thanks the standard curve (optical density in function of the standard
concentration).
1.3.3 Cell surface LDLR expression measurement
After 4 hours of incubation, the monolayer of cells are washed with PBS, detached from the
matrigel with accutase (Sigma-Aldrich, Saint Louis, USA) (50 µL per well, 10 min at 37°C) and
transferred to wells from a 96-well plate with v-bottom.on ice The accutase mimics the action of
trypsin but it has a more gentle action, preserving most of the epitopes for further cytometry
analysis. Cells are subsequently washed two times with cold PBS 1% BSA (200 µL/well) : PBS 1%
BSA is added to each well, a centrifugation during 1 minute at 1000g (2500 rpm) is performed and
supernatants are eliminated. Then, cells are incubated in 40 µL of PBS-1% BSA containing an
antibody anti-human LDLR conjugated to a fluorochrome of allophycocyanine (APC) (clone
#472413) (R&D Systems, Lille, France) (at a concentration 1/40), for the control, isotypic control
IgG1 conjugated with fluorochrome APC (clone #11711) (R&D Systems, Lille, France), for 20
minutes at room temperature and protected from the light. Finally, cells are washed twice with cold
PBS-1% BSA (200 µL/well), one time in cold PBS and fixed in PBS-0.5% paraformaldehyde
(PFA) before being analyzed by flow cytometry (as described in section 1.6).
1.4 LDL uptake measurement
The day 3, cells are treated as described in the section 1.3.1. However, LDL-BODIPY (Life
Technologies, Invitrogen, Saint Aubin, France) is added into wells at a final concentration of 10

28. 21
µL/mL for the last 3 hours of incubation (Figure 6a). Controls without LDL-Bodipy are keep in
parallel to determine the autofluorescence of cells. Then:
- For confocal microscopy analysis, cells are subsequently washed two times with cold PBS
1% BSA (200 µL/well) and one time with cold PBS before to be fixed in PBS containing
4% PFA (Sigma Aldrich) for 15 min at room temperature. Next, cells are washed one time
in PBS and slides were mounted with cover slides in Prolong antifade reagent containing
DAPI (Life Technologies) and visualized on a confocal A1 N-SIM microscope (Nikon,
Melville, New York).
- For flow cytometry analysis, cells are subsequently washed with PBS, detached from the
matrigel using accutase and transferred to wells from a 96-well plate with v-bottom. Finally,
cells are washed two times with cold PBS 1% BSA (200 µL/well) and one time with cold
PBS before to be analyzed by flow cytometry (as described in section 1.6) in presence of
trypan blue (0.2%) to quench the fluorescence of cell surface bound LDL-BODIPY.
1.5 Glucose stimulated insulin secretion (GSIS) assay
To perform GSIS assay, the cells are seeded and cultured as described in section 1.2.1 and
1.2.2
On day 3 of culture, the cells are gently washed with PBS, then 50 µL of glucose starving
medium supplemented with and without mevastatin (10µg/mL) and with or without 600 ng/mL of
PCSK9 GOF is added to wells. The cells are incubated for 1 hour at 37°C. After this time, LDL is
added to the corresponding wells for a final concentration of 200µg/mL. Once again, the cells are
incubated for 3 more hours at 37°C (Figure 6b). After this incubation, cells are gently washed one
time with Krebs-Ringer solution that is composed by 3 different solutions that are prepared
separately the day before the assay and then mixed. The Krebs-Ringer solution is made out of 25%
of solution 1, 25% of solution 2 and 25% of solution 3 (Table 2), 0.2% of BSA, Hepes 10 mM and
water (added at a maximum of 25%) (Table 2).
Table 2: Components of the different solutions used in the Krebs-Ringer solution
Solution 1 Solution 2 Solution 3
5,38g NaCl
200mL H2O
1.61g NaHCO3
300mg KCl
76mg MgCl2
200mL H2O
117.6mg CaCl2-2H2O
200mL H2O

