SEE COMMENTARY
Insulin signaling in the hippocampus and amygdala
regulates metabolism and neurobehavior
Marion Sotoa,b,1, Weikang Caia,b,1, Masahiro Konishia,b, and C. Ronald Kahna,b,2
a
Section of Integrative Physiology and Metabolism, Joslin Diabetes Center, Boston, MA 02215; and bDepartment of Medicine, Harvard Medical School,
Boston, MA 02215
Contributed by C. Ronald Kahn, January 2, 2019 (sent for review October 17, 2018; reviewed by Suzanne Craft and Sam Gandy)
Previous studies have shown that insulin and IGF-1 signaling in the
brain, especially the hypothalamus, is important for regulation of
systemic metabolism. Here, we develop mice in which we have
specifically inactivated both insulin receptors (IRs) and IGF-1 receptors
(IGF1Rs) in the hippocampus (Hippo-DKO) or central amygdala (CeA-
DKO) by stereotaxic delivery of AAV-Cre into IRlox/lox/IGF1Rlox/lox mice.
Consequently, both Hippo-DKO and CeA-DKO mice have decreased
levels of the GluA1 subunit of glutamate AMPA receptor and display
increased anxiety-like behavior, impaired cognition, and metabolic
abnormalities, including glucose intolerance. Hippo-DKO mice also
display abnormal spatial learning and memory whereas CeA-DKO
mice have impaired cold-induced thermogenesis. Thus, insulin/IGF-1
signaling has common roles in the hippocampus and central amyg-
dala, affecting synaptic function, systemic glucose homeostasis, be-
havior, and cognition. In addition, in the hippocampus, insulin/IGF-1
signaling is important for spatial learning and memory whereas
insulin/IGF-1 signaling in the central amygdala controls thermo-
genesis via regulation of neural circuits innervating interscapular
brown adipose tissue.
insulin | hippocampus | amygdala | metabolism | cognition
I nsulin, insulin-like growth factor 1 (IGF-1), and IGF-2 act
through two cognate receptors to control distinct physiological
processes throughout the body. Insulin binds with highest affinity
to the insulin receptor (IR) and low affinity to the IGF-1 receptor
(IGF1R) and thus regulates systemic metabolism (1, 2). In contrast,
IGF-1 and IGF-2 bind with higher affinity to the IGF1R and act as
growth factors important for tissue development and remodeling
(3–5). Differences between insulin and IGF-1/2 are in part explained
by the different expression patterns of these receptors, and in part
by differences in signals generated by the IR and IGF1R (6).
In the central nervous system, both IR and IGF1R are widely
expressed, and their actions have been implicated in the brain in
control of metabolism and energy homeostasis, as well as brain
development, injury repair, and higher neural processes, in-
cluding cognition and mood (7, 8). At the disease level, altered
insulin/IGF-1 signaling in the brain has been linked to increased
risks for Alzheimer’s disease, premature cognitive decline, and
dementia, as well as depression and anxiety (9–11). Patients with
Alzheimer’s disease show decreased expression of both IR and
IGF1R (12), as well as abnormal distribution and cellular lo-
calization of these receptors (13). These CNS phenotypes have
also been observed in states in which there is insulin resistance in
the brain, such as obesity and diabetes (14, 15).
Loss of IR in the whole brain causes hyperphagia, insulin re-
sistance, central hypogonadism, impaired response to hypoglyce-
mia, and increased depressive-like behaviors (16–18) whereas loss
of IGF1R (or one of its major substrates, IRS-2) has been shown to
impair brain development (19, 20). However, single IR or IGF1R
knockout models do not completely eliminate insulin or IGF-1
signaling since both ligands can elicit signaling through the receptor
that remains intact. Furthermore, up to now, most studies have
focused on either the whole brain or hypothalamus (16, 19, 21, 22).
