NATURE REVIEWS | ENDOCRINOLOGY ADVANCE ONLINE PUBLICATION | 1

Institute for Clinical
Diabetology, German
Diabetes Center,
D-40225 Düsseldorf,
Germany
(J. Szendroedi,
E. Phielix, M. Roden).
Department of
Metabolic Diseases,
University Clinics
Düsseldorf, Heinrich-
Heine University,
D-40225 Düsseldorf,
Germany
(J. Szendroedi,
M. Roden).
Correspondence to:
M. Roden
michael.roden@
ddz.uni-duesseldorf.de
The role of mitochondria in insulin resistance
and type 2 diabetes mellitus
Julia Szendroedi, Esther Phielix and Michael Roden
Abstract | Type 2 diabetes mellitus (T2DM) has been related to alterations of oxidative metabolism in
insulin-responsive tissues. Overt T2DM can present with acquired or inherited reductions of mitochondrial
oxidative phosphorylation capacity, submaximal ADP-stimulated oxidative phosphorylation and plasticity of
mitochondria and/or lower mitochondrial content in skeletal muscle cells and potentially also in hepatocytes.
Acquired insulin resistance is associated with reduced insulin-stimulated mitochondrial activity as the result
of blunted mitochondrial plasticity. Hereditary insulin resistance is frequently associated with reduced
mitochondrial activity at rest, probably due to diminished mitochondrial content. Lifestyle and pharmacological
interventions can enhance the capacity for oxidative phosphorylation and mitochondrial content and improve
insulin resistance in some (pre)diabetic cases. Various mitochondrial features can be abnormal but are not
necessarily responsible for all forms of insulin resistance. Nevertheless, mitochondrial abnormalities might
accelerate progression of insulin resistance and subsequent organ dysfunction via increased production of
reactive oxygen species. This Review discusses the association between mitochondrial function and insulin
sensitivity in various tissues, such as skeletal muscle, liver and heart, with a main focus on studies in humans,
and addresses the effects of therapeutic strategies that affect mitochondrial function and insulin sensitivity.
Szendroedi, J. et al. Nat. Rev. Endocrinol. advance online publication 13 September 2011; doi:10.1038/nrendo.2011.138
Introduction
Large prospective studies have shown that insulin resis-
tance, the inability of cells to efficiently respond to stimu-
lation by insulin, precedes the onset of type 2 diabetes
mellitus (T2DM) by many years. The main feature of
insulin-resistant states, including T2DM, is a reduc-
tion of nonoxidative glucose storage as glycogen in
skeletal muscle during hyperinsulinemia.
1
But humans
with insulin resistance also exhibit impaired fasting fatty
acid oxidation and an inability to switch from fatty acid
to glucose oxidation during hyperinsulinemia.
2,3
The
reduced adaptation of fuel oxidation to altered nutrient
availability is termed ‘metabolic inflexibility’ and has been
associated with the accumulation of lipids within muscle
cells (intramyocellular lipids) and insulin resistance.
2,3

Several studies have addressed the role of mito chondria
in insulin resistance, but with conflicting results.
4–13

Differences in terminology may at least partially explain
the contradictory conclusions, as the term ‘mito chondrial
dysfunction’ has been frequently applied to describe either
alteration in mitochondrial content, mitochondrial activ-
ity and/or submaximal ADP-stimulated oxidative phos-
phorylation under various physiological conditions.
5–7
In
this Review, our group proposes a systema tic termi nology
to define parameters of mitochondrial morpho metry
(Box 1) and of mitochondrial function (Figure 1). Of
note, various methods, including in vivo magnetic reso-
nance spectroscopy (MRS), high-resolution respirometry
and morphometry, are used to assess indivi dual features
of mitochondria, and the suggested definitions critically
depend on the applied methods. These methods have
been extensively reviewed elsewhere
8,9
and are only briefly
summarized (Boxes 2 and 3).
Evidence suggests that mitochondrial content or oxi-
dative capacity of mitochondria are altered in insulin-
responsive tissues of patients with insulin resistance.
5,10–14

Nevertheless, several factors remain uncertain: first,
whether these observations result from acquired or inher-
ited functional mitochondrial abnormalities and/or lower
mitochondrial content; second, which mito chondrial
alterations relate to insulin resistance of glucose disposal;
and, third, whether a causal relationship exists between
tissue-specific abnormalities in mitochondria and insulin
sensitivity. This article reviews the current evidence for
mitochondrial impairment in insulin-resistant states in
skeletal muscle, liver, heart, adipose tissue and the brain,
with a main focus on studies in humans. To assess pos-
sible dynamic relationships, studies of interventions that
affect mitochondrial function and insulin sensitivity in
humans are discussed.
Definition of mitochondrial function
Activity
Mitochondrial activity is defined as resting oxidative
phosphorylation flux in distinct metabolic states, for
example fasting, hyperlipidemia and hyperinsulinemia.
Competing interests
M. Roden declares an association with the following company:
Takeda. See the article online for details of the relationship. The
other authors declare no competing interests.
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The flux rate is primarily determined by the ADP con-
centration and cellular demand for ATP synthesis, that
is, the ADP:ATP ratio.
15
Only substrate availability below
the energetic demand or low O
2
concentrations

