AIMS/HYPOTHESIS: Mitochondrial dysfunction has been postulated to underlie muscular fat accumulation, leading to muscular insulin sensitivity and ultimately type 2 diabetes mellitus. Here we re-interpret previously published data on [(13)C]acetate recovery in breath gas obtained during exercise in type 2 diabetic patients and control individuals. METHODS: When infusing [(13)C]palmitate to estimate fat oxidation, part of the label is lost in exchange reactions of the tricarboxylic acid (TCA) cycle. To correct for this loss of label, an acetate recovery factor (ARF) has previously been used, assuming that 100% of the exogenously provided acetate will enter the TCA cycle. The recovery of acetate in breath gas depends on the TCA cycle activity, hence providing an indirect measure of the latter and a marker of mitochondrial function. RESULTS: Re-evaluation of the available literature reveals that the ARF during exercise is highest in lean, healthy individuals, followed by obese individuals and type 2 diabetic patients. CONCLUSIONS/INTERPRETATION: Revisiting previously published findings on the ARF during exercise in type 2 diabetic patients reveals a reduction in muscular TCA cycle flux, reflecting mitochondrial dysfunction, in these patients. How mitochondrial dysfunction is related to type 2 diabetes mellitus-cause or consequence-requires further study

DE Befroy

G Perseghin

KF Petersen

KS Nair

LB Borghouts

LS Sidossis

M Mensink

M. K. C. Hesselink

P Schrauwen

P. Schrauwen

R Boushel

VB Schrauwen-Hinderling

YW Asmann

English

PubMed

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Springer - Publisher Connector

Reduced tricarboxylic acid cycle flux in type 2 diabetes mellitus?

Schrauwen, P.

Hesselink, M.K.