Hence, the roles of IR/IGF1R in other nuclei controlling higher
neural functions, including mood and cognition, are not known.
www.pnas.org/cgi/doi/10.1073/pnas.1817391116
In the present study, we used stereotactic surgery and AAV-Cre to
induce IR and IGF1R double knockout (DKO) in the hippocampus
and central amygdala. We found that IR/IGF1R deletion specifically
down-regulates the expression of an AMPA receptor subunit, glu-
tamate receptor 1, in synaptosomes from both hippocampus and
amygdala. This is accompanied by multiple metabolic and behavioral
abnormalities, including glucose intolerance and increased anxiety-
like behaviors. In addition, while deletion of both IR and IGF1R in
the hippocampus and central amygdala leads to impaired recogni-
tion memory, IR/IGF1R loss in hippocampus also results in im-
paired spatial memory. Finally, deletion of IR and IGF1R in the
central amygdala impairs cold-induced thermogenesis.
NEUROSCIENCE
Results
Deletion of IR and IGF1R in the Hippocampus and Central Amygdala.
We used bilateral stereotaxic injection to deliver AAV encoding
a Cre-GFP fusion protein into the hippocampus and central
amygdala of IRlox/lox/IGF1Rlox/lox mice to delete both IR and
IGF1R in these nuclei in the brain (Fig. 1 A and C). AAV
encoding GFP alone was injected to generate control groups.
The positions of injection and extent of coverage were confirmed
by GFP expression (Fig. 1A). Western blotting of total tissue
lysates taken 8 wk later revealed about 50% reduction in IR and
IGF1R protein in the hippocampus from AAV-Cre-GFP–in-
jected mice (Hippo-DKO) compared with AAV-GFP–injected
Significance
Loss of insulin receptors in the brain causes metabolic and
behavioral abnormalities whereas loss of IGF-1 receptors in the
brain leads to a developmental defect in the brain and pe-
riphery. However, less is known about the impact of brain in-
sulin and IGF-1 receptor (IR/IGF1R) loss in adult mice, especially
in higher neural processing regions. Here, we show that loss of
IR/IGF1R in the hippocampus and central amygdala of adult
mice results in decrease in glutamate receptors, accompanied
by glucose intolerance, anxiety-like behavior, and impaired
cognition. In addition, we identify an insulin/IGF-1 signaling-
dependent neural circuit originating from the central amyg-
dala, which regulates interscapular brown fat activity and
thermogenesis. Thus, brain insulin/IGF-1 signaling is important
for higher neural processing and systemic metabolism.
Author contributions: M.S., W.C., and C.R.K. designed research; M.S., W.C., and M.K.
performed research; M.S., W.C., and M.K. contributed new reagents/analytic tools; M.S.,
W.C., and M.K. analyzed data; M.S., W.C., and C.R.K. wrote the paper; and C.R.K. super-
vised the work.
Reviewers: S.C., Wake Forest University; and S.G., Mount Sinai School of Medicine.
Conflict of interest statement: C.R.K. and S.G. are coauthors on a 2016 review article.
Published under the PNAS license.
See Commentary on page 5852.
1
M.S. and W.C. contributed equally to this work.
2
To whom correspondence should be addressed. Email: c.ronald.kahn@joslin.harvard.edu.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
1073/pnas.1817391116/-/DCSupplemental.
Published online February 14, 2019.
PNAS | March 26, 2019 | vol. 116 | no. 13 | 6379–6384

Fig. 1. AAV-Cre mediates efficient gene recombination in the hippocampus
(Hippo-DKO) and central amygdala (CeA-DKO) of IR/IGF1Rlox/lox mice. (A)
Bilateral AAV injection sites of the anterior and posterior hippocampus.
Representative images of immunohistochemical staining of GFP and DAPI in
the anterior and posterior hippocampus. (B) Immunoblotting of IR and IGF1R
in the hippocampus of adult IR/IGF1Rlox/lox mice injected with AAV-GFP or
AAV-Cre-GFP. Bottom, densitometry analysis. (C) Bilateral AAV injection sites
of the central amygdala. Representative images of immunohistochemical
staining of GFP and DAPI in the central amygdala. (D) Immunoblotting of IR
and IGF1R in the central amygdala of adult IR/IGF1Rlox/lox mice injected with
AAV-GFP or AAV-Cre-GFP. Bottom, densitometry analysis. *P < 0.05 by un-
paired t test, n = 6. Data are presented as mean ± SEM.
control mice (Hippo-CTR) (Fig. 1B). A similar decrease was
observed in synaptosomes isolated from the hippocampus (SI
Appendix, Fig. S1A). Likewise, there was an ∼50% decrease in
protein levels of IR and IGF1R in the central amygdala of CeA-
DKO mice compared with CeA-CTR mice (Fig. 1D) and syn-
aptosomes (SI Appendix, Fig. S1B). Thus, using stereotaxic
approaches, we were able to significantly reduce IR and IGF1R
in the hippocampus and central amygdala of adult mice.
