Articles in PresS. Am J Physiol Heart Circ Physiol (August 15, 2014). doi:10.1152/ajpheart.00210.2014
1
Title:
2
AMPK activation by glucagon-like peptide-1 prevents NADPH oxidase
3
activation induced by hyperglycemia in adult cardiomyocytes.
4 5
Authors:
6
Magali Balteau1, Anne Van Steenbergen1, Aurélie D. Timmermans1, Chantal
7
Dessy2, Gaetane Behets-Wydemans2, Nicolas Tajeddine3, Diego Castanares-
8
Zapatero1, Patrick Gilon4, Jean-Louis Vanoverschelde1,5, Sandrine Horman1,
9
Louis Hue6, Luc Bertrand1, Christophe Beauloye1,5.
10 11
Affiliation:
12
1
13
et Clinique (IREC), Pôle de Recherche Cardiovasculaire, Brussels, Belgium
14
2
15
et Clinique (IREC), Pôle de Pharmacothérapie et Thérapeutique, Brussels,
16
Belgium
17
3
18
Cell Physiology, Brussels, Belgium
19
4
20
et Clinique (IREC), Pôle d'Endocrinologie, Diabète et Nutrition, Brussels,
21
Belgium
22
5
Cliniques universitaires Saint Luc, Division of Cardiology, Brussels, Belgium
23
6
Université catholique de Louvain (UCL), de Duve Institute, Brussels, Belgium
Université catholique de Louvain (UCL), Institut de Recherche Expérimentale
Université catholique de Louvain (UCL), Institut de Recherche Expérimentale
Université catholique de Louvain (UCL), Institute of Neuroscience, Division of
Université catholique de Louvain (UCL), Institut de Recherche Expérimentale
24 25
Running head :
1 Copyright © 2014 by the American Physiological Society.
26
AMPK-mediated inhibition of ROS production by GLP-1
27 28
Address for correspondence
29
Christophe Beauloye, MD, PhD
30
Université catholique de Louvain (UCL)
31
Institut de Recherche Expérimentale et Clinique (IREC)
32
Pôle de Recherche Cardiovasculaire
33
55, Avenue Hippocrate
34
1200 Brussels
35
Belgium
36
Phone: +32 2 764 55 66
37
Fax: +32 2 764 5536
38
e-mail: [email protected]
39 40 41
2
42
Abstract:
43
Exposure of cardiomyocytes to high glucose concentrations (HG) stimulates
44
ROS production by NADPH oxidase (NOX2). NOX2 activation is triggered by
45
enhanced glucose transport through a sodium-glucose co-transporter (SGLT)
46
but not by a stimulation of glucose metabolism. The aim of this work was to
47
identify potential therapeutic approaches to counteract this glucotoxicity.
48
In cultured adult rat cardiomyocytes incubated with 21 mM glucose (HG), AMP-
49
activated protein kinase (AMPK) activation by A769662 or phenformin nearly
50
suppressed ROS production. Interestingly, GLP-1, a new anti-diabetic drug,
51
concomitantly induced AMPK activation and prevented the HG-mediated ROS
52
production (maximal effect at 100 nM). α2AMPK, the major isoform expressed in
53
cardiomyocytes (but not α1AMPK), was activated in response to GLP-1. Anti-
54
ROS properties of AMPK activators were not related to changes in glucose
55
uptake or glycolysis. Using in situ proximity ligation assay, we demonstrated that
56
AMPK activation prevented the HG-induced p47phox translocation to caveolae,
57
whatever the AMPK activators used. NOX2 activation by either α-methyl-D-
58
glucopyranoside, a glucose analog transported through SGLT, or angiotensin II
59
was also counteracted by GLP-1. The crucial role of AMPK in limiting HG-
60
mediated NOX2 activation was demonstrated by overexpressing a constitutively
61
active form of α2AMPK using adenoviral infection. This overexpression
62
prevented NOX2 activation in response to HG, whereas GLP-1 lost its protective
63
action in α2AMPK deficient mouse cardiomyocytes. Under HG, the GLP-
64
1/AMPK pathway inhibited PKCβ2 phosphorylation, a key element mediating
65
p47phox translocation.
