© 2001 American Heart Association, Inc.
Integrative Physiology |
No-Flow Ischemia Inhibits Insulin Signaling in Heart by Decreasing Intracellular pH
From the Division of Cardiology (C.B., J.-L.V.), and Hormone and Metabolic Research Unit (C.B., L.B., U.K., A.-S.M., L.H.), Christian de Duve Institute of Cellular Pathology, Université catholique de Louvain, Brussels, and Biomedical Nuclear Magnetic Resonance Unit (T.D., F.V.), Department of Radiology, Katholieke Universiteit Leuven, Belgium.
Correspondence to Prof L. Hue, HORM Unit, 75, Ave Hippocrate, ICP-UCL 7529, B-1200 Brussels, Belgium. E-mail hue{at}horm.ucl.ac.be
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Abstract |
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Abstract—Glucose-insulin-potassium solutions exert beneficial effects on the ischemic heart by reducing infarct size and mortality and improving postischemic left ventricular function. Insulin could be the critical protective component of this mixture, although the insulin response of the ischemic and postischemic myocardium has not been systematically investigated. The aim of this work was to study the insulin response during ischemia by analyzing insulin signaling. This was evaluated by measuring changes in activity and/or phosphorylation state of insulin signaling elements in isolated perfused rat hearts submitted to no-flow ischemia. Intracellular pH (pHi) was measured by NMR. No-flow ischemia antagonized insulin signaling including insulin receptor, insulin receptor substrate-1, phosphatidylinositol 3-kinase, protein kinase B, p70 ribosomal S6 kinase, and glycogen synthase kinase-3. These changes were concomitant with intracellular acidosis. Perfusing hearts with ouabain and amiloride in normoxic conditions decreased pHi and insulin signaling, whereas perfusing at pH 8.2 counteracted the drop in pHi and the inhibition of insulin signaling by ischemia. Incubation of cardiomyocytes in normoxic conditions, but at pH values below 6.75, mimicked the effect of ischemia and also inhibited insulin-stimulated glucose uptake. Finally, the in vitro insulin receptor tyrosine kinase activity was progressively inhibited at pH values below physiological pHi, being abolished at pH 6.0. Therefore, ischemic acidosis decreases kinase activity and tyrosine phosphorylation of the insulin receptor thereby preventing activation of the downstream components of the signaling pathway. We conclude that severe ischemia inhibits insulin signaling by decreasing pHi.
Key Words: ischemia • insulin • signal transduction • glucose • acidosis
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Introduction |
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Experimental and clinical studies have shown that glucose-insulin-potassium (GIK) perfusions exert beneficial effects on the ischemic heart by reducing infarct size and mortality and improving postischemic left ventricular function.1 2 3 4 The protection conferred by GIK could be due to insulin and/or glucose, and each one could act during ischemia and/or reperfusion. The aim of this work was not to confirm the beneficial effects of GIK solutions but rather to focus on the insulin response of the heart during ischemia.
The heart response to insulin is usually studied by measuring glucose uptake and glycolysis, which are known targets of insulin on this tissue. This experimental approach is useless in ischemic hearts. Indeed, a correct balance of glucose metabolism cannot be made in no-flow ischemia. Moreover, the effects of insulin on myocardial glucose metabolism are not easily distinguished from those of ischemia, because both stimulate glucose uptake and glycolysis.5 6 Therefore, metabolic parameters such as glucose uptake and transport are not adequate to specifically measure the insulin response during ischemia. Another method of studying whether the ischemic myocardium is responsive to insulin is to analyze the insulin signaling pathway, which differs from that of ischemia. The stimulation of glycolysis resulting from anaerobic conditions seems to be mediated by AMP-activated protein kinase (AMPK).7 8 This protein kinase responds to the energy state of the cell but has not been reported to participate in insulin signaling.