Decreased Expression of Glutamate Receptor 1 in Synaptosomes of
Hippo-DKO and CeA-DKO Mice. Both insulin signaling and IGF-1
signaling modulate synaptic plasticity, especially in excitatory
glutamatergic neurons (23, 24). Glutamate acts via AMPA and
NMDA receptors, both of which are ionotropic transmembrane
cation channels that mediate fast synaptic transmission (25, 26).
Both Hippo-DKO and CeA-DKO mice displayed 60 to 70%
reductions of the GluA1 (also known as GluR1) subunit of the
AMPA receptor in the isolated synaptosomes from these re-
gions, compared with their controls, whereas the expression of
the other major AMPA receptor, subunit GluA2, was not af-
fected by IR/IGF1R loss (Fig. 2). These occurred with no change
in the phosphorylation of either GluA1 or GluA2 (SI Appendix,
Fig. S1 C and D), suggesting that insulin/IGF-1 signaling was not
important for the phosphorylation of the AMPA receptor sub-
units but did affect protein levels of GluA1. The NMDA re-
ceptor subunits NR2A and NR2B, on the other hand, were not
affected by IR/IGF1R deletion (SI Appendix, Fig. S1 C and D).
Expression of the synaptic marker PSD95 was similar in both the
hippocampus and amygdala between control and DKO mice (SI
6380 | www.pnas.org/cgi/doi/10.1073/pnas.1817391116
Appendix, Fig. S1 E and F), indicating that the defect of GluA1
expression in the DKO mice was not likely due to the impair-
ment of the structural integrity of the synapsis in these mice.
Thus, IR signaling and IGF1R signaling modulate synaptic
plasticity specifically through regulating the expression of GluA1
in both the hippocampus and central amygdala.
Glucose Homeostasis Is Regulated by IR/IGF1R Signaling in the
Hippocampus and Central Amygdala. Central insulin signaling has
been shown to affect systemic energy homeostasis (16, 21, 22).
Following AAV injection, both controls and DKO mice showed
similar body weight gain and food intake (SI Appendix, Fig. S2
A–D). However, 3 wk after injection, Hippo-DKO mice displayed
significantly higher random-fed blood glucose levels (Fig. 3A) and
a strong trend toward decreased plasma insulin levels 9 wk after
injection (SI Appendix, Fig. S2G). This was accompanied with an
impaired oral glucose tolerance test (Fig. 3B). IR/IGF1R deletion
in the hippocampus also resulted in moderate impairment of the
glucose lowering effect of i.p. insulin injection (SI Appendix, Fig.
S2E) and glucose-stimulated insulin secretion (SI Appendix, Fig.
S2F), both of which might contribute to the glucose intolerance in
Hippo-DKO mice. AUC, area under the curve.
CeA-DKO mice also displayed increased random-fed blood glu-
cose levels compared with control mice (Fig. 3C) and glucose in-
tolerance (Fig. 3D), but this did not appear until 3 wk after AAV
injection. However, loss of IR/IGF1R in the central amygdala did not
have any significant impact on plasma insulin levels, insulin sensitivity,
or glucose-stimulated insulin secretion (SI Appendix, Fig. S2 H–J).
Both Hippo-DKO and CeA-DKO mice housed in the meta-
bolic cages under the fed and fasted conditions displayed similar
spontaneous activity, energy expenditure measured by oxygen
consumption (VO2) and CO2 production (VCO2), and re-
spiratory exchange ratio (RER) as their control mice (SI Ap-
pendix, Fig. S3), indicating no major role for hippocampal and
amygdala IR and IGF1R signaling on circadian rhythm, energy
expenditure, and substrate preference.
