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Insulin signalling in the heart

Luc BERTRAND1, Sandrine HORMAN

1, 2, Christophe BEAULOYE

1 and Jean-

Louis VANOVERSCHELDE1

1Université catholique de Louvain, Division of cardiology, Brussels, Belgium.

2Université catholique de Louvain, de Duve Institute, Hormone and Metabolic Research Unit,

Brussels, Belgium.

Send correspondence to: Luc Bertrand, Université catholique de Louvain, CARD Unit, 55,

Avenue Hippocrate, CARD5550, 1200, Brussels, Belgium. Tel: +32 2 764 55 52, Fax +32 2

764 55 36, E-mail: [email protected], website: http://www.card.ucl.ac.be/

Time for primary review: 21

1

Cardiovascular Research Advance Access published April 5, 2008 at Pennsylvania State U

niversity on February 21, 2013http://cardiovascres.oxfordjournals.org/

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Abstract

The main role of insulin in the heart under physiological conditions is obviously the

regulation of substrate utilization. Indeed, insulin promotes glucose uptake and its utilization

via glycolysis. In addition, insulin participates in the regulation of long-chain fatty acid

uptake, protein synthesis and vascular tonicity. Significant advancements have been made

over the last 20 years in the understanding of the signal transduction elements involved in

these insulin effects. Amongst these molecular mechanisms, the phosphatidylinositol 3-

kinase/protein kinase B (Akt) pathway is thought to play a crucial role. Under pathological

conditions, such as type-2 diabetes, myocardial ischemia and cardiac hypertrophy, insulin

signal transduction pathways and action are clearly modified. These molecular signalling

alterations are often linked to atypical crosstalks with other signal transduction pathways. On

the other hand, pharmacological modifications of parallel and interdependent signalling

components, such as the AMP-activated protein kinase pathway, is now considered to be a

good therapeutic approach to treat insulin signalling defects such as insulin resistance and

type-2 diabetes. In this review, we will focus on the description of the molecular signalling

elements involved in insulin action in the heart and vasculature under these different

physiological, pathological and therapeutical conditions.

Key words: Energy metabolism, PKB/Akt, ischemia, protein synthesis, insulin resistance,

AMPK.

2

at Pennsylvania State University on February 21, 2013

http://cardiovascres.oxfordjournals.org/D

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1. Introduction

Insulin plays a key role in the regulation of various aspects of cardiovascular metabolism and

function including glucose and long-chain fatty acid (LCFA) metabolism, protein translation

and vascular tone. Since its discovery in 1921, the effects of insulin have been extensively

studied.1 However, the full identification of the molecular signalling events involved in these

insulin actions is still in progress nowadays. For example, it had been demonstrated for the

first time that insulin induced glucose uptake in 1949,2 whereas the insulin-sensitive glucose

transporter Glut4 was only discovered in the 80s and the tandem formed by two other

proteins, namely the protein kinase B (PKB)/Akt and the Rab GTPase-activating protein

(GAP) Akt substrate 160 (AS160), were only suspected to be part of the missing link of this

insulin-induced glucose uptake in 2003.3 Even if certain insulin-dependent molecular steps

still need to be identified, the recent understanding of the signal transduction mechanisms

involved in insulin action informs us of the possibility to exploit this information to develop

molecular therapeutic approaches to treat cardiac diseases such as insulin resistance, cardiac

hypertrophy or myocardial ischemia.

2. Heart metabolism under physiological conditions

The heart is an energy consuming organ that requires a constant supply of fuel and oxygen in

order to maintain its intracellular ATP level which is essential for the uninterrupted

myocardial contraction/relaxation cycle. The human heart produces and consumes between

3.5 and 5 kg of ATP every day to sustain pumping.4 The way to generate this energy depends

on the cardiac environment including coronary flow, blood substrate supply, hormones and

nutritional status.5-8

Under physiological conditions, this ATP production comes from the

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mitochondrial oxidation of different substrates, LCFAs (60-70%) being predominant over

glucose (20%) and lactate (10%) (Fig.1). This substrate preference is due to the fact that

LCFA oxidation inhibits glucose uptake and catabolism via the Randle cycle.9 Ketone body

circulating levels, which are low under control conditions, rise upon starvation, in the

presence of high blood concentrations of LCFAs, in chronic heart failure or in poorly

controlled diabetes.6,7

In these conditions, ketone bodies become a major substrate for the

heart by inhibiting the oxidation of other substrates. Ketone bodies inhibit glucose oxidation

at the pyruvate dehydrogenase level via acetyl-CoA production, whereas the inhibition of

LCFA oxidation is still poorly understood.6,7

On the other hand, when glucose and insulin

concentration rises, glucose becomes the favoured oxidized substrate of the heart (Fig. 1). The

signal transduction mechanism involved in this insulin-induced adaptation in substrate

utilisation is complex and acts on several cellular targets, including the glucose transporter

Glut4, the LCFA transporter 88kDa FA translocase (FAT)/CD36 and the glycolytic enzyme

6-phosphofructo-1-kinase (PFK-1) (Fig. 1 and 2). The proximal part of the insulin signal

transduction pathway is common to all these metabolic effects.