Insulin signaling is initiated by its binding to the insulin receptor (IR). This activates the tyrosine kinase activity of IR leading to IR autophosphorylation and to subsequent phosphorylation of IR substrates (IRS) (for recent review see Vanhaesebroeck and Alessi9 ). The downstream signaling components involve phosphatidylinositol 3-kinase (PI3K), which is activated by binding to phosphorylated IRS, and protein kinase B (PKB)/Akt, which is activated by phosphorylation of Thr308 and Ser473 by 3-phosphoinositide–dependent protein kinase-1 and -2. PKB mediates some of the metabolic effects of insulin, which include the following: (1) stimulation of glycogen synthesis by phosphorylation and inactivation of glycogen synthase kinase-3 (GSK-3); (2) activation of phosphodiesterase 3B, the enzyme responsible for the anti-cAMP effect of insulin; and (3) recruitment of GLUT4 transporters to the plasma membrane.10 However, PKB does not seem to be required for the insulin-induced activation of 6-phosphofructo-2-kinase, the enzyme responsible for the synthesis of fructose 2,6-bisphosphate, a potent stimulator of glycolysis.11 Nevertheless, it is clear that PKB participates in insulin signaling, even if it does not mediate all its metabolic effects. Moreover, PKB could mediate some effects of insulin involved in cell protection, but which are not related to the metabolic effects.12 Therefore, as a first experimental approach in this study, we resorted to PKB activation to assess insulin action during ischemia.
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Materials and Methods |
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Perfusion Protocol and Preparation of Isolated Cardiomyocytes
Hearts from male Wistar rats (weight 200 to 220 g, anesthetized with 50 mg/kg pentobarbital) were perfused retrogradely13 at 37°C and at a constant pressure of 60 mm Hg with a Krebs Henseleit buffer containing 1.5 mmol/L CaCl2 and 10 mmol/L glucose and in equilibrium with a 95%O2/5%CO2 gas phase (except where otherwise stated). No-flow ischemia was obtained by interrupting the flow. The ischemic hearts were maintained at 37°C by immersion in a thermostated reservoir filled with buffer. The perfusion protocols are described in the legends to the figures. At the indicated times, the hearts were freeze-clamped. Perfusions in "working" conditions were performed as described,14 with 10 cm H2O preload and 60 cm H2O afterload for "low-load" perfusions and with 15 cm H2O preload and 100 cm H2O afterload for "high-load" perfusions.
Isolated cardiomyocytes were
prepared13 and resuspended (
50 mg wet
weight/mL) in a medium containing (in mmol/L) NaCl 95, PIPES 25, MES
25, KCl 4.7, MgSO4 1.25,
H2PO4 1.2,
CaCl2 1, and glucose 10 and 2% (wt/vol) BSA, at
the indicated pH. The effect of 100 nmol/L insulin on the uptake of
2-[2-14C]deoxyglucose (1 µmol/L; 100
mCi/mmol) was measured in 20-minute
incubations.13 For other
measurements, cardiomyocytes were preincubated for 20 minutes and then
incubated for 2 minutes with 100 nmol/L insulin and finally centrifuged
(10 000g for 5 seconds). The
cell pellets were frozen in liquid nitrogen and stored at
-80°C.
NMR Spectroscopy
Hearts were positioned in a home-built thermostated
probe head in a Biospec spectrometer (4.7 T). Intracellular pH was
estimated15 from the shift
of the inorganic phosphate signal in the spectra (81.1 MHz, 90°
pulses, 2.32-second repetition time, 64 scans, or 2.5-minute total
acquisition time).
Analytical Procedures
The frozen hearts or cardiomyocytes were homogenized
(Ultra-Turrax) at 0°C to 4°C in 5 to 10 vol (vol/wt) of
homogenization buffer,16 and
the supernatants (10 000g for
30 minutes) were stored at -80°C. PKB activity was measured in
immunoprecipitates (BAK
antibody).11 The
phosphorylation state of PKB and of p38 mitogen-activated protein
kinase (p38) were monitored by immunoblots (anti–phospho-Thr308PKB,
anti-phospho-Ser473PKB, and anti–phospho-Thr180/phospho-Tyr183p38
antibodies; New England Biolabs). p70 ribosomal S6 kinase
(p70s6k)17
and GSK-318 activity were
measured after immunoprecipitation (anti–
-specific or ß-specific
GSK-3 antibodies, kindly provided by Dr J. Vandenheede, Leuven,
Belgium; anti-p70s6k antibody, Upstate
Biotechnology). AMPK activity was assayed in the presence of 0.2 mmol/L
AMP in a 10% polyethylene glycol 6000
fraction.7 The activity of
PI3K17 associated with IRS-1
was measured in immunoprecipitates (anti–IRS-1 antibody, Upstate
Biotechnology). Tyrosine phosphorylation of IRS-1 or of the ß
subunits of IR was measured after immunoprecipitation (anti–IRS-1
antibody or anti–ß subunit of IR, Santa Cruz Biotechnology) by
immunoblotting with anti-phosphotyrosine antibody (PY20, New England
Biolabs). IR tyrosine kinase activity was measured in
immunoprecipitates using synthetic peptides
(RRLIEDAEYAARG)19 or with
the copolymer polyglutamate-tyrosine (4:1 molar
ratio)20 as substrate. The
blots shown in this work are representative of at least 3 experiments.