IR/IGF1R Signaling in the Amygdala Contributes to Cold-Induced
Thermogenesis. To investigate whether the IR/IGF1R in the
hippocampus or amygdala is required for normal thermogenesis,
mice were challenged to cold (∼6 °C), and their rectal temper-
ature was measured every 30 min for 3 h. Core body temperature
Fig. 2. Decreased expression of glutamate receptor 1 in the synaptosome of
Hippo-DKO and CeA-DKO mice. (A and B) Representative Western blots and
quantification of glutamate receptor 1 and 2 (GluA1 and GluA2) in synap-
tosomes extracted from the hippocampus in Hippo-DKO and control mice
(A) and the central amygdala in CeA-DKO and control mice (B). GAPDH serve
as loading controls. *P < 0.05, **P < 0.01 by unpaired t test, n = 6. Quan-
titative data are presented as mean ± SEM.
Soto et al.
 SEE COMMENTARY
for anxiety-like behavior in rodents. In the dark–light box test,
however, both control and DKO mice showed similar time spent in
the light zone (Fig. 5C). Loss of IR and IGF1R in the central
amygdala also resulted in ∼40% reduction in entries and time in the
center zone in the open field test (Fig. 5D) and an ∼25% increase in
marble-burying activity (Fig. 5E). In these mice, there was also an
∼33% decrease in time spent in the light compartment of a dark–
light box compared with control (Fig. 5F). Thus, IR/IGF1R sig-
naling in both the hippocampus and amygdala is important for
controlling anxiety-like behavior in mice.

Hippo-DKO and CeA-DKO Mice Display Impaired Cognition. The ef-

Fig. 3. Glucose homeostasis is regulated by IR/IGF1R signaling in the hip-
pocampus and central amygdala. (A) Blood glucose levels in the random-fed
state of Hippo-DKO (n = 31) or Hippo-CTR mice (n = 27). (B) Oral glucose
tolerance test (OGTT) performed in Hippo-DKO or Hippo-CTR mice (n =13).
(C) Blood glucose levels in the random-fed state of CeA-DKO (n = 29) or CeA-
CTR mice (n = 28). (D) OGTT performed in CeA-DKO or CeA-CTR mice (n = 6).
*P < 0.05, **P < 0.01, ***P < 0.001 by unpaired t test. Data are presented as
fect of IR/IGF1R loss in the hippocampus and central amygdala
showed differential effects on learning and memory. In the ha-
bituation stage of the novel object recognition test, both control
and Hippo-DKO mice showed equal exploration time with each
of the two identical objects (SI Appendix, Fig. S5A). When one of
the familiar objects was replaced with a novel object in the test
stage 6 h later, control mice spent ∼65% of the time exploring
the novel object (Fig. 6A). By contrast, Hippo-DKO mice
showed equal interacting time between the familiar and novel
objects (Fig. 6A), suggesting impaired recognition memory. In
the novel object location test, one of the familiar objects was

NEUROSCIENCE
mean ± SEM. AUC, area under the curve. moved to a new location in the testing stage 6 h after the ha-
bituation phase. Control mice explored the object in the new
location ∼75% of the total exploration time whereas Hippo-
of Hippo-DKO mice dropped minimally from 38 °C to 37.6 °C DKO mice explored both objects equally regardless of the lo-
following 3-h cold exposure, similar to the drop observed in cation of the objects (Fig. 6B and SI Appendix, Fig. S5B), which is
control mice (Fig. 4A). In contrast, the core body temperature of a sign of a spatial memory deficit.
the CeA-DKO mice decreased by ∼1.0 °C during the 3-h cold
exposure, significantly more than control mice, whose tempera-
ture was decreased by ∼0.5 °C (Fig. 4 B and C). Since brown
adipose tissue (BAT) plays an important role for thermoregu-
lation in rodents by producing heat through uncoupling protein 1
(UCP-1) (27), we assessed the expression of UCP-1 in BAT and
found that both messenger and protein levels of UCP-1 in CeA-
DKO mice were slightly, but not significantly, reduced compared
with control mice (SI Appendix, Fig. S4 A and B).