29. 22
Then, cells are incubated for 1 hour in Krebs Ringer solution supplemented with, 2.8, 5.6 or
15 mM of glucose ±0.1 µM of exendin-4 or phosphodiesterase inhibitor IBMX (500µM) with or
without PCSK9 GOF (600ng/ml) and with or without LDL (200µg/mL). Exendin-4 is a molecule
analogue of Glucagon-Like Peptide 1 (GLP1) that stimulates adenylate cyclase and thus increases
insulin secretion. IBMX is a non-specific inhibitor of cAMP and cGMP phosphodiesterases, which
stimulates insulin release.
1.5.1 Recovering of culture supernatants
At the end of GSIS assay, culture supernatants are recovered on ice, centrifuged for 5
minutes at 1200g, transferred in a new plate and stored at -20°C. These supernatants can be used
afterwards to measure the insulin secreted by the cells by an ELISA test.
1.5.2 Cell Lysates
After the harvesting of the supernatants, a cell lysis is performed using lysis solution
(TETG). This solution’s components are described in the table below (Table 3). Additionally, anti-
protease (04693159001, Roche, Mannheim, Germany) must be added to 10 ml of this solution and
used fresh. From this solution, 50 µL are added to each well. The plate must be put on ice and
incubated for 5 minutes. The cell lysates are then transferred to a 96 well plate “v bottom” and
centrifuge at 1300g or 5 minutes. Then, supernatants of cell lysates are recovered and stored at -
20°C for performing subsequently the measurement of insulin content by ELISA.
Table 3: Components for Lysis cell solution (without protease)
Compound Stock Concentration Volume for a final solution of 50mL
Tris HCl pH8 0.5M 2mL
Triton 100X 1X, 0.5 mL
Glycerol Pure 5mL
NaCl 5M 1.37 mL
EGTA 0.2M 0.250 mL
Sterile water Pure 40.88 mL
1.5.3 Measure of the secreted and produced insulin by ELISA (MERCODIA INSULINE
ASSAY®)
After thawing the samples (culture supernatant and lysate supernatants) they are centrifuged
at 1000g for one minute, and kept on ice all the time. For performing the insulin ELISA test it is
necessary to dilute the samples: 1/21 for the supernatant samples and 1/1040 for the Lysate samples.
For performing this dilution, PBS, Krebs-Ringer solution, or TETG can be used. For generating the

30. 23
standard curve, the kit includes calibrators with different known concentrations of insulin. It is
preferable to make the assay in duplicate: 25 µL of each samples, lysate or supernatant and
calibrator are put into each well. A 100 µL of enzyme conjugate 1X are added to each sample and
calibrator. This reagent must be previously prepared by diluting enzyme conjugate 11X in enzyme
conjugate buffer to obtain a final concentration 1X. The plate is incubated in a shaker (700-900
rpm) for 1 hour at room temperature (18-25°C). Afterwards, the plate is put on the autowasher to
perform a wash with 700 µL of wash buffer 1X solution per well. This process is repeated 6 times.
After the last wash process is finished, the plate is inverted and press firmly against absorbent
paper. Then, 200 µL of TMB solution is added into each well and incubated for 15 minutes at room
temperature. After, 50 µL of stop solution are added into each well. To make sure to stop the
reaction, the reagents must be well mixed. Once the reaction is been stopped, the optical density is
measure in a fluorescence multilabel plate reader Victor® (Perkin Elmer, Waltham, USA) at
450nm.
The data obtained are analyzed through the standardization by the standard curve, which
make it possible to calculate the concentration of insulin in each sample. Then, by determining the
absolute quantity of insulin in the culture supernatants and in cell lysates, the percentage of
secretion of insulin was calculated according to the following formula: absolute quantity of secreted
insulin/(absolute quantity of secreted insulin + absolute quantity of insulin in cell lysates) *100.
1.5.4 Protein measurement content
Protein quantitation is an integral part of the process to obtain the normalized value of a
sample measured by insulin ELISA. To perform this essay, the cell lysate obtained from the cells by
the addition of cell Lysis buffer TETG is used. A standard curve is prepared using BSA fraction V
(BIORAD, Berkeley California, USA), starting with 4 mg/mL diluting successively by 2 (2, 1 0.5,
0.25, 0.125, 0.0625 and finally 0 gm/mL). 5 µL of sample or standard are used for each well. Then,
solution A’ and B are added to the sample or scale. After 15 minutes de reaction can be measured.
The assay is based on the reaction of protein with an alkaline copper tartrate solution and Folin
reagent, making it a colorimetric assay where the absorbance of each sample is read with multilabel
plate reader Victor® (Perkin Elmer, Waltham, USA) at 750nm. Once the optical density is
obtained, it is necessary to create a standard curve to calculate the concentration in each sample and
then see the absolute quantity of protein in each well, according to the mL contained on each.