3. The proximal insulin signalling

The insulin receptor (IR) is a tetrameric enzyme comprising two extracellular α subunits and

two transmembrane β subunits.10

The binding of insulin to the extracellular part of IR induces

the activation of the intrinsic tyrosine kinase activity of the β subunits of the receptor (Fig. 2).

This leads to an autotransphosphorylation of the receptor where one β subunit phosphorylates

the other on several tyrosine residues. IR shares a similar structure with the insulin-like

growth factor (IGF-1) receptor, explaining cross-reaction and partially overlapping functions

between the two receptors and their ligands.11,12

Once activated and phosphorylated, IR binds

via its phosphotyrosine residues, and phosphorylates a series of downstream elements,

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including the IR substrate (IRS) family and Shc.13-16

This recruitment and activation lead to

the activation of two main pathways, the phosphatidylinositol 3-kinase (PI3K) and the

mitogen activated protein kinase (MAPK) pathway, respectively. PI3K (especially class Ia) is

considered to be the main player of the metabolic action of insulin, whereas the MAPK

pathway is principally involved in cell growth and differentiation. The MAPK pathway has

already been well reviewed.17-20

So, we primarily focus the present review on the PI3K

pathway. PI3K is a heterodimeric protein composed of a p110 catalytic and a p85 regulatory

subunit. This regulatory subunit contains SH2 domains that bind to phosphorylated tyrosine

residues of IR or IRS adaptor proteins.21

This insulin-induced PI3K recruitment at the plasma

membrane leads to its activation. PI3K is a lipid kinase that phosphorylates

phosphatidylinositol (PI) phosphates at the 3 position.21

The main product of PI3K is the

PI3,4,5P3 produced from the PI4,5P2. The increase in PI3,4,5P3 at the plasma membrane

induced the recruitment and co-localization of the phosphoinositide-dependent kinase 1

(PDK1) and the protein kinase B (PKB)/Akt. Once recruited, PDK1 phosphorylates and

activates PKB/Akt. Insulin is a very potent PKB/Akt activator in the heart.22

Moreover, it has

been shown in PDK1 muscle-specific knockout (KO) mice that PDK1 is required for the

insulin-induced activation of cardiac PKB/Akt.23

The tandem composed by PDK1 and

PKB/Akt is an important player of the metabolic effect of insulin. For example, PDK1 and the

PKBβ/Akt2 isoform seem to be essential for the regulation of glucose uptake in the heart.24-26

In parallel to PKB/Akt, PDK1 is also able to activate the atypical protein kinase Cs (PKCs) λ

and ζ even if this insulin-induced activation has not yet been actually demonstrated in cardiac

tissue.27

4. Regulation of glucose and long-chain fatty acid metabolism

We will now illustrate the downstream mechanism implicated in the insulin-induced

regulation of glucose and LCFA entry and metabolism (Fig. 2). Glucose has to enter into the

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cardiomyocyte to be oxidized. The uptake of glucose depends on the presence of glucose

transporters at the plasma membrane. Amongst Glut1 and Glut4, the two glucose transporters

expressed in the heart, Glut4 is considered to be the main contributor for the regulation of

glucose uptake by insulin.28,29

Gluts are present into the cardiomyocyte in two different

populations, one located in intracellular compartments, the other being at the plasma

membrane. Insulin induces the translocation of Glut4 from the intracellular storage to the

sarcolemmal membrane and so favou srs glucose entry.28,30

The role of the PI3K/PKB/Akt

signalling cascade in the insulin-induced Glut4 translocation has been well established for

many years.31

Conversely, the distal elements involved in this recruitment have been only

recently partially determined.31-34

Indeed, It has been shown that AS160, the GAP of the small

G proteins Rabs required for membrane trafficking, is a substrate of PKB/Akt. This insulin-

induced phosphorylation inactivates AS160, prevents Rab GAP function and, so, favours

Glut4 translocation in adipocytes3 and muscle.

