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Hypoglycemia-Sensing Neurons of the Ventromedial
Hypothalamus Require AMPK-Induced Txn2 Expression
but Are Dispensable for Physiological Counterregulation
Simon Quenneville,
1
Gwenaël Labouèbe,
1
Davide Basco,
1
Salima Metref,
1
Benoit Viollet,
2
Marc Foretz,
2
and
Bernard Thorens
1
Diabetes 2020;69:2253–2266 | https://doi.org/10.2337/db20-0577
The ventromedial nucleus of the hypothalamus (VMN) is
involved in the counterregulatory response to hypogly-
cemia. VMN neurons activated by hypoglycemia (glucose-
inhibited [GI] neurons) have been assumed to play
a critical although untested role in this response. Here,
we show that expression of a dominant negative form of
AMPK or inactivation of AMPK a1and a2subunit genes
in Sf1 neurons of the VMN selectively suppressed GI
neuron activity. We found that Txn2, encoding a mito-
chondrial redox enzyme, was strongly downregulated in
the absence of AMPK activity and that reexpression of
Txn2 in Sf1 neurons restored GI neuron activity. In cell
lines, Txn2 was required to limit glucopenia-induced
reactive oxygen species production. In physiological
studies, absence of GI neuron activity after AMPK sup-
pression in the VMN had no impact on the counterregu-
latory hormone response to hypoglycemia or on feeding.
Thus, AMPK is required for GI neuron activity by control-
ling the expression of the antioxidant enzyme Txn2. How-
ever, the glucose-sensing capacity of VMN GI neurons is
not required for the normal counterregulatory response to
hypoglycemia. Instead, it may represent a fail-safe system
in case of impaired hypoglycemia sensing by peripherally
located glucose detection systems that are connected to
the VMN.
The brain requires a continuous supply of glucose as
a source of metabolic energy. This imposes that blood
glucose concentrations never fall below the euglycemic
level of ;5 mmol/L. In healthy individuals, hypoglycemia
does not usually occur because multiple counterregulatory
mechanisms rapidly induce the secretion of hormones—
glucagon, epinephrine, glucocorticoids, growth hormone—
which together induce hepatic glucose production, suppress
insulin secretion, and reduce insulin action on peripheral
tissues to restore normoglycemia and glucose availability to
the brain (1). However, in insulin-treated patients with type
1 or type 2 diabetes, iatrogenic hypoglycemia is frequently
observed, and antecedent hypoglycemia increases the risk to
develop subsequent hypoglycemicepisodesofhigherseverity,
due to progressive impairments in counterregulatory hor-
mone secretion (2).
The counterregulatory hormone response to hypogly-
cemia is triggered in large part by glucose-sensing cells of
the nervous system (3). These cells regulate the activity of
the autonomous nervous system, which is involved in the
control of insulin, glucagon, and epinephrine secretion and
of glucose production by the liver. They also activate the
hypothalamo-pituitary-adrenal axis, which controls gluco-
corticoid secretion. Glucose-responsive neurons are pres-
ent in many brain regions, including in the hypothalamus,
the brainstem, and the thalamus (3–5). These neurons fall
into two categories, glucose-excited (GE) neurons, whose
firing rate increases in response to a rise in extracellular
glucose concentration, and glucose-inhibited (GI) neurons,
which are activated by hypoglycemia (6) and are thought to
trigger the counterregulatory response to hypoglycemia.
The ventromedial nucleus (VMN) of the hypothalamus
plays an important role in this counterregulatory response,
as evidenced by the observation that secretion of glu-
cagoninresponsetoinsulin-induced hypoglycemia can
be blocked by intra-VMN injection of glucose and that
1
Center for Integrative Genomics, University of Lausanne, Lausanne, Switzerland
2
Universitéde Paris, Institut Cochin, CNRS, INSERM, Paris, France
Corresponding author: Bernard Thorens, bernard.thorens@unil.ch
Received 28 May 2020 and accepted 18 August 2020
This article contains supplementary material online at https://doi.org/10.2337/
figshare.12827294.
S.Q. and G.L. equally contributed to this work.
© 2020 by the American Diabetes Association. Readers may use this article as
long as the work is properly cited, the use is educational and not for profit, and the
work is not altered. More information is available at https://www.diabetesjournals
.org/content/license.
Diabetes Volume 69, November 2020 2253
METABOLISM
2-deoxy-D-glucose (2DG) injection in the VMN of normo-
glycemic animals is sufficient to stimulate glucagon secre-
tion (7,8). More recently, it has been shown that inactivation
of the vesicular glutamate transporter (vGlut2)genein
Sf1 neurons, which represent most of the VMN neurons,
suppresses glutamatergic synaptic transmission and pre-
vents the normal counterregulatory response to hypogly-
cemia (9). Also, the optogenetic activation of Sf1 neurons
induces, whereas their silencing suppresses glucagon secre-
tion (10). There is, thus, strong support for a role of VMN
neurons in the control of glucagon secretion.
However, VMN neurons are part of a multisynaptic
circuit that includes an afferent and an efferent limb (11).
The afferent limb comprises glucose-sensing neurons lo-
cated outside of the blood-brain barrier, such as those
present in the hepatoportal vein area (3) or in the nucleus
of the tractus solitarius and which respond to small
variations in blood glucose concentration (5,12), and
neurons located within the blood-brain barrier, such as
the GI neurons of the lateral parabrachial nucleus, which
form direct synaptic contacts with VMN neurons (13). The
efferent limb, which regulates pancreatic a-cell secretion,
involves projections from the VMN to the bed nucleus of
the stria terminalis, the periaqueductal gray, and preau-
tonomic regions of the brainstem (14). In this circuit, it is
usually assumed that hypoglycemia sensing by VMN GI
neurons plays an essential role in triggering the counter-
regulatory response. However, supporting evidence is only
circumstantial, based on various, non–cell-specific phar-
macological or gene-silencing approaches (15–17). Thus,
the relative importance in triggering the counterregulation
response of VMN GI neurons and of GI neurons present at
other locations of the afferent limb is not established (11).
Activation of VMN GI neurons by hypoglycemia has
been proposed to require the presence of AMPK, which
recruits a nitric oxide synthase–soluble guanylate cyclase
pathway to amplify AMPK activity; this leads to the closure
of the CFTR chloride channel and neuron firing (18). Here,
we aimed at identifying the role of the AMPK a1 and a2
catalytic subunits in the glucose responsiveness of VMN
Sf1 neurons using combination of genetic, electrophysio-
logical, and physiological studies. We showed that GI
neurons are no longer detected when AMPK is inactivated.
We identified Txn2, encoding a mitochondrial redox pro-
tein, as one of the most downregulated genes when AMPK
is suppressed and showed that reexpression of Txn2 in Sf1
neurons is sufficient to restore the presence of GI neurons.
Finally, we showed that mice with selective inactivation of
VMN GI neurons activity had normal counterregulatory
response to hypoglycemia and feeding behavior, suggest-
ing that the glucose sensitivity of these neurons is dispens-
able for the counterregulatory response.
RESEARCH DESIGN AND METHODS
Mice
AMPKa1
lox/lox
,AMPKa2
lox/lox
mice (19) were crossed with
Sf1-cre mice (9) to generate AMPKa1
lox/lox
a2
lox/lox
and
Sf1-cre;AMPKa1
lox/lox
a2
lox/lox
mice. Mice were on a C57BL/6
background. All studies used littermates as controls. Mice
were age-matched and randomly assigned to experimental
groups. Animals were housed on a 12-h light/dark cycle
and fed with a standard chow (Diet 3436; Provimi Kliba
AG). All procedures were approved by the Veterinary Office
of Canton de Vaud (Switzerland).