31. 24
1.6 Cell staining analysis by Flow Cytometry
The cell staining was analyzed using a flow cytometer LSR II (BD biosciences, San José,
USA). The diffraction of the light by the cells allows to study different parameters like size (FSC)
and granularity (SSC) and to select the live cells inside the sample. Two kind of fluorochromes
were used in our assays (Table 4)
Table 4: Fluorochromes used in flow cytometry.
Optimal wavelength
of excitation (nm)
Optimal wavelength
of emission (nm)
Laser used
Allophycocyanine 633 660 Red laser 640nm
40mW
BODIPY 488 525 Blue laser 488nmm
50mW
The data obtained by measurement of flow cytometry is analyzed with the software
FlowJo©. The variable studied is called ΔMFI and is defined as the difference of mean fluorescence
intensity of the sample with the mean fluorescence intensity of the corresponding control (isotypic
control or autofluorescence). This value corresponds to the specific fluorescence that can be
attributed to the sample.
The data acquired by flow cytometry were subsequently analyzed with the software
FlowJo© (Tree star Inc., Ashland, Oregon, USA). The variable studied is called «ΔMFI» and is
defined as the difference of mean fluorescence intensity between the specific staining and the
controls (Isotype control for anti-LDLR staining and autofluorescence for LDL-Bodipy uptake
assays). This value corresponds to the specific fluorescence that can be attributed to the sample.
1.7 Statistical analysis
All the data obtained can be introduced in the software Graphpad Prism® (GraphPad
Software, Inc., La Jolla, CA 92037 USA) for further data analysis and graphics. Significance
difference between the values obtained for the different assays is calculated using the non-
parametrical test Mann-Whitney. Results are expressed as means with standard error of the mean
(SEM). For p value * p<0.05, ** p<0.01 and ***p<0.001.

32. 25
2. Results
2.1 Statins and PCSK9 modulates LDL receptor expression in EndoC-βH1 cells
First, we measured cell surface LDLR expression levels in EndoC-βH1 cells by flow
cytometry, after treatments with or without mevastatin and supplementation with or without
recombinant PCSK9 gain of function (GOF) or wild type WT (figure 7).
c)
d)
a) b)
Mevastatine 0μg/mL +PCSK9 600 ng/Ml
+LDLR APC
Mevastatine 0μg/mL + LDLR APC
Mevastatin 0µg/ml + IgG APC
SSC-A
Text