35 Phosphorylation of AS160 is also enhanced

by insulin in cardiomyocytes (L. Bertrand, personal communication). However, more recent

studies, principally made in adipocytes and myotubes, show that the PKB/AS160 axis is

probably not the sole mechanism regulating the insulin-induced Glut4 translocation. Indeed,

another Rab GAP, TBC1D1, an additional PKB/Akt substrate, could play a role parallel to

AS160.36,37

Two other PKB/Akt substrates, the phosphoinositide 5-kinase PIKfive38

and the

SNARE-associated protein synip39

have also been proposed to participate in Glut4

translocation. PIKfive may regulate Glut4 vesicles trafficking by acting on F-actin dynamics.

Synip seems to control the fusion of Glut4 vesicles with the plasma membrane. In addition,

Bai and colleagues showed that the preparation of the Glut4 storage compartments for fusion

with the plasma membrane is another key step regulated by insulin independently of the

PI3K/PKB/Akt axis.40

In parallel to PKB/Akt, another PDK1 substrate, the atypical PKC λ/ζ

family, has been implicated in Glut4 transfer to the plasma membrane as well.41

Besides the

PI3K/PKB/Akt,, there is another pathway, located in specific lipid raft microdomains, that

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involves the G protein TC10, coupled to IR and the IR substrate Cbl. This pathway has been

proposed to play a role in the translocation of Glut4 in adipocytes.31,32,34,42,43

In cardiac

muscle, insulin has been shown to increase the phosphorylation of both Cbl and TC10.44

All

these parameters regulated by insulin seem to share the control of Glut4 translocation.

However, the roles of TBC1D1, PIKfive, synip, PKC λ/ζ or Cbl/TC10 in the insulin-induced

stimulation of glucose uptake have not yet been studied in the heart.

Once entered into cardiomyocytes, glucose is metabolized to participate in glycogen synthesis

and glycolysis in an insulin-dependent manner also (Fig. 1 and 2). Insulin is able to stimulate

glycogen synthase (GS) in muscle cells via its dephosphorylation which is dependent of the

PKB-induced inactivation of the GS kinase-3 (GSK-3).26,45-47

However, this insulin-induced

and PKB/Akt-dependent GS stimulation does not seem to be implicated in the regulation of

the glycogen content in the heart.26

A mechanism involving allosteric activation of GS by

glucose-6-phosphate could be the main factor regulating cardiac glycogen level.26

More

importantly, insulin favours the use of glucose by stimulating cardiac glycolysis.48-50

Indeed,

insulin activates the cardiac 6-phosphofructo-2-kinase (PFK-2) isoform which is the enzyme

that synthesizes fructose 2,6-bisphosphate.51

Fructose 2,6-bisphosphate is a potent stimulator

of PFK-1, the main enzyme regulating glycolytic flux.50

PFK-2 activation results from its

phosphorylation which is undoubtedly under the control of a PI3K/PDK1-dependent

pathway.23,48,52-54

However, the identity of the downstream protein kinase responsible for the

direct PFK-2 phosphorylation is still controversial with one study showing a putative role of

PKB53

while others implicate another PDK1 dependent kinase.22,27,52

The role of PFK2 in the

regulation of cardiac function seems to be important because the cardio-specific expression of

a kinase-dead PFK2 decreases glycolytic flux, induces hypertrophy and fibrosis and reduces

cardiomyocyte function.55

In parallel to the stimulation of glucose uptake, insulin also induces LCFA uptake in

cardiomyocytes.56,57

This stimulation comes from the insulin-dependent translocation to the

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plasma membrane of the LCFA transporter FAT/CD36 (Fig. 2). This phenomenon requires

the insulin-induced PI3K activation58

but needs additional studies to be further characterized.

In contrary to glucose, the resulting increase in intracellular LCFA concentration does not

result in the increase in LCFA oxidation but in the storage of this excess into the intracellular

pool of lipids 59

.

5. Regulation of protein synthesis

Insulin can be considered as an anabolic hormone promoting protein synthesis and cell

growth. The control of protein synthesis by insulin involves

phosphorylation/dephosphorylation of several translation factors and ribosomal proteins.60,61

PKB/Akt is a pivotal element of this complex regulation (Fig. 2). PKB/Akt can phosphorylate

and inactivate the tuberous sclerosis factor 2 (TSC2), the GAP of the G protein Rheb.62,63

Active TSC2 promotes the formation of the GDP-bound inactive state of Rheb. By

inactivating TSC2, PKB/Akt induces Rheb activation which, in turn, induces the

phosphorylation and activation of the mammalian target of rapamycin (mTOR) by a

mechanism which is still not well defined.64

Insulin has been clearly shown to regulate the

PKB/Akt/TSC2/mTOR pathway in cardiomyocytes.65

Once activated, mTOR mainly

regulates two targets involved in the regulation of protein translation, the 4E-binding protein-

