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International Journal of
Molecular Sciences
Article
PCSK9 is Expressed in Human Visceral Adipose
Tissue and Regulated by Insulin and Cardiac
Natriuretic Peptides
Marica Bordicchia 1, Francesco Spannella 1,2 , Gianna Ferretti 3, Tiziana Bacchetti 3,
Arianna Vignini 3, Chiara Di Pentima 1,2, Laura Mazzanti 3and Riccardo Sarzani 1,2 ,*
1Internal Medicine and Geriatrics, Department of Clinical and Molecular Sciences,
University “Politecnica delle Marche”, 60126 Ancona, Italy; marica.bordicchia@gmail.com (M.B.);
fspannella@gmail.com (F.S.); chiara.dipentima@live.it (C.D.P.)
2Internal Medicine and Geriatrics, “Hypertension Excellence Centre” of the European Society
of Hypertension, IRCCS-INRCA, 60127 Ancona, Italy
3Department of Clinical Sciences, Section of Biochemistry, Biology and Physics, School of Nutrition,
University “Politecnica delle Marche”, 60126 Ancona, Italy; g.ferretti@univpm.it (G.F.);
t.bacchetti@univpm.it (T.B.); a.vignini@univpm.it (A.V.); l.mazzanti@univpm.it (L.M.)
*Correspondence: r.sarzani@univpm.it; Tel.: +39-071-596-4595
Received: 9 November 2018; Accepted: 4 January 2019; Published: 9 January 2019
Abstract:
Proprotein convertase subtilisin/kexin type 9 (PCSK9) binds to and degrades the
low-density lipoprotein receptor (LDLR), contributing to hypercholesterolemia. Adipose tissue plays
a role in lipoprotein metabolism, but there are almost no data about PCSK9 and LDLR regulation in
human adipocytes. We studied PCSK9 and LDLR regulation by insulin, atrial natriuretic peptide
(ANP, a potent lipolytic agonist that antagonizes insulin), and LDL in visceral adipose tissue (VAT)
and in human cultured adipocytes. PCSK9 was expressed in VAT and its expression was positively
correlated with body mass index (BMI). Both intracellular mature and secreted PCSK9 were abundant
in cultured human adipocytes. Insulin induced PCSK9, LDLR, and sterol-regulatory element-binding
protein-1c (SREBP-1c) and -2 expression (SREBP-2). ANP reduced insulin-induced PCSK9, especially
in the context of a medium simulating hyperglycemia. Human LDL induced both mature and
secreted PCSK9 and reduced LDLR. ANP indirectly blocked the LDLR degradation, reducing the
positive effect of LDL on PCSK9. In conclusion, PCSK9 is expressed in human adipocytes. When the
expression of PCSK9 is induced, LDLR is reduced through the PCSK9-mediated degradation. On the
contrary, when the induction of PCSK9 by insulin and LDL is partially blocked by ANP, the LDLR
degradation is reduced. This suggests that NPs could be able to control LDLR levels, preventing
PCSK9 overexpression.
Keywords: PCSK9; natriuretic peptides; adipose tissue; lipid metabolism; LDL receptor; insulin
1. Introduction
Maintenance of optimal blood lipid levels is central to vascular health. Liver plays a key role in
lipoprotein metabolism, but much less is known about the role of adipose tissue. The adipose tissue is
involved in energy balance and energy storage. It has endocrine functions and plays a fundamental
role in the metabolism of triglyceride-rich lipoproteins. Recent studies suggest that thermogenic
“brown” adipocytes are also involved in lipoprotein metabolism. Brown adipose tissue not only takes
up triglycerides derived from plasma triglyceride-rich lipoproteins, but is also actively involved in the
metabolic flux of high-density lipoprotein (HDL)-cholesterol to the liver [1].