NARCIS 

SHORT COMMUNICATION
Reduced tricarboxylic acid cycle flux in type 2
diabetes mellitus?
P. Schrauwen & M. K. C. Hesselink
Received: 8 May 2008 /Accepted: 14 May 2008 /Published online: 28 June 2008
# The Author(s) 2008
Abstract
Aims/hypothesis Mitochondrial dysfunction has been pos-
tulated to underlie muscular fat accumulation, leading to
muscular insulin sensitivity and ultimately type 2 diabetes
mellitus. Here we re-interpret previously published data on
[13C]acetate recovery in breath gas obtained during exercise
in type 2 diabetic patients and control individuals.
Methods When infusing [13C]palmitate to estimate fat
oxidation, part of the label is lost in exchange reactions of
the tricarboxylic acid (TCA) cycle. To correct for this loss of
label, an acetate recovery factor (ARF) has previously been
used, assuming that 100% of the exogenously provided
acetate will enter the TCA cycle. The recovery of acetate in
breath gas depends on the TCA cycle activity, hence
providing an indirect measure of the latter and a marker of
mitochondrial function.
Results Re-evaluation of the available literature reveals that
the ARF during exercise is highest in lean, healthy
individuals, followed by obese individuals and type 2
diabetic patients.
Conclusions/interpretation Revisiting previously published
findings on the ARF during exercise in type 2 diabetic
patients reveals a reduction in muscular TCA cycle flux,
reflecting mitochondrial dysfunction, in these patients. How
mitochondrial dysfunction is related to type 2 diabetes
mellitus—cause or consequence—requires further study.
Keywords Human .Mitochondria . Mitochondrial function .
Muscle . Stable isotopes . Type 2 diabetesmellitus
Abbreviations
ARF acetate recovery factor
acetyl-CoA acetyl coenzyme A
IGT impaired glucose tolerance
TCA tricarboxylic acid
V
:
O2max maximal aerobic capacity
The prevalence of type 2 diabetes mellitus is increasing
rapidly and is affecting the health of millions of humans
now, and will do so in the near future. Among the factors
that are responsible for the increasing prevalence of this
disease are obesity, consumption of energy-dense diets and
low levels of physical activity. Together, these factors
contribute to fat accumulation in non-adipose tissues, like
heart, liver and skeletal muscle. Indeed, the accumulation of
fat in skeletal muscle is an early hallmark of the
development of type 2 diabetes mellitus, and high muscular
fat levels can already be detected in the prediabetic state
[1]. In the last 5 years, a reduced muscular oxidative
capacity, referred to as mitochondrial dysfunction, has been
postulated to contribute to fat accretion in skeletal muscle.
Thus, reduced in vivo mitochondrial function under resting
conditions as well as during post-exercise recovery has
been observed in type 2 diabetic patients [2] and in first-
degree relatives of type 2 diabetic patients [3], and a
reduced mitochondrial tricarboxylic acid (TCA) cycle flux
was shown to underlie the reduced mitochondrial oxidative
Diabetologia (2008) 51:1694–1697
DOI 10.1007/s00125-008-1069-x
P. Schrauwen (*)
Nutrition and Toxicology Research
Institute Maastricht (NUTRIM),
Department of Human Biology, Maastricht University,
PO Box 616, 6200 MD Maastricht, the Netherlands
e-mail: p.schrauwen@hb.unimaas.nl
M. K. C. Hesselink
Department of Human Movement Sciences,
Maastricht University,
Maastricht, the Netherlands
capacity in insulin-resistant offspring of type 2 diabetic
patients [4]. But other studies have reported no defects in
mitochondrial function in diabetic patients [5, 6]. The
reason for this discrepancy in findings is so far unclear, but
could include differences in individual characteristics, the
severity and duration of the patients’ type 2 diabetes, the
type and duration of the glucose-lowering medication used,
and their habitual physical fitness levels.
Therefore, the current debate on mitochondrial dysfunction
as a contributor to the pathogenesis of type 2 diabetes is
ongoing and has called for more studies towards mitochon-
drial function in patients with type 2 diabetes. These studies
may, however, be biased by prior knowledge of reported
defects inmitochondrial function.We here re-evaluate existing
data that was gathered for other goals and from another
perspective and in a period when mitochondrial dysfunction
had not yet been proposed to be key in the development of
type 2 diabetes. This unbiased approach has led us to conclude
that a reduced TCA cycle flux is part of the defect in
mitochondrial dysfunction observed in type 2 diabetes.