Sympathetic outflow is a key regulator of thermogenic acti-
vation of brown fat upon cold exposure (28). The defective cold-
induced thermogenesis of CeA-DKO mice led us to hypothesize
that some neuronal population expressing IR and IGF1R in the
central amygdala are connected to the neural circuitry that
controls sympathetic nerves innervating the interscapular brown
adipose tissue (iBAT). To test this, we injected pseudorabies
virus PRV-765 encoding red fluorescent protein (RFP) into
iBAT to perform retrograde tracing of neuronal connections
from iBAT to the brain. Seven days after viral injection, RFP-
expressing neurons were detected in the spinal cord, nucleus of
the solitary tract (NTS) of the medulla, and parabrachial nucleus
(PBN) of the pons, as well as several areas in the hypothalamus,
including the paraventricular nucleus (PVN), dorsomedial hy-
pothalamus (DMH), and lateral hypothalamic area (LHA) (SI
Appendix, Fig. S4C and Table S1), highlighting the neuronal
circuit from the hypothalamus to the iBAT. Interestingly, the
amygdala was also highly RFP-labeled (Fig. 4D), demonstrating
previously unrecognized input of the neural circuits in the Fig. 4. IR/IGF1R signaling in the amygdala contributes to cold-induced
amygdala for the regulation of iBAT. thermogenesis. (A) Rectal temperature in Hippo-CTR (n = 4) and Hippo-
DKO mice (n = 5) during a 3-h exposure to a 6 °C environment. (B) Rectal
Hippo-DKO and CeA-DKO Mice Display Increased Anxiety-Like temperature in CeA-CTR (n = 9) and CeA-DKO mice (n = 10) during a 3-h
Behaviors. Behavior of the mice was assessed 8 wk after the de- exposure to a 6 °C environment. *P < 0.05 by unpaired t test. (C) Thermal
letion of IR/IGF1R in the hippocampus and central amygdala. images using a FLIR T300 Infrared Camera showing surface temperature
after 3 h at 6 °C between CeA-CTR and CeA-DKO mice. (D) Fluorescent im-
During an open field test, Hippo-DKO mice exhibited a 40% re-
ages of brain sections of mice 7 d after injection of the PRV-765 virus in the
duction in the number of center zone entries and spent ∼75% less BAT. BLA, basolateral amygdalar area; BMA, basomedial amygdalar nucleus;
time in the center zone compared with controls (Fig. 5A), indicating COA, cortical amygdalar area; ENTl, entorhinal area lateral part; PA,
increased anxiety in these mice. These mice also showed signifi- piriform-amygdalar area; PIA, piriform area; TR, postpiriform transition area.
cantly greater marble-burying activity (Fig. 5B), another indicator Data are presented as mean ± SEM.

Soto et al. PNAS | March 26, 2019 | vol. 116 | no. 13 | 6381
 significantly impairs the recognition memory of the mice but has
only minor effects on spatial memory.

Discussion
Insulin signaling and IGF-1 signaling produce a range of effects
on cellular metabolism, proteostasis, growth, and many other

Fig. 5. Hippo-DKO and CeA-DKO mice display increased anxiety-like be-

haviors. (A) Time spent in the center zone and entries in center zone in open
field test in Hippo-CTR (n = 12) and Hippo-DKO mice (n = 15). (B) Assessment
of anxiety as number of buried marbles over 30 min during a marble-burying
task in Hippo-CTR (n = 15) and Hippo-DKO mice (n = 17). (C) Time spent in
light compartment during light/dark box test in Hippo-CTR (n = 11) and
Hippo-DKO mice (n = 12). (D) Time spent in the center zone and entries in
center zone in open field test in CeA-CTR (n = 13) and CeA-DKO mice (n =
14). (E) Assessment of anxiety as number of buried marbles over 30 min
during a marble-burying task test in CeA-CTR (n = 9) and CeA-DKO mice (n =
9). (F) Time spent in light compartment during light/dark box test in CeA-CTR
(n = 18) and CeA-DKO mice (n = 19). *P < 0.05, **P < 0.01 by unpaired t test.
Data are presented as mean ± SEM.

