Mitochondrial reactive oxygen species are required for hypothalamic glucose sensing.

Abstract

The physiological signaling mechanisms that link glucose sensing to the electrical activity in metabolism-regulating hypothalamus are still controversial. Although ATP production was considered the main metabolic signal, recent studies show that the glucose-stimulated signaling in neurons is not totally dependent on this production. Here, we examined whether mitochondrial reactive oxygen species (mROS), which are physiologically generated depending on glucose metabolism, may act as physiological sensors to monitor the glucose-sensing response. Transient increase from 5 to 20 mmol/l glucose stimulates reactive oxygen species (ROS) generation on hypothalamic slices ex vivo, which is reversed by adding antioxidants, suggesting that hypothalamic cells generate ROS to rapidly increase glucose level. Furthermore, in vivo, data demonstrate that both the glucose-induced increased neuronal activity in arcuate nucleus and the subsequent nervous-mediated insulin release might be mimicked by the mitochondrial complex blockers antimycin and rotenone, which generate mROS. Adding antioxidants such as trolox and catalase or the uncoupler carbonyl cyanide m-chlorophenylhydrazone in order to lower mROS during glucose stimulation completely reverses both parameters. In conclusion, the results presented here clearly show that the brain glucose-sensing mechanism involved mROS signaling. We propose that this mROS production plays a key role in brain metabolic signaling.

Full text

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Original Article
Mitochondrial Reactive Oxygen Species Are Required for
Hypothalamic Glucose Sensing
Corinne Leloup,1Christophe Magnan,2Alexandre Benani,1Emilie Bonnet,1Thierry Alquier,1
Ge ´raldine Offer,1Audrey Carriere,1Alain Pe ´riquet,1Yvette Fernandez,1Alain Ktorza,2
Louis Casteilla,1and Luc Pe ´nicaud1
The physiological signaling mechanisms that link glucose
sensing to the electrical activity in metabolism-regulating
hypothalamus are still controversial. Although ATP pro-
duction was considered the main metabolic signal, recent
studies show that the glucose-stimulated signaling in neu-
rons is not totally dependent on this production. Here, we
examined whether mitochondrial reactive oxygen species
(mROS), which are physiologically generated depending on
glucose metabolism, may act as physiological sensors to
monitor the glucose-sensing response. Transient increase
from 5 to 20 mmol/l glucose stimulates reactive oxygen
species (ROS) generation on hypothalamic slices ex vivo,
which is reversed by adding antioxidants, suggesting that
hypothalamic cells generate ROS to rapidly increase glu-
cose level. Furthermore, in vivo, data demonstrate that
both the glucose-induced increased neuronal activity in
arcuate nucleus and the subsequent nervous-mediated in-
sulin release might be mimicked by the mitochondrial
complex blockers antimycin and rotenone, which generate
mROS. Adding antioxidants such as trolox and catalase or
the uncoupler carbonyl cyanide m-chlorophenylhydrazone
in order to lower mROS during glucose stimulation com-
pletely reverses both parameters. In conclusion, the re-
sults presented here clearly show that the brain glucose-
sensing mechanism involved mROS signaling. We propose
that this mROS production plays a key role in brain meta-
bolic signaling. Diabetes 55:2084–2090, 2006
E
been mainly characterized in two tissues, both in the
pancreas (at the ?-cell level) and in the brain (the so-called
“glucose-stimulated” or “glucose-inhibited” neurons) (1,2).
The cellular and molecular mechanisms underlying such
glucose responsiveness appear to share similarities in the
lucidating the signaling mechanisms by which
cells sense nutrient or metabolic status, a vital
process in energy homeostasis, is of prime im-
portance. Glucose-sensing mechanisms have
two glucose responsive cells (i.e., transport and phosphor-
ylation by GLUT2 and glucokinase, respectively) and the
consequent closure of ATP-sensitive K?channels (KATP
channels) and calcium influx (3–5). Although ATP produc-
tion used to be considered the main metabolic signal,
recent studies show that the glucose-excited signaling in
pancreatic ?-cells and neurons is not totally dependent on
this production. Within the hypothalamus, a previous work
showed that glucose challenge monitors KATPclosure
independently of ATP level (6), and more recent data
demonstrated that glucose-induced depolarization might
occur through a new KATPchannel-independent mecha-
nism, at least in some hypothalamic arcuate neurons (7).