One unit (U) of protein kinase activity corresponds to the formation of
1 nmol of product per minute under the assay
conditions.
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Results |
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Ischemia Inhibited PKB Activation by Insulin
Under control conditions, insulin caused a 10- to 20-fold activation of PKB, which lasted for at least 15 minutes (Figure 1A
), in agreement with previous work on other
tissues.21 Ischemia induced
a biphasic change in PKB activity of insulin-treated hearts. There was
a slight but significant increase in PKB activity at 2 minutes,
followed by a progressive and persistent decrease in PKB activity,
which was maximal after 10 minutes
(Figure 1A). The loss of PKB activity corresponded to the
dephosphorylation of both Thr308 and Ser473
(Figure 1A).
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) or no-flow ischemic (•) episode. B, After a 15-minute equilibration period, hearts were submitted to a 10-minute normoxic (
When insulin was added during reperfusion, PKB activation
and phosphorylation were rapidly recovered, however with a slight delay
compared with hearts not previously submitted to ischemia
(Figure 1B
). Insulin signaling was fully restored after 5
minutes of reperfusion
(Figure 1B).
In normoxic hearts, half-maximal effects of insulin on PKB
and on GSK-3 activity were observed at
7x10-9
mol/L insulin, in agreement with values reported for adipocytes and for
hepatocytes.21 Similar
results were obtained for activation of
p70s6k, another protein kinase
downstream of PI3K and 3-phosphoinositide–dependent protein kinase-1
(Figure 2). In ischemic hearts, PKB activation was inhibited
by 62% at saturating concentrations of insulin, whereas
p70s6k activation and GSK-3 inactivation
were completely inhibited
(Figure 2).
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Lack of Effect of Heart Function and Substrates
on Insulin Signaling
We tested whether heart function and fatty acids, which
are known to affect heart metabolism, also have an effect on PKB
activation by insulin. Inhibition of heart contraction by removal of
calcium or by increasing KCl concentration in the perfusion buffer (30
mmol/L, final concentration) did not modify the insulin response. Under
these conditions, perfusion for 15 minutes with insulin increased PKB
activity to the same extent (controls, 7.6±1.6 mU/g tissue
[mean±SEM]; controls in calcium-free buffer, 10.2±1.7; controls
with 30 mmol/L KCl, 7.2±1.2; insulin, 48.2±2.7; insulin in
calcium-free buffer, 53.1±7.6; insulin with 30 mmol/L KCl, 45.8±1.8;
n=4). Moreover, heart work did not affect insulin signaling. In
low-load hearts, insulin increased PKB activity from 5.7±0.8 to
62.2±3.2 mU/g tissue, and in high-load hearts, from 7.5±0.7 to
57.7±1.9 mU/g tissue (n=4). The hydraulic power increased from
1.9±0.5 mW in low-load hearts to 4.9±1.2 mW in high-load hearts
without insulin and from 2.1±0.3 to 5.4±1.5 mW with insulin
(P<0.01). In the "working"
model, a 10-minute period of no-flow ischemia inhibited the activation
of PKB induced by insulin by 50%, an effect similar to that
reported in
Figure 1A.
Addition of fatty acids, which are known preferred substrates for the heart, did not alter PKB activation by insulin. The activity of PKB after 15-minute perfusion with insulin was 62.6±8.4 and 50.0±6.9 mU/g tissue (n=4) without and with 0.6 mmol/L palmitate, respectively, compared with 6.9±0.4 and 4.8±0.6 mU/g tissue (n=4) in the same conditions, but in the absence of insulin. We verified that the ischemia-induced inhibition of PKB activation persisted in hearts perfused with fatty acids (data not shown). Lactate, another preferred substrate for the heart, also had no effect on the activation of PKB by insulin, as presented in the following paragraph.