will limit
ATP generation by mitochondria. Mitochondrial acti v-
ity can be assessed in vivo by measuring ATP saturation
transfer and depends on mitochondrial plasticity
16
and
could be affected by mitochondrial loss.
17
Oxidative phosphorylation capacity
Mitochondrial oxidative phosphorylation capacity is
defined as maximal ADP-stimulated oxidative phos-
phorylation elicited by high ADP:ATP ratios that reflect
maximal energy demand at saturating concentrations of
substrates
18–20
and unlimited oxygen supply. Oxidative
phosphorylation capacity is assessed from oxygen con-
sumption or ATP synthesis during ex vivo experiments
of isolated mitochondria or in permeabilized fibers
calcu lated per mitochondrial content. Maximal oxida-
tive phosphorylation capacity is obtained with glutamate,
malate and succinate, reconstituting the operation of the
tricarboxylic acid cycle and preventing depletion of key
metabolites from the mitochondrial matrix.
21
Key points
■ Overt type 2 diabetes mellitus is associated with reduced oxidative
phosphorylation capacity, submaximal ADP-stimulated oxidative phosphorylation
and mitochondrial plasticity in insulin-responsive tissues
■ Acquired insulin resistance is associated with reduced insulin-stimulated
mitochondrial plasticity that results in the inability of the organism to switch from
fatty acid to glucose oxidation in skeletal muscle
■ Hereditary insulin resistance can be linked to reduced resting mitochondrial
activity at least partly due to a decreased mitochondrial content
■ Lifestyle and pharmacological interventions can enhance oxidative
phosphorylation capacity and mitochondrial content, and in most cases improve
insulin resistance in (pre)diabetic states
■ Reduced oxidative phosphorylation capacity is unlikely to be the general cause
of all forms of insulin resistance but might accelerate its progression and
subsequent organ dysfunction via increased production of reactive oxygen species
Box 1 | Basic features of mitochondrial morphometry
Mitochondrial content per cell
Mitochondrial content is calculated by the mitochondrial area relative to the
whole cell area as seen via electron microscopic imaging.
39
Biochemical
surrogate markers that can be used to measure mitochondrial content include
the relative copy number of genes encoded by mitochondrial DNA,
71
expression
of cardiolipin
47,131
and enzymatic markers of oxidative phosphorylation, such as
citrate synthase.
71
Mitochondrial localization
Electron microscopic imaging reveals different subcellular compartments of
mitochondria, at least in skeletal muscle (intermyofibrillar and subsarcolemmal
areas).
39
Methods such as standard homogenization and differential
centrifugation with or without protease digestion isolate the distinct mitochondrial
populations to discern their individual functional properties.
43
Mitochondrial dynamics
Mitochondrial fusion and fission are defined as mitochondrial dynamics for
maintenance of the mitochondrial network.
148
These two processes determine
formation and breakdown of mitochondria, as well as translocation into subcellular
regions of increased energy demand, and distribution upon cell division.
Submaximal oxidative phosphorylation
We define submaximal ADP-stimulated oxidative phos-
phorylation as in vivo non-resting mitochondrial activ-
ity stimulated by energy depletion during standardized
in vivo experiments. During in vivo experiments, sub-
maximal ADP-stimulated oxidative phosphorylation can
be assessed from resynthesis rates of ATP or phospho-
creatine elicited by induction of high energy demand.
In vivo, energy demand is increased in skeletal muscle
by exercise, which depletes phosphocreatine levels. In
the liver, administration of ethanol or fructose raises
the energy demand by depleting ATP levels. However,
inducible ADP:ATP ratios in vivo are lower than ex vivo;
therefore, various approaches to calculate oxidative
phosphory lation capacity from extrapolation procedures
were developed.
15,22
In contrast to the ex vivo setting, the
parameters relevant to control oxidative metabolism
9,15,23

include not only the delivery of ADP and inorganic phos-
phate from the functional ATP synthase, but also de livery
of oxygen from the capillary circulation and oxygen
uptake;
24
delivery of substrates to form NADH;
25
and
other flux- controlling factors, such as calcium released
during exercise
26
and lactic acidosis.
9,15,23,27
Moreover,
effective mitochondrial coupling between oxygen con-
sumption and the rate of ATP synthesis, which deter-
mines the yield of ATP,
28
is highly variable. Although the
relative impairment of maximal phosphocreatine resyn-
thesis rates in vivo did not exceed the impairment of sub-
maximal ADP-stimulated oxygen consumption ex vivo in
patients with T2DM compared to healthy humans,
29
the
above-mentioned parameters limit the use of
31
P-MRS for
noninvasive measurement of oxidative phosphorylation
capacity despite linear dependence of phosphocreatine
kinetics on oxidative phosphorylation capacity.
15,23
Thus, a
reduction of submaximal ADP-stimulated oxidative phos-
phorylation indicates that mitochondrial activity is not
able to cover the current energy demand of the cell, which
results from either impaired mitochondrial plastici ty,
reduced oxidative capacity or substrate supply.
30
Plasticity
Mitochondrial plasticity is defined as changes of mito-
chondrial activity, mitochondrial content or oxidative
phosphorylation capacity due to altered metabolic con-
ditions.
31,32
Acute hyperinsulinemia triggers short-term
mitochondrial plasticity, whereas chronic metabolic
changes, such as altered substrate fluxes, exercise train-
ing or even blockade of the respiratory complex I by
metformin
33
induce long-term mitochondrial plastic-
ity. Particularly, chronic metabolic changes depend on
transcriptional regulators such as PGC1α and PGC1β
or AMPK.
34,35
Metabolic plasticity of mitochondria is
determined by regulation of fuel uptake and substrate
selection or coupling of the tricarboxylic acid cycle and
fatty acid oxidation. The concept that the spare electron
transport chain capacity is able to elevate the metabolic
rate from baseline in order to accommodate rapid rises
in metabolic demand fits the concept of short-term mito-
chondrial plasticity.
36
Metabolic inflexibility or incom-
plete fatty acid oxidation can, therefore, compromise
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mitochondrial plasticity.
37
Mitochondrial plasticity
can be assessed on the basis of any parameter of mito-
chondrial function (for example, content, activity, oxida-
tive capacity) that is measured before and after any of the
above interventions.
Coupling
Mitochondrial coupling is defined as the molar ratio of
the yield of ATP per oxygen consumed (P/O ratio) and is
also termed electrochemical coupling.
38
The maximum
yield of ATP during metabolism is lower than classical
P/O ratios suggest, so that ~20–30% of basal metabolic
rates might be devoted to driving a futile cycle of proton
pumping. This feature of mitochondrial function has
been reviewed in detail elsewhere.
28
Mitochondria and insulin resistance
Skeletal muscle
Skeletal muscle accounts for the majority of glucose dis-
posal after meal ingestion and for nearly all the glucose
uptake and nonoxidative storage during hyper insulinemia.
On the other hand, skeletal muscle also determines whole-
body substrate oxidation during exercise and adaptation
to exercise training.
Fasting conditions
Results on the role of mitochondria in insulin-resistant
skel etal muscle have been conflicting. Several studies
reported a reduction in mitochondrial content
39–42
or
reduced oxidative capacity of individual mito chon-
dria
29,43,44
in skeletal muscle. In other studies, how ever,
no alterations in mitochondrial oxidative capacity or
activity in the fasting state were found, but impaired
insulin- stimulated ATP production was observed.
45,46