Mouse Genotyping
Mouse genotyping was performed by PCR analysis (Sup-
plementary Fig. 1). Primers for AMPK a1
lox/lox
were P1a1
(59-ATT AAA CAC CAC TAA TTG GAA AAC ATT CCC-39)
and P2a1(59-GGG CAA GTA AGG CCT GCA GCC CTA
CAC TGA-39), and AMPK a1D:Pa1and P3a1(59-GAC
CTG ACA GAA TAG GAT ATG CCC AAC CTC-39). AMPK
a2
lox/lox
PCR primers were P1a2(59-GTT ATC AGC CCA
ACT AAT TAC AC-39) and P2a2(59-GCT TAG CAC GTT
ACC CTG GAT GG-39), and AMPK a2D:P3a2(59-TTG GCG
CTG TCT AGA TCA GGC TTG C-39) and P4a2(59-GTG CTT
CCT AAC TGC AGA TGC AGT G-39).
Analysis of genetic recombination in brain regions was
performed with DNA extracted (DNeasy Kit, cat: 69504;
Qiagen) from tissue punches (punch, ref. 18036; Fine
Science Tools) prepared from 1-mm-thick brain sections.
Viral Vectors
All recombinant AAV constructs were produced at the Gene
Therapy Center at the University of North Carolina (Chapel
Hill, NC). Viruses were pAAV8-EF1a-Flex-DN-AMPK-K45R-
T2A-mCherry (a2 subunit mutant, AAV8-DIO-DN-AMPK)
(20), pAAV8-EF1a-Flex-CA-AMPK-H150R-T2A-mCherry
(AAV8-DIO-CA-AMPK) (20), pAAV8-FLEX-EGFPL10a (AAV8-
DIO-L10-EGFP) (21), rAAV8-EF1a-DIO-Txn2-P2A-EGFP
(AAV8-DIO-Txn2-EGFP), and rAAV8-EF1a-DIO-P2A-EGFP
(AAV8-DIO-EGFP). The last two virus constructs were
produced in the laboratory. Lentivectors lenti-hPGK-DN-
AMPK-K45R-T2A-mCherry and lenti-hPGK-CA-AMPK-H150R-
T2A-mCherry; lenti-U6-shTxn2-DIO-EGFP were produced
as described (22).
Stereotactic Injection of Viruses
This procedure was performed as described (4) using 6- to
12-week-old mice. Bilateral stereotactic injections in the
VMN used the following coordinates: AP 1.3/ML 60.6/
DV 25.3 mm. A total of 200 nL of the virus preparations
(10
11
–10
14
viral genomes/mL) were injected in each hemi-
sphere at a rate of 0.1 mL/min.
Electrophysiology
Mice (8–12 weeks old) were deeply anesthetized with
isoflurane before decapitation, and 250-mm coronal sec-
tions containing VMN were prepared using a vibratome
(VT1000S; Leica). Electrophysiological recordings were
conducted as previously described (5). Whole-cell record-
ings were performed in current-clamp mode using a Multi-
Clamp 700B amplifier associated with a 1440A Digidata
digitizer (Molecular Devices). Neurons with an access
2254 AMPK Controls Txn2 Expression in GI Neurons Diabetes Volume 69, November 2020
resistance .25 MVor changed by .20% during the
recording were excluded. A hyperpolarization step (220 pA,
500 ms) was applied every 30 s to measure membrane
resistance. Membrane potential and neuronal firings were
monitored over time at different extracellular glucose
concentrations after a 10–15 min baseline. Signals were
digitized at 10 kHz, collected, and analyzed using the
pClamp 10 data acquisition system (Molecular Devices).
Translating Ribosome Affinity Purification
Translating ribosomes affinity purification (TRAP) was
performed as described (23). Sf1-cre mice were injected
in the VMN with AAV8-DIO-L10-EGFP (21). The VMN
were microdissected, and pools of six VMNs were consti-
tuted. Anti-green fluorescent protein (GFP) antibody (cat
11814460001; Sigma-Aldrich) was used to immunoprecip-
itate RNAs from Sf1-positive cells. RNAs were amplified by
single primer isothermal amplification with the Ovation
RNA-Amplification System V2 (NuGEN), providing DNA
libraries for RNA sequencing (RNA-seq). After reads were
aligned, read counts were summarized with htseq-count
(v. 0.6.1) (24) using Mus musculus GRCm38.82 gene anno-
tation. Library sizes were scaled using TMM (trimmed mean
of M) normalization (EdgeR package version 3.16.3). To
evaluate enrichment and depletion of control genes, a mod-
erated ttest was used comparing all six inputs compared
with all six outputs.
GT1-7 Cells
GT1-7 cells were cultured in DMEM containing 25 mmol/L
glucose, 10% FBS, and 5% horse serum. Cells were trans-
duced with lentivectors at a multiplicity of infection of
500, yielding ;95% transduction efficiency. Then, 5 310
5
cells/well were plated in six-well dishes. For superoxide
production measurements, GT1-7 cells were incubated in
0.1 mmol/L or 30 mmol/L glucose for 48 h before adding
MitoSOX (cat: M36008; Thermo Fisher). After 45 min,
cells were trypsinized and analyzed using a FACS (BD
Accuri C6; BD Bioscience). Western blot and real-time
quantitative PCR (qPCR) analysis were performed as pre-
viously described (25). Rabbit antibodies against ACC (cat
3662; Cell Signaling), anti–phospho-Ser79-ACC (cat 3661;
Cell Signaling), anti-Txn2 (cat ab185544; Abcam), and
anti-actin (cat A2066; Sigma Aldrich) were used. The
secondary antibody was a goat horseradish peroxidase-
coupled anti-rabbit antibody (cat: NA934; GE Healthcare).
Real-time qPCR analysis was performed using the follow-
ing primers for Txn2 (59-TTC CCT CAC CTC TAA GAC
CCT-39,59-CCT GGA CGT TAA AGG TCG TCA-39) and
actin (59-CTA AGG CCA ACC GTG AAA AGA T-39,59-CAC
AGC CTG GAT GGC TAC GT-39).
Glucose and Insulin Tolerance Tests, Glucagon
Measurements, and Hypoglycemic Clamps
Glucose tolerance tests (2 g/kg, i.p.) were performed in
15-h fasted mice and insulin tolerance tests (0.8 units/kg,
i.p.) with 6-h fasted mice, as previously described (26).
Plasma glucagon concentrations were measured by ELISA
(Mercodia, Uppsala, Sweden) from blood collected by sub-
mandibular puncture under isoflurane anesthesia into
tubes containing aprotinin/EDTA. Plasma catecholamines
were determined by liquid chromatography-tandem mass
spectrometry (27). Hypoglycemic clamps were performed
as previously described (28) in 5-h fasted mice.
Food Intake
Continuous food consumption was measured in a 12-
chamber Oxymax system (Columbus Instruments, Colum-
bus, OH). Measurements of feeding initiation after a fast
were performed with mice food-deprived for 15 h and
placed in individual cages with weighted food pellets,
which were weighted at the indicated intervals.
Statistics
Values are reported as mean 6SEM. Data were analyzed
with GraphPad Prism software (GraphPad Software, San
Diego, CA). Statistical significance was assessed using
appropriate statistical tests that are mentioned in each
figure’s legend.
