Download AMP-activated protein kinase regulation of fatty acid oxidation in the

AMPK 2002 – 2nd International Meeting on AMP-activated ProteinKinase
AMP-activated protein kinase regulation of fatty
acid oxidation in the ischaemic heart
T. A. Hopkins, J. R. B. Dyck and G. D. Lopaschuk1
Cardiovascular Research Group, 423 Heritage Medical Research Centre, University of Alberta, Edmonton, Canada, T6G 2S2
Abstract
The heart relies predominantly on a balance between fatty acids and glucose to generate its energy supply.
There is an important interaction between the metabolic pathways of these two substrates in the heart. When
circulating levels of fatty acids are high, fatty acid oxidation can dominate over glucose oxidation as a source of
energy through feedback inhibition of the glucose oxidation pathway. Following an ischaemic episode, fatty
acid oxidation rates increase further, resulting in an uncoupling between glycolysis and glucose oxidation.
This uncoupling results in an increased proton production, which worsens ischaemic damage. Since high rates
of fatty acid oxidation can contribute to ischaemic damage by inhibiting glucose oxidation, it is important to
maintain proper control of fatty acid oxidation both during and following ischaemia. An important molecule
that controls myocardial fatty acid oxidation is malonyl-CoA, which inhibits uptake of fatty acids into the
mitochondria. The levels of malonyl-CoA in the heart are controlled both by its synthesis and degradation.
Three enzymes, namely AMP-activated protein kinase (AMPK), acetyl-CoA carboxylase (ACC) and malonyl-CoA
decarboxylase (MCD), appear to be extremely important in this process. AMPK causes phosphorylation and
inhibition of ACC, which reduces the production of malonyl-CoA. In addition, it is suggested that AMPK also
phosphorylates and activates MCD, promoting degradation of malonyl-CoA levels. As a result malonyl-CoA
levels can be dramatically altered by activation of AMPK. In ischaemia, AMPK is rapidly activated and inhibits
ACC, subsequently decreasing malonyl-CoA levels and increasing fatty acid oxidation rates. The consequence
of this is a decrease in glucose oxidation rates. In addition to altering malonyl-CoA levels, AMPK can also
increase glycolytic rates, resulting in an increased uncoupling of glycolysis from glucose oxidation and an
enhanced production of protons and lactate. This decreases cardiac efficiency and contributes to the severity
of ischaemic damage. Decreasing the ischaemic-induced activation of AMPK or preventing the downstream
decrease in malonyl-CoA levels may be a therapeutic approach to treating ischaemic heart disease.
Introduction
The heart has a high energy demand and in general derives
most of this energy (60–80%) from the oxidation of fatty
acids in the mitochondria [1]. However, the use of fatty acids
as a fuel operates at the expense of carbohydrate utilization
of glucose and lactate. This inverse relationship between the
use of fatty acids and the use of carbohydrates becomes
increasingly important in the clinical setting of ischaemia.
Following an ischaemic episode, the heart becomes less
efficient at converting energy into contractile function (see
[2] for review). This decrease in efficiency occurs because
fatty acid oxidation dominates over glucose oxidation as
the main source of mitochondrial metabolism and overall
ATP production. During ischaemia, glucose uptake and
glycolysis also accelerate. Consequently, proton production
increases [3,4] due to the uncoupling of glycolysis and glucose
oxidation. Contractile energy in the form of ATP is then
diverted towards re-establishment of ionic homoeostasis.
Key words: acetyl-CoA carboxylase, carnitine palmitoyltransferase, malonyl-CoA, malonyl-CoA
decarboxylase, mitochondria.
Abbreviations used: AMPK, AMP-activated protein kinase; ACC, acetyl-CoA carboxylase, MCD,
malonyl-CoA decarboxylase; CPT-1, carnitine palmitoyltransferase-1.
1
To whom correspondence should be addressed (e-mail [email protected]).