We further analyzed spatial learning and memory using a

Stone T maze. In this test, a mouse needs to learn the correct
sequence of 13 left/right turns to successfully escape a water-
filled maze and reach the goal box (SI Appendix, Fig. S5E).
During the first 10 trials of learning conducted over 3 d, control
mice displayed continuous improvement of learning, with fewer
errors and shorter latency to reach the goal box (Fig. 6C and SI
Appendix, Fig. S5F). In contrast, Hippo-DKO mice displayed
significantly slower learning (Fig. 6C and SI Appendix, Fig.
S5F). One week after the last learning trial, mice were exposed
to the same maze to assess their memory. Control mice were
able to finish the maze with low numbers of errors and short
latency, similar to how they performed during the last trial of
learning. By contrast, after a 1-wk hiatus, Hippo-DKO mice
made significantly more errors and took a longer time to
complete the maze (Fig. 6C and SI Appendix, Fig. S5F), and
these mice performed even worse on this task after a 1-mo
hiatus (Fig. 6C and SI Appendix, Fig. S5F). In addition, when
the maze was rotated 180° from its original setting, only con-
trols, but not Hippo-DKO mice, showed worsened performance Fig. 6. Hippo-DKO and CeA-DKO mice have impaired cognition. (A) Time
spent exploring the novel object during the test session of the object rec-
in the T maze, indicating that control mice used spatial cues to
ognition task in Hippo-CTR (n = 8) and Hippo-DKO mice (n = 9). (B) Time
finish the maze while Hippo-DKO mice did not (Fig. 6C and SI spent exploring the object that was moved during the test session of the
Appendix, Fig. S5F). object location task in Hippo-CTR (n = 4) and Hippo-DKO mice (n = 5). (C)
Effects on memory and learning were selective and dependent Errors recorded during the 10 acquisition trials, 1-wk, and 1-mo memory
more on the specific task in CeA-DKO mice. Thus, CeA-DKO testing of the Stone T maze in Hippo-CTR (n = 7) and Hipp-DKO mice (n = 8).
mice failed to recognize the novel object compared with controls (D) Time spent exploring the novel object during the test session of the
in the novel object recognition test (Fig. 6D and SI Appendix, Fig. object recognition task in CeA-CTR (n = 9) and CeA-DKO mice (n = 10). (E)
S5C). However, CeA-DKO mice were able to remember the Time spent exploring the object that was moved during the test session of
the object location task in CeA-CTR (n = 6) and CeA-DKO mice (n = 6). (F)
location of the object as well as control mice (Fig. 6E and SI
Errors recorded during the 10 acquisition trials, 1-wk, and 1-mo memory
Appendix, Fig. S5D). In the Stone T maze test, CeA-DKO mice testing of the Stone T maze in CeA-CTR (n = 7) and CeA-DKO mice (n = 9).
showed normal learning and only slightly impaired ability to #
P < 0.05, ##P < 0.01, ###P < 0.001 by unpaired t test. *P < 0.05, **P < 0.01
remember the maze (Fig. 6F and SI Appendix, Fig. S5G). These between DKO and control mice by unpaired t test. Data are presented as
data suggest that loss of IR and IGF1R in the central amygdala mean ± SEM.

6382 | www.pnas.org/cgi/doi/10.1073/pnas.1817391116 Soto et al.


functions (6, 29–32). Recent studies have shown that the brain is
a major target of insulin/IGF-1 signaling. Insulin and IGF-1 are
able to cross the blood–brain barrier (BBB), in part through
receptor-mediated transcytosis, and act on brain tissues (33, 34).
In addition, a significant amount of IGF-1 is produced in the
brain. IR and IGF1R are widely distributed throughout the brain
and are present on both neurons and glial cells (7). Thus, the
brain is able to respond to locally and systemically produced
insulin and IGF-1 and to modulate its activities accordingly. In
the present study, we found that loss of IR and IGF1R in the
hippocampus or central amygdala results in metabolic and be-
havioral abnormalities, including impaired glucose homeostasis
and cognition and increased anxiety-like behavior. We also show
that insulin/IGF-1 signaling in the central amygdala plays a
specific role in thermogenesis while insulin/IGF-1 signaling in
the hippocampus is important for spatial memory.