3
66
In conclusion, GLP-1 induces α2AMPK activation and blocks HG-induced
67
p47phox
68
glucotoxicity.
translocation
to
the
plasma
membrane,
thereby
preventing
69 70
Keywords:
71
Glucose, Heart, Oxidative stress, Glucagon-like peptide-1 (GLP-1), AMP-
72
activated protein kinase (AMPK)
73
4
74
Introduction.
75
Hyperglycemia is a major risk factor for the development of cardiovascular
76
diseases in diabetic patients. Indeed, chronic exposure to high glucose (HG)
77
concentrations damages cardiomyocytes (13). This glucotoxicity involves a
78
number of pathological mechanisms, the most substantial one being the
79
exacerbated production of reactive oxygen species (ROS) (15).
80
Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase plays a critical
81
role in the glucose-induced oxidative stress in the heart (2). Initially described in
82
macrophages, this multimeric enzyme has been later identified in other cell
83
types, including cardiomyocytes (40). NOX2 and NOX4 are the two isoforms
84
found in cardiac cells. They consist of a heterodimeric membrane-bound
85
cytochrome b558 made of either NOX2 or NOX4 and p22phox subunits.
86
Whereas NOX4 is constitutively active and regulated by expression, NOX2
87
activation requires the association at the plasma membrane of four cytosolic
88
partners, p40phox, p67phox, p47phox and Rac1, the two latter being considered
89
as activator subunits (4). p47phox phosphorylation triggers its translocation to
90
the membrane in neutrophils and can be catalysed by five PKCs: α, β, δ, ε and ζ
91
(14).
92
NOX2 is the isoform involved in HG toxicity in cardiomyocytes in culture (39).
93
The mechanism is not solely dependent on glucose metabolism but seems to
94
require a SGLT-type of glucose transporter (2). Increased glucose transport
95
through this transporter triggers a signaling cascade leading to Rac1 activation
96
and p47phox translocation with subsequent ROS generation by NOX2 and
97
eventually cell death. Moreover, PKCβ2 activation in cardiac caveolae was
98
recently shown to mediate glucotoxicity, although the link between PKCβ2
5
99
activation and p47phox translocation leading to NOX2 activation remains to be
100
demonstrated (22).
101
The incretin glucagon-like peptide-1 (GLP-1) is a new therapeutic approach to
102
control glycemia in diabetic patients (12). It is derived from the proglucagon
103
precursor and is secreted by intestinal L-cells following nutrient ingestion. GLP-1
104
(7-36) amide is rapidly degraded to GLP-1 (9-36) by the enzyme dipeptidyl
105
peptidase-4 (DPP4). It acts through a G-coupled receptor (GLP-1R), which is
106
present on pancreatic β-cells where it displays glucoregulatory and insulinotropic
107
effects. This receptor is also expressed in other cell types, including
108
cardiomyocytes, and several GLP-1 cardiovascular beneficial properties have
109
been attributed to a direct effect of GLP-1 on heart, in animal models as well as
110
in humans (34).
111
GLP-1 has been reported to activate AMP-activated protein kinase (AMPK) in
112
liver by a still unknown mechanism (5). AMPK is a serine/threonine kinase made
113
of three subunits: the catalytic α-subunit, and two regulatory β and γ subunits
114
(18). There are seven AMPK genes encoding two α (1 and 2), two β (1 and 2),
115
and three γ (1, 2, and 3) isoforms respectively. AMPK is activated by
116
phosphorylation of Thr172 in the catalytic domain by at least two upstream
117
kinases. The first mechanism senses energy and involves AMP and LKB1,
118
whereas the second implies changes in intracellular calcium ion concentration
119
and is mediated by calmodulin-dependent kinase kinase β (CaMKKβ) (18).
120
AMPK acts as an energy sensor regulating energy homeostasis although its role
121
extends well beyond metabolism (19).
122
In this work, we demonstrate that GLP-1 protects cardiomyocytes against HG-
123
induced NOX2 activation through an AMPK-mediated mechanism.