Several studies indicate that altering energy metabolism
pharmacologically to either decrease fatty acid oxidation
or increase glucose oxidation can improve cardiac recovery
following an ischaemic insult [5–17].
The excessive use of fatty acids as a fuel source during and
following ischaemia is primarily the result of two factors:
(i) circulating fatty acid levels rise dramatically during and
following ischaemia [18–20] and (ii) subcellular changes
occur in the heart that result in a decreased control of
fatty acid metabolism [21–23]. One important subcellular
alteration is a decrease in malonyl-CoA levels (an important
endogenous inhibitor of fatty acid oxidation), which results
in dysregulation of the fatty acid oxidation pathway
[21–24]. The levels of malonyl-CoA are determined by
the rates of the two enzymes involved in its synthesis and
degradation [acetyl-CoA carboxylase (ACC) and malonylCoA decarboxylase (MCD), respectively]. These enzymes are
also controlled by an AMP-activated protein kinase (AMPK),
which is activated during ischaemia, resulting in a decrease in
malonyl-CoA levels and a loss in mitochondrial fatty acid
uptake control.
The purpose of this review will be to discuss the role of
AMPK in controlling the enzymes involved in regulating
malonyl-CoA levels in both the aerobic and ischaemic heart.
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Control of fatty acid oxidation by
malonyl-CoA
Malonyl-CoA tightly regulates fatty acid metabolism by
inhibition at the level of carnitine palmitoyltransferase-1
(CPT-1), which mediates fatty acid transport into the
mitochondria (Figure 1) [25]. This inhibition of CPT-1 by
malonyl-CoA results in decreased mitochondrial fatty acid
uptake and thus reduced fatty acid oxidation rates. In the
heart, malonyl-CoA seems to play an important metabolic
signalling role and consequently is degraded very rapidly
with a half-life of only 1.25 min [26]. Therefore, steadystate levels of malonyl-CoA are controlled by the dynamic
rates of its synthesis and degradation. Malonyl-CoA is
synthesized from acetyl-CoA in the heart by ACC [23,27,28].
Degradation of malonyl-CoA occurs by the reverse reaction
via decarboxylation back to acetyl-CoA by the enzyme MCD
[24,29–31]. Both ACC and MCD are suggested to play an
important role in influencing fatty acid oxidation rates by
altering malonyl-CoA levels.
Synthesis of malonyl-CoA occurs via ACC, which exists
in two isoforms distinguished by molecular mass: ACC-α
(265 kDa) and ACC-β (280 kDa) [32–35]. Both isoforms are
present in the heart [32,34,35], although ACC-β predominates. An association of ACC-β with the mitochondrial
membrane [34,36] led to the suggestion that ACC-β is
directly involved in the regulation of fatty acid oxidation
[23,34,36,37]. This would allow malonyl-CoA production to
occur in close physical proximity to CPT-1 and enable
direct CPT-1 inhibition. Studies by our group and others
have provided direct evidence that ACC-β is an important
regulator of fatty acid oxidation in cardiac myocytes
[1,21–23,38–40]. These studies suggest that ACC-β plays a
very important role in regulating fatty acid oxidation rates
in the heart.
MCD (which decarboxylates malonyl-CoA to acetylCoA) [41–44] is also an important modulator of intracellular malonyl-CoA levels in the cardiac myocyte. We
have previously shown that increased or maintained MCD
activity in conjunction with a decrease in ACC activity is
likely to be responsible for the decrease in malonyl-CoA
levels and increased fatty acid oxidation seen in both the
post-natal heart and the reperfused ischaemic heart [24].
Subsequent to our study, similar work by Goodwin et al. [31]
demonstrated that MCD may control fatty acid oxidation
in the heart during contractile stimulation. Other studies
show an increase in both activity and expression of MCD in
streptozotocin-induced diabetic rat hearts [45] and an increase
in mRNA expression during high-fat feeding, fasting and
streptozotocin-induced diabetes [46]. This group of studies
and several others show a correlation between MCD activity
and myocardial fatty acid oxidation rates, indicating that
MCD may have a role in altering energy metabolism.