These studies suggest that ATP-independent intracellular
signaling mechanisms leading to the stimulation of hypo-
thalamic neurons by glucose might be present.
Transient increase in glucose metabolism generates the
key substrates NADH and FADH2for the mitochondria,
and their use increases electron formation without modi-
fying other complex constraints along the respiratory
chain, ultimately leading to increased superoxide anion
production, also called mitochondrial reactive oxygen
species (mROS) (8). Although the link between substrates,
mROS production, and their deleterious effects has been
fully exemplified, for example, during prolonged hypergly-
cemia (9), emerging data now also demonstrate a physio-
logical role for mROS, including a role as fuel-sensor
components (gene transcription, direct enzyme activation,
or signal transduction activation) (10,11). Therefore, we
investigated whether hypothalamic glucose sensing in vivo
involves the mROS signaling pathway.
RESEARCH DESIGN AND METHODS
Male Wistar rats, weighing 250–300 g, were maintained in an animal quarter
with a constant temperature (21–23°C) and a 12:12-h light/dark cycle. Food
and water were available ad libitum. All rats were treated in accordance with
the European community guidelines, and our local institution approved the
experimentation. Pharmacological agents were all purchased from Sigma-
Aldrich, except carbonyl cyanide m-chlorophenylhydrazone (CCCP), which
was obtained from Calbiochem.
Ex vivo hypothalamic experiments and reactive oxygen species deter-
minations. Brains were rapidly removed from anesthetized animals, the
hypothalamic level was separated by transverse section, and hypothalami
were dissected on an ice-cooled glass plate and immersed in oxygenated (95%
O2/5% CO2) saline solution (300 mOsm, pH 7.4) containing 118 mmol/l NaCl, 25
mmol/l NaHCO3, 3 mmol/l KCl, 1 mmol/l MgCl2, 1.2 mmol/l NaH2PO4, 15
mmol/l sucrose, 1.5 CaCl2, 5 mmol/l HEPES, and 5 mmol/l glucose. After a
20-min recovery period, hypothalamus slices were rinsed two times and
incubated on required milieu, as indicated, for 5 min at 37°C in a final 1-ml
volume. At the end of the incubation, they were rapidly frozen and stored at
?80°C. Tissue treatment for reactive oxygen species (ROS) determination
was performed according to Szabados et al. (12), and hypothalamic pieces
From the1Laboratoire de Neurobiologie, Plasticite ´ Tissulaire et Me ´tabolisme,
Institut L. Bugnard, Toulouse, France; and the2Laboratory of the Physiopa-
thology of Nutrition, Universite Paris, Paris, France.
Address correspondence and reprint requests to Corinne Leloup, UMR
5018-CNRS UPS, Institut L. Bugnard, IFR31, BP 84432, 31 432 Toulouse cedex
4, France. E-mail: coleloup@toulouse.inserm.fr.
Received for publication 19 January 2006 and accepted in revised form 12
April 2006.
CCCP, carbonyl cyanide m-chlorophenylhydrazone; ETC, electron trans-
port chain; KATPchannel, ATP-sensitive K?channel; mROS, mitochondrial
reactive oxygen species; ROS, reactive oxygen species.
DOI: 10.2337/db06-0086
© 2006 by the American Diabetes Association.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked “advertisement” in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
2084DIABETES, VOL. 55, JULY 2006

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were loaded with the fluorescent probe dichlorodihydro-fluorescein diacetate
(H2DCFDA; Molecular Probes), 4 ?mol/l H2CFDA in 1 ml, and incubated for 30
min at 37°C. ROS measurements (on 200 ?l supernatant) were performed in a
Fluorescent Plate Reader (Perkin Elmer). Intensity of fluorescence was
expressed as arbitrary units per milligram of protein.