In 1995, the acetate recovery factor (ARF) was intro-
duced in the field of stable isotope tracer methodology, to
correct tracer estimates of fat oxidation for the loss of label
in exchange reactions of the TCA cycle [7]. Thus, when
[13C]palmitate is used to determine plasma-derived palmi-
tate oxidation, part of the infused label is lost in exchange
reactions of the TCA cycle (Fig. 1). To correct for this loss
of label, the appearance of the 13C label of [13C]acetate into
13CO2 can be used. Acetate is immediately converted into
acetyl coenzyme A (acetyl-CoA), and it is assumed that
almost 100% of the labelled [13C]acetate will enter the TCA
cycle. Under lipogenic conditions, though, it cannot be
excluded that some of the infused acetate is used for de
novo lipogenesis. However, under normal resting condi-
tions only a fraction of the infused [13C]acetate is recovered
as 13CO2 in breath gas, indicating that indeed most of the
13C label is lost in the TCA cycle. Interestingly, the loss of
label in the TCA cycle depends on the flux of the TCA
cycle. Thus, the recovery of acetate as 13CO2 in breath gas
is only about 20% during rest, but increases up to about
100% during high-intensity exercise, when skeletal muscle
mitochondrial TCA activity is accelerated and de novo
lipogenesis is negligible. Indeed, we previously showed
that the recovery of acetate during exercise is highly
dependent on metabolic rate [8]. Especially during exercise
and when measured under standardised conditions, the
ARF can provide a good, albeit indirect, indication of TCA
cycle flux in skeletal muscle mitochondria.
With the above in mind, it is very interesting to note that
the ARF has been previously determined under stand-
ardised conditions in (pre)-diabetic patients compared with
control individuals, albeit not with the original aim of
using this factor as an indirect measure of mitochondrial
TCA cycle flux. Thus, Borghouts et al. [9] reported that
male, overweight, type 2 diabetic patients (see Table 1 for
individual characteristics) had a 23% lower recovery of
acetate during exercise (performed at about 40–45% of
maximal aerobic capacity [V
:
O2max]) when compared with
body weight-matched healthy controls (ARF: 54.5±0.06% vs
70.7±0.07%, p<0.05). Because V
:
O2max was different be-
tween controls and diabetic patients in this study, the latter
group exercised at the same relative but a somewhat lower
absolute workload, which may explain part of the difference
in ARF. However, Mensink et al. [10] measured acetate
recovery during exercise (at 55–58% V
:
O2max) in male obese
type 2 diabetic patients, individuals with an impaired glucose
tolerance (IGT) and healthy controls, all matched for per-
centage body fat and V
:
O2max (Table 1). Again, acetate
[13C]Acetyl-CoA
[13C]Citrate
α-[13C]Ketoglutarate [13C]Glutamate/glutamine
[13C]Succinate
[13C]Oxaloacetate[13C]P-enolpyruvate
[13C]Pyruvate
[13C]Glucose
[13C]Lactate
13CO2
[13C]Aspartate
[13C]Triacylglycerol[13C]Acetate [13C]Palmitate
13CO2
13CO2
TCA
cycle
flux
Fig. 1 Metabolic routes of
[13C]acetate. The 13C label can
be lost in exchange reactions of
the TCA cycle, which will re-
duce the recovery of the label in
13CO2 in breath gas. The loss of
13C label in exchange reactions
depends on the rate of the TCA
cycle flux
Diabetologia (2008) 51:1694–1697 1695
recovery tended to be lower in type 2 diabetic patients and
IGT individuals compared with healthy controls (about 63, 63
and 71%, in type 2 diabetes mellitus, IGT and controls,
respectively, n=7, p=0.07). When jointly examining ARFs
obtained during exercise under standardised conditions in our
laboratory, at on average 50% of V
:
O2max, we previously
demonstrated that the ARF was highest in lean, healthy
individuals (78.5±2.1%, n=21), followed by obese individu-
als (69.9±1.7%, n=31) and type 2 diabetic patients (59.2±
2.1%, n=17) [8]. Remarkably, even after correction for factors
related to mitochondrial function, which actually may differ
between diabetic patients and control individuals, like RQ,
obesity (as defined by body fat%) and metabolic rate
(workload), the ARF tended to be lower in type 2 diabetic
patients compared with obese and lean controls [8]. This
further strengthens the unbiased observation of a reduced
TCA cycle flux in type 2 diabetic patients.
Although our re-evaluation of previously published data
points towards a reduced TCA cycle flux in type 2 diabetic
patients, it cannot answer the question of whether mito-
chondrial dysfunction is the cause or consequence of type 2
diabetes mellitus. The finding that the ARF was reduced