In vivo, patients with T2DM have lower submaximal
ADP-stimulated oxidative phosphorylation in skeletal
muscle than nondiabetic humans matched for age, body
mass and sex,
29,40
which indicates the current energy
demand of the cell is not met owing to either impaired
mitochondrial plasticity, reduced oxidative phosphory-
lation capacity or mitochondrial content. Accordingly,
several studies reported lower oxidative phosphory lation
capacity in muscle biopsy samples from patients with
T2DM compared with healthy individuals.
29,41,42,47
These
reductions remained after controlling for mito chondrial
content in all except one study
42
and were further con-
firmed in isolated mitochondria,
44
corroborating the
finding of compromised mitochondrial capacity in
patients with T2DM. Measures of mitochondrial content
are frequently reduced in patients with T2DM
34,39,41,43,48,49

or insulin resistance.
34,49
However, some studies did
not find differences between patients with T2DM and
humans matched for body mass,
29,42,44
which could, at
least in part, result from a reduction of mitochondrial
content related to obesity rather than insulin resistance.
On the other hand, the observed differences might
be due to the use of surrogate markers of mitochon-
drial content. Measurements of surrogate markers of
mitochondrial content do not generally correlate with
measure ments obtained by electron microscopy.
34,50

Direct subcellular quantification revealed that inter-
myofibrillar mitochondria of overweight or obese,
insulin-resistant patients with T2DM had up to 40%
less content than those of lean, insulin-sensitive indivi-
duals, which correlated positively with glucose disposal,
lipid oxidation and metabolic flexi bility and negatively
with BMI and plasma concentration of free fatty acids
(FFA).
39
Some,
35,51
but not all,
34,52
studies showed reduced
expression of PGC1α and PGC1β, the main regulators
of mitochondrial biogenesis and fatty acid oxidation
in patients with T2DM and their non diabetic relatives.
The PGC1 family of coactivators responds to environ-
mental and nutritional stimuli and is controlled at
post- transcriptional levels; normal protein or mRNA
levels of PGC1, therefore, do not exclude impairments
in protein activity or binding to docking sites.
53
PGC1α
enables the cell to adapt to fluctuations in nutritional
status and could link impaired mitochondrial plasticity
to metabolic inflexi bility, insulin resistance and reduced
mitochondrial content (Figure 2).
Resting mitochondrial activity was found to be
reduced in elderly, insulin-resistant individuals (Figure 3,
Box 2),
46,54
insulin-resistant, nondiabetic offspring of
patients with T2DM
55
and even in previously insulin-
resistant patients treated for acromegaly.
56
ATP synthetic
rates are strongly related to submaximal ADP-stimulated
oxidative phosphorylation,
57
and their reduction
could either reflect impaired mitochondrial plastic-
ity
46,58
or reduced energy demand.
9
However, resting
mito chondrial activity can be normal in patients with
1.5

1.0

0.5

0.0

[ADP]
O
2
, pH, [Ca
2+
], metabolic condition
Activity
K
m
Plasticity
Plasticity
Submaximal
ADP-stimulated
oxidative
phosphorylation
Oxidative
phosphorylation
capacity
V
m
a
x

o
x
y
g
e
n

c
o
n
s
u
m
p
t
i
o
n
P
/
O

r
a
t
i
o
V
m
a
x

A
T
P

s
y
n
t
h
e
t
i
c

r
a
t
e
,
r
e
s
y
n
t
h
e
s
i
s

o
f

p
h
o
s
p
h
o
c
r
e
a
t
i
n
e
/
A
T
P
Figure 1 | Parameters of mitochondrial function. The fraction of maximum oxygen
consumption or ATP synthetic rate (V
max
) is plotted against prevalent ADP
concentration, and other flux-controlling parameters are depicted. Mitochondrial
activity (blue area) is assessed from ATP synthetic rates at low energy demand
(low ADP concentrations) and is determined by prevalent metabolic conditions (for
example, hyperlipidemia). Oxidative phosphorylation capacity (red area) is
assessed in ex vivo experiments from ATP synthetic rates or oxygen consumption
at maximal energy demand (high ADP concentrations) when oxygen and substrate
supply are not flux-controlling. Submaximal ADP-stimulated oxidative
phosphorylation (yellow area) is assessed from resynthesis of ATP or
phosphocreatine at increased ADP concentrations and strongly depends on
mitochondrial plasticity, coupling (P/O ratio), oxygen and substrate supply and
uptake, pH and, in skeletal muscle, on calcium release. Interventions (for example,
insulin) affecting parameters of mitochondrial function allow estimating
mitochondrial plasticity (braces).
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T2DM, by com parison with healthy humans.
46
Thus,
reduced mitochondrial content might be compensated
for by increased mitochondrial activity. However, in
these studies, mitochondrial content, oxidative capacity
and mitochondrial activity in the fasting state did not
corre late with insulin sensitivity within insulin-resistant
groups. This finding points to an important dissociation
between insulin sensitivity and muscular mitochondrial
function, which was confirmed by other studies.
59–62
Hyperlipidemia
Humans with insulin resistance frequently present with
increased plasma concentrations of FFA.
63
A reduced
capacity for muscular lipid oxidation, which results in
the accumulation of lipid intermediates and intramyo-
cellular lipids, might cause the insulin resis tance
observed in lean relatives of patients with T2DM.
34,55,64

Cellular elevation of intermediates, such as diacyl-
glycerols (DAG), acyl-CoAs or ceramides, might
activate atypical protein kinase C (PKC) isoforms, fol-
lowed by serine phosphorylation of insulin receptor
substrate 1 (IRS1), and thereby cause impaired insulin
signaling.
6,65–68
Short-term increases in lipid availability
by triglyceride infusions also induce insulin resistance
in healthy humans, but the under lying cellular mecha-
nisms,
69
and particularly the role of mitochondria,
6
are
yet unclear.
Short-term lipid infusion for 3 h at fasting plasma
insulin concentrations decreased whole-body glucose
disposal but had no effect on mitochondrial activity in
healthy, insulin-sensitive humans.
70
Lipid infusion for 6 h
decreased the inner mitochondrial membrane potential,
which provides the electromotive driving force for ATP
synthesis, but did not affect genes involved in mito-
chondrial function.
71
Lipid infusion for 48 h decreased
the expression of PGC1 and nuclear-encoded mitochon-
drial genes.
72
Fasting for 62 h increased whole-body lipid
oxidation and reduced glucose oxidation and insulin
resistance.
73
Taken together, short-term elevation of FFA
at fasting insulin levels induces insulin resistance in skel-
etal muscle but does not reduce mitochondrial activity.
This finding clearly shows that the physiological insulin
resistance associated with prolonged fasting does not
involve abnormal mitochondrial activity.
In rodent models, insulin resistance can develop
in response to high-fat feeding despite a significant
compensatory increase in mitochondrial content
and enhanced oxidative phosphorylation capacity
for fatty acids.
74–76
Rising FFA levels activate PPARδ,
which increases PGC1α expression, probably via a
post- transcriptional mechanism.
76
These effects could
precede deleterious consequences of lipids on mito-
chondria observed in adult rats fed a high-fat diet.
77