Intracarotid load of glucose or pharmacological agents toward the
brain. Tests were performed on anesthetized animals (pentobarbital, 50
mg/kg, intraperitoneal route [Roche et Dessinge, Vincennes, France]). A
polyethylene catheter was inserted into the carotid artery, pushed 5 mm in the
cranial direction, and secured in place with sutures. Glucose alone (9 mg/kg)
and/or pharmacological agents in 200 ?l physiological saline were injected
through the catheter over 1 min. The concentrations used for the agents were
1 mmol/l trolox, 4,000 units/ml catalase, 1 ?mol/l CCCP, 20 ?mol/l antimycin,
and 2 ?mol/l rotenone. Solutions were all adjusted for their osmolarity (300
mOsm). Some of the agents used required DMSO or ethanol vehicle for
dissolution. When this was the case, appropriate controls were performed. All
of the vehicles used have an effect on insulin secretion on their own (data not
shown). Plasma glucose and insulin were collected from the tail vessels
(before and 1, 3, 5, and 10 min after injection).
Glucose tolerance test. The glucose tolerance test was carried out accord-
ing to the method described previously (13). The glucose load (1 g/kg body wt)
was injected intraperitoneally.
Neuronal activity recordings in vivo. Each rat was initially prepared to
receive the intracarotid load of metabolites or pharmacological agents as
described above. They were then placed in a stereotaxic apparatus. A midline
incision was made. The skull was drilled at 3.1 mm posterior to the bregma on
the midline. The dura was removed to permit electrode insertion. Arcuate
nucleus stereotaxic coordinates were obtained according to Paxinos stereo-
taxic atlas (?3.1 mm posterior to bregma and ?8.7 mm under the brain
surface). Multiunit recordings were made of arcuate nucleus using monopo-
larly platinium electrode. Action potentials were displayed and saved on a
computer after initial amplification through a low-noise amplifier (BIO ampli-
fier; AD Instrument, Rabalot, France). Data were digitized with digitizer
PowerLab/4sp. Signals were monitored with the computer program Chart 4.
Multiunit recordings were made in response to a single injection (200 ?l) of
either saline or tested substances as described above. Baseline unit activity
was recorded for 5 min before infusion of a compound. Neuronal activity was
normalized to reduce variability resulting from differences in background
activity. Specifically, raw data were collected for each recording as the
number of discharges per second. Saline and catalase (results similar to that
of saline, not shown) recording sessions were used as control data and
reflected spontaneous activity.
Lipid peroxidation determinations. Hydroperoxides in biological samples
were estimated using the Lipid Hydroperoxide Assay kit (Cayman; Alexis
Biochemicals). This method is based on lipid extraction into chloroform,
eliminating any interference caused by hydrogen peroxide or endogenous
ferric ions.
Glucose and insulin concentrations. These were measured using a glucose
analyzer (OneTouch II; LifeScan) and a radioimmunoassay kit (Diasorin,
Antony, France), respectively.
ATP determination. ATP contents were determined using a bioluminescent
kit (Sigma-Aldrich).
Statistical analysis. Values are expressed as means ? SE. The statistical
significance of differences between groups was determined by Student’s t test
after testing the normality and homoscedasticity of the groups.
RESULTS
ROS production by hypothalamus. We first aimed to
determine whether transient exposure to increases in
glucose might trigger ROS production in hypothalamic
slices. To that end, hypothalamic slices were subjected to
a 20-mmol/l glucose challenge for 5 min. This stimulating
glucose concentration was chosen since arcuate glucose-
excited neurons responding to a 5- to 20-mmol/l step have
been characterized (7). Under this condition, glucose was
found to significantly increase (by 73%) ROS production as
measured by the oxidation of H2DCFDA in hypothalamic
extracts (Fig. 1B). Adding catalase (a biologically antiox-
idant active enzyme that converts H2O2into H2O and O2)
(14) to the stimulating 20-mmol/l glucose concentration
completely abolished the ROS increase from hypothalamic
slices (Fig. 1B). Consequently, these results indicate that
ROS are produced from hypothalamic cells under tran-
sient glucose stimulation. Hypothalamic slices were sec-
ondly exposed to specific mitochondrial inhibitors or
uncouplers in order to mimic, or reverse, the ROS produc-
tion triggered by glucose increase. First, rotenone, a
classical complex I blocker (Fig. 1A) leads to anion
superoxide production (18), which is reflected by mROS
increase (Fig. 1B). This production was completely abol-
ished when trolox, a vitamin E hydrophilic analog that
scavenges ROS (Fig. 1A), was added to the milieu (Fig.