6
124
Methods
125
Animals and materials
126
Animal handlings were approved by the Animal Research Committee of the
127
Université catholique de Louvain (2012/UCL/MD/003) and conformed to the
128
Guide for the Care and Use of Laboratory Animals published by the US National
129
Institutes of Health (NIH Publication No. 85-23, revised 1996). All biochemicals
130
were from Sigma, unless otherwise stated. SAMS and Exendin 9-39 peptides
131
were kindly provided by V. Stroobant (Ludwig Institute, Brussels, Belgium). The
132
antibodies
133
phosphoAMPK (T172), acetyl-CoA Carboxylase (ACC), eukaryotic elongation
134
factor
135
Hemaglutinin (HA) (Cell Signaling); phosphoACC (S79) (Pierce); caveolin-3
136
(Santa-Cruz); p47phox, Glut-4 (Millipore).
2
used
(eEF2),
were:
α1
c-myc-tag,
and
α2AMPK
phosphoPKC
(Kinasource);
(pan)
(β2
total-αAMPK,
S660),
GAPDH,
137 138
Isolation and culture of adult rat cardiomyocytes
139
Adult male Wistar fed rats were anesthetized with pentobarbital 50 mg/kg IP and
140
sacrificed by cervical dislocation before removing the heart. The cardiomyocytes
141
were isolated by perfusion with collagenase type II (1 mg/ml, Worthington), and
142
cultured as previously described (6). Briefly, cells were plated on laminin-coated
143
dishes and incubated for 24 h in fresh medium 199 (Invitrogen, 5.5 mM D-
144
glucose) containing 100 mg/l glutamine, 2 mM carnitine, 5 mM creatine, 5 mM
145
taurine, 0.1 nM T3 and 0.2% (w/v) BSA (fatty acid free) in the presence of 100
146
U/ml penicillin and 100 µg/ml streptomycin. Primary cultures of cardiomyocytes
147
were incubated with the different concentrations of D-glucose and other
148
additions, for the indicated periods of time and as detailed in the figure legends.
7
149 150
Isolation and culture of adult mouse cardiomyocytes (WT and KO mice for
151
AMPK α2 subunit)
152
Adult male mice were anesthetized with ketamine (80 mg/kg) and xylazine (10
153
mg/kg). Cardiomyocytes were isolated by perfusion with liberase DH enzyme
154
(1.25mg/heart, Roche). Cells were then plated on laminin-coated dishes and
155
incubated for 2 h in fresh medium 199 (Invitrogen, 5.5 mM D-glucose) containing
156
100 U/ml penicillin and 100 µg/ml streptomycin. Primary cultures of mouse
157
cardiomyocytes were treated as adult rat cardiomyocytes.
158 159
Measurement of reactive oxygen species
160
Intracellular ROS production was measured by evaluating oxidation of the cell
161
permeable fluorescent probe 2’,7’-dihydrodichlorofluorescein diacetate (H2DCF-
162
DA, Invitrogen), as previously described (2).
163 164
Metabolic measurements
165
Glucose uptake and glycolytic flux were measured by the rate of detritiation of
166
[2-3H]glucose and [3-3H]glucose, respectively (43). Purine nucleotides were
167
measured in neutralized perchloric acid extracts following their separation by
168
HPLC (26).
169 170
Glut4 translocation to the plasma membrane
171
Adenovirus expressing HA-Glut4-GFP protein was kindly provided by Dr Corley
172
Mastick (Department of Biochemistry and Molecular Biology, University of
173
Nevada, USA) (27). Glut4 is labelled at the C-terminal part with GFP and, at the
8
174
N-terminal part with HA. When Glut4 is translocated to the plasma membrane,
175
HA can be detected at the cell surface. Experiments were performed 48 h after
176
infection (multiplicity of infection 10). After treatment, cardiomyocytes were fixed
177
with paraformaldehyde (4% v/v) on ice and placed in a blocking solution (bovine
178
serum albumin, 5% w/v). Cells were first incubated with a primary antibody (anti-
179
HA) followed by a secondary fluorescent antibody (Alexa Fluor 594). Glut4 at the
180
plasma membrane appears as red fluorescent dots. Nuclei were stained with
181
DAPI. Protein staining was visualized using a Zeiss.Imager.Z1 microscope
182
equipped with an ApoTome device. Red fluorescent dots (HA) were quantified
183
related to green (GFP) by the software Photoshop.