33. 26
Figure 7: Statins and PCSK9 regulate cell surface LDLR expression in EndoC-βH1 cells. EndoC-β cells were
cultured with glucose starving medium with or without mevastatin (10 µg/mL) for 16 hours and subsequently
supplemented with recombinant PCSK9 GOF (150 or 600 ng/ml) of WT (6000ng/ml) for 4 hours before to be stained
for cell suface LDLR expression and analyzed by flow cytometry. a) Representative dot plot showing size (FSC-A) and
granularity (SSC-A) of samples from the experience. The defined window corresponds to the analyzed living cells. b)
Representative histogram showing overlays of anti-LDLR stainings in the conditions without mevastatin, without
mevastatin and with PCSK9 GOF (600 ng/ mL), and of the isotypic control staining. c) and d) Histograms represents
means +/- SEM of results expressed in ΔMFI as difference between the mean fluorescence intensity (MFI) of the LDLR
staining and the isotypic control. (ΔMFI = MFI of cells marked with anti-LDLR APC – MFI of the cells marked with
IgG-APC). c) n=6 and d) n=2 . * p<0.012, ** p<0.002.
We found that EndoC-βH1 cells displayed a basal cell surface LDLR expression level with
an average ΔMFI of 2981 (Figure 7c). This level was significantly increased by the addition of
mevastatin to a ΔMFI = 6407 (+217%) whereas decreased by supplementation with PCSK9 GOF
(600ng/mL) to a ΔMFI = 553 (-81.45%) in condition without mevastatin and ΔMFI = 1424 (-
52.23%) in condition with mevastatin (Figure 7c). In addition, we observed that i) the decreased
LDLR expression level at the surface of EndoC-βH1 cells in presence of PCSK9 GOF was dose-
dependent: ΔMFI = 1423 (-52.78%) for PCSK9 150 ng/mL versus ΔMFI = 639 (-78.81%) for
PCSK9 600 ng/mL in condition without mevastatin (figure 7d), and ii) PCSK9 WT displayed an
inhibitory effect with a ΔMFI = 2063 in condition without mevastatin and ΔMFI = 4303 with
mevastatin, similar to the effects of PCSK9 GOF at 150 ng/mL (Figure 7d). In any case, the
addition of PCSK9 induced a decrease of cell surface LDLR expression, while the addition of
mevastatin significantly increased it (Figure 7).

34. 27
b)
2.2 Statins and PCSK9 modulates LDL uptake by EndoC-βH1 cells
Then, we evaluated the effects of mevastatin and PCSK9 on the capacity of EndoC-βH1
cells to uptake LDL. First, we performed an analysis by confocal microscopy (Figure 8a).
Figure 8: LDL uptake by EndoC βH1 cells and analyzed by confocal microscopy. Representative images of LDL-
bodipy uptake by EndoC βH1 cells obtained by confocal microcscopy. Cells were cultured with glucose starving
medium with or without mevastatin (10 µg/mL) for 16 hours and subsequently supplemented with recombinant PCSK9
GOF (600 ng/ml) and LDL-Bodipy for 3 hours. LDL-BODIPY is shown in red and cell nuclei stained with DAPI are
shown in blue.
We observed a basal level of LDL uptake by the Endoc-βH1 cells, which was further
increased by mevastatin treatment (Figure 8). The addition of PCSK9 reduced LDL uptake by
Endoc-βH1 cells in conditions with or without mevastatin. These observations were confirmed
afterwards by measurement of internalized LDL by flow cytometry (Figure 9).
a)
Mevastatine 0μg/mL
Mevastatine 0μg/mL +PCSK9 600 ng/mL
Autofluorescence
SSC-A

35. 28
c)
Figure 9: Statins and PCSK9 modulate the capacity of Endoc-βH1 cells to internalize LDL. Cells were cultured
with glucose starving medium with or without mevastatin (10 µg/mL) for 16 hours and subsequently supplemented with
recombinant PCSK9 GOF (600 ng/ml) and LDL-Bodipy (10 µg/mL) for 3 hours. LDL-bodipy uptake was analyzed by
flow cytometry after addition of trypan blue to quench the fluorescence of cell surface-bound LDL-BODIPY. a)
Representative dot plot showing size (FSC-A) and granularity (SSC-A) of samples from the experience. The defined
window corresponds to the analyzed living cells. b) Representative histogram showing overlays of LDL-Bodipy
stainings in the conditions without mevastatin, without mevastatin and with PCSK9 GOF and of the autofluorescence of
the cells. c) Histograms represent means +/- SEM of results expressed in ΔMFI as difference between the mean
fluorescence intensity (MFI) of LDL staining and the autofluorescence of cells. (ΔMFI = MFI of LDL-BODIPY – MFI
autofluorescence of the cells) * p<0.02, (n=4).
A basal level of internalized LDL by the Endoc-βH1 cells was observed with a ΔMFI of
44362 (Figure 9c). This level was significantly increased by the addition of mevastatin to a ΔMFI
60 964 (+137.42%) and on the contrary significantly decreased by the addition of PCSK9 GOF
(600 ng/mL) to a ΔMFI of 7652 (-82.75%) in conditions without mevastatin, and to a ΔMFI of
29260 (-34.04%) in condition with mevastatin (Figure 9c).