Potential Mechanisms of Insulin Signaling
Inhibition
No-flow ischemia blocks glucose and oxygen supply, as
well as waste product removal, so that lactate and protons rapidly
accumulate. We tested whether any of these changes could be responsible
for, and thereby mimic, the ischemia-induced inhibition of the insulin
response. No inhibition of PKB activation by insulin was observed when
normoxic hearts were perfused without glucose or with 20 mmol/L lactate
(PKB activity after 15 minutes of insulin in normoxic conditions,
47.3±3.5 mU/g tissue; without glucose, 42.8±4; with 20 mmol/L
lactate, 65.3±12.6; with 10 minutes of ischemia, 6.3±0.25;
n=3).
Moreover, no inhibition of insulin signaling was found in hearts perfused at a normal flow rate but under anoxic conditions, ie, when N2 replaced O2 in the gas phase (PKB activity after 15 minutes of insulin: normoxia, 73.8±3.6 mU/g tissue; 10 minutes of anoxia, 63.9±4; 10 minutes of ischemia, 27.9±5.2; n=3). AMPK was activated to the same extent in both anoxic and ischemic hearts (AMPK activity: in normoxic hearts, 216±13 mU/mg protein; in anoxic hearts, 1595±266; in ischemic hearts, 1223±140), in agreement with previous studies.22 The fact that AMPK activation was the same in anoxia and ischemia rules out AMPK as being responsible for the inhibition of insulin signaling that occurs in ischemia but not in anoxia. Phosphorylation of p38, known to be activated in no-flow ischemia,23 was detectable only after 10 minutes of ischemia (not shown), thus well after the ischemia-induced inhibition of PKB activation.
Inhibition of PKB Activation by
Intracellular Acidosis
By contrast with
anoxia,24 ischemia is known
to cause an important decrease in
pHi.25
This phenomenon was confirmed in our model by NMR
(Figure 3A). No-flow ischemia caused an immediate and rapid
drop in pHi, which was maximal after 15 minutes,
with the half-maximal effect being obtained within 5 minutes of
ischemia, in agreement with data
published.25 Interestingly,
the inhibition of PKB activation
(Figure 1A) was only detectable after 5 minutes of ischemia,
ie, when the pHi was below 6.75. The importance
of acidosis in the inhibition of insulin signaling was evaluated by
changing the extracellular pH (pHe) under
normoxic conditions. Perfusing rat hearts in normoxic conditions and at
pHe 7.1 instead of pHe
7.4 mimicked the ischemia-induced inhibition of PKB activation
(Figure 4A) and decreased pHi
(Figure 3B) to values observed in ischemia. Inhibition of PKB
activation was also observed in hearts perfused with ouabain
(Na+K+ ATPase
inhibitor) and amiloride
(Na+/H+ exchanger
inhibitor), a combination that decreased pHi
(Figure 3B) as
expected26 without affecting
pHe (not shown). This demonstrated that the
change in pHi without change in
pHe was sufficient to inhibit insulin signaling.
Finally, perfusion at pHe 8.2 diminished the
ischemia-induced decrease in pHi
(Figure 3A) and prevented the ischemia-induced inhibition of
PKB activation and phosphorylation by insulin
(Figure 4B).
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). B, Effect of pHe 7.1 (
) or 0.5 mmol/L ouabain+1 mmol/L amiloride (
) on pHi in normoxic conditions. Arrows indicate time at which an inhibition of insulin-induced PKB activation was found (see Figures 1A
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Insulin Resistance Induced by Extracellular
Acidosis in Cardiomyocytes
We used isolated cardiomyocytes incubated at various
pHe values in normoxic conditions to study the
links between PKB activation and a metabolic effect of insulin, namely
the stimulation of glucose transport. Incubation of cardiomyocytes at
pHe values below 6.75 inhibited both
insulin-stimulated glucose transport and PKB activation
(Figure 5B). The insulin response was abolished at
pHe values below 6.25. These data confirm that
inhibition of insulin signaling corresponds to inhibition of the
metabolic effect (ie, stimulation of glucose
uptake).
