Methods
Cell culture
Human umbilical vein endothelial cells (HUVECs), human cardiac microvascular endothelial cells
(HCMECs) and human retinal endothelial cells (HRECs) were maintained in Endothelial Cell
Medium, containing 1 mM glucose (ECM, ScienCell Research Laboratories, Carlsbad, CA) at 37°C
with 5% CO2. Glucosamine (GlcN), D-glucose, Mannitol, Azaserine, Phenyl butyric acid (PBA),
benzyl-2-acetamido-2-deoxy-α-D-galactopyranoside
(BADGP),
O-(2-Acetamido-2-deoxy-
Dglucopyranosylidene-amino-N-phenylcarbamate (PUGNAc), Rapamycin (Sigma-Aldrich, Milan,
Italy), ST045849 (TimTec LLC, Newark, DE, USA) and SRT1720 (Cayman Chemical, Ann Arbor,
MI, USA) were suspended in the appropriate solvent, according to the manufacturer’s instructions,
sterile-filtered and added to complete cell media at the indicated concentrations.
For Adenoviral transduction 50-60% confluent HUVECs were incubated with Ad-U6-h-SIRT1shRNA or Ad-U6 scrambled-shRNA (Vector Biolabs, Malvern, PA, USA) for 8 hours. Cells were
then washed to remove virus, and the culture was continued for 48 hours in the presence or absence
of GlcN and SRT1720 as indicated.
Antibodies
The following primary antibodies were employed: anti-heavy chain-binding protein (Bip) antibody,
anti-inositol requiring enzyme 1 α (IRE1α) antibody, anti-C/EBP homologous protein (CHOP)
antibody, anti-cleaved Poli-ADP-ribose polymerase (PARP) antibody, anti-total and phospho-Jun
N-terminal kinase (Jnk) antibodies, anti-total and phospho-p70S6 kinase 1 (p70S6K1) antibodies,
anti SIRT1 antibody, anti Raptor antibody, anti-acetylated lysine antibody (all from Cell Signaling
Technology, Danvers, MA, USA), anti OGlcNase antibody (Abcam, Cambridge, UK), anti Olinked N-Acetylglucosamine Antibody (RL2) (Thermo Scientific Waltham, MA, USA). Equal
protein loading was confirmed by reblotting the membranes with monoclonal antibody against βactin (Sigma-Aldrich, Milan, Italy); p-p70S6K1/p70S6K1 and p-Jnk/Jnk ratio were calculated to
analyze Jnk and p70S6K1 phosphorylation levels. Acetylated-raptor/raptor ratio, acetylatedOGA/OGA ratio and O-GlcNAcylated-SIRT1/SIRT1 ratio were calculated to analyze raptor and
OGA acetylation and SIRT1 O-GlcNAcylation levels.
Caspase 3 activity assay
HUVECs and HCMECs, treated with GlcN (7.5 mM) for the indicate time were incubated for 1
hour in the presence of a biotinylated caspase inhibitor (biotin-ZVKD-fmk inhibitor, 10 µM) that
specifically binds active caspase 3, while it does not modify the inactive form. Cells were then lysed
with Extraction Buffer and caspase 3 activity was measured with Human Active Caspase 3
immunoassay kit (R&D Systems, Inc., Minneapolis, MN, USA).
NF-kB and SIRT1 activity assay
For both assays HUVECs, treated in the presence of GlcN (7.5 mM) and/or SRT1720 and/or PBA,
as indicated, were pelleted and lysed with a hypotonic buffer containing 10 mM Hepes, 0.1 mM
EDTA and phosphatase inhibitors. Supernatants containing cytosolic fractions were removed and
pellets were resuspended in a Nuclear Extraction Buffer containing 10 mM Hepes, 0.1 mM EDTA,
1.5 mM MgCl2, 420 mM NaCl, 1 mM DTT, and 10% glycerol. NF-kB and SIRT1 activities in
nuclear lysates were then measured, respectively, with NF-kB specific ELISA assay (Cayman
Chemical Company, Ann Arbor, MI, USA) or with Epigenase Universal SIRT activity/inhibition
assay kit (Epigentek Group, Farmingdale, NY, USA). Deacetylated histones were included as an
internal control in the SIRT1 ELISA assay. For both assays, a small fraction of the nuclear lysates
was used to measure protein concentration to normalize ELISA results.