1B). Then, glucose was coinjected with the mitochondrial
uncoupler CCCP, which acts as a channel through the
inner membrane to dissipate the H?gradient and which
accelerates respiration (Fig. 1A), leading to diminished
superoxide anion generation (10). A significant decrease
of mROS production, compared with glucose alone, was
observed (Fig. 1B). Finally, antimycin (a complex III
inhibitor [Fig. 1A] that blocks the electron transport chain
[ETC]) led to mROS production. An identical response to
that induced by glucose alone was observed (Fig. 1B).
Altogether, these results show that a transient glucose
increase elevates mROS production, and this is repro-
duced by manipulating the ETC.
ROS required for cerebral glucose sensing. To evalu-
ate whether ROS production in brain could play a role in
glucose-sensing signaling, we injected a glucose load (9
mg/kg, 30-s injection) toward the brain via the carotid
artery. With this protocol, glucose induced an activation of
hypothalamic arcuate cells, evidenced by immunodetec-
tion of the immediate early gene c-fos protein (15). This
glucose injection does not change the peripheral glucose
level but induces a peak of plasma insulin 1–3 min later
(16), which is shown to be due to a parasympathetic
activation (17). Animals were submitted to the intracarotid
glucose load, as described above, in combination, or not,
with antioxidant molecules (either catalase or trolox).
Control rats received a bolus of NaCl. The antioxidant
doses were carefully chosen to induce no effect them-
selves on the neuronal stimulation of insulin secretion as
well as on glycemia (data not shown). The direct effect of
catalase or trolox toward the pancreas has been excluded.
Thus, when they were infused to the brain during an
intraperitoneal glucose tolerance test (1 g/kg), identical
responses between control and treated rats were found
(results not shown). As previously demonstrated, the
intracarotid glucose load was associated with no change in
peripheral glycemia but stimulated the insulin secretion
(Fig. 2A and B). The coadministration of antioxidant
agents (catalase or trolox) prevented this secretion with-
out change of the glycemia (Fig. 2A and B). This demon-
strates that scavenging the ROS generation in brain
significantly disturbed the insulin secretion induced by the
cerebral glucose load.
Mitochondrial origin of ROS produced in cerebral
glucose sensing. To demonstrate that ROS production
involved in glucose sensing was of mitochondrial origin,
we conducted several complementary experiments in the
same model using specific mitochondrial inhibitors or an
uncoupler. First, glucose was coinjected with the mito-
chondrial uncoupler CCCP, which diminishes superoxide
anion generation (10) (Fig. 1A). A significant decrease of
the insulin peak, compared with glucose alone, was ob-
served (Fig. 2C). Then, antimycin (Fig. 1A), which leads
only to mROS production, triggered an identical response
to that observed with glucose alone (Fig. 2C). These
results strongly suggest that mROS are the effectors of the
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response. Finally, rotenone, which leads also to anion
superoxide production (18) (Fig. 1A), produced a peak
secretion similar to that measured with glucose, as shown
with antimycin (Fig. 2C). This insulin peak was reversed
when trolox was coinjected (Fig. 2C), reinforcing the
hypothesis that mROS act as signaling molecules in the
parasympathetic-controlled insulin secretion. Any modifi-
cation of the glycemia was present whatever the injection
performed (Fig. 2D). Using inhibitors of oxidative phos-
phorylation might block ATP generation. This was evalu-