1 (4E-BP1) and the p70 ribosomal S6 protein kinase (p70S6K).61

The mTOR-mediated

phosphorylation of 4E-BP1 prevents its inhibitory action on the eukaryotic initiation factor 4E

(eIF-4E), allowing this factor to bind the mRNA cap and stimulate the initiation step of

protein synthesis. Activated p70S6K phosphorylates the S6 ribosomal protein involved in the

regulation of the translation of the 5’TOP mRNAs that encode several translation factors and

ribosomal proteins. Therefore, S6 phosphorylation increases both translation factor content

and translational capacity by increasing ribosomal biogenesis. The eukaryotic elongation

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factor-2 (eEF2) kinase (eEF2K) is another p70S6K substrate. eEF2K is a dedicated calcium-

and calmodulin-dependent kinase that controls the phosphorylation and inactivation of eEF2.

The insulin-dependent eEF2K inactivation leads to the stimulation of protein elongation.

Numerous studies already demonstrated the insulin-mediated regulation of 4E-BP1,46,66,67

p70S6K/S646,47,67-70

and eEF2K/eEF246

in the heart or cardiomyocytes.

PKB/Akt controls two other substrates, GSK-3 and the forkhead transcription factor FOXO

family, regulating protein translation and atrophy, respectively. In parallel to the modulation

of GS activity, GSK-3 is also implicated in the regulation of protein translation. GSK-3

phosphorylates and inactivates the initiation factor eIF2B.61

By inhibiting GSK-3, insulin

activates eIF2B and, so, stimulates the initiation of protein synthesis. Even if the relationship

between insulin, PKB, GSK-3 and eIF2B has been demonstrated in various tissues including

skeletal muscle, a similar study still needs to be extensively performed in cardiac tissue.46

To

be complete, GSK-3 also participates in the negative regulation of cardiac hypertrophy by

phosphorylating and inactivating the nuclear factor of activated T cells (NFAT) responsible

for the prohypertrophic gene expression.17

Atrophy, and its associated proteosomal destruction of proteins, can be considered to be the

opposite of protein synthesis. FOXO family members, comprising FOXO1, FOXO3a and

FOXO4, were initially known to promote the atrogene transcriptional program in skeletal

muscle.71

Moreover, it was established that the phosphorylation of FOXOs by PKB/Akt

promotes their nuclear exclusion and prevents atrophy.72

Similarly to what happens in skeletal

muscle, Skurk and colleagues revealed that insulin prevents cardiac muscle atrophy by

inhibiting FOXO3a through a PKB/Akt-dependent pathway.73

More recently, cardiac FOXO1

has been shown to be regulated in the same way.74

6. Insulin and vasculature

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Insulin has important vascular actions which, for most of them, lead to vasodilatation,

increased blood flow and subsequent augmentation of glucose disposal in classical insulin-

target tissues. One of its essential roles is the stimulation of increased production of the potent

vasodilator nitric oxide (NO) from vascular endothelium.75

In endothelial cells, endothelial

NO synthase (eNOS) catalyzes the conversion of the substrate L-arginine to the products NO

and L-citrulline.76

The insulin signalling pathway regulates the activation of eNOS by a

phosphorylation-dependent mechanism. It requires activation of the PI3K/PKB/Akt pathway

(Fig. 2). PKB/Akt directly phosphorylates eNOS, resulting in enhanced eNOS activity77

and

increased production of NO.75,78,79

This pathway is completely distinct and independent from

classical calcium-dependent mechanisms used by G protein-coupled receptors such as

acetylcholine receptor.80,81

PKBα/Akt-1 being the major isoform in the vasculature and

endothelial cells, it is not surprising that PKBα/Akt-1 KO mice have significantly low levels

of active eNOS.82

It is therefore likely that PKBα/Akt-1 mediates insulin-induced activation

of eNOS in these cells. However, activation of PKB/Akt, if necessary, is not sufficient to

activate eNOS. Indeed, although insulin-induced eNOS activation is calcium-independent,

insulin stimulates calmodulin binding to eNOS.83

This requires the prior binding of HSP90

onto the enzyme. The formation of a ternary eNOS/HSP90/PKB/Akt complex facilitates

eNOS phosphorylation by PKB/Akt.80

Rodent models of insulin resistance provide important insights into cardiovascular actions of

insulin. IRS-1 (-/-) and IRS-2 (-/-) deficient mice not only exhibit resistance to the metabolic

actions of insulin, but also demonstrate decreased eNOS activity and elevated blood

pressure.84

In addition, in obese Zucker rat (carrying a recessive mutation in the gene for

leptin) commonly used as a model of insulin resistance, the insulin-mediated attenuation of

vascular contractility85

and increases in limb blood flow86

are substantially reduced. By

contrast, insulin can also have effects which oppose vasodilator actions of NO such as the

stimulation of secretion of the vasoconstrictor endothelin-1 (ET-1) from vascular