184 185
Immunoblot analysis and AMPK activity measurement
186
Cells were lyzed in cold buffer containing 50 mM Hepes (pH 7.5), 50 mM KF, 1
187
mM KPi, 5 mM EDTA, 5 mM EGTA, 15 mM β-mercaptoethanol, 1 µg/ml
188
leupeptin, 1 mM benzamidin, 1 mM phenylmethylsulfonyl fluoride, 1 mM
189
vanadate, and 1 % v/v Triton X-100. Protein content was measured with bovine
190
serum albumin as a standard (9). Immunoblots were performed with total
191
extracts separated on 10% SDS-PAGE and transferred to polyvinylidene
192
difluoride membranes. The membranes were blocked with milk or bovine serum
193
albumin (5% w/v) and then blotted with the corresponding antibodies.
194
incubation with the appropriate secondary antibody, proteins were visualized
195
using electrochemical luminescence (Pierce) and quantified using Image J.
196
Band intensities were normalized relative to those of loading on the same gel.
197
The loading control used was eEF-2 except for PKC (GAPDH). Immunoblotting
After
9
198
with respective anti-total antibodies was performed on a separate gel to verify
199
the absence of any modification in protein expression.
200
Total AMPK activity was measured after PEG fractionation and α1AMPK and
201
α2AMPK activities were measured on respective immunoprecipitates, as
202
previously described (48).
203 204
Proteins co-localisation using in situ proximity ligation assay (PLA)
205
This technique allows visualization of a co-localisation of two proteins (24). After
206
treatment, cardiomyocytes were fixed with paraformaldehyde (4% v/v),
207
permeabilized with triton X-100 (0.2% v/v in PBS), and placed in a blocking
208
solution at 37°C. Cells were then incubated overnight at 4°C with two primary
209
antibodies (from two different species) raised against the two proteins of interest.
210
Secondary antibodies raised against the two different species and coupled with
211
special oligonucleotides dyes were then added. If the proteins are at less than
212
40 nm from each other, a ligase creates a circle-shaped structure
213
(‘concatemer’), which is amplified by a polymerase. This amplified product is
214
finally detected with complementary fluorescently labelled oligonucleotides that
215
hybridize to the concatemers. The result is observed as red fluorescent dots
216
appearing when proteins co-localise. Nuclei were stained with DAPI. Protein
217
staining was visualized using a Zeiss.Imager.Z1 microscope equipped with an
218
ApoTome device. Red fluorescent dots were quantified (AxioVision Rel 4.8.).
219 220
Adenoviral infection
221
Adult rat cardiomyocytes were infected (multiplicity of infection 100) as
222
described (6) with two different adenoviral constructions, one expressing
10
223
constitutively active form of α2AMPK and the other expressing GFP alone.
224
Experiments were performed 48 h after infection.
225 226
Quantitative PCR
227
Total mRNA was extracted from cardiomyocytes using a chloroform/isopropanol
228
procedure (Tripure, Roche). Reverse transcription was performed for 1h at 37°C
229
with 1µg RNA using an iScriptTM cDNA synthesis kit (Bio-Rad laboratories).
230
Quantitative PCR was performed on IQ5 (Bio-Rad) using the qPCR Core kit for
231
Sybergreen (Eurogentec). The mRNA level for each gene in each sample was
232
normalized to the housekeeping gene hypoxanthine guanine phosphoribosyl
233
transferase 1 (Hprt1), after ΔΔCt correction. The nucleotide sequences of
234
primers used were: p47phox: 5’-TCACCGAGATCTACGAGTTC-3’ (sense) and
235
5’-TCCCATGAGGCTGTTGAAGT-3’
(antisense),
236
GTCCCAGCGTCGTGATTAGT-3’
(sense)
237
ACAGAGGGCCACAATGTGAT-3’ (antisense).