Data and Resource Availability
RNA-seq data have been deposited in the Gene Expression
Omnibus database under accession number GSE153872.
The data sets and reagents generated during and/or an-
alyzed during the current study are available from the
corresponding author upon reasonable request.
RESULTS
Glucose-Sensing by Sf1 VMN Neurons
To identify Sf1 neurons in brain slices, we injected an AAV-
DIO-EGFP in the VMN of Sf1-cre mice (Fig. 1A). The
electrical activity of cells expressing EGFP was then char-
acterized by whole-cell patch-clamp recordings in the
presence of 2.5 mmol/L and 0.5 mmol/L glucose. These
concentrations represent those found in the brain paren-
chyma in normoglycemic and hypoglycemic states, respec-
tively (29); these have been routinely used to identify GE
and GI neurons (30). Of the analyzed Sf1 neurons, 43%
were GE (Fig. 1Band C) characterized by their decreased
firing activity during hypoglycemia associated with a hy-
perpolarization (254.9 61.0 mV vs. 265.7 61.1 mV in
2.5 mmol/L and 0.5 mmol/L glucose, respectively; P,
0.001) (Fig. 1D) and lower membrane resistance (957.7 6
81.8 MVvs. 535.9 654.3 MVin 2.5 mmol/L and
0.5 mmol/L glucose, respectively; P,0.001) (Fig. 1E).
GI neurons comprised 23.3% of the recorded neurons (Fig.
1B). At 0.5 mmol/L glucose, firing activity was increased,
membrane potential was reduced (268.9 61.3 mV
vs. 258.7 61.7 in 2.5 mmol/L and 0.5 mmol/L glucose,
respectively; P,0.001), and membrane resistance was
increased (601.7 674.8 MVvs. 846.4 694.3 MVin
2.5 mmol/L and 0.5 mmol/L glucose, respectively; P,
0.01) (Fig. 1H). Of note, the membrane potential and mem-
brane resistance of GE and GI neurons in normoglycemic
diabetes.diabetesjour nals.org Quenneville and Associates 2255
Figure 1—VMN Sf1 neurons are glucose responsive. A: rAAV8-DIO-EGFP was bilaterally injected in the VMN of Sf1-cre mice. Inset shows
EGFP expression in the VMN. Scale bar, 100 mm. B: Distribution of GE (n513 neurons in nine mice), GI (n57 neurons in five mice), and NR
(n510 neurons in nine mice) Sf1 neurons in the VMN. C: GE neurons show decreased activity upon lowering the extracellular glucose to
0.5 mmol/L. This is accompanied by a significant reduction in membrane potential (D) and membrane resistance (E). GI neurons display
increased activity during hypoglycemia (F), with a significant rise in their membrane potential (G) and membrane resistance (H). Other neurons
were defined as nonresponder neurons (NR; panel I) because neither their activity nor their membrane potential (J) or membrane resistance
(K) varied upon extracellular glucose variations. Before-after graphs show individual values. Two-tailed paired ttest was used. *P,0.05,
**P,0.01, and ***P,0.001. Under normoglycemia, GI and GE neurons exhibit significantly different membrane potentials and membrane
resistances. GI neurons have a more negative membrane potential than GE neurons (L) and a lower membrane resistance (M). Two-tailed t
test was used. *P,0.05, **P,0.01, and ***P,0.001. N: Sf1-negative neurons of the VMN contain fewer glucose-sensing neurons than Sf1
neurons. The Fisher exact test was used. P50.0031 for GI 1GE proportion comparison.
2256 AMPK Controls Txn2 Expression in GI Neurons Diabetes Volume 69, November 2020
conditions were significantly different, suggesting differ-
ent complements of ion channels involved in glucose-
sensing (Fig. 1Land M)(P,0.01). Finally, 33.3% of
the recorded neurons did not change their membrane
potential and/or membrane resistance upon changes in
glucose concentrations and were classified as nonrespond-
ing (NR) neurons (Fig. 1I–K). We also characterized the
glucose responsiveness of non-Sf1 neurons of the VMN.
Only 21% were glucose responsive, with a predominance of
GE neurons (Fig. 1N). Thus, the Sf1 neurons of the VMN,
which form most of the VMN neurons, comprise two-
thirds of glucose responsive neurons, and ;40% of those
are GI neurons.
AMPK a1and a2Subunits Are Required for GI Neuron
Response
In a first approach to test the role of AMPK in the glucose
responsiveness of the VMN neurons, we expressed a dom-
inant negative form of AMPK in Sf1 neurons. This was
achieved by stereotactic injection of an AAV-DIO-DN-
AMPK-mCherry in the VMN of Sf1-cre mice (Fig. 2A
and B). Electrophysiological recordings of the transduced
neurons, identified by their red fluorescence, revealed the
presence of GE and NR neurons, but no GI neurons could
be detected (Fig. 2C–I).
In a second approach, we generated Sf1-cre;AMPKa1
lox/lox
a2
lox/lox
mice, which displayed efficient recombination
of the AMPKa1and AMPKa2genes only in the VMN
(Supplementary Fig. 1). To identify Sf1 neurons, an AAV-
DIO-EGFP was injected in the VMN (Fig. 2J), and electro-
physiological recordings of these neurons confirmed that
inactivation of AMPKa1and AMPKa2suppressed the GI
neurons (Fig. 2K). GE and NR neurons were still present in
the same proportions as found using the DN-AMPK ap-
proach. In addition, the reexpression of AMPKa1and
AMPKa2genes in VMN of those mice allowed the reap-
pearance of GI neurons (Supplementary Fig. 2).
We next tested which of the a1 and a2 AMPK subunits
was required for the GI response. We generated Sf1-
cre;AMPKa1
lox/lox
and Sf1-cre;AMPKa2
lox/lox
mice, injected
an AAV-DIO-EGFP in their VMN, and performed electro-
physiological analysis of EGFP-labeled Sf1 neurons (Fig.
3A–D). Inactivation of the AMPKa1or AMPKa2gene did
not suppress the presence of GI neurons, and the distri-
bution of GE, GI, and NR neurons was not significantly
different from that of control mice (Fig. 3A–D)(P.0.05).
Finally, we investigated whether overexpression of
a constitutively active form of AMPK in Sf1 neurons,
using an AAV-DIO-AMPK-CA-mCherry, would modify
the proportion of GE and GI neurons. However, when
overexpressed in Sf1 neurons, it did not affect the pro-
portion of GE, GI, and NR neurons (Fig. 3Eand F)(P.
0.05).
Taken together, the above data showed that the re-
sponse of Sf1 GI neurons to hypoglycemia depends on
AMPK but that the a1 and the a2 subunits have redundant
roles. Furthermore, the expression of a constitutively active
form of AMPK in Sf1 neurons does not change the distri-
bution of GE, GI, and NR neurons, indicating that activation
of AMPK is not sufficient to convert NR or GE neurons into
GI neurons. This further implies that GI neurons have
specific glucose-signaling mechanisms in which AMPK ac-
tivity must play a specificrole.
Thioredoxin 2 Is an AMPK-Regulated Gene Required
for the GI Response
To search for potential regulators of the GI neuron re-
sponse to hypoglycemia, we used a TRAP approach to
selectively characterize the transcriptome of Sf1 neurons.