Although this concept has not been tested in humans,
(pre)diabetic individuals could represent a human
model of prolonged hyperlipidemia.
Oxidative phosphorylation capacity for fatty acids is
not reduced in mitochondria of patients with obesity
and T2DM.
78,79
However, ‘normal’ lipid oxidation
capacity might be insufficient to prevent lipid accumu-
lation at prolonged increased lipid availability. In lean,
insulin-sensitive humans, PGC1α, mitochondrial content
and expression of fatty acid translocase (also known as
platelet glycoprotein 4 or CD36) are related to ex vivo
capacity of lipid oxidation. In humans with obesity,
these correlations are diminished and PGC1α levels are
reduced. One explanation for this observation could be
that prolonged elevation of plasma FFA levels impairs
the lipid-induced PPAR–PGC1 interaction that regulates
fatty acid oxidation in insulin-sensitive humans.
80
Box 3 | Assessment of mitochondrial function ex vivo
Mitochondrial oxidative capacity ex vivo is assessed on the basis of absolute
rates of oxygen consumption or ATP production. Oxygen consumption rate
is measured by high-resolution respirometry with the Clark electrode
157
and
the Oroboros®oxygraph
21
(Oroboros Instruments, Innsbruck, Austria), which
employ a temperature-controlled respiration chamber connected to oxygen-
sensitive sensors. ATP production can be monitored bioluminometrically.
45

Although these methods can be applied to measure maximal oxidative
phosphorylation capacity of the respiratory chain complexes in permeabilized
cells or isolated mitochondria, they do not take into account the physiological
environment surrounding mitochondria in vivo. The Seahorse®extracellular flux
analyzer (Seahorse Bioscience, Billerica, MA, USA) allows for high-throughput
measurements to examining cells or isolated mitochondria and takes into
account the physiological environment surrounding mitochondria in vivo, as it
allows measurements of mitochondrial activity in intact attached cells.
158
Enzyme
activities can be determined by spectrophotometry.
8
Box 2 | Assessment of mitochondrial function in vivo
Phosphocreatine and ATP resynthesis
Submaximal ADP-stimulated oxidative phosphorylation is assessed from
resynthesis of energy-rich phosphates, continuously measured with in vivo
31
P magnetic resonance spectroscopy, upon maximal depletion due to an
energy-consuming intervention. Muscular phosphocreatine recovery is
monitored after exhaustive exercising,
149
whereas hepatic ATP resynthesis is
measured after fructose or ethanol challenges.
150
Tissues rapidly consuming
ATP, such as muscle and brain, generate phosphocreatine as an energy
buffering system for rapid ATP regeneration. Tracing phosphocreatine kinetics
during recovery after one bout of exercise was developed as a tool to estimate
submaximal ADP-stimulated oxidative phosphorylation, whereas other
energy-consuming and energy-producing processes are maintained.
151
During
exercising, phosphocreatine concentrations decrease transiently and recover
rapidly thereafter, yielding a time constant of the recovery rate independent
of work or power output.
152
This tool is of valuable sensitivity and could serve
to identify patients with mitochondriopathies
153
and monitoring therapeutical
interventions.
154
Of note, individual exercise performance and compliance can
influence the result of this test.
ATP saturation transfer
Mitochondrial activity is assessed by evaluating ATP saturation transfer with
31
P magnetic resonance spectroscopy (MRS), which yields the unidirectional
flux through ATP synthase in resting skeletal muscle and liver.
38,155,156
This
measurement reflects mainly ATP synthetic rates resulting from basal energy-
consuming and energy-producing processes (transmembrane transportation,
protein synthesis, glycolysis, etc.) but can be also performed at various metabolic
conditions, including insulin stimulation to assess mitochondrial plasticity.
22

Combination of
31
P-MRS with
13
C-MRS to assess tricarboxylic acid cycle flux as
an index of mitochondrial substrate oxidation yields a measure of mitochondrial
energy coupling.
56
Of note, regional perfusion and concentrations of hormones
and metabolites may influence the results of these tests. ATP synthetic rates
are strongly related to submaximal ADP-stimulated oxidative phosphorylation
57

and a reduction could either reflect mitochondrial loss,
17
impaired mitochondrial
plasticity
46,58
or reduced energy demand.
9
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Previous studies in rodent models show that insulin
resistance and metabolic flexibility are related to
increased levels of incompletely oxidized fatty acids that
are exported as acylcarnitines to the cytosol, plasma and
urine. According to this concept, lipid uptake and rates of
fatty acid oxidation exceed those of the tricarboxylic acid
cycle in the insulin-resistant state.
37
Supplementation of
carnitine, to facilitate export of carbon atoms, improved
completeness of fatty acid oxidation, glucose toler-
ance and metabolic flexibility in rodents fed a high-
fat diet.
81
Acylcarnitine levels in skeletal muscle and
plasma remained unchanged, suggesting that acyl-
carnitines do not mediate insulin resistance per se but
serve as biochemical markers of incomplete fatty acid
oxidation. Accordingly, patients with T2DM and obesity
accumulate plasma acylcarnitines that arise from
increased rates of inefficient fatty acid oxidation.
82–84
In
healthy humans, muscle acyl carnitine levels remained
unchanged after prolonged fasting, independent of their
baseline plasma levels, fuel oxidation rates or insulin
resistance and, therefore, acylcarnitines in muscle prob-
ably do not mediate lipid-induced insulin resistance.
73