At a molecular level, loss of IR and IGF1R results in a sig-
nificant reduction of the GluA1 subunit of the AMPA receptor
present in the synaptosomal fraction. Unlike NMDA receptors,
which are ligand-gated channels permeable to both calcium and
sodium, AMPA receptors are ligand-gated sodium channels re-
sponsible for the rapid depolarization of neuron membrane po-
tential (25, 26). The AMPA receptor is a tetrameric channel,
composed mainly of GluA1/GluA2 or GluA2/GluA3 hetero-
dimers (25). A large proportion of GluA1 subunit-containing
AMPA receptors localize in the endosomes close to the post-
synaptic membrane (35), which can undergo rapid recruitment to
the synaptic membrane upon stimulation by insulin or by NMDA
receptor-mediated calcium influx (36). This is a key molecular
mechanism of long-term potentiation (LTP) (37) and is impor-
tant for learning and memory. Consistent with this, mice with
both IR and IGF1R deleted in the hippocampus display im-
paired recognition and spatial memory, possibly due to the
limited GluA1 subunit-containing AMPA receptor pools in the
synapses of these neurons. In agreement with this, lentiviral-
mediated deletion of IR in the hippocampus results in im-
paired spatial memory due to impaired LTP (38). Also, knockout
of SCAP, a cholesterol sensing protein downstream of insulin
action, also results in impaired LTP (39). Intriguingly, IR/IGF1R
deletion has no major effect on the phosphorylation of the
GluA1 subunit of the AMPA receptor. Consistent with this,
calmodulin kinase and cAMP/PKA pathways, which are re-
sponsible for GluA1 phosphorylation (40, 41), are not generally
considered downstream signaling pathways regulated by IR/
IGF1R. The mechanism by which IR/IGF1R regulates GluA1
protein levels will require further investigation but might include
transcriptional or posttranscriptional alterations.
Insulin is the key hormone regulating glucose handling and
energy homeostasis. Thus, it is not surprising that the majority of
research on brain insulin action has focused on the hypothala-
mus, which controls many metabolic responses (21, 22, 42). Our
study clearly shows that metabolic control by central insulin
signaling is not limited to the hypothalamus since IR/IGF1R
deletion in both hippocampus and amygdala leads to impaired
glucose tolerance. In the hippocampus, this appears to be due to
a combination of systemic insulin resistance and a decrease in
insulin secretion. The causal factor for impaired glucose toler-
ance in mice lacking IR and IGF1R in the amygdala, on the
other hand, is less clear. These mice show normal insulin sensi-
tivity and glucose-stimulated glucose secretion, suggesting a de-
fect in some peripheral insulin-independent pathway involved in
glucose disposal. The sympathetic nervous system (SNS) inner-
vates the liver and controls hepatic glucose production. Our data
indicate that the amygdala may play a role in the regulation of
hepatic glucose metabolism and, eventually, systemic glucose
levels, directly or through other brain regions with which it has
projections, such as the hypothalamus (43). Indeed, it has been
shown that insulin injected in the central amygdala activates
Soto et al.
SEE COMMENTARY
neuronal populations in regions of the hypothalamus (44) known
to affect the autonomic output to the liver. Interestingly, it has
been noted that mice with diet-induced obesity develop insulin
resistance in the central amygdala (44) similar to that reported in
the hypothalamus (45), and thus it is possible that insulin sig-
naling in the central amygdala participates in abnormal glucose
homeostasis in the development of obesity.
Interscapular brown adipose tissue (iBAT) is a major ther-
mogenic tissue in rodents and is important in the regulation of
core body temperature, as well as systemic glucose and lipid
metabolism (27). Brown adipose tissue is also present in humans
where it has both thermogenic and metabolic functions (46).
Regulation of BAT is coordinated by the brain. Thus, when
temperature changes, warm- and cold-sensitive neurons signal to
the preoptic area of the hypothalamus (POA), which, through a
circuit involving the dorsal medial hypothalamus (DMH) and the
rostral raphe pallidus nucleus (rRPa), controls sympathetic
nervous system (SNS) outflow to BAT and thus its thermogenic
activity (47). Our retrograde transsynaptic tracing demonstrates
that the amygdala is directly involved in the neural circuit in-
nervating BAT. This neural pathway is important for cold-
induced thermogenesis, and, more importantly, it is regulated
by insulin/IGF-1 signaling since IR/IGF1R deletion in the cen-
tral amygdala specifically leads to cold intolerance in mice.