Sf1-cre mice were injected in the VMN with an AAV-DIO-
mCherry or an AAV-DIO-AMPK-DN-mCherry, and both
groups of mice received at the same time an AAV-DIO-
L10-EGFP encoding a L10 ribosomal protein-GFP fusion
protein that integrates in Sf1 neurons ribosomes. The day
before the experiment, the mice were fasted overnight.
Their VMNs were then dissected out, lysed, and the
ribosomes immunoprecipitated with an anti-GFP antibody.
RNA-seq was then performed on the immunoprecipi-
tated ribosomal fraction (output) and on the non-
immunoprecipitated (input) material. RNA-seq data from
control and AMPK-DN overexpressing VMN were first
combined and analyzed to confirm that the immunopre-
cipitated fractions were enriched in mRNAs known to be
expressed in the VMN (Nr5a1 [Sf1], Fezf1,Sox14,Gpr149)
(31)anddepletedinmRNAsexpressedbynon-VMN
neurons (Agrp,Npy,Scl16a11 [Gat-3], Th), glial cells
(ApoE,Gfap,Opalin), and oligodendrocytes (Olig1,Plp1,
Mal)(Fig.4A). Then, for each mRNA, we measured their
enrichment in the immunoprecipitated fraction (ratio of
output vs. input) and calculated how these ratios differ in
Sf1 neurons expressing or not the AMPK-DN. These data
arepresentedinthevolcanoplotofFig.4B.
Among the dysregulated genes, we focused on the
downregulation of mitochondrial thioredoxin 2 (Txn2).
This downregulation was observed in the three RNA
samples from neurons expressing the AMPK-DN (Fig.
4C)andwasconfirmed by real-time qPCR analysis of
mRNAs immunoprecipitated from a second TRAP exper-
iment (Fig. 4D). Txn2 is a mitochondrial enzyme involved
in redox reactions (32,33), which can modulate mitochon-
drial respiration (34) and apoptosis (35). It also partic-
ipates in the regulation of reactive oxygen and nitrogen
species that are increased after insulin-induced hypogly-
cemia and which may control the response of glucose-
sensitive neurons (16,18,36,37).
To assess whether Txn2 participates in the response
of GI neurons to hypoglycemia, we transduced Sf1 neurons
of Sf1-cre;AMPKa1
lox/lox
a2
lox/lox
mice with an AAV-DIO-
Txn2-EGFP virus (Fig. 5Aand B). Electrophysiological
analysis of the transduced neurons revealed that Txn2
overexpression restored the presence of GI neurons (Fig.
5G–I), which displayed membrane depolarization (59.9 6
2.7 mV vs. 65.6 62.3 mV in 2.5 mmol/L and 0.5 mmol/L
glucose, respectively; P,0.01) and increased membrane
diabetes.diabetesjour nals.org Quenneville and Associates 2257
Figure 2—Loss of GI neurons after suppression of AMPK activity. A: rAAV8-DIO-AMPK-DN-mCherry was bilaterally injected in the VMN of
Sf1-cre mice. B: Schematic representation of the rAAV8-DIO-AMPK-DN-mCherry viral construct. C: Comparison of the distribution of
glucose responsive Sf1 neurons in control animals (CTRL from Fig. 1) and mice injected with the rAAV8-DIO-AMPK-DN-mCherry. GI neurons
are no longer present when AMPK-DN is expressed (Fisher exact test; P50.0148 for GI neurons proportion comparison). GE neurons (D–F)
(n512 neurons in seven mice) and NR neurons (G–I)(n511 neurons in six mice) subpopulations are still present and display similar features
as GE and NR neurons from control animals (see Fig. 1). J: Experimental approach. rAAV8-DIO-EGFP was bilaterally injected in the VMN of
Sf1-cre;AMPKa1
lox/lox
a2
lox/lox
mice. K: Distribution of the glucose-responsive Sf1 neurons of the VMN (n512 neurons in 8 mice and n5
16 neurons in 10 mice for GE and NR, respectively). GI neurons were no longer detected in the mutant mice (Fisher exact test; P50.0107 for
GI neurons proportion compared with control conditions). Before-after graphs show individual values. Two-tailed paired ttest was used. *P,
0.05; **P,0.01, and ***P,0.001.
2258 AMPK Controls Txn2 Expression in GI Neurons Diabetes Volume 69, November 2020
resistance (692.3 690.9 MVvs. 888.8 6108.2 MVin
2.5 mmol/L and 0.5 mmol/L glucose, respectively; P,0.01),
similar to those measured in control GI neurons. The electro-
physiological properties of GE (Fig. 5D–F)andNR(Fig.5J–L)
neurons were not affected by Txn2 overexpression. The pro-
portion of GE, GI, and NR neurons was also restored by Txn2
overexpression (Fig. 5C).RestorationofGIneuronactivitywas
also obtained when using a lentiviral instead of an AAV vector
for Txn2 reexpression (data not shown).
Thus, Txn2 expression is strongly reduced when AMPK
activity is suppressed, and overexpression of Txn2 in Sf1
neurons is sufficient to restore the presence GI neurons.
Overexpression of Txn2, however, does not modify the
number of GE or NR neurons, suggesting that its role is
specific for the response of GI neurons to hypoglycemia.
AMPK Regulation of Txn2
To investigate, in a cellular system, the link between AMPK
and Txn2 expression, we used the GT1-7 neuronal cell line.
These cells were transduced with recombinant lentiviruses
encoding the dominant negative or the constitutively
active form of AMPK and incubated in the presence of
0.1 mmol/L glucose. Real-time qPCR (Fig. 6A) and Western
blot analysis (Fig. 6B–D) showed that overexpression of
AMPK-DN reduced Txn2 mRNA and protein expression. A
reduction of acetyl-CoA-carboxylase (ACC) phosphoryla-
tion confirmed that AMPK-DN overexpression effectively
reduced endogenous AMPK activity. In contrast, overex-
pression of the constitutively active form of AMPK did not
impact Txn2 expression or ACC phosphorylation, indicat-
ing that the endogenous AMPK was maximally active in
the presence of 0.1 mmol/L glucose.
Hypoglycemia induces reactive oxygen species (ROS)
production, and Txn2 may be required to detoxify ROS
to preserve hypoglycemia detection by GI neurons. To
test this hypothesis, GT1-7 cells were first exposed to
0.1 mmol/L or 30 mmol/L glucose, and superoxide pro-
duction was assessed by measuring MitoSOX red fluores-
cence intensity (Fig. 6E). We could confirm that exposure
to low glucose levels induced ROS production (Fig. 6F). We
then transduced GT1-7 cells with a recombinant lentivirus
encoding a control or a Txn2-specific shRNA, which led to
a very strong reduction of Txn2 mRNA (Fig. 6G). Exposing
these cells to 0.1 mmol/L glucose induced a significantly
higher increase in MitoSOX red staining when Txn2 ex-
pression was silenced (Fig. 6H).
Thus, AMPK controls the level of Txn2 expression; this
was also observed when AMPK-DN was transduced in the
Hepa1-6 mouse hepatoma cell line and therefore seems to
be a general mechanism (Supplementary Fig. 3). In addi-
tion, suppressing Txn2 expression led to increased ROS
production in the presence of low glucose concentrations.
Whether exaggerated ROS production negatively impacts
GI neurons activity is, however, to be further explored
experimentally.
Counterregulatory Response to Hypoglycemia in
Sf1-cre;AMPKa1
lox/lox
a2
lox/lox
Mice
We analyzed glucose homeostasis and counterregulatory
hormone secretion in control and Sf1-cre;AMPKa1
lox/lox
a2
lox/lox
mice. Glycemic levels were identical in both
groups of mice in the fed and 24-h fasted states (Fig.
