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1
Increased hepatic PDGF-AA signaling mediates liver insulin resistance in obesity
associated type 2 diabetes
Amar Abderrahmani1,2*, Loïc Yengo
1*, Robert Caiazzo
3*, Mickaël Canouil
1*, Stéphane
Cauchi1, Violeta Raverdy
3, Valérie Plaisance
1, Valérie Pawlowski
1, Stéphane Lobbens
1,
Julie Maillet1, Laure Rolland
1, Raphael Boutry
1, Gurvan Queniat
1, Maxime Kwapich
1,
Mathie Tenenbaum1, Julien Bricambert
1, Sophie Saussenthaler
4, Elodie Anthony
5,
Pooja Jha6, Julien Derop
1, Olivier Sand
1, Iandry Rabearivelo
1, Audrey Leloire
1, Marie
Pigeyre3, Martine Daujat-Chavanieu
7, Sabine Gerbal-Chaloin
7, Tasnim Dayeh
8,
Guillaume Lassailly9, Philippe Mathurin
9, Bart Staels
10, Johan Auwerx
6, Annette
Schürmann4, Catherine Postic
5, Clemens Schafmayer
11, Jochen Hampe
12, Amélie
Bonnefond1,2, François Pattou
3*, Philippe Froguel
1,2*
1Univ. Lille, CNRS, Institut Pasteur de Lille, UMR 8199 - EGID, F-59000 Lille, France;
2Department of Medicine, Section of Genomics and Common Disease, Imperial College
European Research Council GEPIDIAB - 294785. We are grateful to Ms Estelle Leborgne for
helping in the illustrations of the manuscript. The present manuscript has been posted in the
preprint ArXiv server.
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Legends of Figures
Fig. 1. a) Quantile-quantile (qq-) plot showing the residual inflation of test statistics before
and after genomic-control correction. b) Manhattan plot centered on PDGFA cg14496282
methylation site showing association signal within PDGFA bounds. Hepatic Pdgfa expression
in c) 6 weeks old male B6 mice that were diet-induced obese (DIO) responder (Resp, black
circle, n= 12) and DIO-non-responder (nResp, white circle, n=10) and in d) BXD mice (n=
45) fed on Chow diet (CD, green circle) or a HFD (black circle) fed for 21 weeks. ***
indicates p value < 0.0001 by unpaired t test with Welch’s correction.
Fig. 2. a) PDGF-AA secretion from IHH cells and primary human hepatocytes was measured
by ELISA kit. b) Measurement of insulin-induced AKT phosphorylation in response to
human insulin (NovoNordisk) for the indicated times. IHH cells were incubated in a culture
medium containing 5 mM Glucose, 2 % FCS with or without 100 nM human insulin for the
indicated times. AKT phosphorylation was stimulated by 200 nM insulin for one hour.
Immunoblotting for phospho-AKT (p-AKT) was done using the anti phospho-AKT (Serine
473) antibodies. The Fig. shows the result of a representative experiment out of three. c)
Effect of insulin on the glycogen production. Insulin-induced glycogen production was
measured by ELISA in IHH cells that were pre-cultured with 100 nM insulin for 16 h and 24
h. d) Increase of PDGFA mRNA by insulin. IHH cells were cultured with 100 nM human
insulin for 16 h and 24 h. The PDGFA mRNA level was quantified by qRT-PCR and
normalized against RPLP0. The expression levels from untreated cells were set to 100 %.
Data are the mean ± SEM (**: p < 0.001). e) PDGF-AA abundance in IHH cells cultured with
insulin. IHH cells were cultured with 100 nM human insulin for the indicated times. PDGF-
AA content was quantified by Western Blotting experiments. The blot is one representative
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out of three independent experiments. f) PDGF-AA secretion in response to insulin. IHH cells
were cultured with insulin for the indicated times. The measurement of PDGF-AA by ELISA
from the supernatant that was retrieved of IHH cells cultured . g) Methylation levels at
PDGFA cg14496282 in response to insulin. IHH cells were cultured in a culture medium
containing 5 mM Glucose, 2 % FCS with or without 100 nM human insulin for 24 h.
Methylation level at the cg14496282 was quantified by the Infinium HumanMethylation450
BeadChip. h) Effect of chronic insulin in insulin-induced AKT activation in HepG2 cells.
HepG2 cells were incubated in a culture medium with 100 nM human insulin or without (Ctl)
for 24 h. AKT phosphorylation was stimulated by 200 nM insulin for one hour.
Immunoblotting for phospho-AKT (p-AKT) was done using the anti phospho-AKT (Serine
473) antibodies. The Fig. shows the result of a representative experiment out of three. i)
Increase of PDGFA mRNA by insulin in HepG2 cells. The PDGFA mRNA level was
quantified by qRT-PCR in HepG2 and IHH cells that were cultured with insulin for 24 h. The
PDGFA mRNA was normalized against RPLP0. The expression levels from untreated cells
were set to 100 %. Data are the mean ± SEM (**: p < 0.001).