Int. J. Mol. Sci. 2019,20, 245; doi:10.3390/ijms20020245 www.mdpi.com/journal/ijms
Int. J. Mol. Sci. 2019,20, 245 2 of 15
Besides its role in triglyceride storage, adipose tissue contains a very large pool of free cholesterol,
and adipocytes are known to support cholesterol efflux to HDL and apoA-I
in vitro
[
2
,
3
]. In fact,
the main functional receptors for HDL, such as the ATP-binding cassette subfamily A member 1
(ABCA1) and the scavenger receptor class B type I (SR-BI), are expressed in mature adipocytes.
Zhang et al. demonstrated that these receptors control cholesterol efflux. Furthermore, they suggested
that adipose dysfunction caused by “inflammation”, as seen in the insulin-resistance conditions,
may impair HDL lipidation in the adipocytes, reducing circulating HDL-C levels [4].
On the contrary, very little is known about the adipocyte role in low-density lipoprotein (LDL)
handling and about the involvement of proprotein convertase subtilisin kexin type 9 (PCSK9) on LDL
receptor (LDLR) regulation. About forty years ago, some landmarking studies were carried on LDLR
and adipocytes. In 1979, Angel et al. demonstrated that isolated human adipose cells contain a
high-affinity receptor which can bind, internalize, and degrade LDL, suggesting that adipose tissue
is an important site of LDL and HDL interactions [
5
]. From that time, the role of adipose tissue in
lipoprotein metabolism has been largely forgotten or not widely studied.
PCSK9, a member of the proprotein convertase family, behaves mainly as a chaperon and it is
highly expressed in human liver [
6
]. A PCSK9 gain-of-function mutation was identified as a cause
of autosomal dominant familial hypercholesterolemia [
7
]. Indeed, PCSK9 plays a critical role in
the regulation of cholesterol homeostasis. Many studies have demonstrated its involvement in the
regulation of LDL cholesterol (LDL-C) levels by controlling the recyclable LDLR on hepatocyte [
8
,
9
].
When PCSK9 is released from the Golgi apparatus into the circulation, it is able to induce the
degradation of LDLR after its binding with LDLR-LDL-C hepatocyte surface complex [
7
,
10
,
11
].
The LDLR, the hydroxy-methyl-glutaryl CoA (HMG-CoA) reductase, and the PCSK9 are co-regulated
by the sterol regulatory element binding protein-2 (SREBP-2), to prevent excessive cholesterol uptake
and preserve cholesterol homeostasis [
12
]. Therefore, pharmacological activation of the SREBP
pathway by HMG-CoA reductase inhibitors (statins) induce PCSK9 expression in experimental and
clinical settings [
13
,
14
]. Furthermore, SREBP-1c also appeared to be involved in the induction of PCSK9
by insulin [
12
,
15
,
16
]. The role of SREBP-1c in the regulation of PCSK9 levels has also been observed
in humans, where PCSK9 is positively correlated with insulin resistance, liver steatosis, and very
low-density lipoprotein-triglyceride (VLDL-TG) levels [
17
]. This evidence suggests that PCSK9 may
be also implicated in the metabolism of TG-rich lipoproteins, such as VLDL and intermediate-density
lipoprotein (IDL), that can be uptaken by the LDLR via apoB and apoE binding.
Taken together, we suggest a role of adipose tissue in PCSK9-mediated lipoprotein metabolism,
although there are almost no data about PCSK9 in human adipocytes. Recently, PCSK9 was detected
in mice perigonadal fat [
18
,
19
]. Mice lacking PCSK9 (PCSK9
−
/
−
)exhibit normal weight but increased
visceral adipose tissue (VAT) due to adipocyte hypertrophy, independently from the LDLR. PCSK9
−
/
−
mice increased both fatty acid uptake and triglyceride synthesis in the adipose tissue, together with an
increased surface density of VLDL receptor [
18
,
19
]. Insulin resistance, a condition found in PCSK9
−
/
−
mice, is linked with an impaired expression of natriuretic peptides (NPs) receptors in human VAT.
Moreover, opposite effects of insulin and NPs have been documented regarding lipid storage in
adipocytes, with NPs antagonizing insulin-stimulating TG storage [20,21].