7A), and their fasted plasma glucagon levels were also
identical (Fig. 7B). Their response to an insulin tolerance
tests was also identical (Fig. 7C), indicating the same
insulin sensitivity and counterregulatory response to
Figure 3—AMPK asubunits have redundant role in GI neurons
activation. A: rAAV8-DIO-EGFP was bilaterally injected in the
VMN of Sf1-cre;AMPKa1
lox/lox
mice. B: Distribution of the glu-
cose-responsive Sf1 neurons in the absence of AMPK a1subunit
(n510 GE, 5 GI, and 15 NR neurons in 7, 4, and 10 mice,
respectively). The distribution does not differ from that observed
in control mice (Fisher exact test; P.0.05). C: rAAV8-DIO-EGFP
was bilaterally injected in the VMN of Sf1-cre;AMPKa2
lox/lox
mice. D: Distribution of the glucose-responsive Sf1 neurons in
the absence of AMPK a2subunit (n516 GE, 3 GI, and 16 NR
neurons in eight, three, and nine mice, respectively). The distri-
bution does not differ from that observed in control mice (Fisher
exact test; P.0.05). E: rAAV8-DIO-CA-AMPK-mCherry was
bilaterally injected in the VMN of Sf1-cre mice. F: Distribution
of the glucose-responsive Sf1 neurons in the presence of an
AMPK-CA (n59GE,5GI,and12NRneuronsinsix,four,and
eight mice, respectively). The distribution does not differ from
that observed in control mice (Fisher exact test; P.0.05). *P,
0.05, **P,0.01, and ***P,0.001.
diabetes.diabetesjour nals.org Quenneville and Associates 2259
normalize hypoglycemia. We next assessed plasma gluca-
gon, epinephrine, and norepinephrine levels in response to
insulin-induced hypoglycemia. Figure 7Dshows that the
blood glucose levels in control and mutant mice were the
same 60 min after saline injection and that hypoglycemia
induced by insulin injections reached the same levels. The
basal plasma levels of glucagon, epinephrine, and norepi-
nephrine were identical between control and mutant mice,
as were their levels after induction of hypoglycemia (Fig.
7E–G); insulin-induced glucagon secretion was also not
different between female control and mutant mice
(data not shown). To confirm these results, we measured
plasma glucagon at the end of a hyperinsulinemic-
hypoglycemic clamp. The same rates of glucose infusion
were required to maintain hypoglycemia in control and
Sf1-cre;AMPKa1
lox/lox
a2
lox/lox
animals, and their plasma
glucagon levels measured at the end of the clamp were
similar (Fig. 7H–J).
Figure 4—Txn2 expression is strongly suppressed in the absence of AMPK activity. Sf1-cre mice received intra-VMN administration of an
AAV8-DIO-L10-EGFP (control) or of the same virus and an rAAV8-DIO-AMPK-DN-mCherry (DN). After transcribing ribosome affinity
purification was performed on VMN lysates and RNA sequencing analysis, gene enrichment in the immunoprecipitated fraction over total
lysate was calculated. A: Heat map shows enrichment for VMN neuron mRNAs in the immunoprecipitated fraction (output) comparedwith the
total lysate (input) and specific depletion in mRNAs expressed in non-VMN neurons, in glial cells or oligodendrocytes. B: Volcano plot shows
the mRNAs whose expression is significantly increased or downregulated in VMN Sf1 neurons expressing AMPK-DN. The x-axis shows
logtwofold-changes in expression and the y-axis the log odds of a gene being differentially expressed (two-way ANOVA). C: Relative
expression of Txn2 in control (CTRL) and AMPK-DN–expressing Sf1 neurons (DN); data from the TRAP analysis of (B). D: Real-time (RT) qPCR
analysis of Txn2 expression levels in CTRL and AMPK-DN–expressing Sf1 neurons from a second TRAP experiment.
2260 AMPK Controls Txn2 Expression in GI Neurons Diabetes Volume 69, November 2020
Figure 5—Expression of Txn2 in Sf1-cre;AMPKa1
lox/lox
a2
lox/lox
restores GI neuron activity. A: rAAV8-DIO-Txn2-EGFP was bilaterally injected
in the VMN of Sf1-cre;AMPKa1
lox/lox
a2
lox/lox
mice. B: Schematic representation of the rAAV8-DIO-Txn2-EGFP viral construct. C: Distribution
of glucose responsive Sf1 neurons from Sf1-cre;AMPKa1
lox/lox
a2
lox/lox
mice (from Fig. 2K) and from the same mice having been injected with
the Txn2 virus (n511 GE, 5 GI and 10 NR neurons in seven, four, and seven mice, respectively). Fisher exact test; P.0.05 for GI neurons
proportion comparison. Txn2-expressing GE neurons (D–F), GI (G–I), and NR (J–L) neurons exhibit similar electrophysiological characteristics
as GE, GI, and NR subpopulations monitored in control animals (see Fig. 1). Before-after graphs show individual values. Two-tailed paired
ttest. *P,0.05, **P,0.01. and ***P,0.001.
diabetes.diabetesjour nals.org Quenneville and Associates 2261
TheVMNandAMPKexpressedinSf1neuronshave
also been involved in the control of feeding (38). We
thus tested feeding of CTRL and Sf1-cre;AMPKa1
lox/lox
a2
lox/lox
mice over a 48-h period (Fig. 7K), and no dif-
ference in the cumulative food absorption over time
couldbeobserved.Similarly,therateofrefeedingafter
an 18-h fast was not different between both groups of
mice (Fig. 7L).
DISCUSSION
In the current study, we used a combination of genetic and
electrophysiological approaches to dissect the role of
AMPK a1 and a2 subunits in glucose sensing by VMN
Sf1 neurons. We found that suppressing AMPK activity led
to the selective depletion of GI neurons, without affecting
the presence of GE neurons. These observations allowed us
to further investigate two aspects of hypoglycemia sensing.
Figure 6—Txn2 expression is controlled by AMPK and reduces ROS accumulation. GT1-7 cells were transduced with a control lentivector or
a lentivector encoding AMPK-DN or AMPK-CA. The cells were incubated in 0.1 mmol/L glucose for 48 h before RNA and protein extraction. A:
Txn2 expression in GT1-7 cells transfected with a control vector or a vector encoding AMPK-DN or AMPK-CA (ttest, P,0.02). B: Western
blot analysis of Txn2, phosphorylated (p)-ACC, total ACC, and actin from cells transduced with the mentioned vectors. Cand D: Quantitation
of the Western blots of (B). E: MitoSOX red staining of GT1-7 cells incubated in the presence of 0.1 mmol/L or 30 mmol/L glucose for 48 h.
Unstained cells were used as control. Incubation in low glucose increased superoxide production. F: Quantitation of the MitoSOX red staining
of figure (E). G:Txn2 mRNA levels in GT1-7 cells transduced with a control or a Txn2-specific shRNA. H: MitoSOX red staining of GT1-7 cells
transduced with a control or Txn2-specific shRNA and incubated in 0.1 mmol/L glucose for 48 h. For all panels, ttest, *P,0.05, **P,0.01,
and ***P,0.001.