36. 29
2.3 Basal secretion of PCSK9 by EndoC-βH1cells and its modulation by statins
Moreover, we evaluated the capacity of Endoc-βH1 cells to secrete PCSK9 after treatment with
or without mevastatin and in presence or not of a physiological concentration of LDL (20mg/dL)
(Figure 10).
Figure 10: Basal secretion of PCSK9 by EndoC-βH1cells and its modulation by statins. Cells were cultured with
glucose starving medium with or without mevastatin (10 µg/mL) for 16 hours and subsequently supplemented with
LDL (200 µg/mL) for 3 hours. PCSK9 was measured in the supernatants of the cell culture by ELISA. Histograms
represent means with SEM of PCSK9 concentration in ng per millions of cells (n=8). *** p<0.0002; ns: No significant
difference with p>0.05
Interestingly, our results shows that (i) EndoC-βH1 cells present a basal secretion level of
PCSK9 with 5.27 ng/million of cells, (ii) this secretion can be significantly increased by the
addition of mevastatin (16.43 ng/million of cells, 3.11 fold), with a p<0.0002 and (iii) there is no
significant difference on the secretion of PCSK9 in the conditions with LDL, with a p>0.05 (Figure
10).
2.4 Statins and PCSK9 do not apparently alter GSIS
We measured the glucose-stimulated insulin secretion (GSIS) by the EndoC-βH1 cells to see
their ability to secrete insulin in a glucose-dependent manner, with or without the addition of
agonist as IBMX and Exendin 4, a glucagon-like protein. We assayed GSIS by EndoC-βH1 cells in
conditions with or without mevastatin, with or without PCSK9 and with or without a physiological
concentration of LDL (20mg/dL) (Figure 11).

37. 30
a)
b)
Figure 11: Statins and PCSK9 do not apparently alter GSIS. Cells were cultured in culture medium with glucose
with or without mevastatin (10 µg/mL) for 16 hours. Then, medium was changed for glucose starving medium with or
without mevastatin and supplemented with recombinant PCSK9 GOF (600 ng/ml) and LDL (200 µg/mL) for 3 hours
before to perform GSIS. a) Histograms represent mean +/- SEM of percentage of insulin content per well in function of
the different doses of glucose (gluc 2.8, 5.6 or 15 mM) in conditions with or without mevastatin, with or without
PCSK9 GOF and with or without LDL (n=3). The percentage of insulin was calculated using the absolute quantity of
secreted insulin divided into total of insulin (absolute insulin secreted + absolute quantity of insulin in cell lysate).
Results with SEM (n=2). b) Histograms represent mean +/- SEM of the percentage of insulin secretion relative to the
control 15mM glucose without mevastatin (10 µg/mL) (n=4).
The insulin secretion seemed to increase in a glucose-dependent manner (Figure 11a). The
addition of PCSK9 and or mevastatin had no effect on GSIS (Figure 11a and b). No significant
difference was observed between all the conditions with or without PCSK9 and with or without the
presence of LDL at physiological concentration (Figure 11b).