Cardiac NPs, including type-A (ANP) and type-B (BNP), play a crucial role in maintaining
cardiovascular homeostasis, given their impact not only on blood pressure regulation, but also on
glucose and lipid metabolism [
22
,
23
]. They exert several actions on adipocytes through the activation
of cGMP-dependent pathway, including activation of lipolysis [
24
,
25
] and lipid oxidation [
26
], together
with thermogenic program [
27
]. An important inverse association has been found between plasma
LDL-C and circulating NPs in subjects with a wide range of NT-proBNP levels [28].
In our study, the first step was to verify PCSK9 expression and secretion in human VAT and human
adipocytes. Once confirmed, we studied the reciprocal role of insulin and ANP in the regulation of
PCSK9 and LDLR expression, as well as their respective regulatory genes in human adipocyte cell
Int. J. Mol. Sci. 2019,20, 245 3 of 15
model. We hypothesized that NP activity might influence adipose tissue lipoprotein metabolism,
by regulating those proteins (PCSK9 and LDLR) that play a significant role in atherogenic dyslipidemia.
2. Results
2.1. PCSK9 Expression in Human VAT
General characteristics of the studied population are summarized in Table 1. It is known that
PCSK9 is abundantly expressed in liver, small intestine, and kidney. Our present data show that PCSK9
is abundantly expressed in human adipose tissue as well. Figure 1A shows PCSK9 gene expression
in VAT, even if highly variable among patients. The protein analysis of PCSK9 in human adipose
tissue and liver revealed that the pre-form of PCSK9 is easily detectable (72 kDa; Figure 1B). In fact,
human PCSK9 is synthesized as a precursor that undergoes autocatalytic cleavage of its N-terminal
prosegment in the endoplasmic reticulum (ER) necessary for its activation and function [
6
]. The mature
form (63 kDa) is also detectable, but it is clearly weaker than pre-form, as expected. As shown in
Figure 1C, PCSK9 expression levels are significantly and positively correlated with the body mass
index (BMI) of the 26 patients studied (p= 0.024), even after adjustment for gender and age (
β
= 0.429;
95% CI 0.023–1.224; p= 0.016). No significant correlation emerges between PCSK9 in VAT and LDL
cholesterol, according to linear regression model adjusted for gender and age (p= 0.654).
Table 1. General characteristics of studied population.
Variables NMean ±SE
Gender (male/female)
26
15/11
Age (y)
26
66.9 ±1.4
BMI (kg/m2)
26
25.6 ±0.8
Waist (cm)
26
96.9 ±1.9
SBP (mmHg)
24
139.1 ±3.1
DBP (mmHg)
24
78.9 ±2.0
Triglycerides (mg/dL)
17
125.4 ±9.7
Total Cholesterol (mg/dL)
17
176.1 ±11.7
HDL Cholesterol (mg/dL)
17
39.5 ±2.7
LDL Cholesterol (mg/dL)
17
118.2 ±10.1
Non-HDL Cholesterol (mg/dL)
17
139.7 ±9.9
BMI: body mass index; SBP: systolic blood pressure; DBP: diastolic blood pressure; HDL: high-density lipoprotein;
LDL: low-density lipoprotein.
Figure 1. Cont.
Int. J. Mol. Sci. 2019,20, 245 4 of 15
Figure 1.
(
A
)PCSK9 gene expression in VAT. Cycle threshold of PCSK9 during real-time gene expression
analysis for adipose tissue were between 21 and 24. (
B
)PCSK9 levels in differentiated human adipocytes.
(C)PCSK9 in VAT according to BMI. # number of sample.
2.2. PCSK9 and LDLR Regulation in Human Adipocytes by Insulin
Differentiated adipocytes in multiple wells were treated for 1, 2, or 4 h with 10 nM of insulin.
As shown in Figure 2A, PCSK9 gene expression is 20-fold increased by insulin after 4 h, but a
significant increase is already seen after 2 h. Similarly, LDLR (Figure 2B), SREBP-1c (Figure 2C),
and SREBP-2 (Figure 2D) are significantly increased by insulin with an earlier time course for the
regulatory protein SREBP-1c. Protein analysis of PCSK9 confirms the induction of PCSK9 after insulin
treatment. Interestingly, we analyzed the cell lysate as well as supernatant, and we observed that
PCSK9 after 4 h treatment is increased, especially in the mature form secreted by adipocytes into the
media (Figure 2E). LDLR protein is also induced by insulin in human adipocytes (Figure 2E).