In summary, incomplete fatty acid oxidation can occur
upon prolonged, increased FFA availability in (pre)dia-
betic states (Figure 2). Whether improved coupling of
β-oxidative and tricarboxylic acid cycle rates would raise
insulin sensitivity in humans remains uncertain.
Hyperinsulinemia
During hyperinsulinemic, normoglycemic clamps
in humans without diabetes mellitus, mitochondrial
activ ity increases by about 10–90% after 1–2 h and oxi-
dative capacity by about 32–42% after 8 h, reflecting
mito chondrial plasticity.
45,46,58,85,86
The increase in mito-
chondrial activity after stimulation with insulin is most
probably caused by increased energy demand (increas-
ing ADP:ATP ratios) or activation and/or expression of
key enzymes of the respiratory chain. Insulin-stimulated
inorganic phosphate transport increases intra mycellular
levels of inorganic phosphate,
58,86
but is unlikely to
control resting ATP synthetic rates.
15
Humans with insulin resistance fail to increase their
mitochondrial activity or oxidative phosphorylation
capacity during normoglycemic, hyperinsulinemic
clamps. This finding indicates impaired mitochondrial
plasticity and was reported for patients with overt T2DM,
GLUT
FAT/CD36
G6P
IRS
AKT
FFA Glucose Insulin
Acyl-
carnitines
Glycogen
Nucleus
fATP II I IV III V
Mitochondrion
Biogenesis
DAG
Triglycerides
PKC
δ
PGCs
Ceramides PPARs
Pyruvate
CPT1
LCA-CoA
ROS
Plasticity
TCA cycle
?
?
?
?
?
?
?
β-oxidation
Figure 2 | Role of mitochondria in metabolic inflexibility. In the insulin-resistant state, availability of FFA is increased, which
raises triglyceride storage and intracellular concentrations of lipid metabolites (DAG, ceramides, LCA-CoA). DAG and
ceramides induce impairment of the insulin signaling pathway via activation of inflammatory messengers (for example,
PKC
δ
), which leads to inhibitory serine phosphorylation of IRS. Glucose transport and phosphorylation is reduced.
Stimulation of PGC1α and PGC1β, the main regulators of mitochondrial biogenesis and fatty acid oxidation, is induced by
insulin in skeletal muscle. FFA activate PPARγ and PPARδ. Stimulation of oxidative capacity, mitochondrial biogenesis and
mitochondrial lipid uptake is impaired in the insulin-resistant state. Thus, whole-body lipid oxidation decreases in humans
with obesity and insulin resistance as a result of impaired mitochondrial plasticity. It is yet unknown if insulin has direct,
rapidly acting effects on mitochondrial function. These defects might reflect dysregulation of the lipid-induced PPAR–PGC1
interaction after prolonged hyperlipidemia, which could lead to reduced lipid uptake into mitochondria to compensate for
lower mitochondrial content and increased lipid availability; lipid-induced uncoupling of the respiratory chain; reduced
oxidation of glycolytic substrates, which uncouples fatty acid oxidation rates from TCA cycle rates; and metabolic
inflexibility. Abbreviations: CPT1, carnitine O-palmitoyltransferase 1; DAG, diacylglycerol; IRS, insulin receptor substrate;
FAT, fatty acid translocase (also known as CD36); fATP, ATP flux; FFA, free fatty acids; GLUT, glucose transporter; G6P,
glucose-6-phosphate; LCA-CoA, long-chain acyl-CoA; PKC
δ
, protein kinase C
δ
; PPAR, peroxisome proliferator-activated
receptor; PGC, PPAR coactivator; ROS, reactive oxygen species; TCA, tricarboxylic acid.
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their first-degree relatives, those with type 1 diabetes
mellitus (T1DM)
45,46,58,85–87
and for healthy humans after
a 6 h lipid infusion.
85
As diminished glucose transport
and phosphorylation are the hallmark of insulin resis-
tance, the levels of myocellular glucose-6-phosphate
increase slower and to a lesser extent in these groups
than in control individuals.
88
Thus, reduced substrate
supply could prevent the increase in ATP synthesis to
meet insulin-stimulated energy demand.
30
However, even
the doubling of glucose-6-phosphate levels in hyper-
glycemic, hyperinsulinemic clamps did not raise mito-
chondrial activity in patients with T2DM.
46
Accordingly,
insulin-stimulated oxidative phosphory lation capacity
assessed ex vivo during substrate excess was reduced,
arguing against substrate limitation as a cause of
impaired in sulin-stimulated mitochondrial activity.
45

Although insulin-stimulated transport of inorganic
phosphate was impaired in insulin-resistant relatives of
patients with T2DM,
58
it increased during euglycemic
hyper insulinemia in patients with overt T2DM.
46
This
finding further excludes abnormal inorganic phosphate
transport as the explanation for impaired mitochondrial
plasticity in all forms of insulin resistance. Myotubes
from lean individuals and from those with obesity, but
not from those with T2DM, responded to 4 h insulin
incubation with a ~30% increase in mitochondrial
oxidative phosphorylation capacity.
89
Addition of palmi-
tate diminished this effect in controls, which suggests
that lipid-induced insulin resistance might interfere with
mitochondrial plasticity.
89
Taken together, these data suggest that impaired
in sulin-stimulated mitochondrial plasticity is not simply
due to altered or lower availability of substrates or inorga-
nic phosphate, but is related to impaired sub maximal
ADP-stimulated oxidative phosphorylation in vivo.
Thus, impaired increase of ATP synthetic rates to cover
increased energy demand could be an early feature of
muscular insulin resistance and metabolic inflexibility.
Liver
The liver is the primary organ responsible for endogenous
glucose production in the fasting state and stores glucose
as glycogen after meal ingestion. Hepatic insulin resis-
tance is defined as impairment of glycogen synthesis and
suppression of endogenous glucose production during
hyperinsulinemia, and strongly relates to hepatocellular
lipid content.
90,91
Elevation of hepatocellular lipid content
to 5.5% fat or more, also termed nonalcoholic fatty liver
(NAFL), precedes the manifestation of T2DM. Systemic
insulin resistance inhibits glucose uptake and stimulates
the release of FFA and gluconeogenic precursors, thereby
augmenting the lipid and carbohydrate flux to the liver
and the accumulation of lipids within hepatocytes. As in
skeletal muscle, intracellular lipid metabolites
92
appear
to mediate inhibition of insulin signaling in the rodent
liver. Accumulating hepatocellular lipids are thought to
simulta neously stimulate mitochondrial fatty acid oxi-
dation and the production of reactive oxygen species,
thereby promoting lipid peroxidation and damage of
mitochondrial DNA and proteins.
93
Progression of NAFL to nonalcoholic fatty liver disease
(NAFLD), starting with nonalcoholic steato hepatitis
(NASH), involves intrahepatic inflammation. This
process is associated with dysmorphologies,
94
crystal-
line inclusions
95
and increased amount of mutations in
mitochondrial DNA.
96
Moreover,
31
P-MRS experiments
indicate that intrahepatic ratios calculated on the basis of
levels of phosphomonoesters and NADPH, a marker of
inflammation, are higher in patients with severe NAFLD
than in healthy humans without steatosis and correlate
with disease stage.
97
Despite increased fatty acid oxida-
tion,
94,98
patients with severe NAFLD exhibit diminished
submaximal ADP-stimulated oxidative phosphorylation,
as assessed by the rate of ATP resynthesis—reflecting
either reduced oxidative phosphorylation capacity or
mitochondrial plasticity
99
—but hepatic insulin sen-
sitivity has not been measured. Of note, even within
the normal range, hepatic lipid content can be higher
in slightly insulin-resistant humans at increased risk of
T2DM compared with insulin-sensitive individuals.
100
To
examine the role of energy metabolism in individuals
with marked insulin resistance, our group studied meta-
bolically well-controlled nonobese patients with T2DM
without clinical evidence of NAFLD. They had lower
absolute concentrations of hepatic inorganic phosphate
and ATP, which were strongly correlated with hepatic
140 –
120