Hprt1 and
5’5’-
238 239
Statistical analysis
240
All the values are expressed as mean ± SEM. Comparisons were performed
241
using ANOVA followed by a Bonferroni post-hoc correction. p
1
Title:
2
AMPK activation by glucagon-like peptide-1 prevents NADPH oxidase
3
activation induced by hyperglycemia in adult cardiomyocytes.
4 5
Authors:
6
Magali Balteau1, Anne Van Steenbergen1, Aurélie D. Timmermans1, Chantal
7
Dessy2, Gaetane Behets-Wydemans2, Nicolas Tajeddine3, Diego Castanares-
8
Zapatero1, Patrick Gilon4, Jean-Louis Vanoverschelde1,5, Sandrine Horman1,
9
Louis Hue6, Luc Bertrand1, Christophe Beauloye1,5.
10 11
Affiliation:
12
1
13
et Clinique (IREC), Pôle de Recherche Cardiovasculaire, Brussels, Belgium
14
2
15
et Clinique (IREC), Pôle de Pharmacothérapie et Thérapeutique, Brussels,
16
Belgium
17
3
18
Cell Physiology, Brussels, Belgium
19
4
20
et Clinique (IREC), Pôle d'Endocrinologie, Diabète et Nutrition, Brussels,
21
Belgium
22
5
Cliniques universitaires Saint Luc, Division of Cardiology, Brussels, Belgium
23
6
Université catholique de Louvain (UCL), de Duve Institute, Brussels, Belgium
Université catholique de Louvain (UCL), Institut de Recherche Expérimentale
Université catholique de Louvain (UCL), Institut de Recherche Expérimentale
Université catholique de Louvain (UCL), Institute of Neuroscience, Division of
Université catholique de Louvain (UCL), Institut de Recherche Expérimentale
24 25
Running head :
1 Copyright © 2014 by the American Physiological Society.
26
AMPK-mediated inhibition of ROS production by GLP-1
27 28
Address for correspondence
29
Christophe Beauloye, MD, PhD
30
Université catholique de Louvain (UCL)
31
Institut de Recherche Expérimentale et Clinique (IREC)
32
Pôle de Recherche Cardiovasculaire
33
55, Avenue Hippocrate
34
1200 Brussels
35
Belgium
36
Phone: +32 2 764 55 66
37
Fax: +32 2 764 5536
38
e-mail: [email protected]
39 40 41
2
42
Abstract:
43
Exposure of cardiomyocytes to high glucose concentrations (HG) stimulates
44
ROS production by NADPH oxidase (NOX2). NOX2 activation is triggered by
45
enhanced glucose transport through a sodium-glucose co-transporter (SGLT)
46
but not by a stimulation of glucose metabolism. The aim of this work was to
47
identify potential therapeutic approaches to counteract this glucotoxicity.
48
In cultured adult rat cardiomyocytes incubated with 21 mM glucose (HG), AMP-
49
activated protein kinase (AMPK) activation by A769662 or phenformin nearly
50
suppressed ROS production. Interestingly, GLP-1, a new anti-diabetic drug,
51
concomitantly induced AMPK activation and prevented the HG-mediated ROS
52
production (maximal effect at 100 nM). α2AMPK, the major isoform expressed in
53
cardiomyocytes (but not α1AMPK), was activated in response to GLP-1. Anti-
54
ROS properties of AMPK activators were not related to changes in glucose
55
uptake or glycolysis. Using in situ proximity ligation assay, we demonstrated that
56
AMPK activation prevented the HG-induced p47phox translocation to caveolae,
57
whatever the AMPK activators used. NOX2 activation by either α-methyl-D-
58
glucopyranoside, a glucose analog transported through SGLT, or angiotensin II
59
was also counteracted by GLP-1. The crucial role of AMPK in limiting HG-
60
mediated NOX2 activation was demonstrated by overexpressing a constitutively
61
active form of α2AMPK using adenoviral infection. This overexpression
62
prevented NOX2 activation in response to HG, whereas GLP-1 lost its protective
63
action in α2AMPK deficient mouse cardiomyocytes. Under HG, the GLP-
64
1/AMPK pathway inhibited PKCβ2 phosphorylation, a key element mediating
65
p47phox translocation.