Int. J. Mol. Sci. 2019,20, 245 5 of 15
Figure 2.
(
A
)PCSK9 induction by insulin in differentiated human adipocytes. (
B
)LDLR induction by
insulin in differentiated human adipocytes. (
C
)SREBP-1c induction by insulin in differentiated human
adipocytes. (
D
)SREBP-2 induction by insulin in differentiated human adipocytes. (
E
) PCSK9, LDLR,
and GAPDH levels after treatment with insulin. * p< 0.05, ** p< 0.01 and *** p< 0.001 vs control.
2.3. PCSK9/LDLR Modulation by Cardiac Natriuretic Peptides
To understand whether PCSK9 and LDLR respond not only to insulin but also to NPs, that are
physiologic antagonists of insulin effects on lipid metabolism in adipose tissue, adipocytes were treated
for 4 h with insulin (10 nM), or ANP (100 nM), or insulin together with ANP. Gene expression analysis
shows that PCSK9 and LDLR are significantly induced by insulin, as described above, but also that
ANP is able to partially block the insulin effect (Figure 3A,B). Considering the main genes involved in
the regulation of cholesterologenesis, we found that SREBP-2 is induced by insulin and ANP is able
to block the insulin effect (Figure 3C). On the contrary, ANP is not able to reduce the induction of
Int. J. Mol. Sci. 2019,20, 245 6 of 15
SREBP-1c by insulin (Figure 3D). Similar results for PCSK9 and LDLR were obtained using Western
blot analysis (Figure 3E).
Figure 3.
One-way ANOVA. (
A
) Effects of insulin and ANP on PCSK9 in differentiated human
adipocytes. (
B
) Effects of insulin and ANP on LDLR in differentiated human adipocytes. (
C
) Effects of
insulin and ANP on SREBP-2 in differentiated human adipocytes. (
D
) Effects of insulin and ANP on
SREBP-1c in differentiated human adipocytes. (
E
) PCSK9, LDLR, and GAPDH levels after treatment
with insulin and ANP. *** p< 0.001 vs control; * p< 0.05 insulin 10nM vs insulin+ANP treatment.
2.4. The Influence of LDL from Human Plasma
To study the physiological mechanism that links adipocyte PCSK9 with circulating LDL, human
adipocytes were treated with increasing concentrations of isolated LDL from human plasma (from 25 to
100 ng/mL) for 4 and 18 h. Gene expression analysis shows that at the first time point (4 h, Figure 4A–D)
the treatment with LDL induces a significant increase of all the target genes: PCSK9, LDLR, SREBP-1c,
and SREBP-2. Interestingly, at the second time point (18 h) it is evident that human LDL decreases LDLR
but, at the same time, significantly increases PCSK9 together with SREBP-1c and SREBP-2 (Figure 4F–I).
Protein analysis clearly shows that, during the incubation times, PCSK9 initially increases as a mature
form into the cells (Figure 4E) and then, after 18 h, increases as the mature form secreted into the
media (Figure 4J). At the same time, LDLR increases after 4 h treatment (Figure 4E) and, thereafter,
is significantly reduced after 18 h (Figure 4J). These time-dependent responses suggest that, at the
Int. J. Mol. Sci. 2019,20, 245 7 of 15
beginning, the presence of LDL induces all the physiological pathways involved in cholesterol uptake,
but after 18 h, the strong induction of PCSK9, especially for the secreted mature form, may induce
LDLR reduced function by degradation.
Figure 4.
One-way ANOVA. (
A
) Effects of LDL treatment for 4 h on LDLR in differentiated human
adipocytes. (
B
) Effects of LDL treatment for 4 h on PCSK9 in differentiated human adipocytes.