38. 31
3. Discussion
Our in vitro data indicates that human pancreatic β-cells express functional LDLR. As
expected, we found that the level of expression of this receptor at the surface of human pancreatic
β-cells can be significantly decreased with the addition of PCSK9 and increased with mevastatin
(Figure 7). We have confirmed that LDLR present on human pancreatic β-cells is functional as
shown by its capacity of LDL internalization. This capacity is modulated by the addition of
recombinant PCSK9, which can significantly decrease the absorption of LDL. The addition of
mevastatin, on the other hand, can significantly increase it (Figure 8).
According to the results we obtained, human pancreatic β-cells seem able to secrete
PCSK9 in a basal level and this secretion can be increased with mevastatin treatment (Figure 10).
This increased secretion is concordant with what is described in the literature, which states that
PCSK9 expression increases in response to statins, by its induction over SREBP-2 (Horton et al.
2007). Secretion of PCSK9 has been described before in other organs besides liver, like small
intestine and kidneys, but it is the first time it was observed in pancreatic β-cells (Mbikay et al.
2013). In our experimental conditions, the addition of LDL in the culture seems to have no
significant effect in the PCSK9 secretion, but it was possible to observe that the addition of LDL
can produce a change in β-cells morphology (data not shown), that we observed by optical
microscopy. In a study conducted by Bogan et al., this condition was further analyzed using
pancreatic mice islets. Using confocal microscopy, they observed a change in insulin granule size
that increased up to 80% higher, due to accumulation of LDL, showing that LDL can alter cell
morphology and membrane remodeling (Bogan et al. 2012). These findings suggest a potential
importance of LDL on impaired insulin secretion.
Additionally, we observed inhibitory effects of both recombinant PCSK9 WT and GOF
(D374Y) on LDLR expression. However differences are noted. The GOF mutation is described as
one of the main causes of FH due its higher affinity for LDLR and consequent higher rate of LDLR
degradation in comparison to WT (Schulz et al. 2015). In agreement with these characteristics, we
observed higher efficiency of PCSK9 GOF to reduce LDLR expression in β-cells than PCSK9 WT.
Because of the higher affinity of PCSK9 GOF for LDLR, we obtained a similar inhibitory effect
between the lower concentration of PCSK9 GOF at 150 ng/ml and PCSK9 WT at 6000 ng/ml
(Figure 7c).
Interestingly, we confirmed in our experimental conditions that EndoC-βH1 cells are able
to secrete insulin in response to increasing glucose concentrations, as described already (Ravassard
et al. 2011). Our preliminary data indicates that LDL overloading in EndoC-βH1 cells does not

39. 32
seem to significantly alter GSIS (Figure 11b). This differs from the in vitro published data from
Rütti et al., in which LDL in medium culture of human and mice isolated islets seems to decrease
their insulin secretion and proliferation (Rütti et al. 2009). In this study, the cells were incubated
with LDL for 4 days, while our time of culture with LDL was for 4 hours. The concentration used
by Rütti et al. was around 223 mg/dL, while our concentration was ten times lower. Nevertheless,
the use of statins in our experiments, unlike Rütti et al. experiences, means an overload of LDL for
cells, due to the significant increased LDL uptake with mevastatin shown in Figure 9. This use of
statin could compensate our shorter period of culture. Further analysis using different periods of
incubation could be useful for future research. Likewise, we observed that the addition of PCSK9
with or without LDL does not seem to produce any alteration on GSIS of β-cells. No significance
difference in the insulin secretion was found with the addition of mevastatin (Figure 11b). A study
published by Mbikay et al, performed in PCSK9-null male mice suggests that PCSK9 deficiency
could have an effect on glucose homeostasis since morphological abnormalities in mice pancreatic
islets are visible as signs of inflammation and early apoptosis with diminished pancreatic content
and reduced plasma levels of insulin (Mbikay et al. 2010). On the contrary, an in vitro assay
indicated that PCSK9 deficiency did not alter basal GSIS in mouse islets (Langhi et al. 2009). It
would be necessary to elucidate whether repeated action of PCSK9 can play a different role in
insulin secretion in β-cells. For now, we are not able to conclude on PCSK9 effect on insulin
secretion.
A recent study from Lorza-Gil et al., showed that pravastatin treatment has a negative
effect over insulin secretion in isolated pancreatic islets, in case of chronic treatment (Lorza-Gil et
al. 2016). In our case, we used mevastatin, a drug that has not being previously studied on its effect
on pancreatic β-cells. In our experience, no significant difference was found with the addition of
mevastatin. This discrepancy may be linked to the type of statins used in each assay that may reveal
a difference between mevastatin and pravastatin, and show the importance of the time of exposure
to the drug. In both cases, statins have shown to be a hypolipidemic drug that increases β-cells
capacity to internalized LDL.
It is important to point out that, compared to other tissues such as liver or kidney,
pancreatic islets have low activity of free radical detoxifying enzymes such as catalase, superoxide
dismutase (SOD) and glutathione peroxidase, therefore islets can be more sensitive to damage
caused by oxidative stress (Acharya & Ghaskadbi 2010). This must be taking into consideration
when comparing results from pancreatic islets and EndoC-βH1 cells.