3
66
In conclusion, GLP-1 induces α2AMPK activation and blocks HG-induced
67
p47phox
68
glucotoxicity.
translocation
to
the
plasma
membrane,
thereby
preventing
69 70
Keywords:
71
Glucose, Heart, Oxidative stress, Glucagon-like peptide-1 (GLP-1), AMP-
72
activated protein kinase (AMPK)
73
4
74
Introduction.
75
Hyperglycemia is a major risk factor for the development of cardiovascular
76
diseases in diabetic patients. Indeed, chronic exposure to high glucose (HG)
77
concentrations damages cardiomyocytes (13). This glucotoxicity involves a
78
number of pathological mechanisms, the most substantial one being the
79
exacerbated production of reactive oxygen species (ROS) (15).
80
Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase plays a critical
81
role in the glucose-induced oxidative stress in the heart (2). Initially described in
82
macrophages, this multimeric enzyme has been later identified in other cell
83
types, including cardiomyocytes (40). NOX2 and NOX4 are the two isoforms
84
found in cardiac cells. They consist of a heterodimeric membrane-bound
85
cytochrome b558 made of either NOX2 or NOX4 and p22phox subunits.
86
Whereas NOX4 is constitutively active and regulated by expression, NOX2
87
activation requires the association at the plasma membrane of four cytosolic
88
partners, p40phox, p67phox, p47phox and Rac1, the two latter being considered
89
as activator subunits (4). p47phox phosphorylation triggers its translocation to
90
the membrane in neutrophils and can be catalysed by five PKCs: α, β, δ, ε and ζ
91
(14).
92
NOX2 is the isoform involved in HG toxicity in cardiomyocytes in culture (39).
93
The mechanism is not solely dependent on glucose metabolism but seems to
94
require a SGLT-type of glucose transporter (2). Increased glucose transport
95
through this transporter triggers a signaling cascade leading to Rac1 activation
96
and p47phox translocation with subsequent ROS generation by NOX2 and
97
eventually cell death. Moreover, PKCβ2 activation in cardiac caveolae was
98
recently shown to mediate glucotoxicity, although the link between PKCβ2
5
99
activation and p47phox translocation leading to NOX2 activation remains to be
100
demonstrated (22).
101
The incretin glucagon-like peptide-1 (GLP-1) is a new therapeutic approach to
102
control glycemia in diabetic patients (12). It is derived from the proglucagon
103
precursor and is secreted by intestinal L-cells following nutrient ingestion. GLP-1
104
(7-36) amide is rapidly degraded to GLP-1 (9-36) by the enzyme dipeptidyl
105
peptidase-4 (DPP4). It acts through a G-coupled receptor (GLP-1R), which is
106
present on pancreatic β-cells where it displays glucoregulatory and insulinotropic
107
effects. This receptor is also expressed in other cell types, including
108
cardiomyocytes, and several GLP-1 cardiovascular beneficial properties have
109
been attributed to a direct effect of GLP-1 on heart, in animal models as well as
110
in humans (34).
111
GLP-1 has been reported to activate AMP-activated protein kinase (AMPK) in
112
liver by a still unknown mechanism (5). AMPK is a serine/threonine kinase made
113
of three subunits: the catalytic α-subunit, and two regulatory β and γ subunits
114
(18). There are seven AMPK genes encoding two α (1 and 2), two β (1 and 2),
115
and three γ (1, 2, and 3) isoforms respectively. AMPK is activated by
116
phosphorylation of Thr172 in the catalytic domain by at least two upstream
117
kinases. The first mechanism senses energy and involves AMP and LKB1,
118
whereas the second implies changes in intracellular calcium ion concentration
119
and is mediated by calmodulin-dependent kinase kinase β (CaMKKβ) (18).
120
AMPK acts as an energy sensor regulating energy homeostasis although its role
121
extends well beyond metabolism (19).
122
In this work, we demonstrate that GLP-1 protects cardiomyocytes against HG-
123
induced NOX2 activation through an AMPK-mediated mechanism.