(
C
) Effects of LDL treatment for 4 h on SREBP-1c in differentiated human adipocytes. (
D
) Effects of
LDL treatment for 4 h on SREBP-2 in differentiated human adipocytes. (
E
) PCSK9, LDLR, and GAPDH
proteins levels after LDL treatment for 4 h. (
F
) Effects of LDL treatment for 18 h on LDLR in
differentiated human adipocytes. (
G
) Effects of LDL treatment for 18 h on PCSK9 in differentiated
human adipocytes. (
H
) Effects of LDL treatment for 18 h on SREBP-1c in differentiated human
adipocytes. (
I
) Effects of LDL treatment for 18 h on SREBP-2 in differentiated human adipocytes.
(
J
) PCSK9, LDLR, and GAPDH proteins levels after LDL treatment for 18 h. * p< 0.05, ** p< 0.01 and
*** p< 0.001 vs. control.
2.5. LDL and ANP Effects on Human Adipocytes
PCSK9 and LDLR expression were also analyzed in presence of isolated LDL (50 ng/mL) and ANP
(100 nM). As shown in Figure 5, protein analysis reveals that 4 h of treatment with LDL significantly
Int. J. Mol. Sci. 2019,20, 245 8 of 15
induces PCSK9, as well as LDLR, and that this effect is blocked by ANP. After 18 h, it is still evident
that, while PCSK9 is clearly induced also in the secreted form, LDLR is reduced, as described in
Figure 4. Interestingly, ANP is still able to block the effect of LDL on target genes after 4 and 18 h
treatments (Figure 5).
Figure 5. PCSK9, LDLR, and GAPDH levels after treatment with LDL and ANP for 4 and 18 h.
Int. J. Mol. Sci. 2019,20, 245 9 of 15
3. Discussion
The focus of this study was to investigate the expression and regulation of PCSK9 in
human adipose tissue and adipocytes, using insulin and ANP, two opposing hormones on lipid
metabolism [
20
]. It is known that the active form of PCSK9 is derived from three different steps:
the cleavage of the signal sequences, the intramolecular proteolysis of proPCSK9, and trafficking
through the trans-Golgi network before the secretion into extracellular space, where it reaches its target,
LDLR [
6
,
29
,
30
]. Gene expression and protein analysis revealed that the precursor form of PCSK9 is
expressed and easily detectable in human visceral perirenal fat, even if there are wide differences
among subjects. The concentrations of PCSK9 in adipose tissue positively correlated with BMI values,
the most used clinical index of adiposity. Interestingly, PCSK9 plasma concentrations were also related
to carotid artery intima-media thickness (cIMT) in overweight and obese individuals, in comparison
with the normal weight group, suggesting that PCSK9 could be an indicator as well as a player of
cardiometabolic and vascular changes induced by excessive adiposity [31].
Therefore, we investigated the regulation of PCSK9 using the Simpson–Golabi–Behmel syndrome
(SGBS) human adipocyte cell line. Differentiated SGBS adipocytes were used to verify the ability
of insulin, ANP, and LDL to regulate PCSK9. First, we showed that 10 nm of insulin strongly
induced PCSK9 and LDLR after 2 and 4 h of treatment. It has been reported that LDLR and PCSK9
share SREBP-2 as a common regulatory pathway, and that PCSK9 expression is also regulated
by insulin via the SREBP-1c in primary mouse and rat hepatocytes, as well as
in vivo
, during
hyperinsulinemic-euglycemic clamps. [
32
]. These SREBPs seem to be active also in adipocytes. Indeed,
SREBP-1c is activated by insulin earlier than SREBP-2, being already significantly increased after 1 h.
This suggests that SREBP-1c is involved in PCSK9 regulation in human adipocytes.