Histone acetyltransferase (HAT) activity assay
To assess HAT activity HUVECs incubated for 48 hours in the presence or absence of GlcN 7.5
mM and/or SRT1720 were lysed with RIPA buffer. Fifty µg of each sample were then incubated in
96 well U-shaped plates for 2 hours at 37 oC with an assay mix containing HAT substrates and
NADH generating enzyme, according to the manufacturer’s instructions (HAT activity assay kit,
Sigma-Aldrich, Milan, Italy).
Interleukin-6 (IL-6) release Elisa assay
To assess IL-6 release from HUVECs, cell media were collected, diluted 1:25 with Assay Diluent
buffer and analyzed with the Quantikine High Sensitivity human IL-6 (R&D Systems, Inc.,
Minneapolis, MN, USA). The kit detection range is 10-0.016 pg/ml. Since the kit is not specifically
designed to measure IL-6 in cell culture medium we tested the inter and intra assay precision CV in
our experimental setting and obtained the following values: inter-assay CV 7.2%; intra-assay CV
7.8% .
Results
GlcN induces ER stress in a dose-dependent manner
To confirm that the concentration of GlcN (7.5 mM) employed in this study induced the maximal
activation of ER stress in HUVECs, dose-response experiments were performed treating HUVECs
with increasing doses of GlcN (0.1-15 mM) for 48 hours and protein levels of the ER stress marker
Bip were analyzed. We observed that the lower GlcN doses (0.1, 0.5 and 1 mM) were unable to
significantly induce Bip expression; while a 3.4-fold increase was achieved maintaining HUVECs
in the presence of 2.5 mM GlcN (Figure S1 A). The higher GlcN doses had an even more evident
effect on Bip expression, with a maximal 8.5-fold induction observed at 7.5 mM GlcN. Increasing
GlcN concentration to 15 mM did not result in a further increase in Bip expression (Figure S1 A).
Hyperglycemia induces ER stress in HUVECs through the glucosamine pathway
To confirm that GlcN was mimicking high glucose effects in our experimental model, we repeated
the time-course experiments maintaining HUVECs in a medium containing 30 mM glucose (HG)
for 24-48-72-96-120-144 hours. Expression levels of Bip were only marginally significantly
increased after 48 hours incubation with HG (Figure S1 B). Notably, after 120-144 hours incubation
with HG, Bip expression reached induction levels comparable with those observed after 48 hours
incubation with GlcN (8.35±0.87 and 8.59±0.61 fold increase, at 120 and 144 hours respectively,
Figure S1 B). To exclude that ER stress induction was a consequence of the increased osmolarity of
cell media, HUVECs were maintained in the presence of the non-metabolizable sugar mannitol as
an osmotic control. Bip levels after 144 hours incubation with Mannitol (30 mM) remained
significantly lower than those induced by HG 30 mM at the corresponding time (Figure S1 B).
Finally, to confirm that HG ability to induce ER stress was mediated by the HBP, we incubated
HUVECs with HG for 144 hours in presence of the inhibitor of HBP activation, Azaserine (5.8
μM). Notably Azaserine was able to completely abolish HG-induced increase of Bip protein levels
(Figure S1 C).
The inhibition of ER stress hampers GlcN-induced apoptosis, pro-inflammatory and prothrombotic state in HUVECs
In order to evaluate whether the effect of GlcN on activation of pro-inflammatory, pro-thrombotic
and pro-apoptotic phenotype in HUVECs is mediated by ER stress induction we incubated
HUVECs with GlcN in the presence of a known inhibitor of ER stress, PBA at 10 mM. Initially, we
measured Bip levels as evidence that ER stress was inhibited in our experimental model, and
observed that GlcN-induced increase of Bip expression was completely abolished by the addition of
PBA (Figure S3 A). We then proceeded to evaluate the downstream responses to ER stress and
observed that cleaved PARP levels were significantly reduced in HUVECs treated with GlcN in the
presence of PBA as compared to cells treated with GlcN only (Figure S3 B). The GlcN-induced
increase of Jnk phosphorylation and NF-kB activity was also significantly reduced by PBA
treatment (Figure S3 C, D) and a decrease in PAI-1, VCAM-1 and ICAM-1 expression was
observed (Figure S4 A, B, C). In addition, mRNA levels of TNFα and CRP were reduced by 20%
and 25% respectively in HUVECs cultured in the presence of PBA in comparison to cells treated
with GlcN only (Figure S4 D, E). Moreover, GlcN-induced increase of IL-6 expression and release
was completely abolished by PBA (Figure S4 F, G).