6
124
Methods
125
Animals and materials
126
Animal handlings were approved by the Animal Research Committee of the
127
Université catholique de Louvain (2012/UCL/MD/003) and conformed to the
128
Guide for the Care and Use of Laboratory Animals published by the US National
129
Institutes of Health (NIH Publication No. 85-23, revised 1996). All biochemicals
130
were from Sigma, unless otherwise stated. SAMS and Exendin 9-39 peptides
131
were kindly provided by V. Stroobant (Ludwig Institute, Brussels, Belgium). The
132
antibodies
133
phosphoAMPK (T172), acetyl-CoA Carboxylase (ACC), eukaryotic elongation
134
factor
135
Hemaglutinin (HA) (Cell Signaling); phosphoACC (S79) (Pierce); caveolin-3
136
(Santa-Cruz); p47phox, Glut-4 (Millipore).
2
used
(eEF2),
were:
α1
c-myc-tag,
and
α2AMPK
phosphoPKC
(Kinasource);
(pan)
(β2
total-αAMPK,
S660),
GAPDH,
137 138
Isolation and culture of adult rat cardiomyocytes
139
Adult male Wistar fed rats were anesthetized with pentobarbital 50 mg/kg IP and
140
sacrificed by cervical dislocation before removing the heart. The cardiomyocytes
141
were isolated by perfusion with collagenase type II (1 mg/ml, Worthington), and
142
cultured as previously described (6). Briefly, cells were plated on laminin-coated
143
dishes and incubated for 24 h in fresh medium 199 (Invitrogen, 5.5 mM D-
144
glucose) containing 100 mg/l glutamine, 2 mM carnitine, 5 mM creatine, 5 mM
145
taurine, 0.1 nM T3 and 0.2% (w/v) BSA (fatty acid free) in the presence of 100
146
U/ml penicillin and 100 µg/ml streptomycin. Primary cultures of cardiomyocytes
147
were incubated with the different concentrations of D-glucose and other
148
additions, for the indicated periods of time and as detailed in the figure legends.
7
149 150
Isolation and culture of adult mouse cardiomyocytes (WT and KO mice for
151
AMPK α2 subunit)
152
Adult male mice were anesthetized with ketamine (80 mg/kg) and xylazine (10
153
mg/kg). Cardiomyocytes were isolated by perfusion with liberase DH enzyme
154
(1.25mg/heart, Roche). Cells were then plated on laminin-coated dishes and
155
incubated for 2 h in fresh medium 199 (Invitrogen, 5.5 mM D-glucose) containing
156
100 U/ml penicillin and 100 µg/ml streptomycin. Primary cultures of mouse
157
cardiomyocytes were treated as adult rat cardiomyocytes.
158 159
Measurement of reactive oxygen species
160
Intracellular ROS production was measured by evaluating oxidation of the cell
161
permeable fluorescent probe 2’,7’-dihydrodichlorofluorescein diacetate (H2DCF-
162
DA, Invitrogen), as previously described (2).
163 164
Metabolic measurements
165
Glucose uptake and glycolytic flux were measured by the rate of detritiation of
166
[2-3H]glucose and [3-3H]glucose, respectively (43). Purine nucleotides were
167
measured in neutralized perchloric acid extracts following their separation by
168
HPLC (26).
169 170
Glut4 translocation to the plasma membrane
171
Adenovirus expressing HA-Glut4-GFP protein was kindly provided by Dr Corley
172
Mastick (Department of Biochemistry and Molecular Biology, University of
173
Nevada, USA) (27). Glut4 is labelled at the C-terminal part with GFP and, at the
8
174
N-terminal part with HA. When Glut4 is translocated to the plasma membrane,
175
HA can be detected at the cell surface. Experiments were performed 48 h after
176
infection (multiplicity of infection 10). After treatment, cardiomyocytes were fixed
177
with paraformaldehyde (4% v/v) on ice and placed in a blocking solution (bovine
178
serum albumin, 5% w/v). Cells were first incubated with a primary antibody (anti-
179
HA) followed by a secondary fluorescent antibody (Alexa Fluor 594). Glut4 at the
180
plasma membrane appears as red fluorescent dots. Nuclei were stained with
181
DAPI. Protein staining was visualized using a Zeiss.Imager.Z1 microscope
182
equipped with an ApoTome device. Red fluorescent dots (HA) were quantified
183
related to green (GFP) by the software Photoshop.