We recently described that higher insulin levels, together with higher glucose concentration,
simulate insulin resistance found in obese patients, and reduced the ability of NPs to induce lipolysis
and the thermogenic pathway [
20
]. Some interactions between the PCSK family and NPs have been
recently discovered, such as for PCSK6, that activates the corin, a key enzyme in the activation of
ANP precursors [
33
,
34
]. Here, we show that ANP is able to reduce the insulin-mediated induction
of PCSK9 and LDLR in human adipocytes. Our data also show that ANP is able to reduce the
regulation of SREBP-2, but not of SREBP-1c. SREBP-1c preferentially activates genes involved in
fatty acid biosynthesis or carbohydrate metabolism, including fatty acid synthetase, acetyl-CoA
carboxylase, or glucokinase (GK) [
32
]. GK converts glucose into glucose 6-phosphate and, therefore,
SREBP-1c is thought to have a permissive action on glucose-dependent gene regulation [
35
]. Moreover,
SREBP-1c is induced by LXRalpha, that is stimulated by insulin independently by glucose concentration,
as we previously described [
20
]. In the liver, SREBP-2 processing is also enhanced by peroxisome
proliferator-activated receptor gamma (PPAR-gamma) activation, affecting both PCSK9 and LDLR
expression [
36
]. It is known that PPAR-gamma has an important role in both cell differentiation and
energy metabolism in adipocytes [
37
,
38
]. The role of PPAR-gamma activation on these proteins in
human adipocytes could be an interesting aspect to be explored in future studies.
Given the close relation between circulating PCSK9 and LDL levels in human plasma [
39
–
41
],
we tested the role of different concentrations of LDL on PCSK9 and LDLR expression in our human
adipocyte cell model. We used LDL isolated from human plasma to test the functionality of the
LDLR/PCSK9 system, and our data indicate a fully functional system. The object of the present study
was only to evaluate the expression and regulation of PCSK9 mediated by insulin and ANP. Future
research, focused on the direct role of PCSK9 and LDLR in VAT and the relationship between PCSK9
and the morphology of adipocytes (i.e., lipid droplet formation or differentiation status), is warranted.
Published data reported that expression of PCSK9 in cultured cells has variable effects on LDLR.
In some cell types, such as human hepatoma cells (HepG2 and HuH7) or human embryonic kidney
cells (HEK-293 cells), PCSK9 expression dramatically reduces LDLR levels [
29
,
42
,
43
]. In other cells
types, including fibroblasts, Chinese hamster ovarian (CHO-K1), monkey kidney cells (COS7) and rat
liver cells (McArdle RH7777), PCSK9 is not likely to affect LDLR expression [
42
–
44
]. Here, we found
Int. J. Mol. Sci. 2019,20, 245 10 of 15
that human LDL, added to the cells culture media, initially—after 4 h treatment—induces LDLR and
pre-form PCSK9. After 18 h of treatment, the high levels of mature PCSK9 are secreted into the media,
and are able to induce the degradation of LDLR. In fact, after 18 h, LDLR protein levels are reduced
compared with LDLR after 4 h LDL treatment.
Moreover, when we tested the effect of LDL combined with ANP, we observed that LDL induced
LDLR and the non-secreted form of PCSK9 after 4 h. At the same time, ANP is able to partially block
the LDL-induced regulation. After 18 h, we confirmed that the LDL-mediated induction of PCSK9 was
still present, and the PCSK9 secreted form was also significantly induced.
The elevated secretion of PCSK9 could represent the mechanism for the reduction of LDLR. Indeed,
in the cells treated with ANP, where PCSK9 is reduced, the levels of LDLR are stable, suggesting again
that ANP, by blocking the induction of PCSK9, indirectly reduced the degradation of LDLR.
Although in our research the regulatory effect of ANP appears to be modest, to the best of our
knowledge, this is the first study demonstrating that PCSK9 is expressed in adipose tissue, and ANP is
able to interact with PCSK9 pathways. Several studies showed the role of NPs on lipid metabolism
and adipose tissue, but the interaction of ANP with cholesterol metabolism through PCSK9 and LDLR
regulation is absolutely new. ANP partially blocked the effect of insulin and LDL in adipocytes, but it
was more effective in culture conditions simulating hyperglycemia. Overall, NPs are likely to reduce
triglycerides and to increase cholesterol in human adipocytes, opposing insulin effects. Such activities
may also have systemic consequences on blood lipid levels (Figure 6). We believe that this study could
be the first of future studies on human adipose tissue, in order to better explain which mechanisms are
involved in lipid metabolism.