Interestingly, both enzymes involved in the control of
cardiac malonyl-CoA levels have been suggested to be
Figure 1 Uptake of fatty acids into the mitochondria through the carnitine shuttle is inhibited by malonyl-CoA at the level of CPT-1
The rate of fatty acid uptake influences the rates of mitochondrial fatty acid oxidation.
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Figure 2 Phosphorylation control of ACC and MCD by AMPK
alters the levels of malonyl-CoA and subsequently influences
rates of fatty acid oxidation
under phosphorylation control by AMPK. Phosphorylation
of ACC by AMPK has been well documented [47–49].
More recently it has been suggested that MCD is also a
phosphorylation target of AMPK [50,51]. AMPK therefore
plays a distinct and important role in regulating both malonylCoA levels and fatty acid oxidation in the heart (Figure 2).
AMPK and regulation of malonyl-CoA levels
AMPK is a serine/threonine kinase which consists of three
subunits, α, β and γ (see [52] for a comprehensive review
of AMPK). The α catalytic subunit includes the kinase
domain and is responsible for the transfer of a high-energy
phosphate from ATP to specific protein targets [52]. The β
and γ subunits act as regulatory components of the AMPK
heterotrimer and each subunit has several distinct isoforms,
including α1, α2, β1, β2, γ 1, γ 2 and γ 3 [52]. AMPK is
activated by metabolic breakdown products such as the
AMP/ATP ratio and the creatine/phosphocreatine ratio [53].
However, cardiac AMPK activity can be changed under
conditions where significant alterations in the AMP/ATP
ratio do not occur [54,55]. In particular, insulin inhibits
AMPK activity in isolated perfused hearts under conditions
where the AMP/ATP and creatine/phosphocreatine ratios
do not change [7,55,56], which may indicate an involvement of an upstream kinase AMPK kinase. AMPK kinase
phosphorylates AMPK on Thr-172 and causes a significant
increase in AMPK activity (greater than 50-fold; reviewed in
[52]). Increased AMPK activity has been shown to increase
phosphorylation of several targets including 3-hydroxy3-methylglutaryl-CoA (HMG-CoA) reductase, hormonesensitive lipase, glycogen synthase [52] and endothelial nitric
oxide synthase [57].
In the heart, AMPK is as an important metabolic sensor,
which is activated under conditions of metabolic stress such as
hypoxia, or under conditions where ATP is depleted. AMPK
is an important modulator of fatty acid metabolism in the
myocardium by accelerating fatty acid oxidation when ATP
is low, and decreasing fatty acid oxidation when ATP supply
is high. The modulation of fatty acid metabolism occurs by
potential phosphorylation of two key enzymes involved in
the control of malonyl-CoA levels: ACC and MCD.
Cardiac ACC phosphorylation by AMPK appears to
involve the regulation of fatty acid oxidation at the level of
CPT-1. A close correlation exists between increased AMPK
activity, decreased ACC activity and increased fatty acid
oxidation in isolated working rat hearts [21,22,39,56]. It has
been well established that AMPK is able to phosphorylate
both isoforms of ACC [47–49] and we have shown that
cardiac ACC co-purifies with the α2 isoform of the catalytic subunit of AMPK [58]. These studies suggest a tight
association of AMPK with ACC in the heart and phosphorylation/dephosphorylation control of ACC. Recent work by
Park et al. [59] shows a strong correlation between AMPK
activity, ACC activity and phosphorylated ACC protein
in electrically stimulated gastrocnemius muscle, providing
further proof that AMPK phosphorylates and inactivates
ACC. This inactivation results in a decrease in the synthesis
of malonyl-CoA and thus relieves the inhibition of CPT-1mediated uptake of mitochondrial fatty acids.