NEUROSCIENCE
Several groups, including us, have previously observed the
thermogenic effects of central insulin/IGF-1 signaling using
whole brain IR knockout mice (7, 48, 49). The current study
pinpoints the amygdala as a critical nucleus where insulin/IGF-1
signaling exerts a major role in thermogenesis. How this con-
verges with the POA → DMH → rRPa pathway requires
further studies.
We have also identified a crucial role for IR and IGF1R in
the hippocampus and amygdala on mood and cognition. Both
Hippo-DKO and CeA-DKO mice displayed significantly in-
creased anxiety-like behavior and impaired cognition, indicating
a beneficial role of insulin signaling on mood and cognition.
Intranasal insulin delivery has been shown to improve mood and
cognition in humans (50, 51). Our studies suggest that the ion-
otropic glutamate AMPA receptor and glutamate signaling may
be a molecular link between brain insulin/IGF-1 signaling de-
ficiency and altered neurobehavior. Other studies have shown
that inhibition of metabotropic glutamate receptors mGluR2/3
can improve mood behavior, and this is accompanied by in-
creased adult hippocampal neurogenesis (52). Both insulin/IGF-
1 and BDNF have been shown to contribute to adult hippo-
campal neurogenesis and have potential beneficial effects in
Alzheimer’s disease and psychotic disorders (53, 54). Whether
insulin/IGF-1 signaling and BDNF signaling share a common
mechanism for their antidepressive and antidementia effects
awaits further investigation. In addition, future studies are
needed to explore the electrophysiological mechanism of the
amygdala GluA1-associated defects in cognition and mood in
CeA-DKO mice.
Interestingly, the phenotypes of the region-specific IR/IGF1R
double knockout mice are more severe than those of the mice
with single IR-only knockout throughout the whole brain, since
mice with Nestin-Cre-dependent IR deletion (NIRKO) display
no apparent spatial memory deficit, and the anxiety-like behav-
iors develop only with aging (18, 55). On the other hand, IR
knockout in astrocytes does have significant effects on mood and
behavior in young mice (56). To what extent the phenotype of
these region-specific IR/IGF1R knockouts involves astrocytes vs.
neurons is uncertain. Combined IR and IGF1R deletion in pe-
ripheral tissues, like muscle and adipose tissue, leads to more
severe phenotypes than IR-only knockout (29, 32), suggesting
that IGF1R might partially compensate for IR in peripheral
tissues, and this could be also true in the brain. Previous reports
have shown higher expression of IGF1R than IR in the brain (7),
PNAS | March 26, 2019 | vol. 116 | no. 13 | 6383
indicating a potential important role for this insulin-IGF1R
cross-activation in the brain. Further studies will be necessary
to dissect the specific contributions of IR and IGF1R or the
potential role of IR/IGF1R hybrids in these phenotypes.
In summary, we have shown important roles for hippocampal
and amygdala IR and IGF1R signaling in systemic glucose ho-
meostasis and thermogenesis. At least a part of this effect may be
through modulation of an amygdala-dependent neural circuit to
control sympathetic outflow to liver and brown adipose tissue,
thus regulating peripheral glucose handling and cold-induced
thermogenesis. In addition, we demonstrate that IR and IGF1R
in the hippocampus and central amygdala are important for
mood and cognition. These defects in IR/IGF1R-deficient mice
may be the result of a reduction of AMPA receptor subunit
GluA1 in the synapse, which impairs synaptic plasticity in DKO
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6384 | www.pnas.org/cgi/doi/10.1073/pnas.1817391116
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Methods
All animal studies were conducted in compliance with the regulations and
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ACKNOWLEDGMENTS. We thank Dr. Lynn W. Enquist (Princeton University)
for sharing the PRV-765 virus for the retrograde tracing studies. This work
was supported by NIH Grants R01 DK031036 and R01 DK033201 (to C.R.K.).
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