FIG. 1. ROS are produced in hypothalamic slices after a glucose challenge. A: Effects of pharmacological agents used to modulate mROS
generation. Approximately 1–5% of the O2consumed by the ETC is incompletely metabolized and reduced into anion superoxide (O2
complexes I and III (a). O2
leading to enhanced mROS generation (b). Adding antioxidants scavenges the free radicals generation. The mitochondrial uncoupler CCCP
increases electron flux into the ETC, which are less “exposed” in time to oxygen (c). Antimycin inhibits complex III and electrons accumulate
and react with O2, leading to enhanced mROS generation (d). Glucose oxidation leads to increased NADH and FADH2formation which enter ETC
at complexes I and II, respectively (e). They then activate the ETC, resulting in ATP generation. It was recently proposed that this activation
increases mROS formation. Adding antioxidants scavenges free radical generation. B: Relative ROS fluorescence (DCFDA intensity, arbitrary
units, adjusted per milligram of protein) from hypothalamic pieces in 5 or 20 mmol/l glucose, or 20 mmol/l glucose ? catalase (4,000 units/ml) and
after treatment with rotenone (2 ?mol/l), rotenone ? trolox (1 mmol/l), 20 mmol/l glucose ? CCCP (1 ?mol/l), or antimycin (20 ?mol/l). In the
experiments with rotenone and antimycin, 5 mmol/l glucose was in the milieu. n ? 8–10 hypothalami per group, 3-min incubation. *P < 0.05 vs.
5 mmol/l glucose, ##P < 0.01 vs. 20 mmol/l glucose, ***P < 0.001 rotenone vs. 5 mmol/l glucose, and §§§P < 0.001 rotenone ? trolox vs. rotenone
alone.
?) at the
?is then diverted into other ROS as H2O2. Rotenone inhibits complex I and electrons accumulate and react with O2,
ROS SIGNALING AND CEREBRAL GLUCOSE SENSING
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FIG. 2. mROS are required for cerebral glucose sensing. A: Insulin secretion 1–3 min after the carotid load (200 ?l) toward the brain of saline, glucose (9 mg/kg), glucose coinjected
with trolox (10?3mol/l), or glucose coinjected with catalase (4,000 units). Osmolarity has been adjusted (300 mosM). n ? 6–9 male Wistar rats per group. **P < 0.01 vs. NaCl, ##P <
0.01 vs. glucose alone, and #P < 0.05 vs. glucose alone. B: Respective glycemia for each group; any modification of the glycemia was present whatever the brain treatment. C: Insulin
secretion as described in A, except the agents injected were glucose (9 mg/kg), glucose coinjected with CCCP (10?6mol/l), antimycin alone (20 ?mol/l), rotenone alone (2 ?mol/l),
and rotenone coinjected with trolox (10?3mol/l). n ? 6–9 male Wistar rats per group. #P < 0.05 vs. glucose and §§§P < 0.001 vs. rotenone. D: Respective glycemia for each group
described in C; any modification of the glycemia was present whatever the brain treatment.
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ated for rats injected with the mitochondrial complexes
blockers, either rotenone or antimycine. ATP content was
measured and compared with NaCl controls and glucose-
injected rats. A significant difference was detected be-
tween hypothalamic ATP concentrations in NaCl and
glucose-injected rats when compared with rotenone- and
antimycin-injected rats (n ? 6–8 for each group, data
expressed as 10?10mol/l ATP/?g cytosolic protein: 9.15 ?
1.64 in NaCl, 8.39 ? 1.7 in glucose-injected rats, and 2.83 ?
1.22 in rotenone- and 2.68 ? 0.97 in antimycin-injected
rats). These data confirm the inhibitory effect of rotenone
and antimycine on oxidative phosphorylation and ATP
synthesis.
Taken together, these coherent and convergent results
demonstrate that mROS production is required for brain
glucose sensing, and in counterpart, that mROS mimic
brain glucose sensing.