100

80

60

40

20

0

Young
healthy
controls
Middle-aged
healthy
controls
*
*
1
st
degree
relatives of
patients with
T2DM
M
i
t
o
c
h
o
n
d
r
i
a
l

a
c
t
i
v
i
t
y

(
%

o
f

c
o
n
t
r
o
l

g
r
o
u
p
)
T1DM T2DM
Patients
MELAS
Insulin stimulated
Basal
Figure 3 | Basal and insulin-stimulated flux through the ATP synthase in humans.
ATP synthetic flux rates (mitochondrial activity) of various insulin-resistant groups
are depicted as percentages of their respective controls. Mitochondrial activity was
measured in vivo under fasting conditions and hyperinsulinemic conditions in
nondiabetic participants of different ages: young
46
and middle-aged (~57 years)
controls,
46
insulin-resistant offspring of patients with T2DM,
58
patients with
T1DM,
87
those with T2DM
56
and in one patient with MELAS, a syndrome caused by
a single point mutation in the mitochondrial genome. Data are given as
mean ± SEM. *P <0.05 fasting versus insulin-stimulated. Abbreviations: MELAS,
mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes; T1DM,
type 1 diabetes mellitus; T2DM, type 2 diabetes mellitus.
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insulin resistance independent of hepato cellular lipid
content.
101
The similar reductions in inorganic phos-
phate and ATP argue against an energetic imbalance as
the cause of lower mitochondrial activity, but suggest
that hepatic mitochondrial content could be decreased
in patients with overt T2DM.
Patients with T2DM also showed lower hepatic mito-
chondrial activity at rest,
102
which correlated positively
with peripheral and hepatic insulin sensitivity, but nega-
tively with hepatic inorganic phosphate and ATP concen-
trations, waist circumference, BMI and fasting glycemia.
Similarly, age-matched nondiabetic persons showed no
evidence of diminished hepatic mitochondrial acti vity
despite comparable hepatocellular lipid contents.
102

Taken together, these findings suggest that even a low
degree of chronic glucose toxicity, along with gradual
abdominal (visceral) adiposity, contributes to perturbed
hepatic energy metabolism in overt T2DM.
Heart
Insulin resistance in myocardium has also been reported
after impaired insulin-stimulated cardiac glucose uptake
in humans with obesity
103
and T2DM.
104
Lipid accumu-
lation in cardiomyocytes has been related to contrac-
tile dysfunction in these patients.
105
Insulin-resistant
humans exhibit markedly increased cardiomyocellular
lipid content despite normal left ventricular ejection
fraction (the amount of blood pumped out of the ven-
tricle with each heart beat)
106,107
but impaired diastolic
function,
107
which suggests that lipid accumulation
in human cardio myocytes could be an early feature
of impaired cardiac function in the insulin-resistant
state. How ever, patients with left ventricular diastolic
dys function and decreased myocardial glucose uptake
showed increased myocardial FFA uptake and oxida-
tion.
108
No association was found between left ven-
tricular diastolic function and cardiac substrate or
phosphorus metabolism.
108
Furthermore,
31
P-MRS
studies provided evidence for altered energy balance,
as reflected by decreased phosphocreatine:ATP ratios in
patients with T1DM, T2DM, obesity and/or NAFLD.
109–
113
The ratio of phosphocreatine to ATP is an unspecific
in vivo marker of energy metabolism. Decreased capacity
of lipid oxidation, increased tri glyceride accumulation
and oxidative stress were found in heart biopsy samples
from humans with T2DM.
114
Ceramides and increased
ROS production during hyperglycemia stimulate mito-
chondrial fission (mitochondrial dynamics), which
induces ac tivation of cardiomyocyte apoptosis.
115
Brain
Although cerebral glucose uptake occurs indepen-
dent of insulin, insulin receptors are abundant in the
brain, including in regions that have been implicated
in appetite and satiety regulation, such as the hypo-
thalamus.
116
In mice, central insulin action regulates
glucose levels by lowering hepatic gluconeogenesis
and fat metabolism via stimulation of lipogenesis in
white adipose tissue (WAT).
117
In healthy humans, an
inverse correlation between body mass and cerebral
phosphocreatine and ATP content was found, suggest-
ing an interaction between cerebral energy supply and
body weight regu lation.
118
Of note, patients with T1DM
had a normal phospho creatine to ATP ratio, which did
not change during hypo glycemia.
119
In contrast to non-
diabetic humans, these patients maintain normal energy
metabo lism during hypoglycemia, which might reflect
an increased brain glucose uptake that contributes to
hypoglycemia-associated autonomic failure.
120
However,
no direct evidence exists for altered cerebral mito-
chondrial activity or content related to insulin re sistance
in patients with T2DM.
Adipose tissue
WAT not only serves as the most important energy store
of the body but also releases FFA and cytokines and con-
tributes to whole-body energy homoeostasis and insulin
resistance.
121
T2DM is associated with insulin resistance
in WAT that results in incomplete suppression of lipo-
lysis during hyperinsulinemia. Reduced mitochondrial
content has been reported in WAT biopsy samples from
individuals with T2DM.
122
Unlike WAT, brown adipose
tissue (BAT) cells oxidize lipids to generate heat as a
result of a transmembrane proton leak mediated by
uncoupling protein 1.
123
Previous studies showed that
BAT, detected by fluorodeoxyglucose PET, is present
in humans depending on outdoor temperature. The
size of the BAT depot is further determined by age, sex,
body mass and diabetes status.
124,125
Individuals with low
amounts of BAT were suggested to be prone to obesity,
insulin resistance and cardiovascular disease.
123
Of note,
cold exposure drastically accelerated triglyceride uptake
into BAT, thereby lowering plasma triglyceride levels.
126
Treating mitochondrial impairment
Holloszy demonstrated that exercise training increases
oxidative phosphorylation capacity in rat skeletal mus-
cle.
127
Regardless of the temporal sequence of deteri ora-
tion of mitochondrial function and insulin sensitivity
in T2DM, the question arises of whether these abnor-
malities are reversible upon lifestyle intervention or
pharmacologic al intervention (Table 1).
Lifestyle intervention
In obesity and T2DM, lifestyle intervention with
diet,
50,128,129
combined diet and exercise
50,130
or exercise
only
131,132
improved whole-body insulin sensitivity. Exer-
cise intervention increases muscular mitochondrial
con tent and thereby oxidative capacity (as assessed by
measuring the activities of enzymes of the tri carboxylic
acid cycle
130,132,133
), activity of the electron tran sport
chain
50,130,133
and β-oxidation.
132
In vivo sub maximal
ADP-stimulated oxidative phosphorylation and mito-
chondrial content were restored to values seen in
control indivi duals upon a 12-week exercise interven-
tion in patients with T2DM.
131,134
Dietary interventions
alone improved insulin sensitivity without parallel ame-
lioration of oxidative phosphorylation capacity,
50,129
or
even caused a reduction, possibly due to rapid weight
loss (Table 1).
128
Weight loss resulted in decreased
50
or
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unchanged
128
intramyocellular lipid content, which
indicates that increased energy demand but not reduced
levels of intramyocellular lipids or insulin resistance
trigger mitochondrial plasticity. Furthermore, these
results show that improvement of oxidative phosphory-
lation capacity is not a precondition for exercise-induced
amelioration of glucose disposal and thus argue against a
causal role of impaired mitochondrial activity or capacity
in the develop ment of insulin resistance. Of note, moder-
ate weight loss in patients with poorly controlled T2DM
mobilized a fairly small pool of hepatocellular lipids,
which reversed hepatic insulin resistance and normal-
ized rates of fasting glucose production, independent
of any changes in insulin-stimulated peripheral glucose
metabo lism and levels of intramyocellular lipids.
135