184 185
Immunoblot analysis and AMPK activity measurement
186
Cells were lyzed in cold buffer containing 50 mM Hepes (pH 7.5), 50 mM KF, 1
187
mM KPi, 5 mM EDTA, 5 mM EGTA, 15 mM β-mercaptoethanol, 1 µg/ml
188
leupeptin, 1 mM benzamidin, 1 mM phenylmethylsulfonyl fluoride, 1 mM
189
vanadate, and 1 % v/v Triton X-100. Protein content was measured with bovine
190
serum albumin as a standard (9). Immunoblots were performed with total
191
extracts separated on 10% SDS-PAGE and transferred to polyvinylidene
192
difluoride membranes. The membranes were blocked with milk or bovine serum
193
albumin (5% w/v) and then blotted with the corresponding antibodies.
194
incubation with the appropriate secondary antibody, proteins were visualized
195
using electrochemical luminescence (Pierce) and quantified using Image J.
196
Band intensities were normalized relative to those of loading on the same gel.
197
The loading control used was eEF-2 except for PKC (GAPDH). Immunoblotting
After
9
198
with respective anti-total antibodies was performed on a separate gel to verify
199
the absence of any modification in protein expression.
200
Total AMPK activity was measured after PEG fractionation and α1AMPK and
201
α2AMPK activities were measured on respective immunoprecipitates, as
202
previously described (48).
203 204
Proteins co-localisation using in situ proximity ligation assay (PLA)
205
This technique allows visualization of a co-localisation of two proteins (24). After
206
treatment, cardiomyocytes were fixed with paraformaldehyde (4% v/v),
207
permeabilized with triton X-100 (0.2% v/v in PBS), and placed in a blocking
208
solution at 37°C. Cells were then incubated overnight at 4°C with two primary
209
antibodies (from two different species) raised against the two proteins of interest.
210
Secondary antibodies raised against the two different species and coupled with
211
special oligonucleotides dyes were then added. If the proteins are at less than
212
40 nm from each other, a ligase creates a circle-shaped structure
213
(‘concatemer’), which is amplified by a polymerase. This amplified product is
214
finally detected with complementary fluorescently labelled oligonucleotides that
215
hybridize to the concatemers. The result is observed as red fluorescent dots
216
appearing when proteins co-localise. Nuclei were stained with DAPI. Protein
217
staining was visualized using a Zeiss.Imager.Z1 microscope equipped with an
218
ApoTome device. Red fluorescent dots were quantified (AxioVision Rel 4.8.).
219 220
Adenoviral infection
221
Adult rat cardiomyocytes were infected (multiplicity of infection 100) as
222
described (6) with two different adenoviral constructions, one expressing
10
223
constitutively active form of α2AMPK and the other expressing GFP alone.
224
Experiments were performed 48 h after infection.
225 226
Quantitative PCR
227
Total mRNA was extracted from cardiomyocytes using a chloroform/isopropanol
228
procedure (Tripure, Roche). Reverse transcription was performed for 1h at 37°C
229
with 1µg RNA using an iScriptTM cDNA synthesis kit (Bio-Rad laboratories).
230
Quantitative PCR was performed on IQ5 (Bio-Rad) using the qPCR Core kit for
231
Sybergreen (Eurogentec). The mRNA level for each gene in each sample was
232
normalized to the housekeeping gene hypoxanthine guanine phosphoribosyl
233
transferase 1 (Hprt1), after ΔΔCt correction. The nucleotide sequences of
234
primers used were: p47phox: 5’-TCACCGAGATCTACGAGTTC-3’ (sense) and
235
5’-TCCCATGAGGCTGTTGAAGT-3’
(antisense),
236
GTCCCAGCGTCGTGATTAGT-3’
(sense)
237
ACAGAGGGCCACAATGTGAT-3’ (antisense).
Hprt1 and
5’5’-
238 239
Statistical analysis
240
All the values are expressed as mean ± SEM. Comparisons were performed
241
using ANOVA followed by a Bonferroni post-hoc correction. p