2262 AMPK Controls Txn2 Expression in GI Neurons Diabetes Volume 69, November 2020
Figure 7—Normal counterregulation and feeding in Sf1-cre;AMPKa1
lox/lox
a2
lox/lox
mice. A: Random fed and 24-h fasted glycemia in Sf1-
cre;AMPKa1
lox/lox
a2
lox/lox
knock-out (KO) and control (CTRL) mice (n522, 11 CTRL, 11 KO). B: Plasma glucagon levels in CTRL and KO mice
after 24-h fast. C: Insulin tolerance test in CTRL and KO mice (n520, 10 CTRL, 10 KO). Glycemia (D), plasma glucagon (E), epinephrine (F),
and norepinephrine (G) 60 min after i.p. saline or insulin (0.8 units/kg) injections (n520, 11 CTRL, 9 KO). H: Hyperinsulinemic-hypoglycemic
clamps were performed to maintain glycemic levels at ;2.5 mmol/L glucose. The glucose infusion rates were identical to maintain
hypoglycemia (I), and the plasma glucagon levels were the same at the end of the clamp in CTRL and KO mice (J)(n523, 11 CTRL, 12 KO). K:
Identical food intake between CTRL and KO mice in ad libitum condition monitored for 48 h (n510, 5 CTRL, 5 KO; two-way ANOVA, P5
0.7521). L: Refeeding experiment after overnight fast shows no difference between CTRL and KO mice in food intake (n518, 7 CTRL, 11 KO)
(two-way ANOVA, P50.4760). For all panels, two-tailed ttest was used. *P,0.05, **P,0.01, and ***P,0.001.
diabetes.diabetesjour nals.org Quenneville and Associates 2263
First, we investigated the molecular components participating
in hypoglycemia-induced neuron activation, and second, the
impact of suppressing GI neurons activity in physiological
regulations. We found that Txn2 expression was strongly
decreased when AMPK was inactivated, and we showed
that reexpression of Txn2 in Sf1 neurons of Sf1-
cre;AMPKa1
lox/lox
a2
lox/lox
mice restored GI neuron activity.
We further showed that silencing Txn2 led to exaggerated
ROS production in cells exposed to low glucose concen-
trations. This suggests that the role of AMPK is to main-
tain sufficient levels of Txn2 expression to prevent the
deleterious effect of ROS on GI neuron function. Impor-
tantly, in physiological studies, we found that suppression
of VMN GI neuron activity had no impact on the counter-
regulatory hormone response or on feeding.
AMPK is an evolutionary conserved, ubiquitously
expressed energy-sensing kinase that is activated in con-
ditions such as fasting, hypoglycemia, or hypoxia (39,40).
In the central nervous system, in particular in the VMN
plus arcuate nucleus, it has been reported that its activa-
tion by hypoglycemia or 2DG-induced neuroglucopenia is
involved in the control of feeding (41) and the secretion of
counterregulatory hormones (15). The VMN, however,
consists of several neuronal subpopulations, characterized
by the expression of different transcription factors, neu-
rotransmitter receptors, or neuropeptides (42,43) and by
their differential glucose responsiveness (30,44). Thus,
expression of AMPK in different subpopulations may
have different physiological impacts that are not revealed
in the studies mentioned above. Here, we investigated the
role of the AMPK a1 and a2 subunits specifically in glucose
sensing in Sf1 neurons of the VMN. Overexpression of
a dominant negative form of the kinase or genetic in-
activation of both AMPK a1and a2genes suppressed GI
activity. These observations are in agreement with pub-
lished data showing that AMPK is required for hypogly-
cemia detection by GI neurons of the VMH (17). They,
however, extend these previous data by showing that both
a1and a2isoforms similarly contribute to GI neurons
glucose sensing. They also show that the loss of AMPK
activity does not impact GE neuron response. Thus, AMPK
is selectively required for the activity of GI neurons.
AMPK regulates multiple cellular functions by direct
protein phosphorylation (45) or transcriptional regulation
(46). Here, we identified Txn2 as an AMPK-regulated
mRNA. Txn2 is part of a redox system that detoxifies
ROS and reactive nitrogen species (32); it is distinct from
Txn1, a cytosolic enzyme (47). Hypoglycemia is known to
induce ROS production in the brain and in the VMN
(37,48,49), whereas hyperglycemia reduces ROS produc-
tion in a UCP2-dependent manner in GE neurons of the
VMN (50). ROS levels have also been shown to have
antagonistic effects on NPY/Agrp and POMC neurons,
reducing the activity of the first ones and stimulating
the activity of the second ones (51). Thus, depending
on the cellular context and also on their intracellular
concentrations, ROS can have positive signaling effects
on neuron activity and gene expression or toxic effects
leading to cell apoptosis (52). The concept of mitohormesis
describes that ROS can shift from positive regulators of
cellular function to inducers of cell death (53).
In GT1-7 cells shifted from 30 to 0.1 mmol/L glucose,
there is a strong induction of superoxide production, in
agreement with the fact that hypoglycemia increases ROS
production (49) by shifting metabolism to b-oxidation
(48). If increased ROS production is associated with a nor-
mal firing activity of neurons, the loss of GI response when
Txn2 is suppressed, and restoration of such activity by its
reexpression, suggests that Txn2 plays a protective role
against a toxic effect of ROS. How ROS production can
prevent activation of GI neurons is not known. However,
one possibility is that superoxide ions produced in the
mitochondria can be transported into the cytosol, where
they can react with NO to produce peroxynitrite (ONOO
2
)
(52). Because NO production is required for GI neuron
activity (18), this reaction may dampen the GI neuron
response.
The fact that Txn2 restores GI neuron activity in the
absence of AMPK activity suggests that the role of this
kinase is not in the acute signaling of hypoglycemia. In-
stead, its role may be to protect against the deleterious
effects of ROS on signal transduction by controlling the
level of expression of antioxidant proteins, in particular,
Txn2. In support of this hypothesis is the observation that
among the other genes that were downregulated when
AMPK-DN was overexpressed was Pdss2 (or Coq1B), which
codes for an enzyme involved in the biosynthesis of the
prenyl side chain of coenzyme Q. This cofactor transports
electrons from complex I to complex II of the electron
transport chain and, in its reduced form, also acts as an
antioxidant (54). Decreased expression of Pdss2 and co-
enzyme Q deficiency have been linked to increased ROS
production and cell death (55,56). Thus, AMPK activity
may protect cells against ROS toxicity also by increasing
the expression of this biosynthetic enzyme. Along the
same line, a previous study performed in a rat model of
hypoglycemia-associated autonomic failure showed that
the loss of VMN GI neurons could be prevented by over-
expressing the cytosolic form of thioredoxin, Txn1 (57).
One of the initial goals of our study was to identify the
physiological functions controlled by VMN GI neurons, in
particular whether selective impairment in the counter-
regulatory response to hypoglycemia could be demon-
strated. However, plasma glucagon in fasted mice and
insulin tolerance tests were similar in control and Sf1-
cre;AMPKa1
lox/lox
a2
lox/lox
mice. Similarly, glucagon, epi-
nephrine, and norepinephrine secretion in response to
insulin-induced hypoglycemia, as well as plasma glucagon
levels at the end of a hyperinsulinemic-hypoglycemic
clamp, were identical. Thus, suppression of GI activity
in the VMN does not reduce hypoglycemia-induced coun-
terregulatory hormone secretion. Another aspect of the
response to hypoglycemia is the stimulation of food intake,
but we could not observe any difference in food intake
2264 AMPK Controls Txn2 Expression in GI Neurons Diabetes Volume 69, November 2020
measured over a 48-h period or in the rate of refeeding
after an overnight fast.