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Inhibition of the First Steps in the Insulin
Signaling Cascade
We measured the activity of several components of the
insulin signaling pathway, upstream of PKB. Changes in PKB were usually
found to be concomitant with changes in the upstream PI3K: (1) the
insulin-induced activation of PI3K and PKB was antagonized by ischemia
(compare
Figure 6B with
Figure 1A), (2) this inhibition was not observed when the
hearts were perfused at pHe 8.2 (not shown), and
(3) reperfusion with insulin after an ischemic episode caused a slight
delay in both PKB
(Figure 1B) and PI3K (not shown) activation. However, these
changes were not always concomitant; after 2 minutes of ischemia, PKB
activity was further increased, whereas PI3K activity remained
unaffected (compare
Figures 1A and 6B).
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Changes in PI3K activity were in turn related to a decreased
phosphorylation of the upstream IRS-1 to which it is bound and to a
decreased tyrosine phosphorylation of the ß subunit of IR
(Figure 6A).
In cardiomyocytes incubated at
pHe values below 6.75, the inhibition of PKB
activation correlated with a decreased tyrosine phosphorylation of both
IRS-1 and IR
(Figure 5A), as in ischemic hearts. It also correlated with
an inhibition of the insulin-induced stimulation of glucose uptake
(Figure 5B).
Inhibition of the IR Tyrosine Kinase
Activity
Inhibition of the first step of insulin signaling,
namely the phosphorylation of IR, by ischemia or low pH could result
from inhibition of insulin binding to its receptor, as already reported
for isolated adipocytes incubated at pHe
6.727 and/or from the
inhibition of the IR tyrosine kinase activity itself. Removal of
insulin from the medium by washout procedures is known to decrease
insulin binding to its
receptor.28 However, we
found that PKB activation persisted for 6 and 10 minutes after washing
out insulin by perfusing with insulin-free medium
(Figure 7). This suggests that an element(s) other than the
inhibition of insulin binding should be involved to explain the
ischemia-induced inhibition of insulin signaling. Therefore, the effect
of pH on the IR tyrosine kinase activity was studied in vitro.
Incubation of IR from control or insulin-treated hearts at pH values
below physiological pHi inhibited its activity,
which was completely abolished at pH 6.0
(Figure 8). We conclude that intracellular acidosis inhibits
the activation of IR and so prevents its further phosphorylation and
the activation of the downstream components of the insulin signaling
pathway. Our data do not allow us to rule out a concomitant activation
of tyrosine phosphatases.
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Discussion |
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No-flow ischemia inhibits insulin signaling in the heart. All of the components of the insulin signaling cascade that we tested were inhibited (IR, IRS-1, PI3K, PKB, p70s6k, and GSK-3). The decreased IR tyrosine kinase activity is sufficient to explain the inhibition of the downstream components. However, it is not excluded that ischemia could influence other intracellular components. For instance, the discrepancy between the activity of PKB (Figure 1A
) and PI3K
(Figure 6B) found at 2 minutes of ischemia indicates that
factors other than PI3K could modulate the phosphorylation state and
the activity of PKB and in this case cause a transient activation of
PKB. This suggests that ischemia-dependent events control PKB
activation.
Inhibition of insulin signaling may occur at different
levels of the signaling pathway, and several examples are known. For
instance, tumor necrosis factor- decreases the phosphorylation state
of IRS in cultured muscle
cells.29 Osmotic shock
induces PKB dephosphorylation in cultured hepatocytes without IR
dephosphorylation and PI3K
inactivation.30 In adipose
tissue, isoproterenol and cAMP inhibit IR tyrosine
phosphorylation31 and
tyrosine kinase activity.20
These mechanisms reflect the inherent complexity of a system that
involves numerous control steps.