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endothelium. These effects have been shown to depend exclusively of the MAPK

pathway.87,88

Under insulin resistance, PI3K-dependent pathways are impaired whereas

MAPK-dependent pathways are intact. Therefore, a shift in the balance between

vasoconstrictor and vasodilator actions of insulin could be an important factor in the vascular

pathophysiology of insulin resistance and endothelial dysfunction. Human studies in

overweight89

, obese90

, hypertensive91

and diabetic subjects90

support this notion.

In the vasculature, NO originates mostly from the endothelium and diffuses into vascular

smooth muscle cells (VSMC) where it activates guanylate cyclase to increase cGMP levels

that evoke relaxation. But NO production may also act in an autocrine fashion to regulate

vasodilator functions in VSMC. Indeed, expression of eNOS and inducible NOS (iNOS) has

been detected in these cells and insulin increases their activity and the resulting NO-

dependent cGMP production92-95

via a PI3K-dependent mechanism.92,94

Moreover, the

NO/cGMP-dependent kinase (PKG) pathway attenuates contractility by regulating the

calcium-dependent RhoA/Rho kinase (ROK) pathway stimulated in response to contractile

agonists (Fig. 2).96

Activation of RhoA-dependent pathways is involved in excessive

contraction and thereby increases blood pressure. All Rho proteins are prenylated at their

carboxy-terminus and this prenylation is involved in the translocation of the active GTP-

bound form of the Rho protein to the cell membrane.96

Active RhoA recruits and stimulates

ROK-α, which then acts, in part, by inactivating myosin phosphatase by phosphorylation and

also directly phosphorylating myosin light chains to enhance contraction.97,98

In VSMC,

phosphorylation of RhoA by PKG relocalizes the Rho protein to the cytosol and also inhibits

its affinity for its downstream effector ROK-α.99

These effects are abrogated by wortmannin,

a PI3K inhibitor, and small interfering RNA against PKB/Akt, indicating that, in VSMC, the

PI3K/PKB/Akt insulin signalling pathway is likely to mediate the inhibition of ROK-

mediated RhoA-dependent contraction.100,101

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7. Insulin action under ischemia : crosstalk between insulin and AMPK signalling

Insulin signalling and action are greatly altered under myocardial ischemia.49,102-106

Insulin

signalling can be modified by myocardial ischemia via mainly two pathways, one dependent

of the AMP-activated protein kinase (AMPK), the other being unrelated to this protein kinase.

Firstly, it has been shown that the acidosis produced by myocardial ischemia provokes the

inhibition of the tyrosine kinase activity of IR (Fig. 2).47

This inhibition is directly correlated

to the decrease in phosphorylation of different downstream elements including PKB/Akt,

p70S6K and GSK-3. Secondly, ischemia, by affecting oxidative ATP production, and, so,

increasing AMP concentration, induces AMPK activation.107

Once activated, AMPK is

considered to regulate different components of the insulin signalling pathway that control

glucose metabolism and protein synthesis (Fig. 2).49,102-105,107-109

Similarly to insulin, AMPK

stimulates cardiac glucose uptake and glycolysis by regulating the same targets, namely Glut-

4 translocation (see later in the section dedicated to insulin resistance) and PFK-2

activation.110,111

AMPK also stimulates LCFA uptake.112

In contrast, AMPK is known to

antagonize the stimulating effect of insulin on protein synthesis by inhibiting the

TSC2/mTOR/p70S6K113-116

and eEF2 pathways.68,117

However, the exact role of AMPK on

the regulation of protein synthesis in the ischemic heart still needs to be clearly defined.

Indeed, the pharmacological118

or genetic119

activation of AMPK clearly counteracts the

PKB/Akt-mediated activation of p70S6K, phosphorylation of eEF2 and stimulation of protein

synthesis in cardiomyocytes. However, recent data in our lab showed that the ischemia-

induced phosphorylation of eEF2 is clearly altered by the absence of the AMPKα2 isoform

(the main catalytic isoform in the heart) whereas the inhibition of the mTOR/p70S6K is not

modified (L. Bertrand, personal communication). This last result confirms the role of AMPK

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in the regulation of protein synthesis elongation but questions the implication of this protein

kinase on the inhibition of the (pre)initiation step of protein translation by ischemia.