Since AMPK seems to be indirectly involved in the
production of malonyl-CoA, several studies have also
investigated whether MCD is a substrate for AMPK. The
ability of AMPK to control both the production of malonylCoA (via ACC) and the degradation of malonyl-CoA (via
MCD) is an attractive hypothesis. Our early studies have
shown that purified AMPK does not modify MCD activity
in vitro [30]. However, work by Saha et al. [50] suggests
that 5-amino-4-imidazole carboxamide riboside (AICAR)
activation of AMPK can increase MCD activity 2-fold in rat
extensor digitorum longus muscle. The authors also suggest
that muscle contraction-induced activation of AMPK leads
to phosphorylation and activation of MCD. In contrast, it
has also been shown that neither contraction-induced nor
AICAR-induced AMPK activation of three rat fast twitch
muscle types have any effect on MCD activity [60]. However,
more recently Park et al. [51] have shown an increase in both
AMPK and MCD activity in exercising muscle and liver,
and more importantly that MCD activity could be reduced
when incubated with protein phosphatase 2A. These results
suggest that AMPK does phosphorylate MCD in exercising
rat muscle and liver. The reasons for these discrepancies
are unknown and thus the question of whether AMPK can
phosporylate cardiac MCD remains unclear. Recent studies
from our laboratory have shown that AMPK activation in cardiac cells results in translocation of overexpressed MCD from
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the cytoplasmc space to the mitochondria (N. Sambandam,
J.R.B. Dyck and G.D. Lopaschuk, unpublished work).
Whether this involves the phosphorylation of AMPK has
yet to be established. Phosphorylation control of MCD and
ACC by AMPK becomes especially important in the setting
of ischaemia. AMPK activity dramatically increases during
ischaemia, causing alterations in malonyl-CoA levels through
the phosphorylation of these two key regulatory enzymes.
Figure 3 Ischaemia causes a rapid increase in AMPK activity
AMPK has a dual role of increasing fatty acid oxidation and glycolysis,
which exacerbates the uncoupling of glucose metabolism and may
increase ischaemic damage of the myocardium.
Ischaemia-induced alterations in cardiac
metabolism
There is a dynamic interplay between the oxidation of fatty
acids and glucose metabolism in the heart. Acetyl-CoA
derived from fatty acids can inhibit glucose oxidation at
the level of the pyruvate dehydrogenase complex. Therefore,
increased fatty acid oxidation decreases glucose oxidation.
This occurs even though glycolytic rates in the heart can
remain high, promoting the proton production observed
during and following ischaemia. This is physiologically
relevant since free circulating fatty acid levels increase during
and following ischaemia due to increased hormone-sensitive
lipase activity and breakdown of triacylglycerols [18–20].
Excessive fatty acid levels are detrimental to both the
ischaemic heart [61,62] and the reperfused ischaemic heart
[63,64]. While fatty acids have the potential to have direct
lipotoxic effects on the heart [65–67], excessively high rates
of fatty acid oxidation at the expense of glucose oxidation
appear to be a key contributor to the adverse effects of fatty
acids during and following ischaemia (see [68] for review).
During severe ischaemia, when oxygen is unavailable, the
heart relies on glycolysis to produce ATP. Other energyproducing metabolic pathways are incapable of proceeding
in the absence of oxygen. However, glycolysis results in the
production of protons when uncoupled from the protonconsuming glucose-oxidation pathway. The reduction in
coronary flow during ischaemia causes deleterious byproducts of glycolysis, such as lactate and protons, to accumulate intracellularly. This excessive production of protons
in the heart [3,4] can lead to an accumulation of sodium and
calcium ions [69]. During reperfusion the clearance of these
protons causes a decrease in cardiac efficiency as more energy
is expended to re-establish a normal intracellular pH (see
[68] for a review). If glucose oxidation levels are increased
during reperfusion, the production of protons is lessened and
improved coupling between glycolysis and glucose oxidation
occurs. This in turn promotes improved cardiac recovery by
increasing the efficiency of the heart to convert energy into
contractile function.