Therefore, PBA, blocking ER stress induction, reversed the effects of GlcN on apoptosis, procoagulant and pro-inflammatory pathways activation in HUVECs, sustaining the hypothesis of a
crucial role of ER stress in endothelial dysfunction caused by GlcN.
GlcN induces ER stress and the activation of pro-inflammatory and pro-apoptotic pathways
in HCMECs and HRECs
We evaluated whether GlcN exposure induces ER stress response and cellular dysfunction in two
additional endothelial cell lines involved in the pathogenesis of cardiovascular complications of
diabetes, HCMECs and HRECs. Initially HCMECs were treated with GlcN 7.5 mM for 24, 48, 72
and 96 hours. Bip protein levels were significantly increased in HCMECs cultured with GlcN for
24, 48, 72 and 96 hours in comparison to control cells (6.6-fold, 5-fold, 4.7-fold and 3.7-fold
increase respectively, Figure S5 A). Since the maximal effect of GlcN on Bip expression was
observed after 24 hours we selected this time-point for the subsequent experiments. HCMECs were
treated with increasing concentrations of GlcN (0.1-15 mM) and a dose-dependent ER stress
induction was observed. Bip protein expression raised progressively with increasing GlcN doses
with a the maximal induction at the higher doses of GlcN, 7.5 and 15 mM (Figure S5 B).
Furthermore GlcN induced-Bip expression was accompanied by a significant increase in IRE1α and
CHOP protein levels and, as a consequence, by the activation of pro-apoptotic pathways as
demonstrated by higher levels of cleaved PARP (2-fold increase), Jnk phosphorylation (1.5-fold
increase,) (Figure S5 C-D) and caspase 3 activity (1.5-fold increased, Figure S5 E) observed in
GlcN treated cells.
Subsequently, time-course experiments were performed in HRECs and a significant increase in Bip
expression was observed in GlcN treated cells after 24, 48, 72 and 96 hours (15-fold, 12-fold, 12.6fold and 5-fold increase in comparison to control, Figure S6 A). In order to confirm the dosedependent ER stress induction caused by GlcN treatment HRECs were then exposed to increasing
doses of GlcN for 24 hours and a progressive increase of Bip expression, which achieved the
maximal induction in HRECs treated with 7.5 mM GlcN, was observed (Figure S6 B). As a
consequence of ER stress activation HRETs treated with GlcN showed a pro-inflammatory and prothrombotic phenotype as demonstrated by higher levels of Jnk phosphorylation (1.5-fold increase,
Figure S6 C) and increased mRNA levels of PAI-1, VCAM-1, ICAM-1, TNFα, CRP and IL-6, as
compared to control cells (Figure S6 D).
GlcN decreases SIRT1 expression and promotes its O-GlcNAcylation
To gain insight on the role of SIRT1 in allowing a healthy ER stress response we assessed its
expression in GlcN and GlcN+SRT1720 treated cells. We observed that SIRT1 expression was
reduced by 53% in GlcN treated cells as compared to control cells, while GlcN+SRT1720 treated
cells showed intermediate SIRT1 expression levels (Figure S7 A).
Interestingly, we observed increased SIRT1 O-GlcNAcylation levels in both GlcN-treated and
GlcN+SRT1720 cells (Figure S7 B); we thus hypothesized that this post-translational modification
may be associated with increased SIRT1 activity.
Supplemental Figure Legends
Supplemental Fig.1 Hyperglycemia induces ER stress in HUVECs through the glucosamine
pathway.
A) Expression levels of Bip, a marker of ER stress, were evaluated by immunoblotting, in HUVECs
exposed to increasing concentrations of GlcN for 48 hours; data are means ± SD of 4 independent
experiments, framed p value above the graph refers to multiple comparison by ANOVA;
####p≤0.0001 vs control, ****p≤0.0001 vs GlcN 7.5 mM by Bonferroni test. B) Bip protein levels
in HUVECs treated with GlcN (7.5 mM) for 48 hours or with high glucose (30 mM, HG) for 48, 72,
96, 120, 144 hours. Untreated HUVECs were used as control (c) and HUVECs treated with the nonmetabolizable sugar mannitol for 144 hours (M) were used as an osmotic control; data are means ±
SD of 4 independent experiments, framed p value above the graph refers to multiple comparison by
ANOVA; #p≤0.05, ###p≤0.001, ####p≤0.0001 vs control, ****p≤0.0001 vs GlcN, $p≤0.0001 vs
HG 144 hours by Bonferroni test. C) Bip protein levels in HUVECs treated with HG for 144 hours
in presence or absence of Azaserine (5.8 μM, AZA); data are means ± SD of 3 independent
experiments, framed p value above the graph refers to multiple comparison by ANOVA;
####p≤0.0001 vs control,****p≤0.0001 vs HG only by Bonferroni test.