In parallel to the ischemia-dependent insulin regulation, insulin is known to antagonize

AMPK signalling as well.120,121

Indeed, activated PKB/Akt can phosphorylate AMPK, this

phosphorylation reducing the ischemia-induced AMPK activation (Fig. 2).45,122,123

8. Is insulin protective during ischemia/reperfusion ?

Even if the insulin signalling pathway is blunted during ischemia, insulin is still able to act

during reperfusion. During several decades, the metabolic cocktail glucose/insulin/potassium

(GIK) was considered to be a good approach to reduce infarct size after myocardial

infarcts.124,125

Numerous studies involved the insulin-mediated activation of PKB/Akt to

explain the protective effect of GIK.103,125-127

A first metabolic reason is the shift of the

myocardial metabolism from LCFAs to glucose oxidation during reperfusion (see before),

which is more oxygen efficient and prevents the production of toxic LCFA intermediates.

Secondly, activated PKB/Akt leads to several protective mechanisms. PKB/Akt

phosphorylates, sequesters and/or inactivates several pro-apoptotic proteins including BAD,

BAX and caspase-9 (Fig. 2).128,129

As explained above, PKB/Akt also phosphorylates and

activates eNOS, inducing protection most likely via the NO-dependent PKG activation and

the inhibition of the mitochondrial transition pore.129

Finally, the PKB/Akt/mTOR/p70S6K is

also supposed to be protective by promoting, amongst others, the post-ischemic synthesis of

contractile proteins. It has to be mentioned that, similarly to insulin, IGF-1 exerts a protective

action during ischemia/reperfusion, by acting on the same targets, as for instance the

PKB/Akt pathway.130,131

Despite the existence of all these arguments demonstrating a protective effect of insulin, a

more recent randomized controlled trial, the CREATE-ECLA study, showed a neutral effect

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of GIK on mortality in patients with acute myocardial infarction.132

Several hypotheses can be

presented to understand this apparent divergence. First of all, the time of administration of

GIK could directly interfere with the efficacy of the treatment. The best cardiac recovery

should be obtained if GIK perfusion occurs rapidly, before coronary intervention being

optimal.133

Moreover, a recent study demonstrated that LCFAs attenuate insulin

cardioprotection.134

Even if insulin is known to lower LCFA in the plasma, this new issue has

to be taken into account in the near future. Finally, hyperglycemia, characteristic to GIK

infusion and known to provoke glucotoxicity135

could counteract the beneficial effects of

insulin. In conclusion, a full metabolic monitoring needs to be performed to definitively

prove the beneficial effect of GIK infusion.103

9. Insulin resistance in type 2 diabetes

Obesity as well as myocardial insulin resistance and metabolic alteration are features of type 2

diabetes and are established risk factors for the development of cardiovascular diseases.5,56

The diabetic heart is characterized by a decrease in glucose uptake and oxidation. In contrast,

LCFA uptake and oxidation are clearly enhanced. Nevertheless, the increase in LCFA

oxidation is not sufficient to prevent lipid accumulation in the heart. It is generally accepted

that this metabolic disorder plays an essential role in the establishment of myocardial insulin

resistance despite the fact that the molecular processes leading to this resistance are not yet

fully understood. Insulin resistance is characterized by an alteration of the insulin-induced

activation of the PI3K/PKB/Akt signalling pathway.136-138

Mainly studied in skeletal muscle

and adipose tissue, the decrease in insulin signalling is explained to come from at least three

molecular events. First, LCFA-mediated ceramide accumulation seems to induce atypical

PKC ζ-dependent inhibition of PKB/Akt.139

Second, LCFA-dependent diacylglycerol

accumulation induces conventional and novel PKCs activation that, in turn, could provoke the

serine phosphorylation of IRS1, thereby inhibiting the PIK3/PKB/Akt pathway.140,141

Third,

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insulin signalling by itself could produce a negative feedback loop in which the insulin-

dependent activation of mTOR/p70S6K induces serine phosphorylation and inhibition of

IRS1 (Fig. 2).142

Whatever the mechanism, the insulin signalling alteration explains the

impairment of the insulin-induced Glut4 translocation and glucose uptake stimulation that are

found in diabetic hearts. Ex vivo experimental studies have yielded conflicting results on the

susceptibility of diabetic hearts to ischemia/reperfusion injury.143,144

However, in vivo animal

and human studies have clearly shown that diabetes and insulin resistance aggravate

myocardial ischemic injury143,144

and reduce the beneficial effects of ischemic preconditioning

(IPC). Indeed IPC is known to protect the heart against ischemic injury via activation of the