both in type 2 diabetic patients and in prediabetic
individuals with IGT—comparable with the findings of
reduced in vivo mitochondrial function in first-degree
relatives [4]—could be interpreted as evidence for a causal
relationship between mitochondrial dysfunction and the
development of diabetes. However, in these studies, (pre)
diabetic individuals are characterised by insulin resistance
and associated high insulin levels. In that respect, a recent
study showed that in type 2 diabetic patients, high insulin
levels reduced the expression of peroxisome proliferator-
activated receptor-gamma coactivator-1α, citrate synthase
and cytochrome oxidase subunit 1 and blunted the insulin-
stimulated increase in mitochondrial ATP production [11].
In addition, we observed in patients with type 2 diabetes
that mitochondrial function, measured as post-exercise
phosphocreatine resynthesis rate, correlated inversely with
fasting blood glucose and HbA1c levels [2]. Therefore,
mitochondrial dysfunction could also very well reflect a
consequence of the insulin-resistant state. Alternatively, a
reduced TCA cycle flux in type 2 diabetic patients may be
the consequence of a more sedentary, inactive lifestyle. So
far, most studies that report mitochondrial dysfunction in
type 2 diabetes have not carefully matched for habitual
levels of physical (in)activity. It is well known that physical
endurance training is one of the most powerful strategies to
prevent and treat type 2 diabetes mellitus and it is not
unlikely that training can do so by improving mitochondrial
function.
In conclusion, revisiting previously published findings
on the ARF during exercise in type 2 diabetic patients
reveals a reduction in muscular TCA cycle flux in these
patients. Why ARF is reduced in type 2 diabetic patients
was previously unexplained, but now provides an unbiased
additional indication that indeed mitochondrial function is
reduced in type 2 diabetic patients. How mitochondrial
dysfunction is related to type 2 diabetes mellitus—cause or
consequence—requires further study.
Acknowledgements The research of P. Schrauwen was made
possible by a fellowship of the Royal Netherlands Academy of Arts
and Sciences. The research of M. K. C. Hesselink was made possible
by a Vidi grant for innovative research (no. 917.66.359).
Duality of interest The authors declare that there is no duality of
interest associated with this manuscript.
Table 1 Characteristics of the individuals
Controls Type 2 diabetes IGT
Borghouts et al. [9]
n 8 8 –
Age (years) 47±5 58±5 –
Body weight (kg) 86.3±9.5 86.4±14.1 –
Body fat (%) 29.2±5.3 26.5±8.4 –
V
:
O2max (ml kg
−1min−1) 36.2±2.0 25.8±3.3 –
Exercise intensity (% of V
:
O2max) 44±3 41±6 –
Mensink et al. [10]
n 7 7 7
Age (years) 45.1±4.5 51.3±9.0 58.3±6.3
Body weight (kg) 100.8±9.8 93.0±8.7 110.8±18.8
Body fat (%) 35.0±3.2 36.6±3.4 33.0±3.2
V
:
O2max (ml [kg FFM]
−1 min−1) 41.5±3.2 36.2±7.7 39.6±2.4
Exercise intensity (% of V
:
O2max) 57.8±9.5 55.6±5.6 54.9±12.2
Values are means±SD
FFM, fat-free mass
1696 Diabetologia (2008) 51:1694–1697
Open Access This article is distributed under the terms of the
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A new correction factor for use in tracer estimations of plasma fatty acid oxidation.

Asmann YW et al (2008) Asian Indians have enhanced skeletal muscle mitochondrial capacity to produce ATP in association with severe insulin resistance.

De Cobelli F et al

Determinants of the acetate recovery factor: implications for estimation of [13C]substrate oxidation. Clin Sci (Lond)

Hesselink MK et al (2007) Impaired in vivo mitochondrial function but similar intramyocellular lipid content in patients with type 2 diabetes mellitus and BMI-matched control subjects.

Impaired mitochondrial activity in the insulin-resistant offspring of patients with type 2 diabetes.

Impaired mitochondrial substrate oxidation in muscle of insulin-resistant offspring of type 2 diabetic patients.

Patients with type 2 diabetes have normal mitochondrial function in skeletal muscle.

Plasma free fatty acid uptake and oxidation are already diminished in subjects at high risk for developing type 2 diabetes.

Skeletal muscle mitochondrial functions, mitochondrial DNA copy numbers, and gene transcript profiles in type 2 diabetic and nondiabetic subjects at equal levels of low or high insulin and euglycemia.

Substrate utilization in non-obese Type II diabetic patients at rest and during exercise. Clin Sci (Lond)

Reduced tricarboxylic acid cycle flux in type 2 diabetes mellitus?

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