Moderate weight loss due to a very-low-fat, hypo caloric
diet improved basal and insulin-stimulated hepatic
glucose metabolism, which was associated with ~81%
reduction in hepatocellular lipid levels but no significant
change in intramyocellular lipid levels.
135
Inherited factors could contribute to the mitochon-
drial response to lifestyle intervention (mitochondrial
plasticity). A single nucleotide gene polymorphism
in the NDUFB6 gene (rs540467), which encodes part
of the respiratory chain complex I, was found to modu-
late the response of nonobese, insulin-resistant relatives
of patients with T2DM to a 1-week endurance exercise
training.
16
Carriers of the NDUFB6 single nucleotide
polymorphism rs540467 (G/G) exhibited both increased
insulin sensitivity and mitochondrial activity after
exercise training, which also correlated with maximal
whole-body oxygen consumption, compared with those
carrying the A allele. This finding suggests that long-
term mitochondrial plasticity is in part determined by
inherited factors.
Pharmacological intervention
An early study found that the mitochondrial uncoup-
ling agent dinitrophenol was effective for weight loss
but could not be used clinically due to the risk of liver
failure.
136
At present, few studies reported effects of drug
treatment on both mitochondrial function and insulin
resistance in humans (Table 2). The PPARγ ago nists pio-
glitazone and rosiglitazone improve insulin sensi tivity
by several mechanisms, including increased uptake and
metabolism of FFA, mainly in WAT but also in skeletal
muscle.
137
Furthermore, treatment with pioglita zone
for 12  weeks raised the expression of PGC1α, pos-
sibly via the above-mentioned inter-regulation with
PPARγ, which increased mitochondrial content and
lipid oxidation capacity in parallel with lipid storage in
WAT.
122
Improvement of insulin sensitivity by pioglita-
zone is accompanied by activation of AMPK and
increased mRNA levels of multiple genes involved in
Table 1 | Metabolic end points after lifestyle intervention in patients with T2DM and/or obesity
Reference Intervention n Insulin sensitivity
(% increase)
Mitochondrial function or content
Toledo
et al.
130
16–20 weeks weight
loss and moderate-
intensity exercise
10 (T2DM
plus obesity)
59* Increased mitochondrial content (mitochondrial size,
cardiolipin levels, CS and NADH oxidase activity)
Meex et al.
131
Phielix
et al.
134
12 weeks combined
endurance and
resistance exercise
18 (T2DM) 63* Increased submaximal ADP-stimulated oxidative
phosphorylation (phosphocreatine recovery
half-time), increased oxidative phosphorylation
capacity (maximal ADP-stimulated respiration,
FCCP-stimulated maximal oxidative capacity)
Hansen
et al.
133
6 months low
to moderate and
moderate to
high exercise
50 (T2DM) No change

Increased mitochondrial content (CS, COX,
β-HAD activity)
Unaltered SD activity
Bruce et al.
132
8 weeks exercise 7 (T2DM plus
obesity)
30* Increased mitochondrial content (CS and β-HAD
activity)
Nielsen
et al.
146
10 weeks exercise 12 (T2DM) 20* Increased mitochondrial content (electron
microscopy)
Toledo et al.
50
16 weeks diet or diet
plus exercise
16 (obesity) 38 (diet plus
exercise)
29 (diet only)*
Only in diet plus exercise group:
Increased mitochondrial content (mitochondrial
density and size, NADH oxidase activity, cardiolipin
levels, mtDNA copy number unchanged),
Rabol et al.
128
7.5 weeks diet 9 (obesity) 45

Reduced oxidative phosphorylation capacity
(maximal ADP-stimulated respiration), mitochondrial
content unchanged (mtDNA copy number)
Simoneau
et al.
129
4 months diet 32 (obesity) 15 (women)
30 (men)*
Mitochondrial content unchanged (CS activity)
in men and women
Possibly reduced mitochondrial content
(COX and HADH activity) in women
*Insulin sensitivity was determined as insulin-stimulated GDR.