40. 33
4. Conclusion
To conclude, our work indicates that human pancreatic beta cells in culture are able to
produce and secrete PCSK9 and that statins and PCSK9 can modulate their LDLR expression and
function. Finally, we have observed that the addition of mevastatin and PCSK9 do not apparently
alter GSIS of β-cells. Nevertheless, it is impossible to affirm PCSK9 can protect β-cells against the
noxious effect of LDL. Therefore in vivo and in vitro evidence needs further analysis and discussion
to reconcile them. It will be interesting to measure PCSK9 in plasma from prediabetic individuals
under statins to determine the putative relationship between PCSK9 levels and the development of
T2D. However it is necessary to perform more in vitro and in vivo, studies to evaluate if PCSK9
protects β-cells from an exaggerated LDL uptake, and therefore to be able to propose a PCSK9
protective effect on predisposed individuals from developing T2D.

41. 34
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44. 37
ABSTRACT
Introduction: Statins, common drugs used to treat hypercholesterolemia, slightly increase the risk
of type 2 diabetes (T2D). It is unknown whether new treatments with monoclonal antibodies
directed against Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9), an inhibitor for the
recycling of the LDL receptor (LDLR), may enhance this risk. Here, we wanted to evaluate how
statins and PCSK9 modulate the LDLR pathway of human pancreatic β-cells and how these
modulations can alter their glucose-stimulated insulin secretion (GSIS).
Materials and methods: EndoC-βH1 human pancreatic β cells were starved in glucose and treated
with or without mevastatin and with or without recombinant PCSK9. Bodipy-LDL or unlabeled-
LDL were added in some experiments. Cell surface LDLR expression was measured by flow
cytometry. Bodipy-LDL uptake was analyzed by flow cytometer and confocal microscopy. PCSK9
secretion in culture supernatants was quantified by ELISA. GSIS assays were performed in Krebs-
Ringer buffer with glucose increasing concentrations (2.8 to 15 mM) with or without exendin-4 or
phosphodiesterase inhibitor IBMX, with or without PCSK9, and/or an excess of LDL. Insulin was
measured in supernatants and cell lysates by ELISA.
Results: Cell surface LDLR expression and LDL uptake were significantly increased with
mevastatin whereas decreased with PCSK9. However, mevastastin and PCSK9 treatments did not
seem to alter GSIS of cells supplemented or not with LDL. Interestingly, EndoC-βH1 cells secreted
significant amounts of PCSK9 (5.27 ng/million of cells) that was increased with mevastatin
treatment but not affected by LDL supplementation.
Conclusions: Our in vitro data shows that PCSK9 and mevastatin modulate LDLR function and
expression in human pancreatic β-cells without apparently altering their GSIS, and, for the first
time, that human β-cells are able to secrete PCSK9.
Keywords: Statins, PCSK9, LDL, hypercholesterolemia, type 2 diabetes.