We propose that the inhibition of insulin signaling during
ischemia results from the intracellular acidosis that this condition
causes. This conclusion reflects the parallelism between intracellular
acidosis and inhibition of insulin signaling and is based on the
following observations. First, intracellular acidosis and inhibition of
signaling followed the same time course during ischemia. Second, the
extent of intracellular acidosis was related to the inhibition of
insulin signaling not only in ischemia but also in normoxia at
pHe 7.1 and in normoxia with ouabain and
amiloride. Third, perfusion at pHe 8.2 prevented
the ischemia-induced intracellular acidosis and inhibition of insulin
signaling. Importantly, the parallelism between intracellular acidosis
and inhibition of insulin signaling also holds for the pH profile of
the tyrosine kinase activity of IR, thus indicating that intracellular
acidosis inhibits the whole signaling pathway. However, the
relationship between intracellular acidosis and inhibition of insulin
signaling seems to hold only at pHi values below
6.75 as indicated by the pHi
(Figure 3A) at which point inhibition of PKB activation
becomes detectable in ischemic hearts
(Figure 1A). It is suggested that pHi
6.75 represents a threshold value under which insulin signaling becomes
inhibited. Therefore, inhibition of insulin signaling is expected to
depend on the severity and duration of ischemia and probably reflects
the extent of intracellular acidosis. Our data show side effects of
ischemic acidosis on hormonal signal transduction, whereas others have
emphasized its benefit on postischemic myocardial
function.32
Activation by insulin of the main components of the signaling pathway is transient.16 Hence, short experimental periods had to be used to measure insulin signaling. However, they had to be long enough to allow severe ischemia, and the resulting acidosis, to develop. The experimental protocol used (a 5-minute perfusion period with insulin followed by a 10-minute period of no-flow ischemia) ensured that both insulin signaling and ischemic acidosis would occur simultaneously.
In this simplified model of ischemia, inhibition of insulin signaling is readily demonstrable and specifically evaluates the insulin response. It is not the case for the metabolic effects of insulin, because inhibition of these effects is masked by the stimulation of glucose uptake and glycolysis by AMPK, which is activated under hypoxic conditions.8 Therefore, a stimulation of glucose metabolism persists, even if insulin signaling is impaired in ischemic hearts. This was confirmed by our measurements of lactate concentrations, which were the same in ischemic hearts (10 minutes of no-flow ischemia) whether insulin was present or not (lactate concentration in control hearts, 0.2±0.1 µmol/g tissue without insulin and 0.3±0.1 with insulin; in ischemic hearts, 12.6±0.1 without insulin and 14.2±1.6 with insulin; n=3). In models of low-flow ischemia, increasing insulin concentration did not further stimulate glucose uptake.3 In cardiomyocytes incubated in normoxic conditions but at pHe values below 6.75, such an interference does not exist. In these cells, both insulin signaling and the metabolic effects of insulin (stimulation of glucose uptake) were indeed inhibited, although basal levels of glucose uptake were little affected. We therefore conclude that intracellular acidosis is expected to inhibit insulin response. Several examples of insulin resistance associated with acidosis have already been reported in other tissues.27
Measurement of insulin signaling, and especially changes in PKB activity, could allow one to investigate the insulin effects on cell survival, because PKB is not only mediating metabolic effects but is also involved in cell protection.12 33 Therefore, inhibition of insulin signaling by ischemia may indicate that all insulin effects are abolished. Conversely, insulin signaling was fully restored early on reperfusion suggesting that the hormonal effects could then resume. We speculate that the slight delay in PKB activation corresponds to the time necessary to recover a normal pHi.
Although we demonstrated that insulin signaling was inhibited during ischemia and restored during reperfusion, the implications of this on heart function, glucose metabolism, or other heart responses to insulin remain to be investigated. Moreover, the mechanisms by which insulin protects the postischemic heart are not clear. They could be related to a direct positive inotropic effect on postischemic heart34 or to the well-known metabolic effects of insulin. They could also include other effects of insulin, such as inhibition of apoptosis, control of gene expression, or mitogenic properties.
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Acknowledgments |
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This work was supported by the National Fund for Medical Scientific Research (Belgium), the Fund for Scientific Research—Flanders (G.0360.98), the Belgian Program on Interuniversity Poles of Attraction initiated by the Belgian State, and the Directorate General of Higher Education and Scientific Research (French Community of Belgium). A.-S.M. and C.B. are Research Fellows, and at the time of this study L.B. was a Postdoctoral Researcher, of the National Fund for Scientific Research (Belgium). The expert technical assistance of D. Revets and L. Maisin is gratefully acknowledged. We thank M.H. Rider, J. Deprez, and D. Vertommen for their continued interest and critical reading of the manuscript; Y. Le Marchand-Brustel for help in setting up the IR tyrosine kinase assay; and P. Van Hecke for the building of the NMR coil.
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Footnotes |
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Original received October 2, 2000; revision received December 28, 2000; accepted January 12, 2001.
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