PI3K/PKB/Akt pathway.128

In diabetic hearts, stronger IPC stimuli are required to activate the

PKB/Akt pathway and induce cardioproctection.145

Two types of therapeutic approaches could be performed to re-establish insulin sensitivity : i)

by blocking the upstream source of insulin resistance or ii) by directly modulating insulin

signalling. Indeed, the first solution is to modulate glucose and/or LCFA metabolism to find a

new equilibrium favouring glucose uptake and oxidation in opposition to LCFA oxidation.56

On the other hand, adjustment of insulin signalling can be done via the utilization of the anti-

diabetic drug metformin. It has been shown that metformin induces AMPK activation, which

could explain, at least in part, its insulin sensitizing action.146,147

Metformin-induced AMPK

activation could have a double impact on myocardial metabolism (Fig. 2). AMPK can directly

stimulate glucose uptake by phosphorylating and inactivating AS160, similarly to

PKB/Akt.148

AS160 is therefore another converging point between insulin and AMPK

signalling pathways (Fig. 2) that makes clear the synergistic effect of insulin and metformin

on glucose uptake in cardiomyocytes.149

More importantly, AMPK, by inhibiting the

mTOR/p70S6K axis (see before), could decrease the p70S6K-mediated phosphorylation and

inhibition of IRS1 (Fig. 2). The inhibition of this negative feedback loop should enhance the

insulin-dependent PI3K/PKB/Akt activation and re-establish normal insulin signalling in

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insulin resistant cells. Recently, we demonstrated that AMPK activation by metformin greatly

enhances the insulin-induced PKB/Akt activation in insulin-resistant cardiomyocytes.149

This

PKB/Akt overactivation is correlated to the decrease in serine phosphorylation of IRS1 (L.

Bertrand, personal communication).

10. Insulin signalling and cardiac hypertrophy

Insulin shares signalling pathways with different growth hormones including IGF-1 and

neurohormonal hypertrophic agonists like angiotensin II.17,19

All these hormones can activate

the MAPK and/or the PI3K/PKB/Akt signalling involved in the stimulation of both cell

growth and protein synthesis and in the inhibition of protein breakdown (see before). Under

normal conditions, the stimulation of the PI3K/PKB/Akt pathway by insulin and IGF-1

participates in both embryonic and postnatal cardiac growth.18,150

For example,

cardiomyocyte-restricted IR KO151

and PDK1 KO23

are characterized by a smaller heart and

smaller cardiomyocytes, respectively. It could be considered that IGF-1 acts under the

hypothalamic axis, whereas insulin action reflects nutritional status. On the other hand, in the

adult stage, some physiological (exercise) and pathological (hypertension, valvular

dysfunction) conditions are linked to the chronic stimulation of PI3K/PKB/Akt and/or MAPK

pathways, which undeniably participates in the establishment of myocardial hypertrophy.17-19

The signalling pathways involved in the physiological and pathological hypertrophy are not

similar even if they share some signalling elements. The PI3K/PKB/Akt axis seems more

linked to the physiological hypertrophy whereas MAPK signalling, in collaboration with the

PKC and calcineurin/NFAT pathways, participates in the development of the pathological

hypertrophy typically induced by angiotensin II.18

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By using overexpressing dominant negative PI3K152

or PKBα/Akt1 KO153

mice, it has been

shown that these kinases are required for physiological but not for pathological hypertrophy.

PKB/Akt action requires the mTOR/P70S6K pathway because the use of rapamycin, the

mTOR inhibitor, is sufficient to block the myocardial hypertrophy induced by the cardiac

overexpression of PKB/Akt.154

The important role played by mTOR in the development of

cardiac hypertrophy offers a putative role to AMPK as a potential strategy to prevent

hypertrophic development.155

In parallel to their direct implication in physiological hypertrophy, insulin and insulin

signalling can be indirectly connected to pathological hypertrophy. Indeed, it has been

recently shown that chronic hyperinsulinemia stimulates the angiotensin II signalling which is

involved in pathological hypertrophy.156

Moreover, hypertension-induced hypertrophy is

often found in humans simultaneously with insulin resistance. This insulin resistance appears

firstly in skeletal muscle and adipocytes inducing elevated plasma insulin levels. This

hyperinsulinemia probably facilitates hypertrophy by overactivating cardiac insulin

signalling.157

On the other hand, pathological hypertrophy is characterized by a remodeling of

substrate utilization with a shift to carbohydrate usage mimicking the insulin-stimulated

phenotype.157,158

It seems more and more evident that this metabolic shift participates in the

establishment of hypertrophy via the production of reactive oxygen species.158

11. Summary

A large part of the insulin signalling components involved in the regulation of processes like

myocardial glucose and LCFA utilization, protein translation and vascular activity is now

identified. This knowledge allows us to better understand the connection between insulin and

other signalling pathways as well as under physiological than pathological conditions such as

ischemia, diabetes, hypertension and hypertrophy. The partial elucidation of these signal

17



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transduction pathways is now followed by the development of therapeutical strategies based

on the modulation of certain of these signalling elements. The continuation of the elucidation

of all these multifaceted signalling pathways in the near future should bring new information

that will be used to treat myocardial pathologies with even more efficiency.