Fig. 3. Effects of a) human PDGF-AA recombinant, b) PDGFA blocking antibodies or c) the
PDGFR inhibitor ki11502 on insulin-induced AKT activation. Activation of AKT was
monitored by western blotting using total proteins from IHH cells that were cultured with the
human recombinant PDGF-AA at the indicated concentrations for 24 h, which subsequently
were incubated with 200 nM insulin for stimulating AKT phosphorylation. For a-c, IHH cells
were co-incubated in a culture medium containing 5 mM Glucose, 2 % FCS with or without
100 nM human insulin for 24 hours plus a) PDGF-AA at the indicated concentration, b)
PDGFA antibodies (+; 0.75 µg or ++; 1.5 µg) or c) ki11502 at the indicated concentration.
The Figures show the result of a representative experiment out of three. d) Effect of the
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PDGFR inhibitor ki11502 on the glycogen production. Glycogen was measured by ELISA in
IHH cells that were co-cultured with 5 µM ki11502 and insulin for the indicated times.
Glycogen was monitored after stimulating cells in a KRP buffer without (Ctl) or with insulin
for 1 h and 20 mM glucose. Glycogen was monitored by ELISA.
Fig. 4: a) Volcano plot showing differences in putative serine/threonine kinase activities
between control and insulin-treated IHH cells for 24 h. Specific and positive kinase statistic
(in red) show higher activity in IHH cultured with insulin compared with control samples.
Effects of b) insulin and c) PDGF-AA on the phosphorylation of PKCƟ and PKCε. IHH cells
were cultured with insulin for the indicated times or PDGF-AA (for 24 h). Phosphorylation of
PKCƟ (Ser 676) and PKCε (Ser 729) were measured by western blotting and normalized
against total PKCƟ and PKCε. d) Effect of insulin and PDGF-AA on the tyrosine
phosphorylation (Y972) of INSR (p-IR) and threonine phosphorylation of INSR (T1376),
INSR abundance (IR) and αTubulin. Western Blotting experiments were achieved from total
proteins of IHH cells cultured either with 100 nM insulin of 100 ng/ml PDGF-AA for 24 h.
Phosphorylation of INSR was done by stimulating IHH cells with insulin for 1 h. The Figures
show the result of a representative experiment out of three.
Fig. 5: Effect of insulin on a) IRS1 mRNA level and b) protein. The IRS1 mRNA level and
IRS1 abundance was quantified by qRT-PCR and Western Blotting in IHH cells cultured with
100 nM insulin for 24 h. The IRS1 mRNA was normalized against RPLP0. The expression
levels from untreated cells were set to 100 %. Data are the mean ± SEM (**: p < 0.001). c)
effect of PDGF-AA on the IRS1 content. IHH cells were cultured with PDGF-AA at the
indicated concentrations for 24 h. The Figures show the result of a representative experiment
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out of three. d) Effect of siRNA against IRS1 on the insulin-induced AKT activation.
Duplexes of small interfering RNAs were transfected in IHH cells for 48 h. Thereafter, AKT
phosphorylation on the serine 473 was induced with insulin for 1 h. The Figures show the
result of a representative experiment out of three. Effect of e) and f) the PKC inhibitor
sotraustorin PKC and f) and g) phorbol 12-myristate 13-acetate (PMA) PKC activator on the
expression of IRS1 mRNA and protein. PDGFA mRNA was quantified in IHH cells cultured
with either sotrastaurin at the indicated concentration in the presence of 100 nM insulin for 24
h, or PMA for the indicated times. The IRS1 mRNA was normalized against RPLP0. The
expression levels from untreated cells were set to 100 %. Data are the mean ± SEM of three
independent experiments made in triplicates (***: p < 0.0001).
Fig. 6: Effect of ki11502 PDGFR inhibitor on the PDGFA expression in response to a)
PDGF-AA and b) insulin. IHH cells were cultured for 24 h with 100 ng/ml PDGF-AA or 100
nM insulin in the presence or absence of 5 µM ki11502 for 24 h. Effect of c) PKC activator
phorbol 12-myristate 13-acetate (PMA) and d-e) the PKC inhibitor sotraustorin on the
expression of PDGFA mRNA induced by d) insulin or e) PDGF-AA .The PDGFA mRNA
was quantified by qRT-PCR in IHH cells cultured with either PMA for the indicated times, or
100 nM insulin or 100 ng/ml PDGF-AA in the presence of 1 µM sotrastaurin or at the
indicated concentration for 24 h. The PDGFA mRNA was normalized against RPLP0. The
expression levels from untreated cells were set to 100 %. Data are the mean ± SEM of three
independent experiments made in triplicates (*, p<0.05; ***: p < 0.0001).