Alterations in AMPK activity during
ischaemia/reperfusion
During ischaemia/reperfusion AMPK has a dual role of
increasing both fatty acid oxidation and glycolysis to increase
overall energy production in response to high energy demand
(Figure 3). Ischaemia causes a rapid increase in AMPK
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phosphorylation and activity [21,22,70]. AMPK activation
may occur in order to increase myocyte glucose utilization
by increasing both glucose uptake and glycolysis ([71–73];
Figure 3). For example, AMPK activation promotes the
translocation of GLUT-4 to the sarcolemma of the myocyte
to increase glucose uptake [74]. The glycolytic pathway is
accelerated by the phosphorylation and activation of phosphofructokinase-2 by AMPK [73], which produces fructose
2,6-bisphosphate (a potent activator of glycolysis).
However, as well as accelerating the use of glucose, AMPK
also has a potent effect on fatty acid oxidation rates. Activation
of AMPK during ischaemia results in the use of fatty acids
as the main source of residual oxidative metabolism. The
consequence of this is an accelerated production of deleterious
glycolytic by-products, particularly lactate and protons.
These by-products are formed due to fatty acid inhibition
of glucose oxidation, while glycolytic rates remain high.
AMPK activity persists during reperfusion of the myocardium [21,22] and during this time the heart continues
to be exposed to high circulating levels of fatty acids. Fatty
acid oxidation rates recover and provide the majority of the
tricarboxylic acid cycle acetyl-CoA, again at the expense of
glucose oxidation [13,61,75]. Glycolytic rates remain elevated
[76,77], causing greater uncoupling between glycolysis and
glucose oxidation and resulting in proton production [9,78]
and reduced pH recovery following reperfusion [79].
AMPK 2002 – 2nd International Meeting on AMP-activated ProteinKinase
ACC activity is reduced during reperfusion of ischaemic
hearts, which is accompanied by high AMPK activity and a
drop in malonyl-CoA levels [21]. This decrease in malonylCoA levels is likely to be responsible for the high rates of
fatty acid oxidation that occur during this period [21,22,24].
However, during reperfusion MCD levels are maintained
[24], causing a net decrease in steady-state malonyl-CoA
levels. The balance between malonyl-CoA synthesis and
degradation is shifted towards the breakdown of malonylCoA, which may relieve the inhibition of CPT-1. This
increase in fatty acid transport into the mitochondria, coupled
with a higher concentration of circulating fatty acids may lead
to enhanced ischaemic damage of the myocardium.
Summary
AMPK plays an important role in regulating malonyl-CoA
levels through the phosphorylation of ACC and the proposed
phosphorylation of MCD. The activity of these enzymes
is important in determining the steady-state malonyl-CoA
levels in the myocardium. As levels of malonyl-CoA are
responsible for inhibition of CPT-1 and control of fatty acid
uptake into the mitochondria, malonyl-CoA is an important
metabolic mediator. AMPK is physiologically important
as a sensor of metabolic demand by modulating malonylCoA levels. AMPK accelerates both fatty acid oxidation
and glycolysis in an attempt to replenish ATP production
during ischaemia/reperfusion. Unfortunately, this dual role
of AMPK leads to a greater uncoupling of glycolysis and
glucose oxidation in the heart, leading to excessive proton and
lactate production. AMPK may therefore be an important
pharmacological target for improving cardiac efficiency
following ischaemia.
This work was supported by a grant from the Canadian Institutes for
Health Research (CIHR). T.A.H. is a trainee of the Alberta Heritage
Foundation for Medical Research (AHFMR). J.R.B.D. is a Scholar of the
AHFMR and a CIHR New Investigator. G.D.L. is a Medical Scientist of
the AHFMR.
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