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
Figure1 | 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 exvivo 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.
5962
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 kinaseC (PKC) isoforms, fol-
lowed by serine phosphorylation of insulin receptor
substrate1 (IRS1), and thereby cause impaired insulin
signaling.
6,6568
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.
7476
Rising FFA levels activate PPAR,
which increases PGC1 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, PGC1, mitochondrial content
and expression of fatty acid translocase (also known as
platelet glycoprotein4 or CD36) are related to exvivo
capacity of lipid oxidation. In humans with obesity,
these correlations are diminished and PGC1 levels are
reduced. One explanation for this observation could be
that prolonged elevation of plasma FFA levels impairs
the lipid-induced PPARPGC1 interaction that regulates
fatty acid oxidation in insulin-sensitive humans.
80
Box3 | Assessment of mitochondrial function exvivo
Mitochondrial oxidative capacity exvivo 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 Oroborosoxygraph
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 invivo. The Seahorseextracellular 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 invivo, as it
allows measurements of mitochondrial activity in intact attached cells.
158
Enzyme
activities can be determined by spectrophotometry.
8
Box2 | Assessment of mitochondrial function invivo
Phosphocreatine and ATP resynthesis
Submaximal ADP-stimulated oxidative phosphorylation is assessed from
resynthesis of energy-rich phosphates, continuously measured with invivo
31
Pmagnetic 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 perse 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.
8284
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 (Figure2). Whether improved coupling of
-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 1090% after 12 h and oxi-
dative capacity by about 3242% 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
PGCs
Ceramides PPARs
Pyruvate
CPT1
LCA-CoA
ROS
Plasticity
TCA cycle
?
?
?
?
?
?
?
-oxidation
Figure2 | 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
), which leads to inhibitory serine phosphorylation of IRS. Glucose transport and phosphorylation is reduced.
Stimulation of PGC1 and PGC1, the main regulators of mitochondrial biogenesis and fatty acid oxidation, is induced by
insulin in skeletal muscle. FFA activate PPAR and PPAR. 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 PPARPGC1
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
, protein kinase C
100
80
60
40
20
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
Figure3 | 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 invivo under fasting conditions and hyperinsulinemic conditions in
nondiabetic participants of different ages: young
46
and middle-aged (~57years)
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
meanSEM. *P <0.05 fasting versus insulin-stimulated. Abbreviations: MELAS,
mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes; T1DM,
type1 diabetes mellitus; T2DM, type2 diabetes mellitus.
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NATURE REVIEWS | ENDOCRINOLOGY ADVANCE ONLINE PUBLICATION | 7
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
invivo 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 withT2DM.
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 protein1.
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 (Table1).
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 -oxidation.
132
Invivo 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 (Table1).
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 complexI, 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 (Table2). The PPAR 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 PGC1, pos-
sibly via the above-mentioned inter-regulation with
PPAR, 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
Table1 | Metabolic end points after lifestyle intervention in patients with T2DM and/or obesity
Reference Intervention n Insulin sensitivity
(% increase)
Mitochondrial function or content
Toledo
etal.
130
1620weeks weight
loss and moderate-
intensity exercise
10 (T2DM
plus obesity)
59* Increased mitochondrial content (mitochondrial size,
cardiolipin levels, CS and NADH oxidase activity)
Meex etal.
131
Phielix
etal.
134
12weeks 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
etal.
133
6months low
to moderate and
moderate to
high exercise
50 (T2DM) No change