Supplemental Fig.2 GlcN treatment induces ER stress in HUVECs independently of
intracellular protein O-GlcNAcylation.
HUVECs were treated with PUGNAc, a promoter of O-GlcNAcylation of intracellular proteins, or
with GlcN (7.5 mM) in presence or absence of two inhibitors of O-GlcNAcylation, BADGP (0.5
mM) and ST045849 (ST, 20 mM), or with BADGP or ST045849 only for 48 hours. A)
Representative Western Blot images of O-GlcNAcylated proteins, Bip and β-actin. B) Densitometry
of O-GlcNAcylated proteins; data are means ± SD of 4 independent experiments, framed p value
above the graph refers to multiple comparison by ANOVA; #p≤0.05, ## p≤0.01 vs control, *p≤0.05,
***p≤0.001 vs GlcN by Bonferroni test. C) Densitometry of Bip, data are means ± SD of 4
independent experiments, framed p value above the graph refers to multiple comparison by
ANOVA; ####p≤0.0001 vs control, ****p≤0.0001 vs GlcN by Bonferroni test.
Supplemental Fig. 3 GlcN-induced apoptosis, Jnk phosphorylation and NF-kB activation are
hampered in the presence of the ER stress inhibitor PBA.
HUVECs were treated with GlcN (7.5 mM) for 48 hours in presence or absence of PBA (10mM).
A) Representative Western Blot image and densitometry of Bip protein levels; data are means ± SD
of 4 independent experiments, framed p value above the graph refers to multiple comparison by
ANOVA; ####p≤0.0001 vs control and ****p≤0.0001 vs GlcN by Bonferroni test. B)
Representative Western Blot image and densitometry of cleaved PARP levels; data are means ± SD
of 4 independent experiments, framed p value above the graph refers to multiple comparison by
ANOVA; ##p≤0.01 vs control and **p≤0.01 vs GlcN by Bonferroni test. C) Representative
Western Blot image and densitometry of Jnk phosphorylation levels; data are means ± SD of 4
independent experiments, framed p value above the graph refers to multiple comparison by
ANOVA; #p≤0.05 vs control and *p≤0.05 vs GlcN by Bonferroni test. D) NF-kB activity in nuclear
lysates evaluated using a specific NF-kB ELISA assay; data are means ± SD of 3 independent
experiments, run in triplicate, framed p value above the graph refers to multiple comparison by
ANOVA; #p≤0.01 vs control and **p≤0.01 vs GlcN by Bonferroni test.
Supplemental Fig. 4 GlcN-induced procoagulant factors and cytokines expression is reduced
by PBA treatment.
HUVECs were treated with GlcN (7.5 mM) for 48 hours in presence or absence of PBA (10mM).
A) mRNA levels of PAI-1; data are means ± SD of 7 independent experiments, framed p value
above the graph refers to multiple comparison by ANOVA; ##p≤0.01 vs control and **p≤0.01 vs
GlcN by Bonferroni test. B) mRNA levels of VCAM-1; data are means ± SD of 7 independent
experiments, framed p value above the graph refers to multiple comparison by ANOVA; ##p≤0.01
vs control by Bonferroni test. C) mRNA levels of ICAM-1; data are means ± SD of 7 independent
experiments, framed p value above the graph refers to multiple comparison by ANOVA; #p≤0.05 vs
control by Bonferroni test. D) mRNA levels of TNFα; data are means ± SD of 7 independent
experiments, framed p value above the graph refers to multiple comparison by ANOVA; ##p≤0.01
vs control by Bonferroni test. E) mRNA levels of CRP; data are means ± SD of 7 independent
experiments, framed p value above the graph refers to multiple comparison by ANOVA; ##p≤0.01
vs control by Bonferroni test. F) mRNA levels of IL-6; data are means ± SD of 7 independent
experiments, framed p value above the graph refers to multiple comparison by ANOVA;
###p≤0.001 vs control and **p≤0.01vs GlcN by Bonferroni test. G) Cell media were collected to
evaluate IL-6 release; data are means ± SD of 3 independent experiments, run in duplicate, framed p
value above the graph refers to multiple comparison by ANOVA; ##p≤0.01 vs control and **p≤0.01
vs GlcN by Bonferroni test.