Stimulation of hypothalamic arcuate neuronal activ-
ity by mROS produced in cerebral glucose sensing. As
the arcuate nucleus was described as being critical for
FIG. 3. mROS are required for glucose-stimulated arcuate neuronal activity. A: Extracellular recordings of arcuate nucleus neuronal activity in
male Wistar rats after the carotid load of saline (NaCl), glucose, glucose ? catalase, rotenone, and rotenone ? catalase, from left to right,
respectively. B: Recordings expressed as electrical events per minute. After injection, the recordings were 10 min in duration. Concentrations of
the different solutions injected in the brain through the carotid artery were the same as described in Fig. 2. Data were analyzed by Chart 4
software; n ? 6–8 each case. ***P < 0.001 vs. NaCl, #P < 0.05 vs. glucose, and §§§P < 0.001 vs. rotenone.
ROS SIGNALING AND CEREBRAL GLUCOSE SENSING
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control of insulin secretion by the central nervous system,
we recorded in vivo extracellular activity in this hypotha-
lamic nucleus in some of the key demonstrating experi-
ments. As expected, an intracarotid glucose load triggered
a significant increase in electrical events. Strikingly, the
coinjection of the antioxidant enzyme catalase completely
prevented this response (Fig. 3A and B). Rotenone injec-
tion, which was previously shown to mimic glucose re-
sponse, reproduced a neuronal activation similar to that
observed with glucose (Fig. 3A and B). As seen with
glucose coinjected with catalase, the coinjection of rote-
none with catalase also prevented the neuronal activation
(Fig. 3A and B). Altogether, these data demonstrate that
mROS behave as pivotal signals in brain glucose sensing
by controlling the neuronal activity of arcuate nucleus.
These effects were reversible because extracellular re-
cordings showed a return to basal activity after the stim-
ulation. These activities returned to their minima between
3 (glucose) and 5 (rotenone) min after the injections.
Moreover, the stimulating effect could be reproduced in a
repeated manner (data not shown). These results rein-
force our proposition of a specific effect of rotenone on
transient mROS formation, since the concentration used
for this pharmacological agent allows a reversible effect,
as measured with electrical activity.
Deleterious oxidative stress and physiological re-
sponses. Regarding the toxicity related to the pharmaco-
logical agents used, they have (to our knowledge) never
been used via the carotid route in order to explore brain
function. Nanomolar doses were ineffective in our in vivo
model. Since the load injected into the carotid was first
diluted in the plasma before reaching the brain, it is
reasonable to propose that the effective amount acting at
the target site was far from the initial concentration. To
rule out a putative damaging and nonspecific oxidative
stress of the different mitochondrial agents tested, lipid
peroxidation was evaluated. Hydroperoxide measure-
ments were conducted on hypothalami of NaCl-, glucose-,
and pharmacological-injected rats, dissected 5 min after
injection, which is the maximal time for both the increased
electrical activity and the peak of plasma insulin. No
difference in any of these groups compared with NaCl or
with glucose was observed (Fig. 4). This excludes a
nonspecific toxic effect.
DISCUSSION
Although it is now accepted that small fluctuations in the
steady-state concentration of mROS may play a role in
intracellular signaling (19,20), their pivotal role in brain
glucose sensing had not been considered before. This
study is the first to provide evidence that mROS play an
essential role in glucose sensing in hypothalamus, one
of the main critical systems in maintaining glucose
homeostasis.
Most of the metabolic substrates, and glucose in partic-
ular, lead to the formation of NADH or FADH2, which are
donors of the respiratory chain, activating the ETC and
then ATP generation. It was recently proposed that these
reduced equivalents predisposed to increased mROS for-
mation through a direct effect on the ETC in a number of
cell types (9,21). Taking into account the constraints of the
respiratory chain, the transient increase and oxidation of
these reduced equivalents lead to a small excess of elec-
trons that react with oxygen, increasing superoxide for-
mation. In this case, mROS increase is dependent on
metabolic activation (11). First, we demonstrate that tran-
sient glucose challenge at the hypothalamic level has the
ability to trigger an increased ROS production, reversed
with antioxidant treatment. Second, we demonstrate that
both the neuronal arcuate activation and the consequent
insulin secretion due to neuronal stimulation are moni-
tored by mROS. In both cases, quenching the ROS forma-
tion with antioxidants prevented the glucose response.