Funding

The authors were supported by the Fonds National de la Recherche Scientifique et Médicale

(Belgium) and the Actions de Recherche Concertées (Belgium). L.B. and S.H. are Research

Associate of the Fonds National de la Recherche Scientifique, Belgium.

Conflict of interest: none declared

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Figure legends:

Figure 1: Cardiac metabolism under control (A) and insulin (B) conditions. Under

control conditions, ATP production comes from fatty acids and glucose oxidation. Fatty acid

is the privileged substrate used by the heart, its β-oxidation inhibiting glucose oxidation via

the Randle cycle (dashed lines). When glucose and insulin plasma levels increase, glucose

becomes the main energy-providing substrate. Indeed, insulin induces Glut4 translocation and

6-phosphofructo-2-kinase (PFK-2) activation, leading to the concomitant stimulation of

glucose uptake and glycolysis (see text for further details). Abbreviations : FAT/CD36, LCFA

transporter 88kDa FA translocase (FAT)/CD36; Fru-1,6-P2, fructose 1,6-bisphosphate; Fru-

2,6-P2, fructose 2,6-bisphosphate; Glu-6-P, glucose 6-phosphate; Glut4, glucose transporter 4;

LCFAs, long chain fatty acids; PDH, pyruvate dehydrogenase; PFK-1; 6-phosphofructo-1-

kinase; TCA cycle; tricarboxylic acid cycle. This figure is derived from a previous work.159

Figure 2: Insulin signalling pathways regulating cardiovascular metabolism. The

different insulin signalling elements presented here are described in details in the text. The

effects of insulin on substrate metabolism and protein translation in the cardiomyocyte are

shown in (A) whereas insulin actions on vasodilatation, that occurs in the endothelial/vascular

smooth muscle cell system, on apoptosis and cell growth are in (B). Dotted lines symbolize

translocations and solid lines illustrate direct interactions whereas dashed lines represent

indirect regulation. The insulin-induced negative feedback loop is presented in red.

Interactions between insulin and AMPK signalling are also shown, red lines indicating

inhibiting effects of one pathway to the other, green lines pointing out AMPK effects

analogous to that of insulin. Question marks represent signalling events that have not yet been

established in the heart tissue.

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Cytoplasm

Mitochondria

Glucose(20%)

Glu-6-P

Fru-1,6-P2

Pyruvate

Pyruvate

Glycogen

Citrate

Acetyl-CoA

ATP

Acyl-CoA

Citrate

Acyl-CoA

Glut4Glut4

TCAcycle

LCFAs(60-70%)

PFK-1PFK-1

PDHPDH

Glucose oxidation β-oxidation

ATP

FAT/CD36

Out

A

02

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Cytoplasm

Mitochondria

Glucose(60-70%)

Glu-6-P

Fru-1,6-P2

Pyruvate

Pyruvate

Glycogen

Citrate

Acetyl-CoA

Acyl-CoA

Citrate

Acyl-CoA

Glut4

LCFAs(20%)

Glucose oxidation

ATP

FAT/CD36

Out

Lipid pool

Insulin

Fru-2,6-P2 PFK-2PFK-2

B

ATPTCAcycle

β-oxidation

02

PDHPDH

PFK-1PFK-1

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In

Glucose LCFAsOut

Insulin

Glut4

FAT/CD36

PIP3

IRS-1PI3KPKB/Akt PDK1

Rab

IR

TBC1D1

PKCλ/ζ TC10

PIKfive

synipGSK-3

TSC2

Rheb

mTORp70S6K

4E-BP1

eEF2K

eEF2S6

Glucoseuptake

glycolysis

IschemiaMetformin

AMPK

Translationelongation

Translationinitiation

Ribosomalbiogenesis

LCFAuptake

Negative feedback loop

?

?

?

?

?

IschemiapH

eIF2BAMPK

AS160

PFK-2

PKB/Akt

Glut4 FAT/CD36

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In

Out

Insulin

IRS-1PI3K

IR

GSK-3

PKB/Akt

NFAT

FOXO

MAPK

NOSHSP90

RhoARoK

MLC

Cell growth

Vasodilatation

Bad BaxCaspase

Anti-apoptotic

Shc

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