Supplemental Fig. 5 GlcN induces ER stress and promotes the activation of pro-apoptotic
pathways in HCMECs.
A) HCMECs were treated with GlcN 7.5 mM for 24, 48, 72, 96 hours and expression levels of Bip,
a marker of ER stress activation, were assessed by immunoblotting. Data are means ± SD of 4
independent experiments, framed p value above the graph refers to multiple comparison by
ANOVA; ###p≤0.001, ####p≤0.0001 vs control by Bonferroni test. B) Bip protein levels were
evaluated in HCMECs exposed to increasing concentrations of GlcN for 24 hours. Data are means ±
SD of 4 independent experiments, framed p value above the graph refers to multiple comparison by
ANOVA; ####p≤0.0001 vs control by Bonferroni test. C) Representative Western Blot images of
IRE1α, CHOP, cleaved PARP, phospho-Jnk, Jnk, β-actin in HCMECs cultured with or without
GlcN 7.5 mM for 24 hours. D) Densitometry of IRE1α, CHOP, cleaved PARP and Jnk
phosphorylation in HCMECs cultured with or without GlcN 7.5 mM for 24 hours. Data are means ±
SD of 4 independent experiments; #p≤0.05, ##p≤0.01, ###p≤0.001 vs control by Student’s t-test. E)
Caspase 3 activity was assessed in HCMECs treated with GlcN 7.5 mM for 24 hours in order to
evaluate the activation of apoptosis. Data are means ± SD of 4 independent experiments; ##p≤0.01
vs control by Student’s t-test.
Supplemental Fig. 6 GlcN induces ER stress and promotes the activation of pro-inflammatory
and pro-thrombotic pathways in HRECs.
A) Bip expression levels were assessed by immunoblotting in HRECs treated with GlcN 7.5 mM
for 24, 48, 72, 96 hours. Data are means ± SD of 3 independent experiments, framed p value above
the graph refers to multiple comparison by ANOVA; #p≤0.05, ####p≤0.0001 vs control by
Bonferroni test. B) Bip protein levels in HRECs exposed to increasing concentrations of GlcN for
24 hours. Data are means ± SD of 3 independent experiments, framed p value above the graph
refers to multiple comparison by ANOVA; ##p≤0.01, ####p≤0.0001 vs control and **p≤0.01 vs 7.5
mM GlcN by Bonferroni test. C) HRECs were cultured with or without GlcN 7.5 mM for 24 hours.
Jnk phosphorylation levels were assessed by immunoblotting; data are means ± SD of 3
independent experiments; ##p≤0.01 vs control by Student’s t-test. D) mRNA levels of PAI-1 (n=6),
VCAM-1 (n=11), ICAM-1 (n=12), TNF-α (n=6), CRP (n=13), IL-6 (n=9) were analyzed by RealTime RT-PCR; data are means ± SD of 6-13 independent experiments; #p≤0.05, ##p≤0.01 vs
control by Student’s t-test.
Supplemental Fig. 7 GlcN decreases SIRT1 expression and promotes its O-GlcNAcylation
A) SIRT1 expression in HUVECs treated for 48 hours with Mannitol (M, 7.5mM), SRT1720 (SRT
only, 10mM), GlcN alone (7.5mM) or in the presence of SRT1720 (10 mM) (SRT+GlcN). Data are
means ± SD of 4 independent experiments, framed p value above the graph refers to multiple
comparison by ANOVA; ##p≤0.01 vs control and *p≤0.05, **p≤0.01 vs GlcN by Bonferroni test.
B) O-GlcNAcylated SIRT1 levels, assessed by immunoblotting SIRT1 immunoprecipitates with an
anti O-GlcNAcylated proteins antibody, were evaluated in HUVECs treated with GlcN (7.5 mM) in
presence or absence of SRT1720 (10 mM, SRT). Data are means ± SD of 3 independent
experiments, framed p value above the graph refers to multiple comparison by ANOVA; #p≤0.05 vs
control by Bonferroni test.