Together, these results indicate that the intrinsic glu-
cose sensitivity of GI neurons of the VMN is dispensable
for the physiological response to hypoglycemia. These
neurons are part of a circuit that includes afferent, glu-
cose-sensing neurons directly sensitive to small variations
in blood glucose concentrations, such as those present in
the hepatoportal vein area and of the nucleus of the tractus
solitarius. In this circuit, these peripheral sensing cells
probably have a primary role in triggering the counter-
regulatory response to hypoglycemia. The glucose-sensing
capacity the VMN GI neurons may, thus, represent a fail-
safe system, activated only in case of failure of the pe-
ripheral sensing cells leading to development of brain
hypoglycemia.
In summary, the current study establishes that AMPK
a1and a2subunits play redundant roles in allowing the
hypoglycemia detection capacity of Sf1 GI neurons. It
identifies Txn2 as a necessary element in the GI neurons
response to hypoglycemia, which can restore GI activity
even in the absence of AMPK. This suggests that the
primary role of AMPK in GI neurons is to control the
expression of Txn2, and possibly other enzymes, participat-
ing in the antioxidant protection against ROS produced
during hypoglycemia. Finally, these data show that the
intrinsic hypoglycemia-sensing capacity of VMN Sf1 neu-
rons is dispensable for triggering counterregulatory hor-
mone secretion or feeding and rather represents a fail-safe
system in case of failure of peripheral hypoglycemia sensing
system allowing development of hypoglycemia in the VMN.
Acknowledgments. The authors thank Dr. Sylvain Pradervand and the
members of the Genome Technology Facility of the University of Lausanne for their
help with RNA sequencing and data analysis and Dr. Frédéric Preitner and Anabela
da Costa of the Scientic Service of the Center for Integrative Genomics (SSC),
University of Lausanne, for performing the hypoglycemic clamps. The authors
thank Drs. Brad Lowell and Barbara Kahn (Harvard University) and Dr. Dong Kong
(Tufts University) for the gift of the AMPK-DN and AMPK-CA expression plasmids
and recombinant AAVs.
Funding. The present work was supported by a European Research Council
Advanced Grant (INTEGRATE, No. 694798) and a Swiss National Science Foun-
dation grant (310030-182496) to B.T., and has received funding from the
Innovative Medicines Initiative 2 Joint Undertaking under grant agreement No
777460 (HypoRESOLVE). The Joint Undertaking receives support from the
European Union’s Horizon 2020 Research and Innovation Programme and EFPIA
and T1D Exchange, JDRF, International Diabetes Federation, and The Leona M.
and Harry B. Helmsley Charitable Trust.
Duality of Interest. No potential conflicts of interest relevant to this article
were reported.
Author Contributions. S.Q., G.L., D.B., and S.M. performed the experi-
ments. S.Q., G.L., and B.T. designed the experiments. S.Q., G.L., and B.T. analyzed
the data and wrote the manuscript. B.V. and M.F. provided genetically modified
mice and reviewed the manuscript. B.T. conceived the project. B.T. takes
responsibility for the content of the manuscript. B.T. is the guarantor of this
work and, as such, had full access to all the data in the study and takes
responsibility for the integrity of the data and the accuracy of the data analysis.
References
1. Verberne AJ, Sabetghadam A, Korim WS. Neural pathways that control the
glucose counterregulatory response. Front Neurosci 2014;8:38
2. Cryer PE. Mechanisms of hypoglycemia-associated autonomic failure in
diabetes. N Engl J Med 2013;369:362–372
3. Marty N, Dallaporta M, Thorens B. Brain glucose sensing, counterregulation,
and energy homeostasis. Physiology (Bethesda) 2007;22:241–251
4. Labouèbe G, Boutrel B, Tarussio D, Thorens B. Glucose-responsive neurons
of the paraventricular thalamus control sucrose-seeking behavior. Nat Neurosci
2016;19:999–1002
5. Lamy CM, Sanno H, Labouèbe G, et al. Hypoglycemia-activated GLUT2
neurons of the nucleus tractus solitarius stimulate vagal activity and glucagon
secretion. Cell Metab 2014;19:527–538
6. Routh VH. Glucose-sensing neurons: are they physiologically relevant?
Physiol Behav 2002;76:403–413
7. Borg MA, Sherwin RS, Borg WP, Tamborlane WV, Shulman GI. Local ven-
tromedial hypothalamus glucose perfusion blocks counterregulation during sys-
temic hypoglycemia in awake rats. J Clin Invest 1997;99:361–365
8. Borg WP, Sherwin RS, During MJ, Borg MA, Shulman GI. Local ventromedial
hypothalamus glucopenia triggers counterregulatory hormone release. Diabetes
1995;44:180–184
9. Tong Q, Ye C, McCrimmon RJ, et al. Synaptic glutamate release by ven-
tromedial hypothalamic neurons is part of the neurocircuitry that prevents hy-
poglycemia. Cell Metab 2007;5:383–393
10. Meek TH, Nelson JT, Matsen ME, et al. Functional identification of a neu-
rocircuit regulating blood glucose. Proc Natl Acad Sci U S A 2016;113:E2073–
E2082
11. Bentsen MA, Mirzadeh Z, Schwartz MW. Revisiting how the brain senses
glucose-and why. Cell Metab 2019;29:11–17
12. Dallaporta M, Himmi T, Perrin J, Orsini JC. Solitary tract nucleus sensitivity to
moderate changes in glucose level. Neuroreport 1999;10:2657–2660
13. Garfield AS, Shah BP, Madara JC, et al. A parabrachial-hypothalamic
cholecystokinin neurocircuit controls counterregulatory responses to hypoglyce-
mia. Cell Metab 2014;20:1030–1037
14. Lindberg D, Chen P, Li C. Conditional viral tracing reveals that steroidogenic
factor 1-positive neurons of the dorsomedial subdivision of the ventromedial
hypothalamus project to autonomic centers of the hypothalamus and hindbrain.
J Comp Neurol 2013;521:3167–3190
15. McCrimmon RJ, Shaw M, Fan X, et al. Key role for AMP-activated protein
kinase in the ventromedial hypothalamus in regulating counterregulatory hormone
responses to acute hypoglycemia. Diabetes 2008;57:444–450
16. Fioramonti X, Marsollier N, Song Z, et al. Ventromedial hypothalamic nitric
oxide production is necessary for hypoglycemia detection and counterregulation.
Diabetes 2010;59:519–528
17. Hirschberg PR, Sarkar P, Teegala SB, Routh VH. Ventromedial hypothalamus
glucose-inhibited neurones: a role in glucose and energy homeostasis? J Neuro-
endocrinol 2020;32:e12773
18. Fioramonti X, Song Z, Vazirani RP, Beuve A, Routh VH. Hypothalamic nitric
oxide in hypoglycemia detection and counterregulation: a two-edged sword.