Insulin sensitivity was calculated as the HOMA-IR-index. Abbreviations: β-HAD, β-hydroxyacyl-CoA
dehydrogenase; COX, cytochrome oxidase; CS, citrate synthase; FCCP, p-trifluoromethoxyphenylhydrazone; FFA, free fatty acids; FFM, fat-free mass; GDR, glucose
disposal rate; HADH, hydroxyacyl dehydrogenase; HOMA, homeostatic model assessment; IR, insulin resistance; mtDNA, mitochondrial DNA; SD, succinate
dehydrogenase; TG, triglycerides; T2DM, patients with type 2 diabetes mellitus.
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NATURE REVIEWS | ENDOCRINOLOGY ADVANCE ONLINE PUBLICATION | 9
mitochondrial function and fatty acid oxidation capac-
ity.
138
Amelioration of insulin action could underlie the
positive effect of thiazolidinediones on oxidative phos-
phorylation capacity, although both effects could occur
independently. Accordingly, rosiglitazone- mediated
improvement of glycemic control was a uniform finding
in one study, whereas induction of mitochondrial content
was only evident in a subgroup of indivi duals who pre-
sented with superior baseline aerobic capacity.
31
Those
participants might exhibit preserved mitochondrial plas-
ticity or a specific genetic susceptibility to the positive
treatment effects.
31
Of note, pioglitazone treatment not
only improved whole-body insulin sensitivity but also
reduced hepatocellular lipid content and improved some
histological abnormalities, such as hepatocyte necrosis
and inflammation, in humans with NASH.
139,140
Metformin, the most commonly used glucose-lowering
drug, is known to inhibit hepatic gluconeogenesis at least
partly by activating AMPK.
141
More precisely, metformin
inhibits respiratory chain complex I and thereby decreases
hepatic ATP levels and activates AMPK.
33
In vitro results
suggest that metformin also promotes mitochondrial bio-
genesis via the AMPK–PGC1α pathway.
142
But, to date,
controlled intervention studies testing this concept in
humans are lacking. The observation that combined tro-
glitazone and metformin treatment reduces ectopic lipid
deposition, possibly via enhanced lipid oxidation, might
support such a mechanism.
143
Resveratrol, an activator of SIRT1, delayed the develop-
ment of NAFL in rodents via activation of mitochondrial
biogenesis, in addition to antioxidant and inflamma-
tory activities.
144
New pharmacological approaches that
activate the SIRT1 pathway might, therefore, improve
ox idative capacity and glucose uptake.
145
Conclusions
Under certain insulin-resistant conditions, reduced
oxidative phosphorylation capacity, submaximal ADP-
stimulated oxidative phosphorylation and/or content of
mitochondria have been observed in insulin-responsive
tissues, such as skeletal muscle, liver and heart. Impaired
mitochondrial plasticity, that is, response to metabolic
regulation, seems to be characteristic of many insulin-
resistant patient groups. Whereas inherited mitochondrial
abnormalities have been associated with insulin resis-
tance, acquired metabolic alterations typical of insulin
resistance, such as hyperglycemia, hyper insulinemia and
hypertriglyceridemia, can also promote the production
of ROS and damage mitochondria in insulin-responsive
tissues. Of note, some successful interventions to decrease
insulin resistance also improve features of mitochondria,
making these organelles attractive candidates for novel
therapeutic approaches.
Table 2 | Effects of pharmacological interventions on mitochondrial function in T2DM
Reference Treatment Control n Insulin sensitivity Mitochondrial function or content
Bogacka et al.
122
Pioglitazone 30–45 mg
per day vs placebo,
3 months
Randomized
controlled
48 NA Adipose tissue: increased mitochondrial
content (mtDNA copy number), indirect
surrogate of increased oxidative
phosphorylation capacity for lipid
oxidation (expression of genes involved
in fatty acid oxidation)
Coletta et al.
138
Pioglitazone 45 mg per
day vs diet, 6 months
Randomized
controlled
26 Increased glucose
disposal (30%)
Skeletal muscle: indirect surrogate of
increased oxidative phosphorylation
capacity (expression of genes involved in
oxidative capacity and lipid oxidation)
Pagel-Langenickel
et al.
31
Rosiglitazone 4–8 mg
per day, 3 months
Prospective
open-labeled
23 Increased insulin
sensitivity
Skeletal muscle: no effect on
maximal whole-body oxygen
consumption or mitochondrial content
(mtDNA copy number)
Sreekumar
et al.
147
Insulin treatment for
10 days vs withdrawing
oral treatment for
2 weeks
Randomized
crossover
5 No increase of
insulin sensitivity
(minimal model)
Skeletal muscle: indirect surrogate of
increased oxidative phosphorylation
capacity (expression of genes involved in
oxidative capacity) and increased
mitochondrial content (expression of key
enzymes involved in energy metabolism)
Mathieu-Costello
et al.
143
Troglitazone or
metformin in addition
to glyburide for
3–4 months
Randomized
controlled
18 Increased insulin
sensitivity (glucose
disposal rate)
Skeletal muscle: mitochondrial content
unchanged (fber type, mitochondrial
volume density), decreased
intramyocellular lipid levels
Abbreviations: mtDNA, mitochondrial DNA; NA, not applicable; T2DM, type 2 diabetes mellitus; vs, versus.
Review criteria
The initial studies selected for this Review were from
the authors’ files or identified by a computer search
with PubMed of the scientific literature in English dated
from 2000 using the terms “mitochondrial function
type 2 diabetes”, “mitochondrial function muscle
insulin resistance”, “lifestyle intervention type 2
diabetes insulin resistance”, “intervention mitochondria
randomized”, “brain insulin mitochondria”, “heart
insulin resistance mitochondria”, “liver hepatic insulin
resistance mitochondria” and “brain insulin resistance
mitochondria”. Only randomized controlled trials were
included. Additional references were retrieved from
reviewing the references cited in these articles.
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10 | ADVANCE ONLINE PUBLICATION www.nature.com/nrendo
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Author contributions
All authors researched the data and contributed
equally to writing the article. J. Szendroedi and
M. Roden provided substantial contributions to
discussions of the content, and reviewed and/or
edited the manuscript before submission.
Supplementary information
Supplementary information is linked to the online
version of the paper at www.nature.com/nrendo
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