Third, in contrast, mROS generation by the inhibitors
antimycin and rotenone mimics the glucose effect. In each
case, a toxic effect of the pharmacological agents was
carefully examined and can be excluded. Indeed, no
excessive hydroperoxidation was observed, and the ef-
fects were reversible. The fact that glucose, as well as an
inhibitor of glucose metabolism, produce the same effect
on hypothalamic neurons may first appear as a paradox.
This might be explained if NADH, not ATP, constitutes the
key metabolite mediating the stimulatory effects of 20 vs.
5 mmol/l glucose on hypothalamic neurons, as already
suggested (22). Indeed, because complex I is the main
source of mROS and the production of mROS increases
when NADH is in its more reduced state, blocking com-
plex I also enhances the production of mROS and mimics
the effects of glucose. Other studies have also implicated
NADH in glucose sensing in the pancreas (23,24). Thus,
the mROS mechanism we describe here is consistent with
an NADH mechanism mediating glucose signaling, as
suggested earlier (22). Moreover, it is noteworthy that the
obligatory generation of mROS is dependent on the proton
gradient and the electrons fluxes. These fluxes result from
the balance between the electron supply to the respiratory
chain and the constraints on the ETC exerted by the
control of the dissipation of protons gradient via the ATP
synthesis or uncoupling mechanism (25). Thus, mROS
appear as a signal integrating both the NADH redox state
and the phosphate potential, thus reflecting the whole
mitochondrial metabolism and then the cell metabolism.
FIG. 4. Hydroperoxide measurements on hypo-
thalami of pharmacological-injected rats. To ex-
clude any damaging and nonspecific oxidative
stress of the different mitochondrial agents
tested, lipid peroxidation was evaluated. Hy-
droperoxides were evaluated on hypothalami of
rats injected with NaCl, glucose (9 mg/kg), CCCP
(10?6mol/l), antimycine (20 ?mol/l), or rotenone
(2 ?mol/l) (as described in Fig. 2A and C). Hypo-
thalami are dissected 5 min after injection, which
is the maximal time for both the increased elec-
trical activity and the peak of plasma insulin. No
difference of any of these groups compared with
NaCl or glucose was observed.
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Altogether, these complementary and convergent data
clearly support a critical role for mROS as part of the
hypothalamic glucose-sensing mechanism. Such a role is
consistent with the emerging general view of mROS as a
sensor of the nutrient environment (11).
Although the mechanisms involved in glucose sensing
have been extensively studied, some of the issues are still
controversial. Neurons that respond to glucose challenge,
named glucose-stimulated neurons, possess an KATPchan-
nel that inactivates as the ATP level increases during
glucose metabolism (26), as already described in ?-cells.
More recently, the role of intracellular ATP in the closure
of KATPhas been discussed, since no detectable increase
in cytosolic ATP was present (6). Our results suggest that
hypothalamic KATPshould be sensitive to mROS produc-
tion, which is consistent with a recent study reporting that
KATPchannels control transmitter release in dorsal stria-
tum through a H2O2-dependent mechanism (27) or that the
signaling involving mROS generation is partially, or totally,
KATPindependent, as suggested from recent data (7).
Thus, further speculation should be made since a variety
of ROS-sensitive and nonselective cationic channels have
been already described (28,29).
Whether a similar mechanism might be extended to
?-cells is of considerable interest. The implication of
NADH in glucose sensing in the pancreas has been fully
exemplified (23,24), and there are some strong arguments
to sustain that glucose-induced insulin secretion and
mROS production may be a tightly linked process (21).
Our finding opens new perspectives for investigating the
putative involvement of mROS in metabolic controls,
apart from their already well-known effect as oxidative
stressors.
ACKNOWLEDGMENTS
This research was supported by grants from the Centre
National de la Recherche Scientifique and the Agence
Nationale Pour la Recherche (05-PNRA 004-01).
We are grateful to Michael Iglesias for help in translation
of the manuscript.
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