Antioxid Redox Signal 2011;14:505–517
19. Boudaba N, Marion A, Huet C, Pierre R, Viollet B, Foretz M. AMPK
re-activation suppresses hepatic steatosis but its downregulation does not pro-
mote fatty liver development. EBioMedicine 2018;28:194–209
20. Kong D, Dagon Y, Campbell JN, et al. A postsynaptic AMPK→p21-activated
kinase pathway drives fasting-induced synaptic plasticity in AgRP neurons. Neuron
2016;91:25–33
21. Nectow AR, Schneeberger M, Zhang H, et al. Identification of a brainstem
circuit controlling feeding. Cell 2017;170:429–442.e11
22. Salmon P, Trono D. Production and titration of lentiviral vectors. Curr Protoc
Neurosci 2006;Chapter 4:Unit 4.21
diabetes.diabetesjour nals.org Quenneville and Associates 2265
23. Heiman M, Schaefer A, Gong S, et al. A translational profiling approach for the
molecular characterization of CNS cell types. Cell 2008;135:738–748
24. Anders S, Pyl PT, Huber W. HTSeq–a Python framework to work with high-
throughput sequencing data. Bioinformatics 2015;31:166–169
25. Modi H, Cornu M, Thorens B. Glutamine stimulates biosynthesis and se-
cretion of insulin-like growth factor 2 (IGF2), an autocrine regulator of beta cell
mass and function. J Biol Chem 2014;289:31972–31982
26. Tarussio D, Metref S, Seyer P, et al. Nervous glucose sensing regulates postnatal
bcell proliferation and glucose homeostasis. J Clin Invest 2014;124:413–424
27. Dunand M, Gubian D, Stauffer M, Abid K, Grouzmann E. High-throughput and
sensitive quantitation of plasma catecholamines by ultraperformance liquid
chromatography-tandem mass spectrometry using a solid phase microwell ex-
traction plate. Anal Chem 2013;85:3539–3544
28. Burcelin R, Thorens B. Evidence that extrapancreatic GLUT2-dependent
glucose sensors control glucagon secretion. Diabetes 2001;50:1282–1289
29. Silver IA, Ereci
nska M. Extracellular glucose concentration in mammalian
brain: continuous monitoring of changes during increased neuronal activity and
upon limitation in oxygen supply in normo-, hypo-, and hyperglycemic animals. J
Neurosci 1994;14:5068–5076
30. Song Z, Levin BE, McArdle JJ, Bakhos N, Routh VH. Convergence of pre- and
postsynaptic influences on glucosensing neurons in the ventromedial hypotha-
lamic nucleus. Diabetes 2001;50:2673–2681
31. Kurrasch DM, Cheung CC, Lee FY, Tran PV, Hata K, Ingraham HA. The
neonatal ventromedial hypothalamus transcriptome reveals novel markers with
spatially distinct patterning. J Neurosci 2007;27:13624–13634
32. Netto LES, Antunes F. The roles of peroxiredoxin and thioredoxin in hydrogen
peroxide sensing and in signal transduction. Mol Cells 2016;39:65–71
33. Balsera M, Buchanan BB. Evolution of the thioredoxin system as a step
enabling adaptation to oxidative stress. Free Radic Biol Med 2019;140:28–35
34. Hu C, Zhang H, Qiao Z, Wang Y, Zhang P, Yang D. Loss of thioredoxin 2 alters
mitochondrial respiratory function and induces cardiomyocyte hypertrophy. Exp
Cell Res 2018;372:61–72
35. Matsuzawa A. Thioredoxin and redox signaling: roles of the thioredoxin
system in control of cell fate. Arch Biochem Biophys 2017;617:101–105
36. Patocková J, Marhol P, Tůmová E, et al. Oxidative stress in the brain tissue of
laboratory mice withacute post insulin hypoglycemia. Physiol Res 2003;52:131–135
37. Pauliina Markkula S, Lyons D, Yueh CY, et al. Intracerebroventricular catalase
reduces hepatic insulin sensitivity and increases responses to hypoglycemia in
rats. Endocrinology 2016;157:4669–4676
38. Viskaitis P, Irvine EE, Smith MA, et al. Modulation of SF1 neuron activity
coordinately regulates both feeding behavior and associated emotional states. Cell
Rep 2017;21:3559–3572
39. Carling D. The AMP-activated protein kinase cascade–a unifying system for
energy control. Trends Biochem Sci 2004;29:18–24
40. Viollet B, Horman S, Leclerc J, et al. AMPK inhibition in health and disease.
Crit Rev Biochem Mol Biol 2010;45:276–295
41. Minokoshi Y, Alquier T, Furukawa N, et al. AMP-kinase regulates food intake
by responding to hormonal and nutrient signals in the hypothalamus. Nature 2004;
428:569–574
42. Krause WC, Ingraham HA. Origins and functions of the ventrolateral VMH:
a complex neuronal cluster orchestrating sex differences in metabolism and
behavior. Adv Exp Med Biol 2017;1043:199–213
43. McClellan KM, Parker KL, Tobet S. Development of the ventromedial nucleus
of the hypothalamus. Front Neuroendocrinol 2006;27:193–209
44. Kang L, Routh VH, Kuzhikandathil EV, Gaspers LD, Levin BE. Physiological
and molecular characteristics of rat hypothalamic ventromedial nucleus gluco-
sensing neurons. Diabetes 2004;53:549–559
45. Hardie DG. Minireview: the AMP-activated protein kinase cascade: the key
sensor of cellular energy status. Endocrinology 2003;144:5179–5183
46. Collodet C, Foretz M, Deak M, et al. AMPK promotes induction of the tumor
suppressor FLCN through activation of TFEB independently of mTOR. FASEB J
2019;33:12374–12391
47. Lu J, Holmgren A. The thioredoxin antioxidant system. Free Radic Biol Med
2014;66:75–87
48. Kajihara N, Kukidome D, Sada K, et al. Low glucose induces mitochondrial
reactive oxygen species via fatty acid oxidation in bovine aortic endothelial cells. J
Diabetes Investig 2017;8:750–761
49. Páramo B, Hernández-Fonseca K, Estrada-Sánchez AM, Jiménez N, Hernández-
Cruz A, Massieu L. Pathways involved in the generation of reactive oxygen and
nitrogen species during glucose deprivation and its role on the death of cultured
hippocampal neurons. Neuroscience 2010;167:1057–1069
50. Toda C, Kim JD, Impellizzeri D, Cuzzocrea S, Liu ZW, Diano S. UCP2 regulates
mitochondrial fission and ventromedial nucleus control of glucose responsiveness.
Cell 2016;164:872–883
51. Diano S, Liu ZW, Jeong JK, et al. Peroxisome proliferation-associated control
of reactive oxygen species sets melanocortin tone and feeding in diet-induced
obesity. Nat Med 2011;17:1121–1127
52. Shadel GS, Horvath TL. Mitochondrial ROS signaling in organismal ho-
meostasis. Cell 2015;163:560–569
53. Ristow M. Unraveling the truth about antioxidants: mitohormesis explains
ROS-induced health benefits. Nat Med 2014;20:709–711
54. Wang Y, Hekimi S. The complexity of making ubiquinone. Trends Endocrinol
Metab 2019;30:929–943
55. Quinzii CM, Garone C, Emmanuele V, et al. Tissue-specific oxidative stress
and loss of mitochondria in CoQ-deficient Pdss2 mutant mice. FASEB J 2013;27:
612–621
56. Quinzii CM, López LC, Gilkerson RW, et al. Reactive oxygen species, oxidative
stress, and cell death correlate with level of CoQ10 deficiency. FASEB J 2010;24:
3733–3743
57. Zhou C, Routh VH. Thioredoxin-1 overexpression in the ventromedial nucleus
of the hypothalamus preserves the counterregulatory response to hypoglycemia
during type 1 diabetes in male rats. Diabetes 2018;67:120–130
2266 AMPK Controls Txn2 Expression in GI Neurons Diabetes Volume 69, November 2020