Figure 6.
Interactions between insulin and cardiac natriuretic peptides (NPs) in lipid metabolism and
PCSK9-LDL receptor handling. The potent lipolytic activity of NPs (ANP and BNP secreted from
the heart), mediated by NPRA, is strongly reduced by insulin through the induction of the clearance
receptor (NPRC), which binds and degrades NPs. Insulin also induces PCSK9, but this effect is opposed
by NPs, especially in conditions simulating hyperglycemia. Overall, the NPs are likely to reduce the
triglycerides and to increase cholesterol in human adipocytes, opposing insulin effects. Such activities
may also have systemic consequences on blood lipid levels. T-bar arrow indicates inhibitory activity.
Int. J. Mol. Sci. 2019,20, 245 11 of 15
In conclusion, our data show a potentially relevant role for adipose tissue and adipocytes in the
regulation of PCSK9-LDLR in humans, similar to what is known for human liver. NPs appear to play
a key role in this pathway, with potentially important implications, especially in patients with obesity
and hypertension, in which NPs could be able to regulate not only the blood pressure, but also the
LDLR levels, preventing PCSK9 overexpression.
4. Materials and Methods
4.1. Reagents and Antibodies
Insulin, dexamethasone, isobutylmethylxanthine, tri-iodothyronine, transferrin, and wortmannin
were obtained from Sigma-Aldrich (St. Louis, Missouri, USA). Antisera against PCSK9 and LDLR
were from Abcam (Abcam, Cambridge, MA, USA). GAPDH antibody (cat #SC25778) and secondary
antibody anti-rabbit (cat #SC2054) were from Santa Cruz Biotech; CA, USA. SuperSignal West Femto
Maximum Sensitivity Substrate was from Thermo Scientific, Rockford, IL, USA.
4.2. Human Adipose Tissue
A set of human VAT samples (n= 26) were obtained from patients undergoing radical nephrectomy
for localized clear cell renal carcinoma (without any evidence of local or metastatic cancer spread:
T1/T2, N0, M0) at the “Ospedali Riuniti” University Hospital of Ancona, Italy. All women were in
menopause. Patients with diabetes were excluded from the study, therefore, no patients took insulin
or any other medications. The study was conducted in accordance with the guidelines proposed in
The Declaration of Helsinki and the local Ethics Committee approved the study protocol (ID: 206964,
date: 28 September 2006). All patients gave written informed consent for the collection of clinical data
and tissue samples.
4.3. Adipocyte Cells Culture
Cells from Simpson–Golabi–Behmel syndrome cell line (SGBS) were grown in growth medium
(DMEM/F12 with 10% fetal calf serum). When adipocytes reached 85%–90% confluence, they were
differentiated in differentiation medium that included insulin, as previously described. To test the
acute effect of insulin on NP receptors, we removed insulin from differentiation media at day 7 instead
of day 11/12, as previously described [
20
]. Thus, on the seventh day, the cells were washed and
deprived of insulin at least for three days, and were then treated with insulin and/or atrial natriuretic
peptide (ANP). To assess whether PCSK9 expression and related genes are modulated by insulin,
differentiated adipocytes were treated with 10 nM of insulin for 1, 2, and 4 h. PCSK9 regulation was
also evaluated in response to cardiac NPs. Adipocytes were treated with 100 nM of ANP, as used
in previous work [
20
]. ANP was also used together with insulin to evaluate the relative power of
these two physiological counteracting (lipolytic vs. lipogenic) hormones on PCSK9/LDLR. At least
6 different wells in 3 different experiments were performed for each treatment.
4.4. Isolation of Human Plasma LDL
The role of LDL on PCK9 and LDLR expression was studied using LDL separated from human
plasma. Fresh pooled human plasma from fasting healthy young volunteers (coauthors of these
manuscript) was used for the preparation of LDL. LDL (density between 1.025 and 1.063 g/mL)
were isolated by single vertical spin gradient ultracentrifugation, as described by Chung et al. [
45
].