Fig. 7: Schematic representation of the mechanism linking hyperinsulinemia to hepatic insulin
resistance in T2D. Insulin promotes hypomethylation and the rise of PDGFA expression,
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leading to PDGF-AA secretion. In turn, PDGF-AA inhibits the insulin signaling, in a negative
autocrine feedback loop, via a mechanism involving a decrease in the IRS1 and INSR
abundance and PKC (PKCƟ and PKCε) activation.
Tables
Table 1. Association of liver methylation levels of cg14496282 and liver PDGFA gene expression with multiple
quantitative and binary traits. Methylation levels at cg14496282 and PDGFA gene expression are the
endogenous variable in all linear regressions used to measure associations. SD: Standard Deviation.
Traits(unit)
PDGFA cg14496282 methylation PDGFA
Expression
Effect size in % of methylation / trait unit
(p-value)
Effect size in SD/trait unit
(p-value)
Controls T2D cases Controls T2D cases
cg14496282 methylation (%)
-1.44
(6.27×10-3
) -2.497
(4.94×10-3
)
PDGFA expression (Scaled -SD)
-0.0548 (6.27×10
-3)
-0.0338 (4.94×10
-3)
Fasting glucose (mmol/l)
-0.0112 (0.79)
- 0.292 (0.196)
-
Fasting insulin (pmol/l)
-1.45×10-3
(2.32×10
-3)
- 6.83×10
-3
(9.49×10-3
) -
HOMA2-B (unitless - log)
-0.169 (2.92×10
-3)
- 0.626
(0.038) -
HOMA2-IR (unitless - log)
-0.104 (4.93×10
-3)
- 0.528
(7.47×10-3
) -
QUICKI (unitless)
1.66 (0.01)
- -9.192
(9.78×10-3
) -
Steatosis (%) -2.15×10
-3
(0.01) -4.34×10
-4
(0.42) 0.0136
(2.72×10-3
) 0.020
(2.14×10-6
)
NASH (Yes/ No) -0.17 (0.04)
-0.072 (0.03)
2.115 (9.38×10
-7)
1.447 (3.37×10
-8)
Hepatic fibrosis (Yes/ No)
-0.07 (0.09)
-0.051 (0.04)
0.187 (0.434)
0.631 (2.66×10
-3)
Alanine aminotransferase (UI/L)
-1.44×10-4
(0.89) -1.34×10
-3
(0.03) 0.0106 (0.067)
0.0194 (1.46×10
-4)
Aspartate aminotransferase (UI/L)
-4.72×10-3
(0.06) -1.76×10
-3
(0.04) 0.0342
(7.89×10-3
) 0.0327
(2.56×10-6
)
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254x338mm (300 x 300 DPI)
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Controls (n=96) T2D cases (n=96)
Traits (unit) Mean (SD) or n (%) Mean (SD) or n (%) p-value
Table S3. Association between fasting insulin raising alleles and DNA methylation at cg14496282. Associations were assessed using linear regression with
methylation as response variable. All models were adjusted for age, BMI, total cholesterol, HDL cholesterol, triglycerides and fasting glucose. Effect sizes are
reported as percentage of DNA methylation per allele.
Page 59 of 66 Diabetes
Gene Log2 Fold Change p-value
PDGFRB -0.68 3.2E-10
IL1RN -0.59 3.0E-15
A2M -0.33 2.2E-09
FGF2 -0.31 9.5E-03
VDR -0.25 3.2E-02
PRKACB -0.25 1.7E-03
IRS1 -0.21 1.1E-02
POU5F1 -0.19 6.0E-03
NFATC2 -0.17 2.7E-02
NR3C1 -0.14 4.4E-02
PRKACA -0.13 2.1E-02
PDPK1 -0.13 1.8E-02
HDAC1 -0.12 3.9E-02
RXRB -0.12 4.2E-02
PRKAR1A -0.12 2.4E-02
SP1 -0.11 3.9E-02
NME2 0.11 2.6E-02
SSRP1 0.12 2.2E-02
SPP1 0.12 1.6E-02
NCL 0.12 8.2E-03
TAF4 0.16 8.2E-03
NFKB1 0.17 2.8E-03
FURIN 0.18 4.5E-02
VEGFB 0.20 1.5E-02
PDAP1 0.21 3.0E-03
HIF1A 0.22 6.4E-05
TGFB1 0.23 3.0E-04
MAP2K1 0.23 1.3E-04
MAP2K2 0.23 1.1E-02
DNM2 0.25 1.7E-04
RRAS 0.25 4.4E-02
TNFRSF1A 0.25 7.6E-06
GRB14 0.26 2.2E-02
NFIC 0.27 1.3E-02
NME1 0.28 1.1E-06
IL18 0.29 3.1E-02
PDGFB 0.43 1.2E-04
PCSK5 0.52 8.3E-12
EDN1 0.55 6.4E-05
VEGFA 0.56 5.8E-13
NES 0.64 1.2E-25
DUSP1 0.67 9.7E-11
PDGFA 0.80 1.1E-11
KLF5 0.87 3.6E-13
EGR1 1.13 2.2E-16
Table S4. List of deregulated genes within the network of PDGFA