After dialysis at 4
◦
C for 24 h against 10 mmol/L PBS (pH 7.4), LDLs were concentrated in a SpeedVac
Concentrator and LDL protein concentration was determined by the method of Bradford [46].
The first set of experiments was performed to verify the effect of different concentrations of LDLs,
from 25 to 100 ng/mL, after 4 and 18 h of incubations. Subsequently, PCSK9 and LDLR were analyzed
after 4 and 18 h of treatment with human LDL (50 ng/mL), ANP, or together (ANP + LDL).
Int. J. Mol. Sci. 2019,20, 245 12 of 15
4.5. RNA Isolation and Gene Expression Analysis
Total RNA was extracted using TRIzol (Invitrogen, Carlsbad, CA, USA) and RNA reverse
transcription of 2
µ
g was performed with High-Capacity cDNA Reverse Transcription Kit with
RNase Inhibitor (Applied Biosystems, Warrington, UK). All gene expression experiments in SGBS and
primary VAT adipocytes cultures were analyzed with SYBR Select Master Mix (Applied Biosystems
Darmstadt, Germany). Each single gene expression experiment was performed in triplicate. Differences
in total RNA or different efficiency of cDNA synthesis among samples were normalized using human
GAPDH expression.
4.6. Western Blotting
Treated cells were lysed and sonicated in an appropriate buffer, as previously described [
27
].
Protein concentrations were determined using the Bradford Assay (Biorad, Hercules, CA, USA) and
50
µ
g of total proteins was resolved in 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE), transferred to a PVDF membrane (Immobilon P, Millipore, Burlington, MA, USA),
and probed overnight at 4
◦
C with specific PCSK9 or LDLR primary antibodies. Secondary antisera
against rabbit IgG conjugated with peroxidase was used for specific protein detection. Target proteins
were visualized using an enhanced chemiluminescent substrate (SuperSignal West Femto Maximum
Sensitivity Substrate, Pierce) and were measured in comparison with GAPDH (Santa Cruz Biotech,
Dallas, TX, USA). Image acquisition was performed on an ALLIANCE MINI HD9 (UVITEC, Cambridge
UK). All lines were quantified with UVI-TEC NineAlliance analysis software and each line sample of
Western blot shows the relative number of quantification, compared with control, that was considered
as 1 (100%). In some cases, membranes were ‘stripped’ by incubation in a buffer (0.76 g Tris, 2 g SDS,
700
µ
L
β
-mercaptoethanol in 100 mL) at 37
◦
C for 45 min, in order to be subsequently probed with
additional antibodies.
4.7. Statistical Analysis
Results are presented as mean
±
SEM, unless otherwise indicated. Data were analyzed using
two-tailed Student’s t-test, one-way ANOVA, followed by post hoc Newman–Keuls tests when F
was significant. A non-parametric test for two related samples (Wilcoxon’s signed ranks test) was
used to identify differences between each treated group and controls, differences between more than
2 groups were analyzed by analysis of variance and post hoc Holm-Bonferroni test. Pearson’s correlation
coefficient was used to assess the association between PCSK9 gene expression and BMI. Multiple linear
regression was used to create adjusted models. SPSS 11.0 software was used for statistical analysis
(SPSS Inc., Chicago, IL, USA) and a p< 0.05 was considered significant.
Author Contributions:
Conceptualization, R.S. and M.B.; methodology, L.M. and G.F.; formal analysis, M.B. and
T.B.; investigation, M.B. and A.V.; resources, F.S., G.F. and C.D.P.; writing—original draft preparation, M.B., A.V.
and F.S.; writing—review and editing, L.M. and R.S.; visualization, M.B.; supervision, R.S.; project administration,
R.S. and L.M.; funding acquisition, R.S.
Funding: This research was funded by University “Politecnica delle Marche” (Ricerca di Ateneo to R. Sarzani).
Acknowledgments:
We thank Saverio Cinti for the use of microscopes and Martin Wabitsch for the SGBS cell line.
Conflicts of Interest: The authors declare no conflict of interest.
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